Long-chain non-coding RNA for diagnosing cisplatin resistance of triple negative breast cancer and application of long-chain non-coding RNA
A triple-negative breast cancer, long-chain non-coding technology, applied in the field of tumor molecular biology, can solve the problem of high sensitivity of triple-negative breast cancer tumor cells, achieve the effect of improving treatment effect, avoiding waste, and avoiding side effects
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Embodiment 1
[0037] Screening of long non-coding RNA LINC01778 V2
[0038] Cisplatin drugs were used for induction to establish stable cell lines of triple-negative breast cancer resistant to cisplatin drugs. In this example, a stable drug-resistant cell line of triple-negative breast cancer that is completely resistant to cisplatin was established by using the concentration gradient method, starting from a low concentration, and after two years. Then, through lncRNA chip detection, compare cisplatin-resistant triple-negative breast cancer cell lines and cisplatin-sensitive triple-negative breast cancer cell lines, and screen out a batch of differentially expressed lncRNAs; Cisplatin is not resistant to drugs; specifically, BGI sequencing is used to accurately screen lncRNA high-throughput sequencing based on Illumina's fourth-generation high-throughput sequencing platform. lncRNA high-throughput sequencing includes lncRNA identification, lncRNA quantification and differential analysis, ln...
Embodiment 2
[0040] Sequence analysis of the screened long non-coding RNA LINC01778 V2 using RACE
[0041] RACE (Rapid Amplification of cDNA Ends) is a technique for obtaining the full-length sequence of RNA transcripts. The 5' end (5' RACE-PCR) or 3' end (3' RACE-PCR) sequence of the transcript can be obtained based on a short known sequence in the transcript using RACE technology. In this example, the unknown sequence at the end of the long non-coding RNA (Long non-coding RNA, LncRNA) was amplified by the SMARTer RACE method (patented technology of clontech company).
[0042] Specifically include the following steps:
[0043] 1. Extraction of total RNA from cisplatin-resistant breast cancer cells
[0044] Promega's total RNA extraction kit (SV Total RNA Isolation System Start-Up Kit) is used to efficiently and quickly remove genomic DNA from cell samples by using improved recombinant DNase I, and to separate, purify and enrich DNA including long non Total RNA including coding RNA.
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Embodiment 3
[0053] Prediction of the secondary structure of LINC01778 V2 using structure prediction software
[0054] After obtaining the full-length sequence of LINC01778 V2 based on the RACE experiment in Example 2, the RNA secondary structure of LINC01778 V2 was predicted using the Mfold and RNAfold software packages, and the results are as follows image 3 As shown, LINC01778 V2 has a stem-loop structure.
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