siRNA that specifically inhibits lamb1 gene expression, its recombinant vector and application
A gene expression and specific technology, applied in the fields of molecular biology and biomedicine, to reduce cell migration and invasion, increase cell apoptosis, and reduce cell proliferation
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Embodiment 1
[0063] Example 1. Study on the difference of LAMB1 expression in ovarian cancer cell line A2780 and its paclitaxel-resistant cell line A2780 / Taxol
[0064] 1. Detection of LAMB1 mRNA expression by real-time quantitative RT-PCR (qRT-PCR)
[0065] After culturing for 48 hours, the medium in the 6-well plate was aspirated, 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 of RNase-free DEPC water; reaction conditions: incubate at 70°C for 10min and place on ice. The second step is reverse transcription. Reaction system: reverse transcription according to the instructions of the PrimeScript RTMaster Mix kit; reaction conditions: incubate at 42°C for 60 minutes, inactivate at 85°C for 5 mi...
Embodiment 2
[0075] Example 2. Design and synthesis of LAMB1 siRNA
[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 ID NO.) were designed online with siDirect Ver2.0 software (http: / / sidirect2.rnai.jp / ). 6) shown. In the design process, the sequence that satisfies the three algorithms reported in the literature (Ui-Tei×Reynolds×Amarzguioui) is selected at the same time, and the 23nt length fragment with the highest specificity of siRNA effect is selected. This design can avoid the occurrence of interferon-like immune response in future in vivo experiments. , select 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 23nt siRNAs were selected as the experimental screening interference fragments. The structural features are that there are two bases on the 3' ends of the sense strand and antisense strand. The structural features are as follows...
Embodiment 3
[0083] Example 3. Detection and screening of the interference effect of three pairs of LAMB1 siRNA on LAMB1 gene in ovarian cancer taxol-resistant strain A2780 / Taxol
[0084] 1. Experimental group:
[0085] 1. A2780 / Taxol normal group (no siRNA transfection), 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] To ensure transfection efficiency and reduce cytotoxicity, we used Lipofectamine3000 transfection reagent for siRNA transfection. One day before transfection, cells were trypsinized and counted, and cells were plated in six-well plates at a density...
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