WO2018170622A1 - Method for synchronously down-regulating mirna-152 and mirna-185 expressions - Google Patents

Abstract

Provided is a method for reducing miRNA-152 and miRNA-185 expressions, comprising constructing a eukaryotic expression vector pRI-152-185 targeting to miR-152 and miR-185 over a eukaryotic vector pRI-GFP/Neo that contains an H1 promoter, thereby down-regulating miR-152 and miR-185 expressions.

Description

 Title of Invention: A Method for Simultaneous Down-regulation of Expression of MicroRNA-152 and MicroRNA-185

 [0001] The present invention relates to the field of life sciences, and more particularly to a method for simultaneously down-regulating the expression of microRNA-152 and microRNA-185.

 Background technique

 [0002] MicroRNA-152 (miR-152) is a multifunctional miRNA. It was found that miR-152 is related to methylation, such as methyltransferase DNMT1 content and enzyme activity, miR-152 can be DNA methylation of endometrial cancer becomes a silent gene, and it is associated with the development of various cancers. It is a tumor suppressor microRNA, with preeclampsia, trophoblastic tumor, bladder cancer, gastrointestinal cancer, ovarian cancer. It is related to many diseases; microRNA-185 (miR-185) is a 22nt miRNA, located in human chromosome 22ql l.21, in the occurrence and invasion of colon cancer, gastric cancer, esophageal cancer, lung cancer, liver cancer and other tumors. It plays an important role as a tumor suppressor gene. In addition, it is a methylation-related tumor suppressor miRNA that affects the methylation level of the whole genome by directly targeting the expression of DNMT1, thereby regulating the methylation of certain genes. Modification of the state, affecting gene expression. By controlling the expression of miR-152 and miR-185, the synergy with other drugs can provide new epigenetic ideas for the treatment of cancer.

 technical problem

 [0003] At present, miRNA function research is generally achieved by miRNA overexpression and silencing. miRNA silencing is the presentation of artificially synthesized oligonucleotide small molecules into cells, and endogenous miRNAs.

 The formation of a heteroduplex allows the miRNA to reduce the inhibition of the target gene to achieve regulation of gene function. Silencing of miRNAs, especially long-term stable silencing, is currently difficult to achieve. Commonly used methods for silencing miRNA mainly include anti-miR, antagomiR, miRNA sponge, etc. These methods have short effects such as short inter-turn, poor stability, complex construction, high requirements on instrument conditions, and complicated technical operations, which are difficult to achieve, simple, and effective. The effect of miR-152 and miR-185 expression was down-regulated simultaneously.

 Problem solution

 Technical solution

[0004] The object of the present invention is to provide a method for simultaneously down-regulating the expression of miR-152 and miR-185, which is The method is simple, easy to operate, and can easily and effectively adjust the effects of miR-152 and miR-185 expression to overcome the deficiencies of the prior art.

 [0005] A method for the present invention: a method for down-regulating expression of miR-152 and miR-185, and constructing eukaryotic expression targeting miR-152 and miR-185 on a eukaryotic vector pRI-GFP/Neo containing a HI promoter Vector pRI-152-185, which regulates the expression of miR-152 and miR-185 in eukaryotic cells; after transfecting the vector into eukaryotic cells in vitro, the expression of miR-152 and miR-185 was detected by real-time PCR Level.

 [0006] The construction of the pRI-152-185, specifically, the sequence design of the Tud-152-185 is completed by software.

The sequences of pRI-GFP/Neo and Tud-152-185 were double-digested with Xho I and Bgl II, and the recovered fragments were purified and ligated overnight at 4 °C. After ligation, 6 colonies were picked and inoculated. After 12 hours of LB medium containing 1 (Vg/ml kanamycin), the bacterial solution was extracted and sent to Shanghai Yingjun for sequencing. The correctly sequenced vector was the successfully constructed recombinant plasmid pRI-152-185.

 Advantageous effects of the invention

 Beneficial effect

 [0007] The present invention utilizes a molecular biology technique to construct a eukaryotic expression vector containing Tud-152-185 based on a eukaryotic vector containing a CMV promoter, thereby down-regulating the expression of miR-152 and miR-185; After the vector was transfected into eukaryotic cells in vitro, the expression of miR-152 and miR-185 was detected by a real-time PCR instrument. The invention is not only suitable for transfection of cells, but also for the production of whole horizontal transgenic animals.

 Brief description of the drawing

 DRAWINGS

 1 is a schematic diagram showing the structure of the pRI-GFP/Neo vector; FIG. 2 is a quantitative PCR method for detecting the expression level of miRNA, wherein a. miR-152 expression, b. miR-185 expression.

 BEST MODE FOR CARRYING OUT THE INVENTION

 BEST MODE FOR CARRYING OUT THE INVENTION

The present invention can be better understood from the following examples. However, those skilled in the art will readily understand

The specific material ratios, process conditions, and results of the examples are merely illustrative of the invention and should not be construed as limiting the invention as described in the claims.

Example of a method for downregulating expression of miR-152 and miR-185 [0011] 1 First search for the sequence of hsa-miR-152 and hsa-miR-185 in the miRBase database, according to Tou gh Decoy RNA (Tud

 The design principle of RNA) is to design the Tud RNA targeting hsa-miR-152 and hsa-miR-185, and finally add the Xho I and Bgl II restriction sites at the 5' and 3' ends of the Tud RNA to obtain the desired The sequence of Tud-152-185. The nucleotide sequence 歹 IJ of Tud-152-185 is shown as SEQ ID No. 1.

[0012] 2 using Xho l and 8 _§ 1 11 double-cleavage of the backbone vector 1-0?? 60 and 1 ^ (1-152-185 sequence, the recovered fragment was purified, and then connected overnight at 4 ° C; after the connection Transformation, pick 6 colonies, inoculate into LB medium containing lO g/ml kanamycin, extract the bacterial solution after 12 hours, and send it to Shanghai Yingjun for sequencing. The correct vector is the successfully constructed recombinant plasmid. pRI-152-185.

[0013] 3 The endotoxin-free plasmid extraction kit (Tiangen Biochemical) was used to extract the recombinant plasmid pRI-152-185.

[0014] HepG2 cells were seeded in a 6-well plate at a density of 30,000/ml, and 10 ug of plasmid pRI-152-185 was transiently transfected with Lip ofectamine 2000, and cultured at 5<3⁄4C02, 37 °C; After 48 hours, total RNA was extracted from each group, and the expression of miR-152 and miR-185 was detected by Realtime PCR. The results showed that miR-152 and miR-185 were expressed in pRI-152-185 transfected group. Significant down-regulation (Figure 2) suggests that this vector can be used to down-regulate the expression of miR-152 and miR-185.

 Industrial applicability

 [0015] The present invention utilizes a molecular biology technique to construct a eukaryotic expression vector containing Tud-152-185 based on a eukaryotic vector containing a CMV promoter, thereby down-regulating the expression of miR-152 and miR-185; After the vector was transfected into eukaryotic cells in vitro, the expression of miR-152 and miR-185 was detected by a real-time PCR instrument. The invention is not only suitable for transfection of cells, but also for the production of whole horizontal transgenic animals.

Claims

Claim  [Claim 1] A method for simultaneously down-regulating microRNA-152 and microRNA-185, characterized in that: targeting miR-152 and miR- on a eukaryotic vector pRI-GFP/Neo containing a H1 promoter 185 eukaryotic expression vector pRI-152-185, which regulates the expression of miR-152 and miR-185 in eukaryotic cells; after transfecting the vector into eukaryotic cells in vitro, detection of mi R-152 by fluorescence quantitative PCR And the expression level of miR-185.  [Claim 2] The method for synchronously down-regulating the expression of microRNA-152 and microRNA-185 according to claim 1, wherein: the construction of the pRI-152-185, specifically, the completion of Tud- using software Sequence design of 152-185; using Xho I, Bgl II double-digested backbone vector pRI-GFP/Neo and Tud-152-185 sequences, purified fragments were recovered, and then ligated overnight at 4 °C; after transformation, 6 colonies were picked and inoculated to contain 10 μ _§ /ml In the LB medium of kanamycin, 12 hours later, the bacterial liquid was extracted and sent to Shanghai Yingjun for sequencing. The correctly constructed vector was the successfully constructed recombinant plasmid pRI-152-185.