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Small interfering ribonucleic acid sequence for inhibiting expression of human pard6a gene and its application in resisting proliferation and metastasis of ovarian cancer

A technology of gene expression and ovarian cancer cells, applied in the field of proliferation and metastasis, and inhibition of ovarian cancer growth

Active Publication Date: 2019-01-08
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no patent publication on the application of RNA interference technology, which uses small RNA as a targeted drug to specifically interfere with the post-transcriptional expression of the PARD6A gene, thereby achieving the effect of inhibiting the proliferation and metastasis of ovarian cancer cells

Method used

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  • Small interfering ribonucleic acid sequence for inhibiting expression of human pard6a gene and its application in resisting proliferation and metastasis of ovarian cancer
  • Small interfering ribonucleic acid sequence for inhibiting expression of human pard6a gene and its application in resisting proliferation and metastasis of ovarian cancer
  • Small interfering ribonucleic acid sequence for inhibiting expression of human pard6a gene and its application in resisting proliferation and metastasis of ovarian cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: qRT-PCR experiment detects the mRNA level silencing effect of siPARD6A

[0026]Human ovarian cancer cell line A2780 cells (American Type Culture Collection, American Type Culture Collection, USA) were cultured with Dulbecco's modified Eagle containing 10% fetal bovine serum, 10 U / ml penicillin, and 0.1 mg / ml streptomycin Base (Dulbecco modified Eagle medium, DMEM) (American Life Technologies Company, LifeTechnologies, USA) medium, in 5% CO 2 , and cultivated in a 37°C cell incubator (Thermo Fisher, Thermo Fisher, USA). It is necessary to observe the growth state of the cells once a day. When the cells grow to account for 70% or more of the surface area of ​​the culture flask, they should be passaged in time. When the cells are in good condition and in the logarithmic growth phase, they can be used for experiments.

[0027] Experimental procedure: the six tubes of siPARD6A powder and one tube of siGL2 powder ordered (Shanghai Jima Pharmaceutical Technology...

Embodiment 2

[0033] Embodiment 2: the silencing effect of Western verification PAR1, PAR2 protein level

[0034] Experimental procedure: the reverse transfection experiment was the same as that in Example 1, but only three groups of reverse transfection PAR1, PAR2 and GL2 were performed, and two groups were prepared for protein extraction in each group.

[0035] Western blotting experiment: firstly treat each group of cells with SDS protein lysate, collect and process the corresponding protein samples, then prepare SDS-PAGE gel, load the sample, electrophoresis, then transfer to the membrane, block with 5% skimmed milk, and then wash the membrane with 1xTBS , cut the membrane, reacted with the diluted primary antibody anti-PARD6A (Anti-PARD6A) (Sigma-Aldrich, Sigma-Aldrich, USA) for 1 h, and continued to add the second antibody anti-rabbit immunoglobulin G enzyme-linked antibody (Anti-PARD6A) -rabbit IgGHRP-linked Antibody) (Cell Signaling Technology, USA) and the chemiluminescent agent we...

Embodiment 3

[0037] Example 3: siPARD6A affects cell proliferation activity

[0038] The detection of cell proliferation activity is to detect the relative survival rate of cells after siPARD6A has been treated for 72 hours by MTT staining method, so as to judge the effect of siPARD6A on the proliferation ability of A2780 cells.

[0039] Experimental procedure: The siRNA reverse transfection experiment is similar to Example 1, but the scale is adjusted accordingly. According to each condition, 3 replicate wells are added, and 10 μl of the incubated corresponding siRNA and lipofectamine2000 mixture and 90 μl are added to each well to adjust the cell density to 5×10 5 cells / mL of cell suspension, shake gently, and culture in a cell incubator for 72 hours. After 72 h, the 96-well plate was taken out, and 10 μl of 5 mg / ml 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT solution) (BIOSHARP company, China). Then put it in the incubator and incubate for 1.5h, discard the culture...

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Abstract

The invention discloses a human PARD6A gene expression inhibiting small-interference ribonucleic acid sequence and a use thereof in resisting proliferation and metastasis of ovarian cancer, and belongs to the technical fields of targeted gene medicine research and development and cell biology. Two pairs of siRNAs both can be bonded with the mRNA of the PARD6A gene in an ovarian cancer cell line to effectively interfere with transcription thereof before translation such that the proliferation activity and adsorption capability of cells are inhibited, thus achieving the purposes of controlling the growth and metastasis of ovarian cancer cells and treating ovarian cancer. The two pairs of siRNAs of the human PARD6A gene expression inhibiting small-interference ribonucleic acid sequence is promising in preparation of anti-ovarian caner medicines having high efficiency, specificity, and low side effect.

Description

technical field [0001] The invention belongs to the fields of molecular biology and biomedicine, and in particular relates to small interfering RNA (Small interfering RNA, siRNA) encoding gene (Par-6 Family Cell Polarity Regulator Alpha, PARD6A) for silencing segregation defect protein 6 family cell polarity regulator Alpha ) sequence and its use in inhibiting the growth, proliferation and metastasis of ovarian cancer. Background technique [0002] Ovarian cancer is a malignant tumor that occurs in ovarian tissue. Its incidence rate ranks second among female reproductive malignancies, and its mortality rate ranks first among gynecological malignancies. It is the deadliest type of gynecological malignancies. According to the 2012 global cancer statistics, globally, about 200,000 people are diagnosed with ovarian cancer each year, and 125,000 people die from the disease. Because the ovary is deep in the pelvic cavity, the early symptoms are not obvious, and it is difficult fo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61K31/7088A61P35/00
CPCC12N15/113C12N2310/14
Inventor 刘晗青阮玲玲张佩珊尚东胜赵志聪屠志刚刘莉鲁永金卢子文沈艳婷吴燕芳张雅菲梁智全沈明香
Owner JIANGSU UNIV
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