CN109706146B

CN109706146B - Application of finger print composed of small RNAs in diagnosis and treatment of human cancerous pleural effusion

Info

Publication number: CN109706146B
Application number: CN201711014853.0A
Authority: CN
Inventors: 朱苗骏; 谭若颖; 徐静; 张新
Original assignee: BIOVUE TECHNOLOGY Ltd
Current assignee: BIOVUE TECHNOLOGY Ltd
Priority date: 2017-10-25
Filing date: 2017-10-25
Publication date: 2023-06-06
Anticipated expiration: 2037-10-25
Also published as: CN109706146A

Abstract

The invention relates to application of a fingerprint spectrum composed of small RNAs in diagnosis and treatment of cancerous pleural effusion of a human. By screening nearly two thousand miRNAs, a series of specific miRNA combinations are found that can very effectively distinguish cancerous pleural effusions from benign pleural effusions.

Description

Application of finger print composed of small RNAs in diagnosis and treatment of human cancerous pleural effusion

Technical Field

The invention relates to the technical fields of biomedicine, bioengineering and detection, in particular to application of a fingerprint spectrum composed of small RNAs in diagnosis and treatment of cancerous pleural effusion of human.

Background

Pleural effusion, also known as hydrothorax, is a clinically common disorder of the chest, and various diseases such as tuberculous pleurisy, heart failure, hypoalbuminemia and tumors can lead to hydrothorax (Light RW.; 2002). Although common in clinic, the etiology is complex and variable, and diagnosis of the etiology determines the treatment method. On the other hand, according to the data, about 38% to 52% of the pleural effusions in adults are malignant pleural effusions (Konstlatin A. Dimitriaddis; 2001). Malignant pleural effusion (Malignant pleural effusion, MPE) refers to pleural effusion caused by metastasis of malignant tumors that originate in the pleura or malignant tumors in other parts to the pleura. Statistically, the number of MPE incidences in the united states per year exceeds 15 tens of thousands by 2014. MPE can occur in almost all malignant tumors. Among them, lung cancer is the most common cause, accounting for about 1/3 of MPE, breast cancer is secondary, lymphoma is also an important cause of MPE, and in recent years, patients with ovarian cancer and gastrointestinal cancer having MPE are not rare, and about 5% -10% of MPE patients cannot find the primary tumor focus (Xu Yajun et al; 2011). The appearance of MPE indicates that the tumor has spread or progressed to an advanced stage and that the life expectancy of the patient will be significantly shortened. MPE is calculated from the beginning of the diagnosis and median survival is 3-12 months, which is related to the primary tumor type and stage. Evidence has shown that lung cancer causes MPE patients with the shortest survival time, ovarian cancer patients with the longest survival time, and MPE patients with primary foci cannot be found between the two.

Benign pleural effusion is very important for the differential diagnosis of benign and malignant pleural effusion because it is treated and prognosis is quite different from that of both malignant pleural effusion. For example, tuberculous hydrothorax and malignant hydrothorax can both be shown to be ischemic and are difficult to distinguish from conventional examination in terms of appearance, but the treatment and prognosis of the two are quite different. There are many differential diagnosis methods for pleural effusion, for example, by means of clinical symptoms, routine examination and biochemical examination of pleural effusion to distinguish exudates from effusions, and combining imaging diagnosis with judgment of clinicians to determine properties of pleural effusion. However, these methods are relatively low in specificity; however, the qualitative diagnosis of the pleural effusion property by the imaging method is not enough, and the property of the pleural effusion can be indirectly judged only according to lung, pleura, mediastinum and heart diseases. For MPE diagnosis, current techniques rely on pneumothorax access for cytological analysis. However, cytological analysis of pleural effusions has only 50-70% sensitivity for diagnosis of MPE (Ong K C et al; 2000). In addition, in cases where no cancer cells are found in pleural effusion, thoracoscopic pleural biopsies can also be performed, defining the pathology. But the method is invasive Surgery, and is at risk of complications such as infection, bleeding, and chronic pain ^[7] . For patients with poor physical condition and other complications, the development is difficult.

In view of the above, the identification of benign and malignant pleural effusion remains a clinical problem. Particularly for those tumors combined with heart failure, obstructive pneumonia or atelectasis which are secondary to the tumors, and hydrothorax which is generated by lymphatic return disorder caused by tumor invasion of lymphatic vessels or mediastinal lymph nodes, and the like are more difficult to distinguish between benign and malignant diseases. In addition, the cellular components of pleural effusions are relatively complex, in which mesothelial cells, megakaryocytes, lymphocytes, neutrophils, malignant tumor cells, etc. are mixed. Wounds, inflammation, tumors, etc. can lead to hyperproliferative mesothelial cells, and sometimes the degree of hyperproliferative cells is between benign and malignant, thus making it difficult to distinguish between benign disease, mesothelioma, and metastatic adenocarcinoma cytologically.

Therefore, there is a strong need in the art to explore other more sensitive diagnostic methods (such as deep mining of miRNAs associated with the pathogenesis of cancerous pleural effusions) to aid in the differential diagnosis of pleural effusions, which is of great guiding significance for guiding diagnosis and evaluation prognosis of cancerous pleural effusions.

Disclosure of Invention

The invention aims to provide an effective small RNA fingerprint spectrum for diagnosis, tumor grading and prognosis evaluation of cancerous malignant pleural effusion (preferably, lung cancer type malignant pleural effusion); and/or to differentiate between cancerous and benign pleural effusions (preferably, lung cancer type malignant and benign pleural effusions).

In a first aspect of the invention, there is provided an isolated miRNA:

a miRNA with a sequence shown as SEQ ID NO: n, wherein n is a positive integer selected from 1-32;

(ii) a miRNA complementary to the sequence shown in SEQ ID NO: n;

(iii) a combination of two or more miRNAs of the sequences shown in SEQ ID NOS.1-32; or (b)

(iv) combinations of two or more of miRNAs complementary to the sequences shown in SEQ ID NOS.1-32.

The second aspect of the present invention provides a set or combination of mirnas, the set or combination of mirnas being:

(a) Two or more miRNAs with sequences shown as SEQ ID NO. 1-32;

(b) A combination of two or more miRNAs complementary to the sequences shown in SEQ ID NOs 1 to 32; or (b)

(c) At least one miRNA from the sequences shown in SEQ ID NOS.1-32 and at least one miRNA from the sequences complementary to the sequences shown in SEQ ID NOS.1-32, wherein the sequences from the miRNAs of the sequences shown in SEQ ID NOS.1-32 and the sequences from the miRNAs complementary to the sequences shown in SEQ ID NOS.1-32 are not complementary to each other.

In another preferred embodiment, the collection or combination of miRNAs comprises 4 miRNAs in the sequence as shown in SEQ ID NOs 1-32.

In another preferred embodiment, the collection or combination of miRNAs comprises 10 miRNAs with the sequences shown in SEQ ID NOs 1 and 5-13.

In another preferred embodiment, the collection or combination of miRNAs comprises 7 miRNAs with the sequences shown in SEQ ID NOs 1, 14-15 and 18-21.

In another preferred embodiment, the collection or combination of miRNAs comprises 12 miRNAs having the sequences shown in SEQ ID NOs 1, 4, 9, 13, 16-17, 22-23 and 29-32.

In another preferred embodiment, the miRNA is isolated from a human.

In another preferred embodiment, the sequence may be obtained by expression by chemical synthesis or construction of eukaryotic expression vectors.

In another preferred embodiment, the collection or combination of miRNAs is a small RNA fingerprint consisting of 4 miRNAs for diagnosis, tumor grading and prognostic evaluation of cancerous malignant pleural effusion (preferably, lung cancer malignant pleural effusion); and/or to differentiate between cancerous and benign pleural effusions (preferably, lung cancer type malignant and benign pleural effusions);

the 4 miRNAs are HSA-MIR-141, HSA-MIR-429, HSA-MIR-200a and HSA-MIR-96.

In another preferred embodiment, the collection or combination of miRNAs is a small RNA fingerprint consisting of 10 miRNAs for diagnosis, tumor fractionation and prognosis evaluation of cancerous malignant pleural effusion; and/or distinguishing between cancerous and benign pleural effusions;

the 10 miRNAs are HSA-MIR-141, HSA-MIR-140-5P, HSA-MIR-29c#, HSA-MIR-708, HSA-MIR-98, HSA-MIR-196b, HSA-MIR-106b, HSA-MIR-361-3P, HSA-MIR-412 and HSA-MIR-642a.

In another preferred embodiment, the collection or combination of miRNAs is a small RNA fingerprint consisting of 7 miRNAs for diagnosis, tumor grading and prognosis evaluation of lung cancer type malignant pleural effusion; and/or differentiate lung cancer type malignant pleural effusion from benign pleural effusion;

the 7 miRNAs are HSA-MIR-141, HSA-MIR-145, HSA-MIR-29c, HSA-MIR-133a, HSA-MIR-708, HSA-MIR-149 and HSA-MIR-199b-5p.

In another preferred embodiment, the collection or combination of miRNAs is a small RNA fingerprint consisting of 12 miRNAs for diagnosis, tumor grading and prognosis evaluation of lung adenocarcinoma type malignant pleural effusion; and/or differentiating between lung adenocarcinoma type malignant pleural effusion and benign pleural effusion;

the 12 miRNAs are HSA-MIR-141, HSA-MIR-429, HSA-MIR-98, HSA-MIR-106b, HSA-MIR-29b, HSA-MIR-328, HSA-MIR-20a, HSA-MIR-17, HSA-MIR-140-3p, HSA-MIR-139-3p, HSA-MIR-125b-2# and HSA-MIR-106a.

In a third aspect the invention provides an isolated or artificially constructed precursor miRNA which is capable of cleaving and expressing in a human cell a miRNA according to the first aspect of the invention.

The invention also provides an isolated or artificially constructed precursor miRNA set or combination, wherein the precursor miRNA in the precursor miRNA set or combination can be sheared and expressed in human cells as miRNA in the miRNA set or combination according to the second aspect of the invention.

In a fourth aspect, the invention provides an isolated polynucleotide which is transcribed by a human cell into a precursor miRNA which is sheared in the human cell and expressed as a miRNA according to the first aspect of the invention;

preferably, the polynucleotide has a structure represented by formula I:

seq Forward-X-Seq reverse formula I,

in the formula I, the compound (I),

seq forward is the nucleotide sequence capable of expressing said miRNA in human cells,

the reverse of a Seq is a nucleotide sequence that is substantially complementary or fully complementary to the forward of the Seq;

x is a spacer sequence located between the forward direction of the Seq and the reverse direction of the Seq, and said spacer sequence is non-complementary to the forward direction of the Seq and the reverse direction of the Seq,

and the structure shown in formula I forms a secondary structure shown in formula II after being transferred into human cells:

In formula II, the definition of the forward direction of the Seq, the reverse direction of the Seq and X are as above,

the term "complementary base pairing" refers to the base pairing between the forward and reverse directions of Seq.

The invention also provides an isolated set or combination of polynucleotides comprising polynucleotides capable of being transcribed by a human cell into a precursor miRNA, said precursor miRNA being capable of being sheared and expressed in a human cell as set or combination of mirnas according to the second aspect of the invention.

In another preferred embodiment, one or more polynucleotides of the set or combination of polynucleotides has a structure represented by formula I:

seq Forward-X-Seq reverse formula I,

in the formula I, the compound (I),

In a fifth aspect the invention provides a vector comprising an isolated miRNA according to the first aspect of the invention, or a collection or combination of mirnas according to the second aspect of the invention, or a polynucleotide according to the fourth aspect of the invention.

The sixth aspect of the invention provides the use of an isolated miRNA of the first aspect of the invention, or a collection or combination of mirnas of the second aspect of the invention, for the preparation of a chip or kit; the chip or kit is used for:

(1) Diagnosis, tumor grading and prognosis evaluation of cancerous malignant pleural effusion (preferably, lung cancer type malignant pleural effusion);

(2) Distinguishing cancerous pleural effusion from benign pleural effusion;

(3) Distinguishing lung cancer type malignant pleural effusion and benign pleural effusion;

(4) The lung adenocarcinoma type malignant pleural effusion and benign pleural effusion are distinguished.

The seventh aspect of the present invention provides a miRNA chip, comprising:

a solid phase carrier; and

an oligonucleotide probe which is orderly immobilized on the solid phase carrier, wherein the oligonucleotide probe specifically corresponds to part or all of the sequences shown in SEQ ID NOs 1-32.

In another preferred embodiment, the oligonucleotide probe comprises:

a complementary binding region; and/or

A junction region attached to the solid support.

In another preferred embodiment, the oligonucleotide probe specifically corresponds to the entire sequence shown in SEQ ID NOS.1-4.

In another preferred embodiment, the oligonucleotide probes specifically correspond to all of the sequences shown in SEQ ID NOS 1 and 5-13.

In another preferred embodiment, the oligonucleotide probes specifically correspond to all of the sequences shown in SEQ ID NOS 1, 14-15 and 18-21.

In another preferred embodiment, the oligonucleotide probes specifically correspond to

All sequences shown in SEQ ID NOS 1, 4, 9, 13, 16-17, 22-23 and 29-32.

In an eighth aspect, the invention provides the use of a miRNA chip according to the seventh aspect of the invention for the preparation of a kit for distinguishing between cancerous and benign pleural effusions (preferably, for distinguishing between lung cancer type malignant and benign pleural effusions; more preferably, for distinguishing between lung adenocarcinoma type malignant and benign pleural effusions).

The ninth aspect of the present invention provides a kit, which contains the miRNA chip of the seventh aspect of the present invention and/or the detection reagent for the miRNA assembly or combination of the second aspect of the present invention.

In another preferred embodiment, the kit further comprises a collection or combination of mirnas according to the second aspect of the invention for use in a positive control.

In another preferred embodiment, the kit further comprises a specification, wherein the specification describes a method for testing the sequence shown in SEQ ID NO. 1-32 by using the miRNA chip of the present invention.

In a tenth aspect, the present invention provides a method for screening candidate drugs for lung cancer type or lung adenocarcinoma type malignant pleural effusion, the method comprising the steps of:

(a) In the experimental group, culturing lung cancer cells (or lung adenocarcinoma cells) of lung cancer type or lung adenocarcinoma type malignant pleural effusion in the presence of a substance to be tested; and culturing lung cancer cells (or lung adenocarcinoma cells) of the same lung cancer type or lung adenocarcinoma type malignant pleural effusion in a control group under the same conditions as the experimental group but in the absence of the test substance;

(b) Determining the expression level of one or more mirnas of lung cancer type or lung adenocarcinoma type malignant pleural effusion lung cancer cells (or lung adenocarcinoma cells) in the experimental group and comparing the expression level of the mirnas of lung cancer type or lung adenocarcinoma type malignant pleural effusion lung cancer cells (or lung adenocarcinoma cells) in the control group;

Wherein if the expression level of the miRNA in the experimental group is changed toward the expression level of benign hydrothorax cells as compared with the control group, the substance to be tested is indicated to be a candidate drug against lung cancer type or lung adenocarcinoma type malignant hydrothorax.

In another preferred embodiment, the method further comprises step (c): further treating a non-human mammal having lung cancer type or lung adenocarcinoma type malignant pleural effusion with the candidate drug, thereby determining the effect of the candidate drug on lung cancer type or lung adenocarcinoma type malignant pleural effusion of the non-human mammal.

In another preferred embodiment, the miRNA is an isolated miRNA according to the first aspect of the present invention.

In another preferred embodiment, the miRNA is a miRNA collection or combination according to the second aspect of the present invention.

In another preferred embodiment, the "change in expression level occurs toward benign hydrothorax cells" means that for a certain miRNA, the following formula is satisfied:

Q≤0.6

wherein q=abs (A1-A0)/abs (A2-A0)

Wherein A0 is the expression level of the miRNA in benign hydrothorax cells; a1 is the miRNA expression level of the experimental group; a2 is the miRNA expression level of the control group; abs represents the absolute value.

In another preferred embodiment, when the miRNA is of the lung cancer type (or lung adenocarcinoma type) malignant pleural effusion upregulation type (i.e., A2-A0 > 0), then A1-A0.ltoreq.0 or Q.ltoreq.0.6 (preferably.ltoreq.0.5).

In another preferred embodiment, when the miRNA is of the lung cancer type (or lung adenocarcinoma type) malignant pleural effusion down-regulation type (i.e., A2-A0 < 0), then A1-A0 is greater than or equal to 0 or Q is greater than or equal to 0.6 (preferably greater than or equal to 0.5).

In another preferred embodiment, in the method, in step (a), a positive control group is further included, that is, in the presence of a drug known to treat malignant pleural effusion of the lung cancer type or lung adenocarcinoma type in the same condition as the experimental group in the absence of the test substance, culturing lung cancer cells of the same lung cancer type or lung adenocarcinoma type;

and, in step (b), comparing the expression level of one or more mirnas of the lung cancer cells or lung adenocarcinoma cells in the experimental group with the expression level of mirnas of the lung cancer cells or lung adenocarcinoma cells in the positive control group.

In another preferred embodiment, the miR is selected from: HSA-MIR-141, HSA-MIR-429, HSA-MIR-200a and HSA-MIR-96.

In another preferred embodiment, the miR is selected from: HSA-MIR-141, HSA-MIR-140-5P, HSA-MIR-29c#, HSA-MIR-708, HSA-MIR-98, HSA-MIR-196b, HSA-MIR-106b, HSA-MIR-361-3P, HSA-MIR-412 and HSA-MIR-642a.

In another preferred embodiment, the miR is selected from: HSA-MIR-141, HSA-MIR-145, HSA-MIR-29c, HSA-MIR-133a, HSA-MIR-708, HSA-MIR-149 and HSA-MIR-199b-5p.

In another preferred embodiment, the miR is selected from: HSA-MIR-141, HSA-MIR-429, HSA-MIR-98, HSA-MIR-106b, HSA-MIR-29b, HSA-MIR-328, HSA-MIR-20a, HSA-MIR-17, HSA-MIR-140-3p, HSA-MIR-139-3p, HSA-MIR-125b-2# and HSA-MIR-106a.

In an eleventh aspect, the present invention provides a method for non-diagnostic in vitro determination of whether a cell or tissue is a lung cancer type or lung adenocarcinoma type malignant pleural effusion or tissue thereof, comprising the steps of: determining the expression level of an isolated miRNA of the first aspect of the invention or an miRNA of the collection or combination of mirnas of the second aspect of the invention in said cell or tissue thereof, wherein a significant difference in the expression level of said miRNA compared to normal tissue indicates that the cell or tissue is lung cancer or lung adenocarcinoma malignant pleural effusion or tissue thereof.

In a twelfth aspect, the present invention provides a method for diagnosing a lung cancer type or lung adenocarcinoma type malignant pleural effusion sample, comprising the steps of: determining the expression level of the miRNA in the isolated miRNA of the first aspect of the invention or the miRNA set or combination of the second aspect of the invention in a sample, wherein when the expression level of the miRNA is significantly different from that of a normal sample, the sample is a lung cancer type or lung adenocarcinoma type malignant pleural effusion sample.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

Figure 1 shows cancerous pleural effusions in training sets differentiated by SVM model and leave-one-out cross-validation for 10 selected mirnas (table 2): a is a leave-one-out cross-validation result based on an SVM model graph, the abscissa represents each sample, all samples (n=77) are divided into two parts (53 cases of cancer samples and 24 cases of benign samples), the ordinate represents a probability value of each sample predicted as cancer (> =0.5 is cancer, <0.5 is benign), the black dot represents a sample judged to be correct, and the red dot represents a sample judged to be incorrect; and B is an ROC graph obtained by a method of SVM model and based on a result obtained by leave-one-out cross test.

Figure 2 shows lung cancer type malignant pleural effusion differentiation in training set for 7 selected mirnas (table 2) by SVM model and leave-one-out cross validation: a is a leave-one-out cross-validation result based on an SVM model graph, the abscissa represents each sample, all samples (n=60) are divided into two parts (36 cases of lung cancer samples and 24 cases of benign samples), the ordinate represents a probability value of each sample predicted as cancer (> =0.5 is cancer, <0.5 is benign), the black dot represents a sample judged correctly, and the red dot represents a sample judged incorrectly; and B is an ROC graph obtained by a method of SVM model and based on a result obtained by leave-one-out cross test.

Figure 3 shows the discrimination of adenocarcinoma-type malignant pleural effusions in the training set by SVM model and leave-one-out cross-validation method for 12 selected mirnas (table 2): a is a leave-one-out cross validation result based on an SVM model graph, the abscissa represents each sample, all samples (n=51) are divided into two parts (27 cases of adenocarcinoma samples and 24 cases of benign samples), the ordinate represents a probability value of each sample predicted as cancer (> =0.5 is cancer, <0.5 is benign), the black dot represents a sample judged correctly, and the red dot represents a sample judged incorrectly; and B is an ROC graph obtained by a method of SVM model and based on a result obtained by leave-one-out cross test.

Fig. 4 shows the results of cancerous pleural effusion determinations in the test set for 10 selected mirnas (table 2) by SVM model and leave-one-out cross-validation: to leave-one-out cross-validation results are based on SVM model graphs, the abscissa represents each sample, all samples (n=159) are divided into two parts (94 cancer samples and 65 benign samples), the ordinate represents the probability value of each sample predicted to be cancer (> =0.5 for cancer, <0.5 for benign), the black dot represents the sample judged to be correct, and the red dot represents the sample judged to be incorrect.

Fig. 5 shows the lung cancer type malignant pleural effusion determination results in the test set for 7 selected mirnas (table 2) by SVM model and leave-one-out cross-validation: to leave-one-out cross-validation results are based on SVM model graphs, the abscissa represents each sample, all samples (n=140) are divided into two parts (lung cancer samples 75 and benign samples 65), the ordinate represents probability values of each sample predicted as cancer (> =0.5 for cancer, <0.5 for benign), the black dot represents samples judged to be correct, and the red dot represents samples judged to be incorrect.

FIG. 6 shows the results of lung adenocarcinoma type malignant pleural effusion determination in the test set for 12 selected miRNAs (Table 2) by SVM model and leave-one-out cross-validation: to leave-one-out cross-validation results are based on SVM model graphs, the abscissa represents each sample, all samples (n=122) are divided into two parts (57 lung adenocarcinoma samples and 65 benign samples), the ordinate represents probability values of each sample predicted as cancer (> =0.5 for cancer, <0.5 for benign), the black dot represents samples judged to be correct, and the red dot represents samples judged to be incorrect.

Detailed Description

Through extensive and intensive studies, the inventor screens nearly two thousands of miRNAs, screens out a plurality of specific miRNAs for the first time, and tests prove that cancerous pleural effusions and benign pleural effusions can be very effectively distinguished by combining specific miRNA markers to a certain extent. The inventor also screens out a plurality of specific miRNAs for the first time, so that lung cancer type malignant pleural effusion and lung adenocarcinoma type malignant pleural effusion can be distinguished very effectively. The invention also provides application of the fingerprint spectrum composed of miRNA in diagnosis of cancerous pleural effusion. On this basis, the present invention has been completed.

miRNA and precursor thereof

The present invention provides a novel class of mirnas found in humans. As used herein, the term "miRNA" refers to an RNA molecule that is processed from transcripts that can form a precursor of the miRNA. Mature mirnas typically have 18-26 nucleotides (nt) (more particularly about 19-22 nt), nor are miRNA molecules with other numbers of nucleotides excluded. mirnas are generally detectable by Northern blotting.

Mirnas of human origin can be isolated from human cells. As used herein, "isolated" refers to a substance that is separated from its original environment (i.e., the natural environment if it is a natural substance). If the naturally occurring polynucleotide and polypeptide are not isolated or purified in vivo, the same polynucleotide or polypeptide is isolated or purified from other naturally occurring substances.

mirnas may be processed from Precursor mirnas (Pre-mirnas) that fold into a stable stem-loop (hairpin) structure, typically between 50-100bp in length. The precursor miRNA can be folded into a stable stem-loop structure, and the two sides of the stem-loop structure comprise two sequences which are basically complementary. The precursor miRNA can be natural or synthetic.

The precursor miRNA may be sheared to generate a miRNA that may be substantially complementary to at least a portion of the sequence of the mRNA encoding the gene. As used herein, "substantially complementary" means that the sequences of nucleotides are sufficiently complementary to interact in a predictable manner, such as to form a secondary structure (e.g., a stem-loop structure). Typically, two "substantially complementary" nucleotide sequences are at least 70% complementary to each other; preferably, at least 80% of the nucleotides are complementary; more preferably, at least 90% of the nucleotides are complementary; further preferably, at least 95% of the nucleotides are complementary; such as 98%, 99% or 100%. Typically, there may be up to 40 mismatched nucleotides between two sufficiently complementary molecules; preferably, there are up to 30 mismatched nucleotides; more preferably, there are up to 20 mismatched nucleotides; it is further preferred to have at most 10 mismatched nucleotides, such as having 1, 2, 3, 4, 5, 8, 11 mismatched nucleotides.

As used herein, a "stem-loop" structure, also referred to as a "hairpin" structure, refers to a nucleotide molecule that can form a secondary structure that includes a double-stranded region (stem) formed by two regions of the nucleotide molecule (on the same molecule) that are flanked by double-stranded portions; it also includes at least one "loop" structure comprising a non-complementary nucleotide molecule, i.e., a single-stranded region. The double-stranded portion of the nucleotide can remain double-stranded even if the two regions of the nucleotide molecule are not fully complementary. For example, insertions, deletions, substitutions, etc. may result in the non-complementation of a small region or the small region itself forming a stem-loop structure or other form of secondary structure, however, the two regions may still be substantially complementary and interact in a predictable manner to form a double-stranded region of the stem-loop structure. The stem-loop structure is well known to those skilled in the art, and usually after obtaining a nucleic acid having a nucleotide sequence of primary structure, the skilled person is able to determine whether the nucleic acid is capable of forming a stem-loop structure.

The miRNA has a sequence shown as SEQ ID NO: n, wherein n is a positive integer selected from 1-32.

To improve the stability or other properties of the miRNA, at least one protective base, such as "TT" or the like, may also be added to at least one end of the miRNA.

In this context, miRNA, miRN, small RNA, microRNA, miR have the same meaning.

For the disclosed cancerous pleural effusion-specific mirnas, verification can be performed by conventional miRNA chip techniques, including, for example, extraction of the miRNA with conventional methods or conventional kits, followed by detection. Representative kits include (but are not limited to): the Qiagen or Ambion company's miRNAs extraction kit.

In addition, the cancerous pleural effusion-specific miRNAs of the present invention can also be detected or validated by specifically amplifying and detecting the amplified products (or corresponding detectable signals such as fluorescent signals). Preferred high sensitivity and high specificity techniques include (but are not limited to): the technology disclosed in CN10267663 a. Typically, the specific binding region of the primer used may be designed according to the sequence of the known miRNA to be detected, preferably the specific binding region of the primer used in amplification is typically a complementary sequence that is fully complementary to the miRNA.

Antisense oligonucleotides

According to the miRNA sequences provided by the invention, antisense oligonucleotides of the miRNA sequences can be designed, and the antisense oligonucleotides can down-regulate the expression of corresponding miRNAs in vivo. As used herein, "antisense oligonucleotides (AS-Ons or ASO)" is also referred to AS "antisense nucleotides" and refers to DNA molecules or RNA molecules or analogs thereof that are about 18-26nt in length (more particularly about 19-22 nt).

In the present invention, the "antisense oligonucleotide" also includes modified antisense nucleotides obtained by means such as nucleic acid lock-based or nucleic acid strand backbone modification techniques, wherein the modification does not substantially alter the activity of the antisense oligonucleotide, and more preferably, the modification increases the stability, activity or therapeutic effect of the antisense oligonucleotide. Nucleic acid locks (locked nucleic acid, LNA) generally refer to modification techniques in which the 2 'oxygen atom and the 4' carbon atom of ribose are linked by a methylene bridge. LNA can prolong serum half-life of miRNA, improve affinity to target, and reduce scope and degree of off-target effect. The antisense medicine developed based on the modification technology of nucleic acid chain skeleton has greatly improved solubility, nuclease degradation resistance and other aspects, and is easy to synthesize in great amount. There are various methods for backbone modification of oligonucleotides, including thio methods, such as thio modification of a deoxynucleotide chain to a thio deoxynucleotide chain. The method is to replace oxygen atoms of phosphate bonds on the DNA skeleton with sulfur atoms, and can resist nuclease degradation. It is to be understood that any modification capable of retaining most or all of the activity of the antisense oligonucleotide is encompassed by the present invention.

As a preferred mode of the invention, the antisense oligonucleotide is subjected to nucleic acid lock modification; more preferably also thio-modifications.

After the antisense oligonucleotides are transferred into human body, they can obviously reduce the expression of miRNA.

Polynucleotide constructs

According to the human miRNA sequences provided herein, polynucleotide constructs can be designed that, after being introduced, can be processed into mirnas that can affect the expression of the corresponding mRNA, i.e., the amount of the corresponding miRNA that the polynucleotide construct is capable of up-regulating in vivo. Thus, the present invention provides an isolated polynucleotide (construct) that can be transcribed by a human cell into a precursor miRNA that can be sheared by the human cell and expressed as the miRNA.

As a preferred embodiment of the present invention, the polynucleotide construct comprises a structure represented by formula I:

Seq _{forward direction} -X-Seq _{Reverse direction} The compound of the formula I,

in the formula I, the compound (I),

Seq _{forward direction} To express the nucleotide sequence of the miRNA in cells, seq _{Reverse direction} Is equal to Seq _{Forward direction} A substantially complementary nucleotide sequence; alternatively, seq _{Reverse direction} To express the nucleotide sequence of the miRNA in cells, seq _{Forward direction} Is equal to Seq _{Forward direction} A substantially complementary nucleotide sequence;

x is at Seq _{Forward direction} And Seq _{Reverse direction} A spacer sequence therebetween, and said spacer sequence is identical to Seq _{Forward direction} And Seq _{Reverse direction} Are not complementary;

after the structure shown in the formula I is transferred into cells, a secondary structure shown in the formula II is formed:

in formula II, seq _{Forward direction} 、Seq _{Reverse direction} And X is as defined above;

the expression is shown in Seq _{Forward direction} And Seq _{Reverse direction} Complementary base pairing relationship formed between them.

Typically, the polynucleotide construct is located on an expression vector. Thus, the invention also includes a vector comprising said miRNA, or said polynucleotide construct. The expression vector typically also contains a promoter, origin of replication, and/or marker gene, etc. Methods well known to those skilled in the art can be used to construct the expression vectors required for the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as calicheamicin, gentamicin, hygromycin, ampicillin resistance.

Chip

miRNA expression profiling chips typically contain up to several hundred probes covering a variety of mirnas, and detect the content of each miRNA contained in a sample at the whole genome level using the principle of DNA double strand homologous complementation. Thus, the transcript level of the miRNA in the whole genome range in the sample to be tested can be detected at the same time.

The miRNA sequence can be used for preparing corresponding miRNA chips, so that the expression profile and the regulation mode of miRNAs can be studied.

In another aspect, the invention also provides a chip for analyzing miRNA expression profiles, said chip being useful for differentiating between cancerous and benign pleural effusions.

The miRNA chip of the invention comprises:

a solid phase carrier; and

an oligonucleotide probe which is orderly immobilized on the solid support, said oligonucleotide probe specifically corresponding to at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32) of the sequences shown in SEQ ID NOs 1-32.

Specifically, suitable probes can be designed according to the miRNAs of the present invention and immobilized on a solid support to form an "oligonucleotide array". By "oligonucleotide array" is meant an array having addressable locations (i.e., locations characterized by distinct, accessible addresses), each addressable location containing a characteristic oligonucleotide attached thereto. The oligonucleotide array may be divided into a plurality of subarrays, as desired.

The solid phase carrier can be made of various common materials in the field of gene chips, such as but not limited to nylon membranes, glass slides or silicon wafers modified by active groups (such as aldehyde groups, amino groups and the like), unmodified glass slides, plastic sheets and the like.

The preparation of the miRNA chip can be carried out by adopting a conventional manufacturing method of a biochip known in the art. For example, if a modified slide or a silicon wafer is used as the solid phase carrier, and the 5' -end of the probe contains an amino-modified poly dT string, the oligonucleotide probe can be prepared into a solution, then spotted on the modified slide or the silicon wafer by a spotting instrument, arranged into a predetermined sequence or array, and then fixed by standing overnight, so that the miRNA chip of the invention can be obtained. If the nucleic acid does not contain amino modifications, the preparation method can also be referred to as: wang Shenwu, ind. Infinite Instructions on Gene diagnosis technology-nonradioactive Manual; J.L.erisi, V.R.Iyer, P.O.BROWN.Exploring the metabolic and genetic control of gene expression on a genomic scale.science,1997;278:680 and Ma Liren, jiang Zhonghua Ji, biochip: chemical industry Press 2000,1-130.

In another aspect, the present invention also provides a method for detecting a miRNA expression profile in human tissue by a miRNA chip, comprising the steps of:

(1) Providing an RNA sample isolated from human tissue, and providing a marker on said RNA;

(2) Contacting the RNA obtained in the step (1) with the miRNA chip to enable the RNA to carry out hybridization reaction with the oligonucleotide probe on the solid phase carrier, thereby forming an oligonucleotide probe-RNA binary complex on the solid phase carrier;

(3) Detecting the markers of the binary complex formed in step (2), thereby determining the expression profile of the corresponding miRNA in human tissue.

Methods for extracting RNA from human tissue are well known to those skilled in the art and include the Trizol method.

More preferably, in step (1), after isolation of the RNA sample from human tissue, the RNA sample is suitably treated to enrich for RNA having a length, typically between 10 and 100 (small fragment RNA). After the treatment, the small fragment RNAs are used for subsequent hybridization, so that the accuracy of capturing miRNA by the chip can be improved. The person skilled in the art can conveniently isolate RNA having a certain fragment length, for example by gel electrophoresis.

Labelling of RNA is also a well known method to the person skilled in the art and can be achieved by adding a label, such as a labelling group, which specifically binds to RNA during hybridization. Such labeling groups include, but are not limited to: digoxin molecules (DIG), biotin molecules (Bio), fluorescein and its derivative biomolecules (FITC, etc.), other fluorescent molecules (e.g., cy3, cy5, etc.), alkaline Phosphatase (AP), horseradish peroxidase (HRP), etc. These labels and their labeling methods are all well known in the art.

When hybridizing the RNA with the miRNA chip, the miRNA chip and a prehybridization buffer solution can be prehybridized.

The solid phase hybridization between RNA and miRNA chips according to the present invention is performed according to classical methods in the art, and the person skilled in the art can easily determine the optimum conditions for buffer, probe and sample concentrations, prehybridization temperature, hybridization temperature, time, etc., according to experience. Or may be as described in the guidelines for molecular cloning experiments.

And then obtaining information to be detected according to the position, the intensity and other information of the marking signal on the miRNA chip. If the amplified product is marked by a fluorescent group, a fluorescence detection device (such as a laser confocal scanner Scanarray 3000) can be directly used for obtaining the information to be detected.

Detection kit

The invention also provides a kit, which contains the chip of the invention. The kit can be used for detecting the expression profile of miRNA; or for differentiating between cancerous and benign pleural effusions (preferably, lung cancer type malignant and benign pleural effusions, more preferably, lung adenocarcinoma type malignant and benign pleural effusions).

More preferably, the kit further comprises a marker for marking the RNA sample and a substrate corresponding to the marker.

In addition, various reagents required for extracting RNA, PCR, hybridization, color development, etc. can be included in the kit, including but not limited to: extract, amplification solution, hybridization solution, enzyme, control solution, color development solution, washing solution, antibody, etc. Fluorescent dyes, such as EvaGreen, SYBRGreen, may also be included in the amplification solution. The kit can also comprise a primer.

In addition, the kit can also comprise instructions for use and/or chip image analysis software.

The above-mentioned features of the invention, or of the embodiments, may be combined in any desired manner. All of the features disclosed in this specification may be combined with any combination of the features disclosed in this specification, and the various features disclosed in this specification may be substituted for any alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are merely general examples of equivalent or similar features.

The main advantages of the invention include:

(1) The invention provides a miRNA fingerprint spectrum which can be used for better distinguishing cancerous pleural effusion;

(2) The invention provides a miRNA fingerprint spectrum which can be used for well distinguishing cancerous pleural effusion;

(3) The miRNA fingerprint can effectively distinguish cancerous pleural effusion (such as lung cancer pleural effusion) from normal pleural effusion, and has high sensitivity and strong specificity;

(4) The miRNA fingerprint can effectively distinguish cancerous pleural effusion (such as lung adenocarcinoma type pleural effusion) from normal pleural effusion, and has high sensitivity and strong specificity;

(5) The miRNA fingerprint can be effectively used for diagnosis, typing and prognosis evaluation of cancerous pleural effusion.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

Unless otherwise indicated, materials and reagents used in the description of the present invention are commercially available products.

Example 1

Sample collection and information analysis

The samples were obtained following the Shanghai Zhongshan Hospital ethical committee approval and the procedure for collecting pleural effusions according to Shanghai Zhongshan Hospital pathology with patient informed consent to the bookmark. A total of 236 pleural effusion samples were obtained from the patient's chest and stored at-80 ℃. The sample is divided into two parts, one part is used as a training set for establishing an analysis model (77 cases), the other part is used as a double-blind verification set for double-blind verification (159 cases), and the sample information is shown in Table 1 in detail. The ratio of men to women in the sample was about 1.5:1, and the average age of the sample was about 61.6 years.

Table 1 sample information

Example 2

Total RNA extraction

Collecting the fresh pleural effusion, performing centrifugal pretreatment, and respectively placing the sample precipitate and the supernatant into a-80 ℃ refrigerator for freezing.

Chest samples total RNA was extracted with reference to the MIRNeasy MiniKit (Qiagen, 217004) instructions. The specific operation steps are carried out according to the method for extracting total RNA in chest water by Xiangqiong company.

And detecting the quality by electrophoresis, measuring OD260nm and OD280nm by ultraviolet spectrophotometry, and calculating the concentration of RNA. Preserving at-80 ℃.

Example 3

Adding PolyA tail and reverse transcription

Total RNA extracted in example 2 above was diluted to 125ng/ul with 0.1 XRNA storage buffer (Ambion, USA) containing 0.1% Tween-20 (Sigma).

Produced by Xiangqiong Co

The miRNA cDNA synthesis cassette (product number of Xiangqiong Co. 9000004) is the miRNA plus the tail of PolyA and is reverse transcribed into cDNA. The method comprises the following specific steps:

a1.5 ml centrifuge tube was placed on ice and 66. Mu.l of total RNA at a concentration of 125ng/ul, 33. Mu.l of Xiangqiong Co

miRNA cDNA Synthesis reaction solution I (product number of Xiangqiong Co., ltd.: 9000005) 11. Mu.l Xiangqiong Co., ltd.,/I>

miRNA cDNA synthesis reaction solution II (product number of Xiangqiong Co., ltd.: 9000006)), and nuclease-removed ultra-pure water to 165. Mu.l. The final concentration of total RNA was 50ng/ul, and after gentle mixing, 3 PCR tubes of 0.2ml were dispensed, 50ul each. Centrifugation at 1000rpm for 10s, and then placing the mixture into an ABI9700 PCR instrument for reaction, wherein the reaction procedure is as follows: maintained at 37℃for 15min,25℃for 25min,37℃for 30min,85℃for 5min, and 4 ℃. />

And taking out the PCR tube, combining the RT products of the 3 tubes, and preserving at-20 ℃ after shaking and centrifuging or directly using the RT products for qPCR reaction.

Example 4

Fluorescent quantitative PCR detection

The method disclosed with reference to CN10267663a detects small RNAs.

Into a 15 ml centrifuge tube, 24. Mu.l of the reverse transcription product obtained in example 3 and 600. Mu.l of a fluorescent quantitative PCR enzyme reaction solution (product number: 9000008 of Xiangqiong Co.),

2x Universal qPCR Master Mix High Rox), 216 μl of the nuclease ultrapure water (product number of Xiangqiong Co., ltd.): 9000015 Gently mixing.

Small RNA reaction template for diagnosis of pleural effusion produced by Xiangqiong corporation, sharpvue ^TM Human miRNA Array-chest water (product number: 1100001 of Xiangqiong Co.) is taken out from a refrigerator at-20deg.C, and after returning to room temperature, the packaging bag is opened, and placed on a centrifuge, and centrifuged at 2000g for 5min (Thermo, ST16R, model number: M-20). A total of 93 small RNA reactions were performed with 2 positive controls and 1 blank control on the reaction template. Carefully unpack the film.

Pouring the mixed solution obtained in the previous step into a sample adding groove, and adding the mixed solution into the small RNA reaction template line by using a 12-channel continuous pipettor, wherein each hole is 7ul. After the sample addition, the liquid amount of each hole is checked to be uniform.

The mixture was sealed with a quantitative seal plate membrane (ABI, 4711971) and then inverted and mixed well, and centrifuged at 1000g for 5min at room temperature.

The sample was put into a quantitative PCR apparatus (ABI, 7900Ht Fast) to perform quantitative PCR. The procedure is: after 10min at 95℃for 5s at 95℃and 1min at 58℃for 3 cycles; after 95℃for 5s,60℃for 5s,37 cycles, dissolution profile. The reporter fluorescence was SYBR and the reference fluorescence was Rox.

Data were collected for bioinformatics analysis.

Example 5

Computational analysis of miRNA biometric data

The probability of cancer of a patient is predicted by a support vector machine (support vector machine, SVM for short), which is a classification algorithm, the generalization capability of a learning machine is improved by seeking the minimum structural risk, and the minimization of experience risk and confidence range is realized, so that the aim of obtaining good statistical rules under the condition of less statistical sample size is fulfilled, and the model is a class II classification model.

First we performed further analysis of 1888 mirnas in the 1888 mirnas detected and two internal positive controls (HSA-RNU 6B and HSA-RNU 48) with one negative control (water). miRNA panel (1 96-well plate containing 93 mirnas and 3 internal controls) was determined to detect pleural effusions. The ct value and average value of each miRNA are subtracted from background and normalized, and the unified data ct value is less than or equal to 32.

We then screened 96 mirnas for miRNA markers that better distinguish between cancer and non-cancer patients. Considering that the amount of miRNA added may be different for each sample, the measured miRNA value for each sample needs to be normalized first, and we use the difference between the two miRNA detection values for each sample as a new variable, so that when we select a subset from 96 mirnas, the normalization of the data in this subset will only be related to the variables in this subset. We have C (96,2) +96=4560 new variables in total after normalization. We calculated the significance of these variables between cancer patient samples and non-cancer patient samples using T-test, and then selected the 20 new variables with the most significant differences. We finally hoped to find the number of mirnas within 12 as variables of the model, so all combinations of mirnas within 12 were selected from the set of mirnas contained in these 20 new variables, and the accuracy of each combination prediction training set was calculated with the SVM model. Finally, the miRNA combination with highest accuracy is selected as a final variable set M.

Our SVM model was from the e1071 software package for R, training data from 53 cancer patients and 24 non-cancer patients confirmed by the doctor. In the model training process, because the number of samples of a control group and the number of samples of an experimental group are large, the weight of a non-cancer patient sample is set to be 53/24 in a class.weights variable, and a radial basis function (kernel= "radial") is selected as a kernel function, because probability is to be predicted, and probabiity=true is set. The probability that the sample is cancerous and non-cancerous is predicted using the prediction function, and the accuracy is calculated using a leave-one-out cross-validation method (LLO-CV).

The naming of mirnas is according to the mirnas database of miRBase Version 20, followed in contradictory cases.

The results of 32 mirnas selected as specific biomarkers for diagnosing cancerous pleural effusions are shown in table 2.

TABLE 2

Example 6

4 key miRNAs are selected from miRNA variables, so that cancerous pleural effusion and lung cancer malignant pleural effusion are well diagnosed

When the sequence shown in SEQ ID NO. 1-4 is selected, the AUC obtained by the statistical method of SVM for cancerous pleural effusion in the training set (77 samples) of the 4 miRNAs is 0.897, and the accuracy is 83.1%; sensitivity was 84.9% and specificity was 79.2%; the sensitivity in the test set (only 61 samples tested) was 63.1% and the specificity was 95.7%. AUC of the 4 miRNAs on lung cancer malignant pleural effusion in a training set (77 samples) is 0.898, accuracy is 85%, sensitivity is 77.8%, and specificity is 95.8%; the sensitivity in the test set (only 61 samples, including 31 lung cancer malignant pleural effusions) was 64.5% and the specificity was 95.7%.

Example 7

Further selecting key miRNA from miRNA variables to diagnose cancerous pleural effusion

The miRNAs selected in example 5 are arranged and combined, and the best miRNA combination with the specificity, sensitivity and accuracy reaching the clinical detection level can be screened and tested. According to data statistical analysis, the best 5 groups of miRNA combinations are found, and as a result, the result shows that after partial specific miRNAs are combined, cancerous pleural effusion can be effectively diagnosed, wherein the sensitivity and the specificity are better. The five groups of miRNA combinations are shown in table 3.

TABLE 3 Table 3

Preferred results are as follows: when the sequences shown in SEQ ID NO. 1 and 5-13 are selected, the sequences are marked as cancer groups, the 10 miRNAs are subjected to a statistical method of SVM to obtain an ROC curve of a training set, as shown in figure 1, the AUC is 0.972, the accuracy is 93.5%, the sensitivity of the selected marker for detecting cancerous pleural effusion is 94.3%, and the specificity is 91.7%; the 10 miRNAs perform double-blind verification on the test set, and as shown in FIG. 4, the obtained sensitivity is 71.3%, and the specificity is 87.7%; the test set double blind accuracy was 78.0%.

Example 8

Further selecting key miRNA from miRNA variables to diagnose lung cancer type malignant pleural effusion

The mirnas selected in example 5 were aligned and combined, and miRNA combinations with specificity, sensitivity and accuracy all reaching the clinical detection level were screened and tested. According to data statistical analysis, 5 groups of best miRNA combinations are found, and as a result, after partial specific miRNAs are combined, lung cancer type malignant pleural effusion can be effectively diagnosed, wherein the sensitivity and the specificity are better. The five groups of miRNA combinations are shown in table 4.

TABLE 4 Table 4

Preferred results are as follows: when the sequences shown in SEQ ID NO. 1, 8, 14-15 and 19-21 are selected, the sequences are marked as lung cancer groups, the 7 miRNAs are used for obtaining ROC curves from a training set by using a statistical method of SVM, as shown in figure 2, the AUC is 0.964, and the sensitivity of the selected marker for detecting lung cancer malignant pleural effusion is 91.7%, and the specificity is 100%; the 7 miRNAs perform double-blind verification on the test set, and as shown in FIG. 5, the obtained sensitivity is 84%, and the specificity is 84.6%; the test set accuracy was 84.3%.

Example 9

Further selecting key miRNA from miRNA variables to diagnose lung adenocarcinoma type malignant pleural effusion

Further permutation and combination of the mirnas selected in example 5, screening and testing miRNA combinations with specificities and sensitivities reaching clinical detection levels. According to data statistical analysis, 5 groups of best miRNA combinations are found, and as a result, the combination of 12 miRNAs can be found to be more effective in diagnosing lung adenocarcinoma type malignant pleural effusion. The five groups of miRNA combinations are shown in table 5:

table 5:

preferred results are as follows:

when the sequences shown in SEQ ID NOs 1, 4, 9, 13, 16-17, 22-23 and 29-32 were selected, the sequences were marked as lung adenocarcinoma groups, the 12 miRNAs were statistically processed by SVM to obtain ROC curves for the training set, as shown in FIG. 3, with an AUC of 0.961 and an accuracy of 92.2%, and the selected markers had a sensitivity of 85.2% and a specificity of 100% for detecting cancerous pleural effusions; the 12 miRNAs perform double-blind verification on the test set, and as shown in FIG. 6, the obtained sensitivity is 91%, and the specificity is 89.2%; the test set accuracy was 89.9%.

Conclusion(s)

When SEQ ID NO. 1-4 is selected as the combination of miRNA fingerprint patterns, cancerous pleural effusion can be well diagnosed; after the combination of miRNA in the fingerprint is optimized, when SEQ ID NO. 1 and 5-13 are selected as the combination of the miRNA fingerprint, the diagnosis of cancerous pleural effusion can be achieved to meet the clinical requirements; when SEQ ID NOs 1, 8, 14-15 and 19-21 are selected as the combination of miRNA fingerprint patterns, lung cancer type malignant pleural effusion can be more effectively diagnosed, and the sensitivity and the specificity are better; when SEQ ID NOs 1, 4, 9, 13, 16-17, 22-23 and 29-32 are selected as the combination of miRNA fingerprint patterns, lung adenocarcinoma type malignant pleural effusion can be effectively diagnosed. Based on the above, the miRNA fingerprint can effectively distinguish cancerous pleural effusion from benign pleural effusion.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Reference to the literature

1. Xu Yajun, car wind, cai Xiaodong, etc., the marker of thoracic edema tumor is valuable in the differential diagnosis of benign and malignant pleural effusion [ J ].

2.Konstantin A.DIMITRIADIS:Malignant pleural effusions.Archive of Oncology 2001；9(1):2.

Light RW.Clinical practice.Pleural effusion[J].N Engl J Med,2002,346:1971-1977.

3.Bartel DP(2004)MicroRNAs:genomics,biogenesis,mechanism,and function.Cell 116:281-297.

4.Huang Y,Shen XJ,Zou Q,Zhao QL(2010)Biological functions of microRNAs.Bioorg Khim 36:747-752.

5.Mezzanzanica D,Bagnoli M,De Cecco L,Valeri B,Canevari S(2010)Role of microRNAs in ovarian cancer pathogenesis and potential clinical implications.Int J Biochem Cell Biol 42:1262-1272.

Sequence listing

<110> Shanghai Xiangqiong biotechnology Co., ltd

Application of fingerprint spectrum composed of <120> small RNA in diagnosis and treatment of human cancerous pleural effusion

<130> P2017-0381

<160> 32

<170> PatentIn version 3.5

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Claims

Use of a set or combination of mirnas for the preparation of a chip or kit of 12 mirnas having sequences as shown in SEQ ID NOs 1, 4, 9, 13, 16-17, 22-23 and 29-32, characterized in that the chip or kit is used for:

(1) Diagnosis, tumor grading and prognosis evaluation of lung adenocarcinoma type malignant pleural effusion;

(2) The lung adenocarcinoma type malignant pleural effusion and benign pleural effusion are distinguished.
2. A miRNA chip, characterized in that the miRNA chip comprises:

a solid phase carrier; and

oligonucleotide probes which are immobilized on the solid support in an ordered manner, said oligonucleotide probes corresponding specifically to all of the sequences shown in SEQ ID NOs 1, 4, 9, 13, 16-17, 22-23 and 29-32.
3. The use of the miRNA chip of claim 2 for preparing a kit for differentiating lung adenocarcinoma type malignant pleural effusion from benign pleural effusion.
4. A kit comprising the miRNA chip of claim 2.
5. The kit is characterized by comprising a detection reagent for detecting miRNA sets or combinations, wherein the miRNA sets or combinations are 12 miRNAs with sequences shown as SEQ ID NO. 1, 4, 9, 13, 16-17, 22-23 and 29-32.
6. A method of screening for a candidate drug against lung adenocarcinoma type malignant pleural effusion, comprising the steps of:

(a) In the experimental group, lung adenocarcinoma cells of lung adenocarcinoma type malignant pleural effusion are cultured in the presence of a substance to be tested; and culturing lung adenocarcinoma cells of the same lung adenocarcinoma type malignant pleural effusion in the control group under the same conditions as the experimental group but in the absence of the test substance;

(b) Determining the expression level of a plurality of miRNAs of lung adenocarcinoma cells of lung adenocarcinoma type malignant pleural effusion in the experimental group, and comparing the expression level of the miRNAs of the lung adenocarcinoma cells of the lung adenocarcinoma type malignant pleural effusion with the expression level of the miRNAs of the lung adenocarcinoma cells of the lung adenocarcinoma type malignant pleural effusion in the control group;

Wherein if the expression level of the miRNA in the experimental group changes towards the expression level of benign hydrothorax cells compared with the control group, the substance to be tested is indicated to be a candidate drug for resisting lung adenocarcinoma type malignant hydrothorax, and the miRNA is 12 miRNAs with sequences shown as SEQ ID NO. 1, 4, 9, 13, 16-17, 22-23 and 29-32.
7. The method of claim 6, further comprising step (c): further treating a non-human mammal having lung adenocarcinoma type malignant pleural effusion with the candidate drug to determine the effect of the candidate drug on the lung adenocarcinoma type malignant pleural effusion of the non-human mammal.