Applications of CIZI gene and relevant medicines

A gene and drug technology, applied in the use of CIZ1 gene and related drugs, can solve problems such as low survival rate

Active Publication Date: 2013-12-04
SHANGHAI JI KAI GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although cancer patients have undergone chemotherapy, radiotherapy and comprehensive treatment, their five-year survival rate is s

Method used

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  • Applications of CIZI gene and relevant medicines
  • Applications of CIZI gene and relevant medicines
  • Applications of CIZI gene and relevant medicines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1: Preparation of RNAi lentivirus against human CIZ1 gene

[0072] 1. Screening for effective siRNA targets against the human CIZ1 gene

[0073] Get CIZ1 (NM 012127.2 or NM 001131016.1 or NM 001131017.1 or NM 001131018.1 or NM 001131015.1) gene information from Genbank; design effective siRNA targets for CIZ1 gene (NM 012127.2) using the design software Genechem of Shanghai Genechem Technology Co., Ltd. point. In the coding sequence (CDS) region of the CIZ1 gene, a sequence of 21 bases was obtained starting from every other base, and Table 1 lists 13 effective siRNA target sequences for the CIZ1 gene.

[0074] Table 1 is targeted at the siRNA target sequence of human CIZ1 gene

[0075]

[0076] Synthesize a double-stranded DNA Oligo sequence (Table 2) containing Age I and EcoR I restriction sites at both ends for the siRNA target (take SEQ ID NO: 4 as an example); use Age I and EcoR I restriction endonucleases Act on the pGCSIL-GFP vector (provided by Shan...

Embodiment 2

[0094] Example 2: Real-time fluorescent quantitative RT-PCR method to detect the silencing efficiency of CIZ1 gene

[0095] Human liver cancer SMMC7721, lung cancer H1299 cells, breast cancer MCF-7 cells and glioma U87 cells in logarithmic growth phase were trypsinized to make cell suspension (the number of cells was about 5×10 4 / ml) were seeded in 6-well plates and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, SMMC7721:20, H1299:10, MCF-7:20, U87:10) value, add an appropriate amount of the virus prepared in Example 1, and replace the medium after culturing for 24 hours. After 5 days, cells were harvested. Total RNA was extracted according to the instruction manual of Invitrogen's Trizol. According to Promega's M-MLV operating instructions, RNA was reverse-transcribed to obtain cDNA (see Table 6 for the reverse transcription reaction system, react at 42 °C for 1 h, and then bathe in a water bath at 70 °C for 10 min to...

Embodiment 3

[0102] Example 3: Detection of proliferation ability of tumor cells infected with CIZ1-siRNA lentivirus

[0103] Human liver cancer SMMC7721 cells, lung cancer H1299 cells, breast cancer MCF-7 cells and glioma U87 cells in logarithmic growth phase were digested with trypsin to make cell suspension (the number of cells was about 5×10 4 / ml) were seeded in 6-well plates and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, SMMC7721: 20, H1299: 10, MCF-7: 20, U87: 10), add an appropriate amount of virus, replace the medium after 24 hours of culture, and collect the cells in the right cells after the infection time reaches 5 days. Cells of each experimental group in the growth phase were counted. The complete medium was resuspended into a cell suspension (2×10 4 / ml), inoculate a 96-well plate at a cell density of about 2000 cells / well. 5 replicate wells in each group, 100 μl per well. After laying the board, place at 37°C, ...

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Abstract

The invention discloses applications of CIZI gene and relevant medicines, discloses applications of CIZI gene in preparation or screening of oncological therapeutic medicines of liver cancer, lung cancer, breast carcinoma or glioma, further constructs separated nucleic acid molecules, CIZI gene interference nucleic acid construction bodies and CIZI gene interference lentivirus which can decrease expression of CIZI gene in tumor cells and discloses applications thereof. The nucleic acid molecules, nucleic acid construction bodies or CIZI gene interference lentivirus can inhibit the expression of the CIZI gene specifically. Especially, lentivirus can infect target cells efficiently, the expression of the CIZI gene in the target cells is inhibited efficiently, furthermore the growth of the tumor cells is inhibited, the apoptosis of the tumor cells is promoted, and the applications of CIZI gene and the relevant medicines are significant in oncotherapy.

Description

technical field [0001] The present invention relates to the field of biotechnology, and more specifically relates to the use of CIZ1 gene and related medicines. Background technique [0002] The initiation of DNA replication is a major control point in the mammalian cell cycle and the site of action for many unregulated gene products in cancer (Hanahan D, Weinberg RA. The hallmarks of cancerer. Cell. 2000;100(1) :57-70.). The DNA replication initiation process is regulated by the cyclin-dependent protein kinase (CDK,) family, and this family plays a very important role in the transition from G1 phase to S phase (Bell SP, Dutta A. DNA replication in eukaryotic cells. Annu Rev Biochem.). There are two families of CDK inhibitors that negatively regulate CDK in the regulation of cell cycle proteins in the G1 phase (HunterT, Pines J. Cyclins and cancer. II: CyclinD and CDK inhibitors come of age. Cell. 1994; 79 (4 ):573–582. Sherr CJ, Roberts JM. Inhibitors of mammalian G, cyc...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/113C12N15/867C12N15/11A61K48/00A61P35/00
Inventor 韩海雄孙琴谭畅沈浩金杨晟瞿红花曹跃琼
Owner SHANGHAI JI KAI GENE TECH CO LTD
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