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Applications of human PTP4A3 gene and related drugs thereof

A gene and drug technology, applied in the field of the use of human PTP4A3 gene and related drugs, can solve the problems such as unclear molecular mechanism

Inactive Publication Date: 2015-09-23
SHANGHAI GENECHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The research on PRL-3 and tumor metastasis has achieved some results, but its molecular mechanism is still unclear

Method used

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  • Applications of human PTP4A3 gene and related drugs thereof
  • Applications of human PTP4A3 gene and related drugs thereof
  • Applications of human PTP4A3 gene and related drugs thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1: Preparation of RNAi lentivirus against human PTP4A3 gene

[0088] 1. Screening for effective siRNA targets against the human PTP4A3 gene

[0089] Retrieve PTP4A3 (NM_032611) gene information from Genbank; design effective siRNA targets for PTP4A3 gene. Table 1 lists 13 effective siRNA target sequences for the PTP4A3 gene.

[0090] Table 1 is targeted at the siRNA target sequence of human PTP4A3 gene

[0091]

[0092] 2. Preparation of lentiviral vector

[0093] For the siRNA target (take SEQ ID NO: 2 as an example), synthesize a double-stranded DNA Oligo sequence containing Age I and EcoR I restriction sites at both ends (Table 2); use Age I and EcoR I restriction endonucleases Act on the pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd., figure 1 ), to linearize it, and identify the digested fragments by agarose gel electrophoresis.

[0094] Table 2 Double-stranded DNA Oligo with sticky ends containing Age I and EcoR I ...

Embodiment 2

[0112] Embodiment 2: Real-time fluorescent quantitative RT-PCR method detects the silencing efficiency of PTP4A3 gene

[0113] Human breast cancer MCF-7 cells and lung cancer H1299 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, MCF-7:10, H1299:1) value, an appropriate amount of the virus prepared in Example 1 was added, the culture medium was replaced after 24 hours of cultivation, 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 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 inactivate...

Embodiment 3

[0121] Example 3: Detection of proliferation ability of tumor cells infected with PTP4A3-siRNA lentivirus

[0122] Human breast cancer MCF-7 cells and lung cancer H1299 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, MCF-7:20, H1299:1), add an appropriate amount of virus, and replace the medium after 24 hours of culture. After the infection time reaches 5 days, collect the cells in each experimental group in the logarithmic growth phase . 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, 5% CO 2 Incubator cultivation. From the second day after plating, the plate was detected and read once a...

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Abstract

The invention discloses applications of human PTP4A3 gene and related drugs thereof. Applications of human PTP4A3 gene in tumor treatment, tumor diagnosis, and drug preparation are disclosed. The invention further constructs human PTP4A3 gene micromolecule interference RNA, human PTP4A3 gene interference nucleic acid construct, and human PTP4A3 gene interference slow virus, and discloses the applications thereof. The provided siRNA or nucleic acid construct containing the siRNA and slow virus can inhibit the expression of human PTP4A3 gene specifically, especially the slow virus can infect target cells efficiently and inhibit the expression of PTP4A3 gene in target cells efficiently, thus the tumor cell growth is inhibited, apoptosis of tumor cells is promoted, and thus the provided siRNA, nucleic acid construct, and slow virus have an important meaning in tumor treatment.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to the use of human PTP4A3 gene and related medicines. Background technique [0002] RNA interference (RNA interference, RNAi) is the use of short double-stranded RNA (dsRNA) composed of nucleotides for post-transcriptional gene silencing. It can efficiently and specifically block the expression of a specific gene in the body, leading to its degradation, thereby causing the silencing of a specific gene in the organism, and causing the cells to show the absence of a certain gene phenotype. It is a commonly used research gene emerging in recent years. Functional, laboratory techniques for finding cures for disease. Studies have shown that double-stranded RNA with a length of 21-23nt can specifically cause RNAi at the transcriptional and post-transcriptional levels (Tuschl T, Zamore PD, Sharp PA, Bartel DP.RNAi: double-stranded RNA directs the ATP-dependent cleavage of mR...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/113C12N15/867C12N7/01A61K48/00A61P35/00
CPCA61K48/0016C12N15/1135C12N15/867C12Q1/6886
Inventor 朱向莹孙琴谭畅张晓慧金杨晟瞿红花曹跃琼
Owner SHANGHAI GENECHEM
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