Application of inhibitor of GINS2 genes or protein to preparation of antitumor drugs

A 1. GINS2, inhibitor technology, applied in the field of biomedicine, can solve the problem of less GINS2, and achieve the effect of promoting apoptosis and significant differences in expression

Active Publication Date: 2017-05-10
SHANGHAI SEVENTH PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are few reports on the role of GINS2 in tumor-related fields.

Method used

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  • Application of inhibitor of GINS2 genes or protein to preparation of antitumor drugs
  • Application of inhibitor of GINS2 genes or protein to preparation of antitumor drugs
  • Application of inhibitor of GINS2 genes or protein to preparation of antitumor drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Preparation of RNAi lentivirus against human GINS2 gene

[0039] The technical route is: retrieve the human GINS2 gene sequence from Genbank; predict the siRNA site; synthesize an effective siRNA sequence targeting the GINS2 gene, and double-stranded DNA Oligo with sticky ends with restriction sites at both ends; Strand DNA Oligo connection to construct an RNAi plasmid expressing the siRNA sequence of the GINS2 gene; co-transfect the RNAi plasmid and the auxiliary vector required for lentiviral packaging into human embryonic kidney cell 293T cells to produce ethnic lentiviral particles, which can efficiently silence GINS2 Genetic lentiviruses.

[0040] 1. RNA interference target design and double-stranded DNA oligo preparation

[0041] (1) Design and synthesis of effective siRNA targets against human GINS2 gene

[0042] Retrieve GINS2 (NM_016095) gene information from Genbank; design effective siRNA targets for GINS2 gene. Table 1 is the effective siRNA targ...

Embodiment 2

[0104] Embodiment 2: RT-PCR detects the silencing efficiency of GINS2 gene

[0105] Human papillary thyroid carcinoma K1 cells in the logarithmic growth phase were trypsinized to make a cell suspension (the number of cells was about 5×10 4 / ml), seeded in 6-well plates, and cultured until the cell confluency reached about 30%. An appropriate amount of the virus prepared in Example 1 was added, the culture medium was replaced after 6 hours of cultivation, and the cells were collected after the infection time reached 3 days.

[0106] RNA extraction and cDNA synthesis: ① collect samples, Trizol lysis: collect cells, centrifuge at 2000rpm for 5min, remove the supernatant, add 1mL Trizol to the cell pellet, mix well, let stand at room temperature for 5min, and then transfer to a new 1.5mL EP tube middle. ② Add 200 μL chloroform to each tube, turn the EP tube upside down by hand for 15 seconds, and let it stand at room temperature for 10 minutes. ③ 4°C, 12800rpm, centrifuge for 1...

Embodiment 3

[0111] Example 3: Detection of proliferation ability of tumor cells infected with GINS2-siRNA lentivirus

[0112] Human papillary thyroid carcinoma K1 cells in the logarithmic growth phase were trypsinized to make a cell suspension (the number of cells was about 5×10 4 / ml), seeded in 6-well plates, and cultured until the cell confluency reached about 30%. An appropriate amount of the virus prepared in Example 1 was added, the culture medium was replaced after 6 hours of cultivation, and the cells were collected after the infection time reached 5 days. The complete medium was resuspended into a cell suspension (2×10 4 / ml), seeded in a 96-well plate at a cell density of about 2000 / well. Three replicate wells per group, 100 μl per well. After laying the plates, place them in a cell culture incubator. From the second day after plating, use Celigo to detect and read the plate once a day, and continuously detect and read the plate for 3-5 days; by adjusting the input parameter...

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Abstract

The invention relates to application of an inhibitor of GINS2 genes or protein to the preparation of antitumor drugs. A small-molecular interference RNA sequence aiming at the human GINS2 genes, an RNA interference vector and an RNA interference lentivirus are designed, the silencing efficiency of the GINS2 genes and influence of the GINS2-siRNA lentivirus on the proliferation capacity and apoptosis level of tumor cells are further detected, results show that the proliferation and growth of the tumor cells can be inhibited effectively and the apoptosis of the tumor cells can be promoted after an RNAi method is used to lower the expression of the GINS2 genes, the results indicate that the GINS2 genes are proto-oncogenes and can be used as the targets of tumor treatment, and the RNAi method for silencing the expression of the GINS2 can be used as an effective method to inhibit tumor development. The built siRNA, the RNA interference vector and the RNA interference lentivirus can be used for preparing efficient antitumor drugs.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to the application of GINS2 gene or protein inhibitors in the preparation of antitumor drugs. Background technique [0002] With the changes in human living habits and behavior patterns, as well as environmental pollution and population aging brought about by the development of agriculture and industry, malignant tumors have become the main cause of death for humans. The latest data from the International Cancer Research Center of the World Health Organization (IARC / WHO) shows that the incidence of malignant tumors is on the rise in the world. In 2008, 7.6 million people died of malignant tumors, of which 64% occurred in developing countries. In my country, the situation of cancer prevention and treatment is also extremely severe. The latest report shows that in the past 30 years, the incidence and mortality of malignant tumors in my country have shown an obvious upward trend; i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61K31/713C12N15/113C12N15/867C12N7/01A61P35/00C12Q1/68G01N33/574G01N33/573
CPCA61K31/713A61K45/00C12N7/00C12N15/1137C12N15/86C12N2310/14C12N2740/15021C12N2740/15043C12Q1/6886C12Q2600/158G01N33/573G01N33/57407
Inventor 夏伟叶颖倪晶庄菊花王国玉
Owner SHANGHAI SEVENTH PEOPLES HOSPITAL
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