WO2018170623A1 - Method for reducing mirna-152 and mirna-424 expressions - Google Patents

Abstract

Provided is a method for reducing miRNA-152 and miRNA-424 expressions, comprising constructing a eukaryotic expression vector pRI-152-424 targeting to miR-152 and miR-424 over a eukaryotic vector pRI-GFP/Neo that contains an H1 promoter, thereby down-regulating miR-152 and miR-424 expressions.

Description

Method for reducing expression of microRNA-152 and microRNA-424 Technical field

The present invention relates to the field of life sciences, and more particularly to a method of reducing the expression of microRNA-152 and microRNA-424.

Background technique

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 endometrium. Cancer DNA methylation becomes a silent gene, and it is associated with the development of various cancers. It is a tumor suppressing microRNA, and many diseases such as preeclampsia, trophoblastic tumor, bladder cancer, gastrointestinal cancer, ovarian cancer, etc. Related; microRNA-424 (miR-424) is a miRNA discovered in recent years, which affects the biological effects and development of tumor cells by acting on target genes in various tumors and participating in the signal pathway of target gene regulation. It plays a role similar to oncogenes and tumor suppressor genes, or promotes and inhibits the invasion and metastasis of tumors. Studies have shown that miR-424 is a multifunctional miRNA, which is associated with cell invasion and metastasis of cervical cancer, pancreatic cancer, etc.; it is associated with the expression of inflammatory factors such as IL-6 and TNF-α; since the miR-424 promoter region has CpG islands It is also associated with methylation-induced gene silencing. By controlling the expression of miR-152 and miR-424, and synergistically with other drugs, it can provide new epigenetic ideas for the treatment of cancer.

technical problem

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, forming heteroduplexes with endogenous miRNAs, and reducing miRNAs to target genes. Inhibition to achieve regulation of gene function. Silencing of miRNAs, especially long-term stable silencing, is currently more difficult to achieve. Commonly used methods for silencing miRNA mainly include anti-miR, antagomiR, miRNA sponge, etc. These methods have short acting time, poor stability, complex construction, high instrumental requirements and complicated technical operation, which is difficult to achieve convenient, simple and effective. The effect of miR-152 and miR-424 expression was reduced.

Problem solution

Technical solution

The object of the present invention is to provide a method for reducing the expression of miR-152 and miR-424, which is simple in design, easy to operate, and can easily and effectively down-regulate the expression of miR-152 and miR-424 to overcome the existing The lack of technology.

The present invention is a method for down-regulating the expression of miR-152 and miR-424, and constructing a eukaryotic expression vector pRI- targeting miR-152 and miR-424 on the eukaryotic vector pRI-GFP/Neo containing the H1 promoter. 152-424, the expression of miR-152 and miR-424 in eukaryotic cells was down-regulated; after transfecting the vector into eukaryotic cells in vitro, the expression levels of miR-152 and miR-424 were detected by a real-time PCR instrument.

The construction of pRI-152-424, specifically, the sequence design of Tud-152-424 was performed by software; the backbone vectors pRI-GFP/Neo and Tud-152-424 sequences were double-digested with Xho I and Bgl II. The recovered fragments were purified and then ligated overnight at 4 °C; after transformation, 6 colonies were picked and inoculated into LB medium containing 10 μg/ml kanamycin. After 12 hours, the bacterial liquid was extracted and sent to Shanghai Yingjun. Sequencing. The correctly constructed vector was the successfully constructed recombinant plasmid pRI-152-424.

Advantageous effects of the invention

Beneficial effect

The present invention utilizes a molecular biology technique to construct a eukaryotic expression vector containing Tud-152-424 on the basis of a eukaryotic vector containing a CMV promoter, thereby down-regulating the expression of miR-152 and miR-424; After transfecting eukaryotic cells in vitro, the expression of miR-152 and miR-424 was detected by real-time PCR.

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

Figure 1 is a schematic diagram showing the structure of the pRI-GFP/Neo vector; Figure 2 is a quantitative PCR detection of miRNA expression levels, wherein a. miR-152 expression, b. miR-424 expression.

BEST MODE FOR CARRYING OUT THE INVENTION

BEST MODE FOR CARRYING OUT THE INVENTION

The invention can be better understood in light of the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions, and results described in the examples are merely illustrative of the invention and are not intended to limit the invention as described in the claims. .

Examples of methods for downregulating expression of miR-152 and miR-424

1 Firstly, the sequences of hsa-miR-152 and hsa-miR-424 were searched in the miRBase database, and the Tud RNA targeting hsa-miR-152 and hsa-miR-424 was designed according to the design principle of Tough Decoy RNA (Tud RNA). Finally, Xho I and Bgl II restriction sites were added to the 5' and 3' ends of the Tud RNA to obtain the desired Tud-152-424 sequence. The nucleotide sequence of Tud-152-424 is shown in SEQ ID No. 1.

2 The sequences of pRI-GFP/Neo and Tud-152-424 were double-digested with Xho I and Bgl II, and the fragments were purified and then ligated overnight at 4 °C. After ligation, 6 colonies were picked and inoculated. In LB medium containing 10 μg/ml kanamycin, the bacterial liquid was extracted 12 hours later and sent to Shanghai Yingjun for sequencing. The correctly constructed vector was the successfully constructed recombinant plasmid pRI-152-424.

3 The recombinant plasmid pRI-152-424 was extracted using an endotoxin-free plasmid extraction kit (Tiangen Biochemical).

4 Hela cells were seeded in 6-well plates at a density of 30,000/ml, and 10 ug of plasmid pRI-152-424 was transiently transfected with Lip ofectamine 2000, and cultured at 5% CO2 and 37 °C; 48 hours later Total RNA was extracted from each group, and the expression of miR-152 and miR-424 was detected by Realtime PCR. The results showed that the expression levels of miR-152 and miR-424 were significantly down-regulated in the pRI-152-424 transfection group (Fig. 2), suggesting that the vector can be used to reduce the expression of miR-152 and miR-424.

Industrial applicability

The present invention utilizes molecular biology techniques to construct a eukaryotic expression vector containing Tud-152-424 on the basis of a eukaryotic vector containing a CMV promoter, thereby down-regulating the expression of miR-152 and miR-424; After transfecting eukaryotic cells in vitro, the expression of miR-152 and miR-424 was detected by real-time PCR. The invention is not only suitable for transfection of cells, but also for the production of whole horizontal transgenic animals.

Claims (2)

A method for reducing microRNA-152 and microRNA-424, characterized in that a eukaryotic expression vector pRI targeting miR-152 and miR-424 is constructed on a eukaryotic vector pRI-GFP/Neo containing a H1 promoter -152-424, the expression of miR-152 and miR-424 in eukaryotic cells was down-regulated; after transfecting the vector into eukaryotic cells in vitro, the expression levels of miR-152 and miR-424 were detected by a real-time PCR instrument. The method for reducing the expression of microRNA-152 and microRNA-424 according to claim 1, characterized in that the pRI-152-424 is constructed, specifically, the sequence design of Tud-152-424 is completed by software. The sequences of pRI-GFP/Neo and Tud-152-424 were double-digested with Xho I and Bgl II, and the fragments were purified and then ligated overnight at 4 ° C. After ligation, 6 colonies were picked and inoculated. In LB medium containing 10 μg/ml kanamycin, the bacterial liquid was extracted 12 hours later and sent to Shanghai Yingjun for sequencing. The correctly constructed vector was the successfully constructed recombinant plasmid pRI-152-424.

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