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siRNA for inhibiting growth of epithelial ovarian cancer as well as recombinant vector and application thereof

A recombinant vector, ovarian cancer technology, applied in the field of genetic engineering, can solve the problems of high difficulty of operation, unimproved treatment effect, no in-depth research report on HOST2, etc., and achieve the effect of inhibiting tumor growth.

Inactive Publication Date: 2013-06-12
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because there are often no symptoms before metastasis, most EOCs are diagnosed at an advanced stage, which makes the operation difficult and wide, and the lesions are often not completely removed. In addition, the effect of radiotherapy and chemotherapy is limited, resulting in a high recurrence rate and a very poor prognosis.
According to statistics in the past 20 years, the incidence of EOC is increasing year by year, but the treatment effect has not been improved, and the 5-year survival rate hovers at 30-40%.
However, there is no in-depth research report on HOST2

Method used

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  • siRNA for inhibiting growth of epithelial ovarian cancer as well as recombinant vector and application thereof
  • siRNA for inhibiting growth of epithelial ovarian cancer as well as recombinant vector and application thereof
  • siRNA for inhibiting growth of epithelial ovarian cancer as well as recombinant vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Construction of pLKO.1-HOST2-sh2 eukaryotic expression vector

[0041] 1.1 Required reagents

[0042] Restriction enzymes Age I and EcoR I and corresponding Buffer, T4 ligase and its buffer, PCR product purification kit, and gel recovery kit were all purchased from TaKaRa Company (Dalian Bao Biological Engineering Co., Ltd.). The conventional RNAi vector pLKO.1puro (Plasmid8453) was selected as the eukaryotic expression vector, which originated from Addgene, a non-profit organization for plasmid sharing among scientists around the world.

[0043] 1.2 Design, synthesis and screening of HOST2 specific siRNA.

[0044] The HOST2 cDNA sequence is shown in SEQ ID NO:1.

[0045] Design and synthesize specific siRNA1-3 according to the above sequence (all completed by Shanghai Invitrogen):

[0046]

[0047] From figure 2 It can be seen that siRNA2 can inhibit the expression of HOST2 by more than 70%; siRNA1 can only inhibit the expression of HOST2 by about 20...

Embodiment 2

[0054] Embodiment 2: in vitro cell test

[0055] The changes of the biological function of EOC cells before and after HOST2 expression was inhibited were studied by cell scratch test, Transwell test and CCK-8 assay.

[0056] The specific method is as follows:

[0057] 1. Cell scratch experiment

[0058] The cell migration ability was detected by scratch method, and the average migration distance was determined by Image J software. Experimental supplies: 6-well plate, ruler, serum-free medium, PBS / saline, yellow tip, Mark pen, etc.

[0059] Steps: ① Use a Mark pen to draw horizontal lines evenly on the back of the 6-well plate, about every 0.5-1cm, across the holes, and at least 5 lines through each hole. ②Add about 2.5-5×10 to the hole 5 cells, until they grew to about 50%, leaving three blank wells as a control, and transfecting pLKO.1-HOST2-sh2 into the remaining three experimental wells. ③When the cells grow to complete confluence (about 24 hours), use the tip of the y...

Embodiment 3

[0069] Embodiment 3: Simulated treatment experiment in vivo

[0070] 50 BALB / C-nu female nude mice, 4-6 weeks old, weighing about 20g±2g, were randomly divided into two groups (25 mice / group, experimental group and blank control group). Prepare OVCAR-3 single cell suspension, adjust the cell concentration to 1-5×10 7 / mL, take nude mice and inject 100-200 μl of cells subcutaneously on the back near the hind limbs.

[0071]The pLKO.1-HOST2-sh2 plasmid was extracted with a large amount of plasmid extraction kit (Shanghai Tiangen Biological Co., Ltd.), and the density of the plasmid was determined to be 1437 ng / ml by a spectrophotometer. When the tumor grows to an average diameter of 5 mm, inject about 100 ngpLKO.1-HOST2-sh2 recombinant plasmid into the xenograft tumors of nude mice in the experimental group (the injection volume is about 70 μl according to the density calculation), and spray once every 2 days for a total of 30 days , a total of 15 times. After the injection o...

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Abstract

The invention belongs to the technical field of genetic engineering. The epithelial ovarian cancer (EOC) is the most common ovarian cancer; the morbidity ranks the third in malignant tumors of gynecological reproductive organs and ranks only second to cervical cancer and endometrial cancer, but the death rate can be up to 70%, and ranks the first. An interference sequence for inhibiting the expression of the epithelial ovarian cancer HOST2 gene is screened; and the RNA (ribonucleic acid) sequence of the HOST2 gene is shown in SEQ ID NO:4 or 5. According to the invention, a recombinant vector including the interference sequence is also built; in vivo and in vitro experiments show that the recombinant vector can inhibit the specific high-expression recombinant HOST2 in an EOC cell, so as to inhibit the growth of the tumor. The siRNA and the recombinant vector provided by the invention provide a new gene treatment medicament for clinic treatment of EOC.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an interference sequence capable of inhibiting the expression of specific genes related to epithelial ovarian cancer and its recombinant vector, and its application in the preparation of drugs for treating epithelial ovarian cancer. Background technique [0002] Epithelial ovarian cancer (Epithelial Ovarian Cancer, EOC) is the most common type of ovarian cancer, accounting for about 80-90%. Although it ranks third in the incidence of gynecological reproductive organ malignancies, it is second only to cervical cancer and endometrium Cancer, but the mortality rate is as high as 70%, ranking first. According to statistics, the risk rate of ovarian cancer in a woman's life is about 1.45%, and the risk of dying from ovarian cancer is 1%. Due to the complex anatomical location of the ovary, which is located deep in the pelvic floor, it has caused many difficult...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/66A61K48/00A61P35/00
Inventor 刘善荣惠宁高原刘淑鹏余喜亚胡晶晶
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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