siRNA capable of specially inhibiting expression of LAMB1 gene and recombinant vector and application of siRNA

A gene expression and recombinant vector technology, applied in the fields of molecular biology and biomedicine, to achieve the effect of reversing drug resistance, reducing cell migration and invasion ability, and improving sensitivity

Inactive Publication Date: 2017-11-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Our previous studies have shown that LAMB1 is abnormally elevated in ovarian cancer paclitaxel-resistant cell line A2780 / Taxol, but whether this gene is related to ovarian cancer and ovarian cancer drug resistance remains to be further studied

Method used

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  • siRNA capable of specially inhibiting expression of LAMB1 gene and recombinant vector and application of siRNA
  • siRNA capable of specially inhibiting expression of LAMB1 gene and recombinant vector and application of siRNA
  • siRNA capable of specially inhibiting expression of LAMB1 gene and recombinant vector and application of siRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1. Study on the Difference of LAMB1 Expression in Ovarian Cancer Cell Line A2780 and Its Taxol-resistant Cell Line A2780 / Taxol

[0064] 1. Detection of LAMB1 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, 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, inactivate at 85...

Embodiment 2

[0075] Embodiment 2. LAMB1 siRNA design and synthesis

[0076]The LAMB1 gene mRNA sequence (NM_002291.2) 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.

[0077]

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

Embodiment 3

[0083] Example 3. Detection and screening of three pairs of LAMB1 siRNAs in the interference effect of LAMB1 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 si...

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Abstract

The invention discloses siRNA capable of specially inhibiting expression of an LAMB1 gene, a recombinant vector of the siRNA and application of the siRNA in reversing ovarian cancer taxol resistance, and belongs to the technical field of molecular biology and biological medicine. The siRNA comprises a sense strand and an antisense strand, the sense strand is 5'-UGUUUGAAAGCCGAAUCUGCG-3', and the antisense strand is 5'-CAGAUUCGGCUUUCAAACAAA-3'. According to the siRNA, mRNA and protein expression of the LAMB1 gene in a cancer cell can be inhibited specially and efficiently, proliferation of the cancer cell is reduced, cell apoptosis is increased, the invasiveness and migration capability of the cancer cell is reduced, and moreover, the resistance of an ovarian cancer cell to taxol can be reversed effectively. The invention further provides the application of the siRNA and the recombinant vector thereof in preparing medicines treating an ovarian cancer, a hepatocellular cancer, a colorectal cancer, a malignant glioma, a prostatic cancer or a gastric cancer and medicines reversing the ovarian cancer taxol resistance.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and biomedicine, in particular to a siRNA for specifically inhibiting the expression of LAMB1 gene, its recombinant vector and 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 sequ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61K31/713A61P35/00
CPCC12N15/113C12N2310/14C12N2310/33
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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