SiRNA for specific inhibition of PRV1 gene expression and recombinant vector and application thereof

A technology for gene expression and recombinant vectors, applied in the fields of molecular biology and biomedicine, to achieve the effect of reducing tumor cell proliferation, inhibiting mRNA and protein expression, and reducing cell migration and invasion capabilities

Inactive Publication Date: 2018-01-05
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Our previous studies have shown that PRV1 is abnormally elevated in ovarian cancer paclitaxel-resistant cell line A2780 / Taxol, but whether this gene is related to ovarian cancer and ovarian cancer resistance remains to be further studied

Method used

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  • SiRNA for specific inhibition of PRV1 gene expression and recombinant vector and application thereof
  • SiRNA for specific inhibition of PRV1 gene expression and recombinant vector and application thereof
  • SiRNA for specific inhibition of PRV1 gene expression and recombinant vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1. Study on the difference of PRV1 expression in ovarian cancer cell line A2780 and its paclitaxel-resistant cell line A2780 / Taxol

[0064] 1. Detection of PRV1 gene mRNA expression by real-time fluorescent quantitative RT-PCR (qRT-PCR)

[0065] After culturing for 48 hours, the medium in the 6-well plate was discarded, washed twice with PBS, and the total RNA was extracted with Trizol. The RNA concentration was measured with a ThermoNano Drop2000 spectrophotometer, and the operation was performed according to the instructions of the SYBR Premix Ex Taq (perfect Real time) kit. . The first step is RNA denaturation, reaction system: RNA 0.5ug, RNase-free DEPC water to make up to 6.8ul; reaction conditions: incubate at 70°C for 10min and place on ice. The second step of reverse transcription, reaction system: perform reverse transcription according to the instructions of the PrimeScript RTMaster Mix kit; reaction conditions: incubate at 42°C for 60 minutes, inacti...

Embodiment 2

[0075] Example 2. Design and synthesis of PRV1 siRNA

[0076] The PRV1 gene mRNA sequence (NM_020406.3) was found in Genebank, and 3 pairs of siRNA sequences (such as SEQ ID NO.1-SEQ ID NO. 6) as shown. During the design process, select the sequences that satisfy the three algorithms (Ui-Tei×Reynolds×Amarzguioui) reported in the literature at the same time, and select the 23nt length fragment with the highest siRNA action specificity. This design can avoid interferon-like immune reactions in future in vivo experiments , choose 100nt after the start codon, avoid the 5' and 3' UTR regions, and control the GC content at 30-70%. A total of 3 pairs of siRNAs with a length of 23nt were selected as the experimental screening interference fragments. The structural characteristics are that there are two bases attached to the 3' end of the sense strand and the antisense strand.

[0077]

[0078] BLASTN (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) Homology searches were performed onli...

Embodiment 3

[0084] Example 3. Detection and screening of three pairs of PRV1 siRNAs on PRV1 gene interference in ovarian cancer paclitaxel-resistant strain A2780 / Taxol

[0085] 1. Experimental group:

[0086] 1. A2780 / Taxol normal group (not transfected with siRNA), hereinafter referred to as AR;

[0087] 2. A2780 / Taxol negative control group (transfection negative control siRNA), hereinafter referred to as AR-N;

[0088] 3. A2780 / Taxol experimental group (transfection S1), hereinafter referred to as AR-S1;

[0089] 4. A2780 / Taxol experimental group (transfection S2), hereinafter referred to as AR-S2;

[0090] 5. A2780 / Taxol experimental group (transfection S3), hereinafter referred to as AR-S3.

[0091] 2. Group transfection

[0092] In order to ensure transfection efficiency and reduce cytotoxicity, we used Lipofectamine3000 transfection reagent for siRNA transfection. The day before transfection, cells were trypsinized and counted, and the cells were plated in a six-well plate so ...

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Abstract

The invention discloses siRNA which specifically inhibits PRV1 gene expression and a recombinant vector and application thereof in the reversal of paclitaxel resistance in ovarian cancer, and belongsto the technical field of molecular biology and biomedicine. The siRNA includes sense and antisense strands: the sense strand: 5'-UUCUUAGGGGUCCAUUGCCGG-3'; the antisense strand: 5'-GGCAAUGGACCCCUAAGAACA-3'. The siRNA provided by the invention can specifically and effectively inhibit mRNA and protein expression of PRV1 gene in tumor cells, reduce tumor cell proliferation, increase cell apoptosis, reduce migration and invasion ability of the tumor cells, and can effectively reverse drug resistance of ovarian carcinoma cells to paclitaxel. The invention also provides application of the siRNA andthe recombinant vector thereof in the preparation of a medicament for treating ovarian cancer, gastric cancer, colorectal cancer, neuroblastoma or reversing drug resistance of ovarian cancer.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and biomedicine, in particular to an siRNA for specifically inhibiting the expression of PRV1 gene, its recombinant vector and its application. Background technique [0002] RNA interference (RNA interference, RNAi) is a widespread sequence-specific post-transcriptional gene silencing mechanism in animals and plants. In 1998, American scientist Andrew Fire discovered for the first time in C. Elegans that the gene silencing effect produced by the mixture of sense and antisense strands (ie, dsRNA) was at least 10 times that of antisense nucleotides. Moreover, the same gene suppression phenomenon can be induced in the offspring. Mechanistic studies on the RNAi phenomenon have shown that a small amount of siRNA can silence a large number of target RNAs through post-transcriptional gene silencing, and this key molecule that efficiently and specifically degrades homologous RNAs and leads to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11C12N15/867A61K31/713A61P35/00
Inventor 叶枫陈怀增洪蝶刘佳王浛知程琪余明华周彩云
Owner ZHEJIANG UNIV
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