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shRNA for inhibiting tumor cell growth as well as recombinant vector and application thereof

A technology of tumor cells and expression vectors, applied in the field of genetic engineering, can solve the problems of poor clinical prognosis, blood metastasis, and lack of specificity, and achieve the effects of inhibiting growth, inhibiting proliferation, and promoting apoptosis of PADC cells

Active Publication Date: 2015-05-20
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The clinical manifestations of PADC lack specificity, blood metastasis is prone to occur in the early stage, and metastasis has often occurred when the diagnosis is made, and the clinical prognosis is extremely poor
[0005] At present, there is no literature showing that AXIIR gene is related to PADC, and there is no literature showing that inhibiting the expression of AXIIR gene can inhibit the growth of PADC cells

Method used

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  • shRNA for inhibiting tumor cell growth as well as recombinant vector and application thereof
  • shRNA for inhibiting tumor cell growth as well as recombinant vector and application thereof
  • shRNA for inhibiting tumor cell growth as well as recombinant vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Screening and preparation of AXIIR gene high-efficiency interference lentivirus.

[0055] 1.1 Reagents

[0056] Restriction enzymes Bam H1 and Age I and corresponding Buffer, T4 ligase and its buffer, PCR product purification kit, and gel recovery kit were all purchased from TaKaRa Company. The conventional RNAi vector pFU-GW-RNAi (Addgene Company) was selected as the eukaryotic expression vector.

[0057] 1.2 Screening of effective shRNA targets for AXIIR gene

[0058] The human AXⅡR (NM_001014279.2) gene information was retrieved from Genbank; Wuhan Xima Biotechnology Co., Ltd. designed the software genesilRNAi to design effective shRNA targets for the AXⅡR gene. In the coding sequence (CD) region of the AXIIR gene, a sequence of 9 bases was obtained, and Table 1 lists 9 effective shRNA target sequences for the AXIIR gene.

[0059] Table 1. shRNA target sequences targeting the AXIIR gene

[0060]

[0061]

[0062] Design and synthesize specific si...

Embodiment 2

[0091] Example 2: Detection of AXIIR gene silencing efficiency by real-time fluorescent quantitative RT-PCR

[0092] Human pancreatic cancer Panc-I, SW-1990 and PATU-8988 cells were trypsinized, and the number of cells was about 5X 10 5 / ml of cell suspension was inoculated in a 6-well plate and cultured until the cell confluency reached about 40%. An appropriate amount of AXIIR-shRNA-N lentivirus was added, the medium was changed after 24 hours, and the cells were collected after 5 days of culture. Total RNA was extracted according to the Trizol operating procedure (Invitrogen Company) and the M-MLV operating manual of Promega Company, and the RNA was reverse-transcribed to obtain cDNA. Reaction system 10μl, each tube contains reverse transcription buffer (5×) 3.0μl, dNTPs (10mM) 2μl, RNasin 0.5μl, M-MLV-RTase 1.0μl, DEPC H 2 O 3.5 μl, 42 ° C for 1 h, 70 ° C water bath for 10 min to inactivate reverse transcriptase.

[0093] Quantitative detection with Bio-RAD-IQ5Multicolo...

Embodiment 3

[0095] Example 3: Detection of the proliferation ability of PADC cells infected with AXⅡR-shRNA-LV lentivirus

[0096] Human pancreatic cancer Panc-I, SW-1990 and PATU-8988 cells were digested with trypsin to make a cell suspension (the number of cells was about 5×10 5 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 40%. Add an appropriate amount of AXIIR-shRNA-LV virus, culture for 24 hours, replace the medium, and collect the cells after the infection time reaches 5 days. Complete medium resuspended into cell suspension 10X 10 5 / ml), inoculate a 96-well plate at a cell density of about 2000 / well. 5 replicate wells in each group, 100 μl per well. After laying the board, place it at 37°C, 5%C0 2 Incubator cultivation. The Cellomics Array Scan screening analyzer was used to detect once a day, and the plate was read continuously for 4 days. In the control group, the infection, cultivation and detection methods of N-AXⅡR-shRNA-LV...

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Abstract

The invention belongs to the technical field of genetic engineering and in particular relates to shRNA capable of inhibiting expression of AXIIR genes and further inhibiting tumor cell growth, as well as a recombinant vector of shRNA and application thereof in preparation of antitumor drugs. 19-23 RNAi target sequences aiming at the AXIIR genes are designed, and a corresponding AXIIR shRNA vector is constructed. According to the shRNA vector pFU-AXIIR shRNA constructed by the invention, the expression of the AXIIR genes in mRNA level and protein level can be obviously inhibited, a slow virus further serves as a vector carrying shRNA, the shRNA sequence aiming at the AXIIR genes can be introduced into tumor cells in a targeted mode by virtue of the pFU-AXIIR shRNA, and the tumor cell proliferation capacity is obviously inhibited.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a shRNA capable of inhibiting the expression of AXIIR gene, thereby inhibiting the growth of tumor cells, its recombinant vector, and its application in the preparation of antitumor drugs. Background technique [0002] AXIIR (Annexin II receptor) is a protein molecule with 193 amino acids and a molecular weight of about 26 kDa isolated from bone marrow (BM) mesenchymal cells in 2001. The AXIIR gene is located in the open reading frame 39 of chromosome 5, so it is also called C5orf39. The expression of AXIIR receptor mRNA is detected in most tissues of the human body. According to SOSUI and TMPred analysis, AXIIR is a type I transmembrane protein containing a transmembrane domain. Studies have shown that the expression of AXIIR in MM cell lines and patients' CD138+ positive MM cells is elevated. ANXA2 and AXⅡR expressed in stromal cells / osteoblast-like cel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867A61K31/713A61P35/00
Inventor 王凯慧赵世红朱杰章汝文潘昌霖钱梦星黄丽斌江泓蝶
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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