Application of knockdown lncbcas1-4_1 cell line combined with active vitamin D in the preparation of antitumor drugs
A technology of anti-tumor drugs and vitamins, applied in the field of biomedicine, can solve problems such as high recurrence rate, poor prognosis, and rapid diffusion
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Embodiment 1
[0047] lncBCAS1-4_1 is located on chromosome 20q13.2, with a full length of 380nt (52781087to52779119, Genomeassembly: hg19), which belongs to intronic non-coding RNA, and its sequence is as follows:
[0048] GGGAGGATGATGGTCACTCCAGTCGAGCTGCACAAGAGCCTCAACACCAAGGTCTGGCAGGACCACACTCTGGCCTGGGACACCATTTTCAAATCAGTCAAAGCTTGTATCGACAACCGGTTAGAGAAGTATTCTCAGCAGCCTAGTGCAGATTTCCTTTGTGACATTTATCACCAGAATCGGCTTTCAAAGAAAGAATTGTATGCTGCTGTCACAGAGCTCCAGCTGGCTGCGGTGGAAACGGTAAGGGTGGGCTGGATTTGAGTTTGCTAAATGAACGGTCTGTACCATTTTTAGATGACAGATTTTAAATAGTAATTCATCTCATTCATTTCACTGGGATTCACTAGCATCAACTCCACAAAAATGAAAAGTCTCAT;SEQ ID NO.1。
[0049] lncBCAS1-4_1 contains two exons, Exon1 is between 52780991-52781087, Exon2 is between 52779119-52779401, and the predicted score by CPC is 2.19985, suggesting that lncBCAS1-4_1 may have coding ability, and may encode 85 amino acids, amino acid sequence as follows:
[0050] MMVTPVELHKSLNTKVWQDHTLAWDTIFKSVKACIDNRLEKYSQQPSADFLCDIYHQNRLSKKELYAAVTELQLAAVETVRVGWI; SEQ ID NO. 2.
[...
Embodiment 2
[0053] Example 2 Cell Culture
[0054] (1) Cell culture conditions
[0055] A549 and OVCAR-8 cells were cultured with RPMI-1640 medium containing 10% FBS without double antibody. Calu-1 and SKOV-3 cells were cultured with RPMI-1640 medium containing 10% FBS and 1% penicillin and streptomycin. All cells were placed at 37°C in 5% CO 2 in a sterile culture environment.
[0056] (2) Cell replacement
[0057] Preheat the culture medium and PBS to room temperature, place the petri dish in an ultra-clean bench, discard the original culture medium, add 1ml of PBS, shake the petri dish gently, and discard. After washing with PBS three times, add 5ml of fresh culture base to a 60mm-diameter petri dish, and put it into an incubator to continue culturing. Each cell type was changed individually to avoid cross-contamination between cells.
[0058] (3) Cell passage
[0059] When the cell confluency reaches 80%-90%, preheat the medium, PBS and trypsin to room temperature, discard the ...
Embodiment 3
[0066] Embodiment 3 siRNAs transfection
[0067] (1) For the silencing of lncBCAS1-4_1, three interference sequences were designed, namely si-BCAS1, si-BCAS2 and si-BCAS3.
[0068] Using siRNAs freeze-dried powder and riboFECT developed by Guangzhou Ruibo Biotechnology Co., Ltd. TM CP transfection reagent. The freeze-dried powder of siRNAs was centrifuged briefly, dissolved in DEPC water, prepared into a storage solution with a concentration of 20 μM, aliquoted, and stored at -20°C. Three interference sequences were designed for lncBCAS1-4_1, namely si-BCAS1 (GCTAAATGAACGGTCTGTA; SEQ ID NO.3), si-BCAS2 (GCTGCGGTGGAAACGGTAA; SEQ ID NO.4), and si-BCAS3 (GGATTCACTAGCATCAACT; SEQ ID NO.5). riboFECT TM CP Transfection Reagent contains riboFECT TM CP Buffer(10×) and riboFECT TM CP Reagent two reagents. Among them, riboFECT TMCP Buffer (10×) needs to be diluted to 1× with PBS before use, and it is ready-to-use.
[0069] A549 / OVCAR-8 cells with a cell confluence of 80% and in ...
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