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Small interfering RNA for TRAPPC4 gene target point, and uses thereof

A technology of small interference and target, applied in the field of small interfering RNA, can solve the problem of poor effect of colorectal cancer, achieve significant technical effect and inhibit proliferation

Active Publication Date: 2013-01-30
RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide a kind of small interfering RNA and its application for the TRAPPC4 gene target, and the described small interfering RNA and its application for the TRAPPC4 gene target will solve the surgical treatment, chemotherapy and radiotherapy means in the prior art Technical Problems in Ineffective Treatment of Colorectal Cancer

Method used

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  • Small interfering RNA for TRAPPC4 gene target point, and uses thereof
  • Small interfering RNA for TRAPPC4 gene target point, and uses thereof
  • Small interfering RNA for TRAPPC4 gene target point, and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Interaction between TRAPPC4 and ERK2 and its specific expression in colorectal cancer

[0025] 1 Discovery and verification of the interaction between TRAPPC4 and ERK2

[0026] 1.1 The discovery of the interaction between TRAPPC4 and ERK2

[0027] Amplify the ERK2 gene by PCR method (see attached for primers and reaction conditions); perform ligation reaction to obtain recombinant plasmid after enzyme digestion and gel recovery, transform into competent cells, extract plasmid after inoculation culture, and use membrane detection method to detect The expression of the Laz gene, that is, the self-activation detection of the ERK2 gene. Transform Y190 yeast competent cells containing pGB-ERK2 with 400ug of human Hela cDNA yeast two-hybrid library plasmid DNA, and spread SC / -Trp-Leu-His+3AT screening plate. At the same time, different concentration gradients of 1:1000, 1:10000, and 1:100000 were used to coat 100 μl of bacterial solution on 3 SC / -Trp-Leu culture p...

Embodiment 3

[0056] Example 3 Inhibition of TRAPPC4-siRNA on the expression of TRAPPC4 in human colorectal cancer cell SW1116.

[0057] According to the human transport protein particle complex 4 (TRAPPC4) NM 016146.3 sequence (SEQ ID NO: 3), the template strand and the coding strand were designed (see the appendix for design principles). After screening, the TRAPPPC4 interference fragment sequence of the present invention with the most significant interference effect was obtained as follows:

[0058] 5'-CCGGUGUCCAUUCGAUUUGTT-3' (SEQ ID NO: 1)

[0059] 5'-CAAAUCGAAUGGACACCGGTT-3' (SEQ ID NO: 2)

[0060] Attachment: siRNA Design Principles

[0061] 1. The thermal stability of the double-stranded end of siRNA, namely the value of

[0062] 2. Selection of the last two bases at the end of the antisense strand of the siRNA duplex, namely: the selection of bases at positions 20 and 21

[0063] 3. Selection of bases at positions 1 and 19 of the antisense strand of the siRNA duplex

[0064]...

Embodiment 4

[0079] Example 4 Inhibition of TRAPPC4-siRNA on ERK-MAPK Signaling Pathway Activity in Human Colorectal Cancer Cell SW1116

[0080] The TRAPPC4-siRNA interference fragment was used as DharmaFECT TM Human colorectal cancer cells SW1116 were transfected, and Negtive siRNA that did not affect protein expression was used as a negative control (synthesized by Gemma Biotechnology Company). After 48 hours, the cells of each group were collected to extract the total protein, and the specific antibodies against ERK1 / 2 and phosphorylated ERK1 / 2 (pERK1 / 2) (purchased from Cell Signaling Company) were used for immunoblotting experiments, and GAPDH was used as an internal reference. The results are as follows: Figure 6 as shown in a. The results showed that after 48 hours of transfection with TRAPPC4-siRNA, the total expression of ERK1 / 2, a key protein in the ERK-MAPK signaling pathway, did not change significantly, while the expression of phosphorylated ERK1 / 2 decreased significantly, wh...

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Abstract

The present invention provides small interfering RNA for a TRAPPC4 gene target point, wherein a sense strand nucleotide sequence is represented by SEQIDNO:1, and an antisense strand nucleotide sequence is represented by SEQIDNO:2. The present invention further provides applications of the small interfering RNA for the TRAPPC4 gene target point in preparation of drugs for prevention or treatment of colorectal carcinoma. The present invention further provides a targeting molecule transport protein microparticle complex of the siRNA and applications of the targeting molecule transport protein microparticle complex in preparation of drugs for prevention or treatment of colorectal carcinoma. According to the present invention, experiment results show that: after the small interfering RNA for the TRAPPC4 gene target point is adopted to transfect human colorectal carcinoma cells to slice TRAPPC4 expression so as to inhibit ERK-MAPK signaling pathway activation, inhibit colorectal carcinoma proliferation and induce apoptosis, such that the small interfering RNA can be used for colorectal carcinoma treatment.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a small interfering RNA, in particular to a small interfering RNA targeting the TRAPPC4 gene target and its application. Background technique [0002] At present, the treatment of colorectal cancer mainly adopts surgical treatment, chemotherapy and radiotherapy, but the proportion of patients who can receive surgery is relatively small, and the latter two have large side effects. RNA interference (RNA interference, RNAi), also known as post transcriptional gene silencing (PTGS), is a small fragment of double-stranded RNA (20-30bp) (small interfering RNA, siRNA) molecule blocking or reducing the expression of homologous genes phenomenon that can involve the shutting down and regulation of gene expression at various stages of life. At present, RNAi has become an independent technology, with high target specificity, low cytotoxicity, and relatively safe. It is mainly used in reve...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K48/00A61P35/00C12N15/85C12N15/12
Inventor 赵树靓房静远
Owner RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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