Application of microRNA-7 for inhibiting prostate tumor growth and prostate tumor progress

A technology for prostate tumor and process, applied in the field of microRNA, can solve problems such as unclear mechanism of action, achieve the effect of inhibiting proliferation ability, broad application prospects, and promoting nuclear entry

Inactive Publication Date: 2014-11-12
RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether miR-7 plays the role of a tumor suppressor gene in prostate cancer, whether it participates in the regulation of prostate cancer tumor progression by regulating the "stemness maintenance" of prostate cancer stem cells, and the related mechanisms are still unclear

Method used

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  • Application of microRNA-7 for inhibiting prostate tumor growth and prostate tumor progress
  • Application of microRNA-7 for inhibiting prostate tumor growth and prostate tumor progress
  • Application of microRNA-7 for inhibiting prostate tumor growth and prostate tumor progress

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Using the dual luciferase reporter system to identify the interaction between miRNA and target gene mRNA 3'UTR: PC3 cells (purchased from the Cell Bank of Chinese Academy of Sciences, as an example, but not limited to this) were completely cultured in DMEM in a 24-well plate Cell culture medium (containing 10% fetal bovine serum, purchased from Gibco, USA) was adhered to the wall, and when the cell density reached 70% to 80%, use lipofectamine2000 transfection reagent (purchased from Life Technology, USA) to perform the following operations: 1) take Cells in 3 wells were used as a parallel sample, and the positive control reporter vector (the positive control here is a sequence that is completely complementary to miR-7, i.e. sequence 4) and the internal reference vector pRL-cmv expressing the Renilla luciferase gene (purchased In the United States promega company) and control scramble RNA (purchased in the United States Life Technology company, the data see figure 1 Whi...

Embodiment 2

[0032] (1) Construction of an expression vector for stably expressing miRNA in eukaryotic cells: with the pEGP-miR plasmid as the backbone (purchased from Cell BioLab, USA), the plasmid was linearized by conventional restriction endonuclease BamHI / NheI double digestion. change. By artificially synthesizing the desired miRNA precursor molecule sequence (here three different precursor molecules pri-miR-7-1, pri-miR-7-2 and pri-miR-7-3 of miR-7 are taken as examples , but not limited to this, see sequence 1, 2, 3), and introduce restriction enzymes BamHI and NheI (also can be XbaI, which is the same tail enzyme of NheI) at the 5' end and 3' end of the sequence respectively recognition site. The obtained fragment is subjected to conventional restriction endonuclease BamHI / NheI (also BamHI / XbaI) double digestion, and conventional DNA ligation operations to construct an expression vector for eukaryotic cells to stably express miRNA (herein pEGP -miR-7-1, pEGP-miR-7-2, and pEGP-miR...

Embodiment 3

[0036] Stable overexpression of miR-7 can achieve cell cycle arrest in prostate cancer cell lines (take PC3-miR-7 cells as an example, but not limited to): Pass PC3-miR-7 and control PC3-null through conventional cell lines The culture method is adherent culture in a 12-well plate, and each type of cell needs to repeat 3 replicate wells at each time point of detection. In this embodiment, the ratio of cell numbers in each cell phase (G0 / G1 phase, S phase, G2 / M phase) was detected every 2 hours, and the detection was continued for 48 hours, with a total of 24 time points. When the cell density reaches 60%-70%, the complete medium is replaced with serum-free basic medium, and the cell cycle is synchronized to the G0 phase through serum starvation for 30-36 hours, and is in a resting state. The detection starts after the serum supply is restored, and the starting point of the serum supply is 0 hour, and the time is sequentially counted. At the detection time point, use the conve...

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Abstract

The invention relates to application of microRNA-7 for inhibiting prostate tumor growth and prostate tumor progress. According to the application, by establishing a prostate cancer cell subcloning cell system for stable over-expression of miR-7 and by establishing a corresponding naked mouse subcutaneous bearing cancer animal model, the function of miR-7 in inhibiting prostate tumor growth and'dryness maintenance' capability of prostate cancer stem cells are verified in vitro and in vivo. After the stable over-expression of MiR-7, targeted inhibition of expression of a PI3K / Akt signal path can be achieved, the activity of Akt is degraded, the downstream period of Akt can be remarkably reduced to regulate and control phosphorylation level of protein p21, and nuclear localization of non-phosphorylated p21 protein is prompted, so that inhibition on periodic cell progress and tumor progress in tumor cells is achieved, and the multiplication capacity of tumor cells is inhibited. The functions of miR-7 in habiting the prostate tumor growth and the prostate tumor progress are verified both in vitro and in vivo. The result shows that natural nucleic acid molecules such as miR-7 as a novel small molecular medicine has wide application prospect in clinical treatment on prostate.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a microRNA, microRNA-7 (miR-7). Stable overexpression of miR-7 in prostate cancer cells can effectively inhibit the growth of tumor cells. Meanwhile, stable overexpression of miR-7 can effectively inhibit the tumor progression of prostate cancer by inhibiting the ability of prostate cancer stem cells to maintain stemness. It is suggested that miR-7 as a natural nucleic acid small molecule drug can be applied in targeted gene therapy for prostate cancer. Background technique [0002] Prostate cancer is the most common malignant tumor in men, 95% of which occur in the elderly population over 60 years old. With the deepening of the aging population in our country, there are about 70,000 to 80,000 new cases of prostate cancer every year, and the incidence rate is increasing at a rate of 25%-30% per year, becoming the malignant tumor with the fastest increasing incidence rate....

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P35/00A61P13/08
Inventor 高维强方煜翔常云丽
Owner RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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