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DNA fragment for peculiarly inhibiting Rab27B gene expression and application thereof

A technology in fragments and sequence lists, applied in DNA/RNA fragments, recombinant DNA technology, gene therapy, etc., can solve problems such as tumors that have not yet been seen

Active Publication Date: 2010-07-28
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there have been no reports related to tumors

Method used

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  • DNA fragment for peculiarly inhibiting Rab27B gene expression and application thereof
  • DNA fragment for peculiarly inhibiting Rab27B gene expression and application thereof
  • DNA fragment for peculiarly inhibiting Rab27B gene expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Comparison of the mRNA and protein expression levels of Rab27B in hepatocellular carcinoma cell lines with different metastatic abilities, and the intracellular localization of Rab27B

[0039] 1. Collect cells, extract RNA and protein

[0040] Hepatocellular carcinoma cell lines HCC97L, HCC97H and HCCLM6 were purchased from the Liver Cancer Institute of Fudan University, and their metastatic ability was enhanced sequentially. 2 The cells were cultured in DMEM high-glucose medium (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS, PAA, Australia). When the cells grew to 80% confluence, the cells were collected.

[0041] The total RNA of the three cell lines was extracted, and its cDNA was synthesized by reverse transcription. The collected cells were lysed with cell lysate (20mM Tris[pH7.5], 150mM NaCl, 1mM EDTA, 1mM PMSF, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate), and then heated at 4°C Centrifuge at 10,000 g for 15 minutes to collect the supe...

Embodiment 2

[0047] Example 2, Construction of a cell line stably knocking down Rab27B and its phenotypic detection

[0048] 1. Construction of a cell line that stably knocks down Rab27B

[0049] 1. Construction of recombinant expression vector

[0050] 1) Chemically synthesized fragment ABC

[0051] Fragment ABC is formed by sequentially connecting fragment A, fragment B and fragment C, wherein the nucleotide sequence of fragment A is shown in sequence 2 in the sequence listing, the nucleotide sequence of fragment B is shown in sequence 3 in the sequence listing, and fragment The nucleotide sequence of C is reverse complementary to fragment A. Add restriction sites EcoR I and BamH I at both ends of fragment ABC.

[0052] 2) Insert fragment ABC into pGCsilencer TM Between the restriction site EcoR I and BamH I of the U6 / Neo / GFP vector (purchased from Shanghai Jikai Gene Technology Co., Ltd., Cat. No. 20106), the recombinant expression vector pGCsilencer TM U6 / Neo / GFP / RNAi.

[0053] 2...

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Abstract

The invention discloses a DNA fragment for peculiarly inhibiting Rab27B gene expression and the application thereof. A small interfering RNA provided by the invention is of a double-stranded RNA consisting of nucleotide sequences indicated by sequences 4 and 5 in a sequence table. The DNA fragment is shown in formula I, and SEQ forward nucleotide sequence is shown as sequence 2 in the sequence table; the SEQ reverse is complementary with the SEQ forward in opposite directions; and X is of an intron. The experiment approves that: when the DNA fragment is introduced to hepatic cell cancer cell, the Rab27B expressed protein is remarkably reduced, and the vitro proliferation capacity, vitro adhesion capacity, vitro migration capacity and vitro invasive capacity of HCC97H cell are remarkably reduced after the Rab27B is reduced. SEQ FORWARD - X - SEQ REVERSE (I).

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a DNA fragment for specifically inhibiting the expression of Rab27B gene and its application. Background technique [0002] Hepatocellular carcinoma (hepatocellular carcinoma, HCC) is a common malignant tumor with a high lethality rate. About 650,000 people die from HCC every year, and its incidence rate tends to increase. The disease mostly occurs in Southeast Asia and Africa, and more than 50% of it occurs in China, ranking second in my country's cancer mortality rate. Early diagnosis of HCC is difficult, the disease progresses rapidly, and the prognosis is low. Surgical resection has always been considered the best way to achieve a radical cure for HCC, but only 10-20% of patients are suitable for surgical intervention; in addition, even with radical resection, 60-70% of patients still remain within 5 years Metastatic recurrence can be life-threatening, and the rate of metastati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63A61K48/00A61P35/02
Inventor 姜颖贺福初齐菲菲孙薇魏汉东
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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