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Application of human UAP1L1 gene and related product

A gene and application technology, applied in the application of human UAP1L1 gene and related products, can solve the problem of lack of UAP1L1 gene

Pending Publication Date: 2020-11-13
上海市静安区中心医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report on the use of UAP1L1 gene in the treatment of gastric cancer

Method used

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  • Application of human UAP1L1 gene and related product
  • Application of human UAP1L1 gene and related product
  • Application of human UAP1L1 gene and related product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1 Preparation of RNAi lentivirus against human UAP1L1 gene

[0108] 1. Screening for effective siRNA targets against the human UAP1L1 gene

[0109] Retrieve UAP1L1 (NM_207309) gene information from Genbank; design effective siRNA targets for UAP1L1 gene. Table 1-1 lists the screened effective siRNA target sequences against the UAP1L1 gene.

[0110] Table 1-1 is targeted at the siRNA target sequence of human UAP1L1 gene

[0111] SEQ ID NO TargetSeq(5'-3') 1 CCTTCTTACTGCAAACCAT

[0112] 2. Preparation of lentiviral vector

[0113] 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.

[0114] Table 1-2 Double-stranded DNA Oligo with sticky...

Embodiment 2

[0133] Example 2 Detection of gene silencing efficiency of tumor cells infected with UAP1L1-siRNA lentivirus

[0134] Human gastric cancer AGS and MGC80-3 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, AGS:10, MGC80-3:20), an appropriate amount of the lentivirus prepared in Example 1 was added, and the culture medium was replaced after 24 hours of culture. After the infection time reached 5 days, the cells were collected.

[0135] a) Real-time fluorescent quantitative RT-PCR method

[0136] 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, an...

Embodiment 3

[0170] Example 3 Detection of proliferation ability of tumor cells infected with UAP1L1-siRNA lentivirus

[0171] Human gastric cancer AGS and MGC80-3 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, AGS:10, MGC80-3:20), 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 of 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 15,000 / 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 Celig...

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Abstract

The invention belongs to the field of biomedical research, and particularly relates to application of a human UAP1L1 gene as a target in preparation of gastric cancer treatment drugs or gastric cancerdiagnosis drugs. It is found through extensive and intensive researches that after the expression of the human UAP1L1 gene is down-regulated by adopting an RNAi method, the proliferation of gastric cancer cells can be effectively inhibited, the apoptosis can be promoted, and the growth process of gastric cancer can be effectively controlled. The siRNA or the nucleic acid construct containing thesiRNA sequence and the lentivirus provided by the invention can specifically inhibit the proliferation rate of gastric cancer cells, promote apoptosis of the gastric cancer cells, inhibit tumor formation of the gastric cancer cells, inhibit cloning of the gastric cancer cells and inhibit growth of the gastric cancer, so that the gastric cancer is treated, and a new direction is opened up for treatment of the gastric cancer.

Description

technical field [0001] The invention belongs to the field of biomedical research, and specifically relates to the use of human UAP1L1 gene and related products. Background technique [0002] UAP1L1 is a protein-coding gene. Its related pathways include the metabolism and metabolism of amino sugars and nucleotide sugars. GO (gene Ontology) annotation analysis showed that the gene was related to uridine acyltransferase activity. An important homologue of this gene is UAP1. UAP1-like-1 (UAP1L1) is a protein with approximately 59% sequence homology to UAP1, and the methylation status of this gene is associated with the recurrence-free survival rate of breast cancer patients [Genome-wide DNA methylation profiling of CpG islands in breast cancer identifies novel genes associated with tumorigenicity. Hill VK, Ricketts C, Bieche I, Vacher S, Gentle D, Lewis C, Maher ER, Latif F. Cancer Res. 2011 Apr 15; 71(8):2988-99. doi: 10.1158 / 0008 -5472. CAN-10-4026. Epub 2011 Mar 1. Erratu...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867A61K31/713A61K48/00A61P35/00
CPCC12N15/1135C12N15/86A61K31/713A61P35/00C12N2310/141C12N2310/14C12N2310/531C12N2740/15043
Inventor 顾而立黄耀王虹唐峰车智慧周磊
Owner 上海市静安区中心医院
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