Use of long-chain non-coding RNA in the preparation of preparations for inhibiting tumor cell metastasis
A long-chain non-coding, multi-purpose technology, applied in the field of genes
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preparation example Construction
[0168] Preparation of competent bacteria:
[0169] (1) Inoculate the Top 10 strains on LB plates without ampicillin and culture for 8h-12h.
[0170] (2) Pick a single clone into LB liquid medium without ampicillin, and culture overnight at 37° C. with shaking at 220 r / min.
[0171] (3) Inoculate the above-mentioned culture solution into 200 mL of fresh LB liquid medium at a ratio of 1:100, culture at 37°C and shake at 220 r / min until the logarithmic growth phase (OD value is about 0.6).
[0172] (4) Divide the cultured bacterial solution into 50mL centrifuge tubes and place in ice bath for 30min. Centrifuge at 2400rmp for 10min, discard the supernatant.
[0173] (5) Add 10 mL of 0.1 mol / L pre-cooled CaCl2 to suspend the bacteria. 2400rmp, discard the supernatant.
[0174] (6) Add 10 mL of 0.1 mol / L pre-cooled CaCl2 to suspend the bacteria, place on ice for 30 min, 2400 rpm, and discard the supernatant.
[0175] (7) Add 1 mL of 0.1 mol / L CaCl2 to pre-cool the suspended bac...
Embodiment 1
[0331] Example 1 Long non-coding RNA BC009639 inhibits lung cancer cell migration
[0332] 1.1 Lung cancer cell long non-coding RNA expression microarray data
[0333] A pair of lung cancer cell lines 95C and 95D with different metastatic potentials were selected as comparative research objects, and the metastatic ability of 95D was higher than that of 95C. The long non-coding RNA expression in the pair of cell lines was obtained by using a long non-coding RNA chip Data, in the obtained data, by means of bioinformatics analysis, the long-chain non-coding RNA with relatively high expression abundance and high expression difference in this pair of cells was screened, and the long-chain non-coding RNA RNA BC009639 has a relatively high expression level, and more importantly, its expression level difference between 95C and 95D is very significant, so it was selected as a further research object;
[0334] 1.2 RT-PCR verification of the expression of BC009639 in 95C and D
[0335]...
Embodiment 2
[0346] Example 2 Functional experiment of LncRNA BC009639 affecting MiR-96 and MiR-155
[0347] Sequence analysis of lncRNA BC009639
[0348] The sequence of lncRNA BC009639 was analyzed, and the BLAST results showed that in the human gene database, lncRNA BC009639 highly matched an mRNA sequence named IMPAD1, and the complete sequence of lncRNA BC009639 showed the characteristics of overlapping with some regions of the IMPAD1 mRNA sequence, and their matching regions Mainly in the 3'UTR region of the IMPAD1 mRNA sequence;
[0349] Correlation between IMPAD1 and lncRNA BC009639
[0350] Based on the important role of the IMPAD1 gene in physiological development, its expression level in various tumor cells was further studied, and its expression level in several different tumor cells such as lung cancer, liver cancer, and colorectal cancer was detected by real-time PCR. The results show that it has a relatively high expression level in various tumor cells. Based on the correl...
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