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Application of human CCDC154 gene and related product

A technology of genes and uses, applied in medical preparations containing active ingredients, gene therapy, genetic engineering, etc.

Active Publication Date: 2020-03-06
THE FIRST AFFILIATED HOSPITAL OF MEDICAL COLLEGE OF XIAN JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are few reports on the function of this gene

Method used

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  • Application of human CCDC154 gene and related product
  • Application of human CCDC154 gene and related product
  • Application of human CCDC154 gene and related product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Example 1 Preparation of RNAi Lentivirus for Human CCDC154 Gene

[0102] 1. Screening of effective siRNA targets for human CCDC154 gene

[0103] Get CCDC154 (NM_001143980) gene information from Genbank; design effective siRNA targets for CCDC154 gene. Table 1-1 lists the screened effective siRNA target sequences for CCDC154 gene.

[0104] Table 1-1 siRNA target sequence targeting human CCDC154 gene

[0105] SEQ ID NO TargetSeq(5’-3’) 1 ACAAGTGCCTGCTTCATAA

[0106] 2. Preparation of lentiviral vector

[0107] For siRNA target (take SEQ ID NO:1 as an example), synthesize double-stranded DNA Oligo sequence (Table 1-2) with Age I and EcoR I restriction sites on both ends (Table 1-2); within the restriction of Age I and EcoR I Dicer acts on pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd.) to linearize it, and agarose gel electrophoresis to identify the digested fragments.

[0108] Table 1-2 Double-stranded DNA Oligo with sticky ends containing Age...

Embodiment 2

[0127] Example 2 Real-time fluorescence quantitative RT-PCR method to detect gene silencing efficiency

[0128] Human liver cancer BEL-7404 cells and SMMC-7721 cells in the logarithmic growth phase were trypsinized to prepare a cell suspension (the number of cells is about 5×10 4 / ml) inoculate in a 6-well plate and culture until the cell confluence reaches about 30%. According to the multiplicity of infection (MOI, BEL-7404: 10, SMMC-7721: 10), add an appropriate amount of the lentivirus prepared in Example 1, culture for 24 hours and then change the medium. After the infection time reaches 5 days, collect the cells . According to Invitrogen's Trizol operating instructions, total RNA was extracted. According to the M-MLV operating instructions of Promega, reverse transcription of RNA to obtain cDNA (see Table 2-1 for the reverse transcription reaction system, react at 42°C for 1 hour, and then inactivate the reverse transcriptase in a water bath at 70°C for 10 minutes).

[0129]...

Embodiment 3

[0135] Example 3 Detection of the proliferation ability of tumor cells infected with CCDC154-siRNA lentivirus

[0136] Human liver cancer BEL-7404 cells and SMMC-7721 cells in the logarithmic growth phase were trypsinized to prepare a cell suspension (the number of cells is about 5×10 4 / ml) inoculate in a 6-well plate and culture until the cell confluence reaches about 30%. According to the multiplicity of infection (MOI, BEL-7404: 10, SMMC-7721: 10), add an appropriate amount of virus, culture for 24 hours and then change the medium. After the infection time reaches 5 days, collect each experiment in the logarithmic growth phase Group of cells. Resuspend in complete medium into a cell suspension (2×10 4 / ml), inoculate a 96-well plate with a cell density of approximately 3000 cells / well. Each group has 5 duplicate wells, each well is 100μl. After laying the board, place it at 37℃, 5% CO 2 Culture in an incubator. Beginning on the second day after plating, use Celigo instrume...

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Abstract

The invention belongs to the field of biomedical research, and particularly relates to an application of a human CCDC154 gene as a target in preparation of a liver cancer treatment drug. Through the wide and deep research, proliferation of liver cancer cells can be effectively inhibited after expression of the human CCDC154 gene is reduced by adopting an RNAi method, apoptosis is promoted, and thegrowth process of liver cancer can be effectively controlled. The siRNA or a nucleic acid construct containing the siRNA sequence and the lentivirus provided by the invention can specifically inhibitthe proliferation rate and proliferation activity of liver cancer cells, promote apoptosis of the liver cancer cells, inhibit cloning of the liver cancer cells and inhibit growth of liver cancer, sothat liver cancer is treated, and a new direction is opened up for liver cancer treatment.

Description

Technical field [0001] The invention belongs to the field of biomedicine research, and specifically relates to the use of the human CCDC154 gene and related products. Background technique [0002] Coiled-Coil Domain Containing 154 (CCDC154, Coiled-Coil Domain Containing 154) is a new coding gene, which is widely expressed in human and mouse tissues. The sub-cells are mainly located in endosomes. [0003] The 5kb sequence deletion in the 1-6 exon region of CCDC154 gene is associated with spontaneous autosomal recessive osteosclerosis mutant mice (ntl mutation). There are few reports on the related functions of this gene. [0004] It has been reported that CCDC154 is related to the pathogenesis of bone sclerosis. Overexpression of CCDC154 inhibits the proliferation activity and soft agar cloning ability of HEK293 cells by inducing G2 / M cycle arrest, but has no effect on the apoptosis ability. It shows that CCDC154 is a new type of osteopetrosis-related gene involved in cell cycle re...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/113C12N15/867C12N7/01A61K31/713A61P35/00A61K45/00A61K48/00
CPCC12Q1/6886C12N15/1135C12N15/86C12N7/00A61K31/713A61P35/00A61K45/00C12Q2600/136C12Q2600/158C12N2310/141C12N2310/531C12N2740/15043C12N2740/15021
Inventor 石磊孟磊
Owner THE FIRST AFFILIATED HOSPITAL OF MEDICAL COLLEGE OF XIAN JIAOTONG UNIV
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