Applications and correlated medicament of human STIM1 gene

A gene and drug technology, applied in the use of human STIM1 gene and related drug fields, can solve the problem of lack of in-depth reports on functional research and other problems

Active Publication Date: 2012-05-02
SHANGHAI GENBASE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(Yang SY, Zhang JL, Huang XY. Orai1 and STIM1 Are Critical for Breast Tumor Cell Migration and Metastasis, Cancer Cell, 2009; 15(2): 124-134) In summary, STIM1 plays a role in tumo...

Method used

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  • Applications and correlated medicament of human STIM1 gene
  • Applications and correlated medicament of human STIM1 gene
  • Applications and correlated medicament of human STIM1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Preparation of RNAi lentivirus against human STIM1 gene

[0068] 1. Screening for effective siRNA targets against the human STIM1 gene

[0069] Get the STIM1 (NM 003156) gene information from Genbank; use the design software Genechem of Shanghai Jikai Gene Chemical Technology Co., Ltd. to design effective siRNA targets for the STIM1 gene. In the coding sequence (CDS) region of the STIM1 gene, a sequence of 21 bases was obtained starting at every other base, and Table 1 lists 54 effective siRNA target sequences for the STIM1 gene.

[0070] Table 1 is targeted at the siRNA target sequence of human STIM1 gene

[0071]

[0072]

[0073]

[0074] Aiming at the siRNA target (taking SEQ ID NO: 40 as an example), synthesize a double-stranded DNA Oligo sequence (Table 2) with AgeI and EcoRI restriction sites sticky ends at both ends; act on pGCSIL- GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd., figure 1 ), to linearize it, and i...

Embodiment 2

[0093] Embodiment 2: Real-time fluorescent quantitative RT-PCR method detects the silencing efficiency of STIM1 gene

[0094] Human lung cancer H1299 cells, liver cancer SMMC-7721 cells, breast cancer MCF-7 cells, prostate cancer PC-3 cells and human pancreatic cancer Panc-1 cells in logarithmic growth phase were digested with trypsin to make cell suspension (cell The number is about 5×10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (the MOI of H1299 is 10, the MOI of Panc-1, SMMC-7721, MCF-7 and PC-3 is 20) value, add the virus prepared in Example 1 of appropriate amount, culture medium is replaced after 24h , after the infection time reached 5 days, the cells were collected. Total RNA was extracted according to the instruction manual of Invitrogen's Trizol. According to the M-MLV instruction manual of Promega Company, RNA was reverse-transcribed to obtain cDNA (see Table 7 for...

Embodiment 3

[0101] Example 3: Detection of proliferation ability of tumor cells infected with STIM1-shRNA lentivirus

[0102] Human lung cancer H1299 cells, liver cancer SMMC-7721 cells, breast cancer MCF-7 cells, prostate cancer PC-3 cells and human pancreatic cancer Panc-1 cells in logarithmic growth phase were digested with trypsin to make cell suspension (cell The number is about 5×10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (the MOI of H1299 is 10, the MOI of Panc-1, SMMC-7721, MCF-7 and PC-3 is 20), add an appropriate amount of STIM1-shRNA virus, replace the medium after culturing for 24 hours, and wait for the infection After the staining time reached 5 days, the cells of each experimental group in the logarithmic growth phase were collected. 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 / well. 5...

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Abstract

The invention discloses applications a correlated medicament of a human STIM1 gene. The invention discloses applications of the human STIM1 gene in tumor treatment, tumor diagnosis and medicament preparation. The invention also further constructs a human STIM1 gene small-interfering ribonucleic acid (siRNA), a human STIM1 gene interfering nucleic acid construct, a human STIM1 gene interfering slow virus and applications thereof. The siRNA or nucleic acid construct and slow virus comprising the siRNA sequence can specifically inhibit the expression of the human STIM1 gene, particularly, the slow virus can infect a target cell efficiently so as to effectively inhibit the expression of STIM1 gene in the target cell, thus further inhibiting the growth of tumor cells and promoting apoptosis of the tumor cells. Therefore, the human STIM1 gene has important significance in tumor treatment.

Description

technical field [0001] The present invention relates to the field of biotechnology, and more specifically relates to the use of human STIM1 gene and related medicines. Background technique [0002] RNA interference (RNA interference, RNAi) uses double-stranded RNA to mediate sequence-specific post-transcriptional gene silencing, and has become an emerging and effective method for studying gene function, and is expected to become a tool for gene therapy of tumors and other diseases (Izquierdo M. Short interfering RNAs as a tool for cancer gene therapy. Cancer Gene Ther. 2005; 12(3): 217-27.). Lentivirus (lentivirus) vector is an efficient gene transduction tool, which has a wider host range than retrovirus vector, and can stably introduce exogenous hairpin RNA (short hairpin RNA, shRNA) sequences into aperiodic and mitotic cells cells after. shRNA will be automatically processed into small interfering RNA (small interfering RNA, siRNA) in cells, causing the degradation of m...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12N15/113C12N15/867A61K48/00A61P35/00
CPCC12Q1/6886C12Q2600/136C12Q2600/158
Inventor 韩海雄孙琴顾雪峰沈浩金杨晟瞿红花曹跃琼
Owner SHANGHAI GENBASE BIOTECH CO LTD
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