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Application and related products of human EME1 gene

A gene and application technology, applied in the application of human EME1 gene and related products, can solve the problem of no report of EME1 function

Inactive Publication Date: 2020-03-06
CANCER CENT OF GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

All of the above suggest that EME1 may be a potential marker of cisplatin resistance in human tumors, but the role of EME1 in nasopharyngeal carcinoma has not been reported yet

Method used

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  • Application and related products of human EME1 gene
  • Application and related products of human EME1 gene
  • Application and related products of human EME1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Example 1 Preparation of RNAi lentivirus against human EME1 gene

[0120] 1. Screening for effective siRNA targets against the human EME1 gene

[0121] Retrieve EME1 (NM_152463) gene information from Genbank; design effective siRNA targets for EME1 gene. Table 1-1 lists the screened effective siRNA target sequences against the EME1 gene.

[0122] Table 1-1 is targeted at the siRNA target sequence of human EME1 gene

[0123] SEQ ID NO TargetSeq(5'-3') 1 GATAAAGAACGCCAGAATT 2 CTGAGAAGACAGGAAAGAA

[0124] 2. Preparation of lentiviral vector

[0125] For siRNA targets (take SEQ ID NO: 1 and 2 as examples), synthesize double-stranded DNA Oligo sequences (Table 1-2) containing Age I and EcoR I enzyme cleavage sites at both ends; Restriction endonuclease 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 fragments.

[0126] Table ...

Embodiment 2

[0144] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency

[0145] Human nasopharyngeal carcinoma CNE-2Z cells in logarithmic growth phase were digested with trypsin 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 value (MOI=10, the following examples are all 10), 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 mi...

Embodiment 3

[0164] Example 3 Celigo assay to detect the proliferation ability of tumor cells infected with EME1-shRNA lentivirus

[0165] Human nasopharyngeal carcinoma CNE-2Z cells in logarithmic growth phase were digested with trypsin 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, 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 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 plate was detected and read once...

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Abstract

The invention belongs to the field of biological medicine research, and particularly relates to an application of a human EME1 gene serving as a target in preparing a treatment medicament of nasopharyngeal carcinoma. According to extensive and deep research, after expression of the human EME1 gene is downregulated by adopting an RNAi method, proliferation of nasopharyngeal carcinoma cells can be effectively inhibited, apoptosis is promoted, and thus a growth process of nasopharyngeal carcinoma can be effectively controlled. An siRNA provided by the invention or a nucleic acid construct containing the siRNA sequence, and lentivirus can specifically inhibit the proliferation rate of nasopharyngeal carcinoma cells, promote apoptosis of the nasopharyngeal carcinoma cells, inhibit metastasis ofthe nasopharyngeal carcinoma cells, inhibit invasion of the nasopharyngeal carcinoma cells and inhibit growth of the nasopharyngeal carcinoma, so that nasopharyngeal carcinoma is treated, and a new direction is opened up for treatment of nasopharyngeal carcinoma.

Description

technical field [0001] The invention belongs to the field of biomedical research, and specifically relates to the use of human EME1 gene and related products. Background technique [0002] EME1 is an endonuclease identified as a member of the XPF endonuclease family. EME1 plays an important role in DNA repair following topoisomerase 1 inhibition and its upregulation confers resistance to multiple DNA damaging agents (doi:10.1016 / j.dnarep.2007.02.019). This protein cooperates with the methanolsulfonate-sensitive UV-sensitive 81 protein to form an endonuclease complex. The Mus81-Eme1 complex induces DNA double bond breaks near the ICL (interstrandcrosslinks, ICL) through structure-specific endonuclease activity, suggesting that the complex plays an important role in ICL repair (doi:10.1038 / sj.emboj. 7601344). Consistent with this finding, normal cells from mice deficient in the Mus81 or Eme1 genes were hypersensitive to mitomycin and cisplatin (doi.org / 10.1093 / emboj / cdg580;...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/113C12N15/867A61K31/713A61K45/00A61K48/00A61P35/00C12N7/01C12R1/93
CPCA61K31/713A61K45/00A61P35/00C12N7/00C12N15/1135C12N15/86C12N2310/141C12N2320/30C12N2740/15021C12N2740/15043C12Q1/6886C12Q2600/136C12Q2600/158
Inventor 齐斌陈冬平王梦瑶
Owner CANCER CENT OF GUANGZHOU MEDICAL UNIV
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