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siRNA capable of specifically inhibiting expression of Eya2 gene as well as recombinant vector and application of siRNA

A gene expression and recombinant vector technology, applied in the fields of molecular biology and biomedicine, to reduce tumor cell migration and invasion ability, increase cell apoptosis, and inhibit mRNA and protein expression.

Active Publication Date: 2017-10-24
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 Eya2 is abnormally elevated in the ovarian cancer paclitaxel-resistant cell line A2780 / Taxol, but whether this gene is related to the pathogenesis and drug resistance of ovarian cancer remains to be further studied

Method used

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  • siRNA capable of specifically inhibiting expression of Eya2 gene as well as recombinant vector and application of siRNA
  • siRNA capable of specifically inhibiting expression of Eya2 gene as well as recombinant vector and application of siRNA
  • siRNA capable of specifically inhibiting expression of Eya2 gene as well as recombinant vector and application of siRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1. Study on the difference in expression of Eya2 in ovarian cancer cell line A2780 and its paclitaxel-resistant cell line A2780 / Taxol

[0064] 1. Detection of Eya2 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, ina...

Embodiment 2

[0075] Embodiment 2.Eya2siRNA design and synthesis

[0076] The Eya2 gene mRNA sequence (NM_005244.4) 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 23 nt were selected as the experimental screening interference fragments. The structural characteristics are as follows:

[0077]

[0078] BLASTN (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) Homology searches were performed online to exclude sequences with homology, and to avoid the influence of non-specific ...

Embodiment 3

[0083] Example 3. Detection and screening of three pairs of Eya2 siRNAs in interference effect on Eya2 gene in ovarian cancer paclitaxel-resistant strain A2780 / Taxol

[0084] 1. Experimental group:

[0085] 1. A2780 / Taxol normal group (not transfected with siRNA), hereinafter referred to as AR;

[0086] 2. A2780 / Taxol negative control group (transfection negative control siRNA), hereinafter referred to as AR-N;

[0087] 3. A2780 / Taxol experimental group (transfection S1), hereinafter referred to as AR-S1;

[0088] 4. A2780 / Taxol experimental group (transfection S2), hereinafter referred to as AR-S2;

[0089] 5. A2780 / Taxol experimental group (transfection S3), hereinafter referred to as AR-S3.

[0090] 2. Group transfection

[0091] 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...

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Abstract

The invention discloses a siRNA capable of specifically inhibiting expression of an Eya2 gene as well as a recombinant vector and an application of siRNA in reversing drug resistance of ovarian cancer to paclitaxel and belongs to the technical field of molecular biology and biological medicine. The siRNA comprises a sense strand 5'-UAGUUCUACCAUUUCCUUGUA-3' and an antisense strand 5'-CAAGGAAAUGGUAGAACUAGU-3'. The siRNA can specifically and efficiently inhibit mRNA and protein expression of the Eya2 gene, reduce tumor cell proliferation, accelerate tumor cell apoptosis, reduce tumor cell migration and invasion ability and effectively reverse drug resistance of ovarian cancer cells to paclitaxel. The invention further provides the application of Eya2 siRNA and recombinant vector thereof in preparation of drugs for treating ovarian cancer, prostate cancer, breast cancer, adenocarcinoma cancer, squamous-cell carcinoma, urethra cancer and cervical cancer 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 a siRNA specifically inhibiting the expression of Eya2 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 seque...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61K31/337A61P35/00C12N15/63
CPCA61K31/337A61K48/005C12N15/113C12N2310/141C12N2310/33C12N2310/352A61K2300/00
Inventor 陈怀增叶枫刘佳洪蝶王浛知程琪余明华周彩云
Owner ZHEJIANG UNIV
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