Application and correlation product of human IMPA2 gene

A gene and application technology, applied in the field of application of human IMPA2 gene and related products, can solve problems such as the absence of IMPA2 gene

Inactive Publication Date: 2020-03-31
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

[0003] There is no relevant report about IMPA2 gene being used in the treatment of cervical cancer

Method used

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  • Application and correlation product of human IMPA2 gene
  • Application and correlation product of human IMPA2 gene
  • Application and correlation product of human IMPA2 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1 Preparation of RNAi lentivirus for human IMPA2 gene

[0103] 1. Screening for effective siRNA targets against the human IMPA2 gene

[0104] Retrieve IMPA2 (NM_014214) gene information from Genbank; design effective siRNA targets for IMPA2 gene. Table 1-1 lists the screened effective siRNA target sequences against the IMPA2 gene.

[0105] Table 1-1 siRNA target sequence targeting human IMPA2 gene

[0106] SEQ ID NO TargetSeq(5'-3') 1 CTTACAGACGATTAACTAT

[0107] 2. Preparation of lentiviral vector

[0108] 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.

[0109] Table 1-2 Double-stranded DNA Oligo with sticky ends containing Age I...

Embodiment 2

[0128] Example 2 Detection of silencing efficiency of genes infected with IMPA2-siRNA lentivirus

[0129] Human cervical cancer SiHa cells in logarithmic growth phase were trypsinized to make cell suspension (the number of cells was 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 (MOI, SiHa: 20), 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.

[0130] a) Detection of gene silencing efficiency by real-time fluorescent quantitative RT-PCR

[0131] 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, ...

Embodiment 3

[0168] Example 3 Detection of proliferation ability of tumor cells infected with IMPA2-siRNA lentivirus

[0169] Human cervical cancer SiHa cells in logarithmic growth phase were trypsinized to make cell suspension (the number of cells was 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 (MOI, SiHa: 20), 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 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 1000 / well. 5 replicate wells in each group, 100 μl per well. After laying the board, place at 37°C, 5% CO 2Incubator cultivation. From the second day after plating, the plate was detected and read once a day with a Celi...

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PUM

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Abstract

The invention belongs to the field of biopharmaceutical research, and particularly relates to an application of a human IMPA2 gene as a target to preparation of drugs for treating cervical cancer or drugs for diagnosis of cervical cancer. Through wide and deep research, the inventor finds that after an RNAi method is adopted for reducing the expression of the human IMPA2 gene, proliferation of cervical carcinoma cells can be effectively restrained, apoptosis is promoted, and growth process of the cervical cancer can be effectively controlled. siRNA or a nucleic acid constructor containing thesiRNA sequence, and a slow virus provided by the invention can specially restrain proliferation rate of the cervical carcinoma cells, can promote the apoptosis of the cervical carcinoma cells, can restrain cloning of the cervical carcinoma cells, can influence the period of the cervical carcinoma cells, and can restrain growth of the cervical cancer, so as to treat the cervical cancer and open upa new direction for treatment of the cervical cancer.

Description

technical field [0001] The invention belongs to the field of biomedical research, and specifically relates to the use of human IMPA2 gene and related products. Background technique [0002] The IMPA2 gene encodes inositol monophosphatase, an enzyme that catalyzes the dephosphorylation of inositol monophosphate to free inositol, an enzymatic pathway that is critical in cellular function because inositol is the synthetic membrane lipid phosphatidylinositol (PI) substrate. Dephosphorylation of inositol monophosphate is the rate-limiting step of PI regeneration [Ohnishi T., Ohba H., Seo K.C., Im J., SatoY., Iwayama Y. Spatial expression patterns and biochemical properties distinguish a second myo-inositol monophosphate IMPA2 from IMPA1. J BiolChem. 2007; 282(1):637–646., Harwood A.J. Lithium and bipolar mood disorder: theinositol-depletion hypothesis revisited. Mol Psychiatry. 2005; 10(1):117–126.]. When cells are stimulated, PI 4,5-bisphosphate is hydrolyzed by phospholipase ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/113C12N15/867A61K45/00A61K31/713A61P35/00
CPCA61K45/00A61P35/00C12N15/1137C12N15/86C12N2310/14C12N2740/15043C12N2800/107C12Q1/6886C12Q2600/136C12Q2600/158C12Y301/03025C12N2310/531
Inventor 王敏李先平杨敏张衎刘蕾
Owner THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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