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Use of human gsdmb gene and related products

A technology of use and product, applied in the field of use of human GSDMB gene and related products, can solve the problems that GSDMB has not been reported in bladder cancer research, and achieve the effect of inhibiting bladder cancer cell metastasis, inhibiting proliferation, and promoting cell apoptosis

Active Publication Date: 2022-02-08
THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The research of GSDMB in bladder cancer has not been reported

Method used

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  • Use of human gsdmb gene and related products
  • Use of human gsdmb gene and related products
  • Use of human gsdmb gene and related products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Example 1 Preparation of RNAi lentivirus for human GSDMB gene

[0120] 1. Screening for effective siRNA targets against the human GSDMB gene

[0121] Retrieve GSDMB (NM_018530) gene information from Genbank; design effective siRNA targets for GSDMB gene. Table 1-1 lists the screened effective siRNA target sequences against the GSDMB gene.

[0122] Table 1-1 is targeted at the siRNA target sequence of human GSDMB gene

[0123] SEQ ID NO TargetSeq(5'-3') 1 GGAGAGTTGATAGTGAGAT

[0124] 2. Preparation of lentiviral vector

[0125] Aim at the siRNA target (take SEQ ID NO: 1 as an example) to synthesize a double-stranded DNA Oligo sequence (Table 1-2) with Age I and EcoR I restriction sites at both ends; Dicer acts on the pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd.) to linearize it, and agarose gel electrophoresis identifies the digested fragment.

[0126] Table 1-2 Double-stranded DNA Oligo with sticky ends cont...

Embodiment 2

[0143] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency

[0144] The human bladder cancer 5637 and T24 cells in the logarithmic growth phase were respectively trypsinized to make a cell suspension (the number of cells was about 5×10 4 / ml) were inoculated in 6-well plates, and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, 10 for both cell lines), an appropriate amount of the lentivirus prepared in Example 1 was added, the culture medium was replaced after 24 hours of culture, and the cells were collected after the infection time reached 5 days. 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 2-1 for the reverse transcription reaction system, react at 42°C for 1 hour, and then bathe in a water bath at 70°C for 10 minutes to in...

Embodiment 3

[0151] Example 3 Celigo detects the proliferation ability of tumor cells infected with GSDMB-shRNA lentivirus

[0152] The human bladder cancer 5637 and T24 cells in the logarithmic growth phase were respectively trypsinized to make a cell suspension (the number of cells was about 5×10 4 / ml) were inoculated in 6-well plates, and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, 10 for both cell lines), an appropriate amount of virus was added, and the culture medium was replaced after 24 hours of culture. After the infection time reached 5 days, the cells in 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 replicate wells in each group, 100 μl per well. After laying the board, place at 37°C, 5% CO 2 Incubator cultivation. From the second day after plating, the...

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Abstract

The invention belongs to the field of biomedical research, and specifically relates to the use of human GSDMB gene as a target in the preparation of bladder cancer treatment drugs. After extensive and in-depth research, the present invention finds that the RNAi method can effectively inhibit the proliferation of bladder cancer cells, promote cell apoptosis, and effectively control the growth process of bladder cancer after down-regulating the expression of human GSDMB gene. The siRNA provided by the present invention or the nucleic acid construct containing the siRNA sequence, lentivirus can specifically inhibit the proliferation rate of bladder cancer cells, promote the apoptosis of bladder cancer cells, inhibit the metastasis of bladder cancer cells, inhibit the invasion of bladder cancer cells, and inhibit bladder cancer growth, thereby treating bladder cancer and opening up a new direction for bladder cancer treatment.

Description

technical field [0001] The invention belongs to the field of biomedical research, and specifically relates to the use of human GSDMB gene and related products. Background technique [0002] GSDMB is a member of the GSDM protein family. Members of this protein family are relatively conserved in structure, and are divided into N-terminal region, hinge region and C-terminal region. [0003] Gasdermin B (GSDMB) gene is related to various immune diseases (such as asthma, diabetes, enteritis, etc.), and it is highly expressed in leukocytes of patients with sepsis, and there is a large amount of N-terminal GSDMD protein in the blood of patients with sepsis. Through experimental verification, it is found that GSDMB can specifically activate caspase-4 enzyme, and the GSDMB-N-terminal (1-83AA) domain binds to the CARD domain of caspase-4, prompting caspase-4 to cut GSDMD, thereby releasing GSDMD-N-terminal protein , release a large number of inflammatory factors, non-classical pyrop...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K45/00A61K48/00A61K31/713A61P35/00A61P35/04C12N15/113C12N15/867C12N7/01
CPCA61K45/00A61K31/713A61K48/005A61P35/00A61P35/04C12N15/113C12N15/86C12N7/00C12N2310/14C12N2740/15021C12N2740/15043
Inventor 熊伟朱梁钟朝晖王荫槐赵晓昆
Owner THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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