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SiRNA [small interfering RNA(ribonucleic acid)] for specifically suppressing expression of PADI2 (protein-arginine deiminase type-2) genes, recombinant vector with siRNA and application thereof

A technology for gene expression and recombinant vectors, applied in the fields of molecular biology and biomedicine, to reduce cell migration and invasion, increase cell apoptosis, and reduce cell proliferation

Active Publication Date: 2017-10-17
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Our previous research showed that PADI2 is also highly expressed in ovarian cancer cells, and it was found to be abnormally elevated in the ovarian cancer paclitaxel-resistant cell line A2780 / Taxol, but whether this gene is related to the pathogenesis of ovarian cancer and ovarian cancer drug resistance yet to be further studied

Method used

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  • SiRNA [small interfering RNA(ribonucleic acid)] for specifically suppressing expression of PADI2 (protein-arginine deiminase type-2) genes, recombinant vector with siRNA and application thereof
  • SiRNA [small interfering RNA(ribonucleic acid)] for specifically suppressing expression of PADI2 (protein-arginine deiminase type-2) genes, recombinant vector with siRNA and application thereof
  • SiRNA [small interfering RNA(ribonucleic acid)] for specifically suppressing expression of PADI2 (protein-arginine deiminase type-2) genes, recombinant vector with siRNA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1. Study on the difference in expression of PADI2 in ovarian cancer cell line A2780 and its paclitaxel-resistant cell line A2780 / Taxol

[0063] 1. Detection of PADI2 gene mRNA expression by real-time fluorescent quantitative RT-PCR (qRT-PCR)

[0064] 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, make up to 6.8ul with RNase-free DEPC water; reaction conditions: incubate at 70°C for 10min and place on ice. The second step is reverse transcription. Reaction system: perform reverse transcription according to PrimeScript RTMaster Mix kit instructions; reaction conditions: incubate at 42°C for 60 minutes, inactiva...

Embodiment 2

[0074] Embodiment 2.PADI2siRNA design and synthesis

[0075] The PADI2 gene mRNA sequence (NM_007365.2) was found in Genebank, and 3 pairs of siRNA sequences (such as SEQ ID NO.1-SEQ IDNO. 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.

[0076]

[0077] BLASTN (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) Homology searches were performed onl...

Embodiment 3

[0082] Example 3. Detection and screening of three pairs of PADI2 siRNA on PADI2 gene interference effect in ovarian cancer paclitaxel-resistant strain A2780 / Taxol

[0083] 1. Experimental group:

[0084] 1. A2780 / Taxol normal group (not transfected with siRNA), hereinafter referred to as AR;

[0085] 2. A2780 / Taxol negative control group (transfection negative control siRNA), hereinafter referred to as AR-N;

[0086] 3. A2780 / Taxol experimental group (transfection S1), hereinafter referred to as AR-S1;

[0087] 4. A2780 / Taxol experimental group (transfection S2), hereinafter referred to as AR-S2;

[0088] 5. A2780 / Taxol experimental group (transfection S3), hereinafter referred to as AR-S3.

[0089] 2. Group transfection

[0090] 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 p...

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Abstract

The invention discloses siRNA [small interfering RNA(ribonucleic acid)] for specifically suppressing expression of PADI2 (protein-arginine deiminase type-2) genes, a recombinant vector with the siRNA and application of the siRNA to reversing taxol resistance of ovarian cancer, and belongs to the technical field of molecular biology and biomedicines. The siRNA comprises positive-sense strands 5'-AAUGUACAGAACUAUCUCGUA-3' and antisense strands 5'-CGAGAUAGUUCUGUACAUUUC-3'. The siRNA, the recombinant vector and the application have the advantages that expression of mRNA (messenger RNA) and proteins of the PADI2 genes can be specifically and efficiently suppressed by the siRNA, cell proliferation can be reduced, cell apoptosis can be increased, the migration and invasion capacity of cells can be deteriorated, and the taxol resistance of ovarian cancer cells can be effectively reversed; the PADI2 siRNA and the recombinant vector can be applied to preparing medicines for treating ovarian cancer, breast cancer, cervical squamous cell carcinoma, colon cancer, liver cancer and lung cancer or reversing medicine 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 PADI2 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): C12N15/113C12N15/867A61K31/713A61K48/00A61P35/00
CPCA61K31/713C12N9/78C12N15/1137C12N15/86C12N2310/141C12N2740/15043C12Y305/03015C12N2310/321C12N2320/31
Inventor 叶枫陈怀增洪蝶刘佳王浛知程琪
Owner ZHEJIANG UNIV
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