Construction of SRSF6-targeting lentivirus interference vector and applications of SRSF6-targeting lentivirus interference vector in colorectal cancer treatment

A lentiviral, colorectal technology applied in the field of molecular biology

Active Publication Date: 2015-10-14
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current research on the role of SRSF6 in colorectal cancer metastasis is still blank.

Method used

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  • Construction of SRSF6-targeting lentivirus interference vector and applications of SRSF6-targeting lentivirus interference vector in colorectal cancer treatment
  • Construction of SRSF6-targeting lentivirus interference vector and applications of SRSF6-targeting lentivirus interference vector in colorectal cancer treatment
  • Construction of SRSF6-targeting lentivirus interference vector and applications of SRSF6-targeting lentivirus interference vector in colorectal cancer treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction and verification of SRSFR6-shRNA interference vector

[0034] Materials and methods

[0035] 1. Materials

[0036]Colorectal cancer cell lines SW620, HT29, HCT116 and lentiviral packaging cell line HEK293T were provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; RPMI1640, DMEM, 0.05% Trypsin were purchased from Boster; fetal bovine serum was purchased from Siji Green Company. SRSF6 antibody was purchased from SIGMA; ACTIN antibody was purchased from CST; Lipo2000 transfection reagent and puromycin were purchased from Invitrogen; Age I, EcoR I endonuclease and T4 DNA ligase were purchased from TaKaRa. Escherichia coli DH5α, PLKO.1, psPAX2, and pMD2.G vectors were all preserved in our laboratory; primers were entrusted to Shanghai Sunny Co., Ltd. to synthesize; plasmid extraction kits were purchased from QIGEN; other drugs were of domestic analytical grade.

[0037] 2. Method

[0038] 2.1. Design of shRNA seq...

Embodiment 2

[0064] Example 2 Inhibition of the proliferation ability of colorectal cancer cells by SRSFR6-shRNA

[0065] Materials and methods

[0066] 1. Materials

[0067] Colorectal cancer cell lines SW620, HT29, and HCT116 were provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. RPMI1640, DMEM, 0.05% Trypsin, and CCK8 reagents were purchased from Boster; fetal bovine serum was purchased from Sijiqing Company. Puromycin was purchased from Invitrogen. Other medicines were domestic analytically pure.

[0068] 2. Method

[0069] 2.1 Take SW620 as an example for the screened stable cell line, count 5*10^5 cells and spread them on two wells of a six-well plate. At this time, the medium is free of puromycin

[0070] 2.2 About two days later, lay a 96-well plate for CCK8 proliferation experiment.

[0071] 1) Digest the cells of SW620‐SRSF6‐shRNA group and SW620‐scramble group with trypsin, collect the cells, and count them so that the final concentra...

Embodiment 3

[0076] Example 3 Inhibition of SRSFR6-shRNA on migration and invasion of colorectal cancer cells

[0077] Materials and methods

[0078] 1. Materials

[0079] Colorectal cancer cell lines SW620, HT29, and HCT116 were provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. RPMI1640, DMEM, 0.05% Trypsin, reagents were purchased from Boster Company; fetal bovine serum was purchased from Sijiqing Company. Puromycin was purchased from Invitrogen. Transwell double-layer board was purchased from COSTAR Company, Matrigel glue was purchased from BD Company, and crystal violet was purchased from Biyuntian Company. Other medicines were domestic analytically pure.

[0080] 2. Method

[0081] 2.1 Take SW620 as an example of the screened stably transfected cell line, count 5*10^5 cells and spread them on two wells of a six-well plate. At this time, the medium is free of puromycin.

[0082] 2.2 After about two days, spread the Transwell chamber.

[008...

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PUM

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Abstract

The present invention discloses a shRNA inhibiting SRSF6 gene expression, and a lentivirus expression vector and a construction method thereof, wherein the shRNA sequence is F:5'-CCGGCGTACAGAATACAGGCTTATTCTCGAGAATAAGCCTGTATTCTGTACGTTTTTG-3' and R:5'-AATTCAAAAACGTACAGAATACAGGCTTATTCTCGAGAATAAGCCTGTATTCTGTACG-3'. According to the lentivirus expression vector, the shRNA is inserted into vector plasmid PLKO.1 to construct PLKO.1-SRSF6-shRNA, the PLKO.1-SRSF6-shRNA and packaging plasmids PSPAX2 and PMD2. G are transfected into HEK293T cells after vector sequencing verifying is correct to obtain a lentivirus suspension, the lentivirus suspension is used to infect colorectal cancer cells, and then puromycin is used to screen and verify whether the virus packaging is successful. According to the present invention, the Western Blot results verify that the expression level of SRSF6 can be significantly reduced; the proliferation ability and the migration invasion ability of the colorectal cancer cells can be significantly reduced; and the research on the effect of SRSF6 on tumor cell migration and invasion ability is firstly provided.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to the construction and application of shRNA. Specifically, the present invention designs a synthetic shRNA for the mRNA sequence of the human SRSF6 gene, and the shRNA can inhibit the proliferation, migration and invasion of the tumor cells after being transferred into colorectal cancer cells. Background technique [0002] RNA splicing (RNA splicing): The process of removing introns from the initial transcript transcribed from the DNA template strand and joining the exons to form a continuous RNA molecule. The resulting mature mRNA also includes capping at the 5' end (plus m7‐Gppp), and polyadenylation at the 3' end. In eukaryotes, each cell gene contains about 8 introns on average, and the length of pre-mRNA is usually 4-10 times that of mature mRNA, most of which are excised during processing. [0003] Members of the SR protein family play a crucial role in the process of RNA spli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867A61K48/00A61P35/00
Inventor 张红河孔建鲁来茂德
Owner ZHEJIANG UNIV
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