Application of human EXOSC2 gene and related product

A gene and application technology, applied in the field of application of human EXOSC2 gene and related products, can solve problems such as the lack of EXOSC2 gene

Pending Publication Date: 2020-06-19
唐蔚 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] So far, there are no related reports about the u

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  • Application of human EXOSC2 gene and related product
  • Application of human EXOSC2 gene and related product
  • Application of human EXOSC2 gene and related product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Example 1 Preparation of RNAi Lentivirus for Human EXOSC2 Gene

[0110] 1. Screening of effective siRNA targets against human EXOSC2 gene

[0111] Retrieve EXOSC2 (NM_014285) gene information from Genbank; design an effective siRNA target for EXOSC2 gene. Table 1-1 lists the screened effective siRNA target sequences for EXOSC2 gene.

[0112] Table 1-1 siRNA target sequences targeting human EXOSC2 gene

[0113] SEQ ID NO TargetSeq(5'-3') 1 GAAGCTCATTGCATCTGTT

[0114] 2. Preparation of Lentiviral Vectors

[0115] Synthesize a double-stranded DNA Oligo sequence (Table 1-2) with sticky ends containing Age I and EcoR I restriction sites at both ends against the siRNA target (taking SEQ ID NO: 1 as an example); The enzyme acted on the pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd.) to linearize it, and agarose gel electrophoresis was used to identify the digested fragments.

[0116] Table 1-2 Double-stranded DNA Ol...

Embodiment 2

[0134] Example 2 Detection of gene silencing efficiency by real-time fluorescence quantitative RT-PCR

[0135] Human colorectal cancer HCT116 cells and colorectal cancer RKO cells in logarithmic growth phase were digested with trypsin to prepare cell suspensions (the number of cells was about 5×10 4 / ml) were seeded in 6-well plates and cultured until the cell confluence reached about 30%. According to the multiplicity of infection (MOI, HCT116:10, RKO:10), an appropriate amount of lentivirus prepared in Example 1 was added, and the medium was replaced after culturing for 24 hours. After the infection time reached 5 days, the cells were collected. Total RNA was extracted according to Invitrogen's Trizol operating instructions. According to the M-MLV operating instructions of Promega company, reverse transcription of RNA to obtain cDNA (reverse transcription reaction system is shown in Table 2-1, 42 °C reaction for 1 h, and then in a 70 °C water bath for 10 minutes to inactiva...

Embodiment 3

[0142] Example 3 Detection of proliferation ability of tumor cells infected with EXOSC2-siRNA lentivirus (MTT assay)

[0143] Human colorectal cancer HCT116 cells and colorectal cancer RKO cells in logarithmic growth phase were digested with trypsin to prepare cell suspensions (the number of cells was about 5×10 4 / ml) were seeded in 6-well plates and cultured until the cell confluence reached about 30%. According to the multiplicity of infection (MOI, HCT116:10, RKO:10), an appropriate amount of virus was added, and the medium was changed after 24 hours of culture. The cells of each experimental group in the logarithmic growth phase were collected and digested with trypsin, and the complete medium was resuspended. The cell suspension was formed and counted. Determine the cell density (2500cell / well) according to the speed of cell growth. Repeat 3-5 times for each group. After uniform plating, after the cells are completely settled, observe the cell density of each experiment...

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Abstract

The invention belongs to the field of biomedical research, and particularly relates to application of a human EXOSC2 gene as a target in preparation of a drug for treating colorectal cancer. Through wide and deep research, it is found that: after expression of the human EXOSC2 gene is down-regulated by adopting an RNAi way, proliferation of colorectal cancer cells can be effectively inhibited, apoptosis is promoted and the growth process of colorectal cancer can be effectively controlled. The siRNA or a nucleic acid construct and lentivirus containing the siRNA sequence provided by the invention can specifically inhibit the proliferation rate of the colorectal cancer cells, promote apoptosis of the colorectal cancer cells, inhibit cloning of the colorectal cancer cells, inhibit invasion and metastasis of the colorectal cancer cells, and improve the survival rate of the colorectal cancer cells. Therefore, colorectal cancer is treated, and a new direction is opened up for colorectal cancer treatment.

Description

technical field [0001] The invention belongs to the field of biomedical research, and specifically relates to the use of human EXOSC2 gene and related products. Background technique [0002] RNA exosome complex (RNA exosome complex), referred to as exonuclease, is a complex composed of at least five kinds of exonuclease. [0003] RNA exosome complex 2 (EXOSC2), also known as ribosomal RNA processing protein 4 (RRP4), located on chromosome Xp11.23, missense mutations in EXOSC2 cause a unique syndrome including retinitis pigmentosa, hearing loss decline and mild intellectual disability. Pathogenic G198D mutations prevent EXOSC2 from binding to other RNA exosome components, resulting in instability of constituent proteins and complexes and altered expression and / or activity of key genes in the autophagy pathway. [0004] EXOSC2 is a non-catalytic component of the RNA exosome complex with 3'→5' exonuclease activity and is involved in RNA processing and degradation in cells. U...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/02C12N15/113C12N15/63C12N7/01A61K45/00A61K48/00A61K31/713A61P35/00A61P35/04
CPCC12Q1/6886G01N33/5011C12N15/1135A61K45/00A61K31/713A61P35/00A61P35/04C12N2310/141C12Q2600/158
Inventor 唐蔚宋程潘博蒋益兰付剑锋曾瀚罗星
Owner 唐蔚
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