| Objectives:1.Screening differential expression of microRNAs in plasma of small cell lung cancer patients by small RNA sequencing;2.Real-time quantitative PCR was performed on the differentially expressed microRNAs in the small cell lung cancer parental cell line H69 and its multi drug-resistant strain H69AR.;3.Predict differentially expressed target genes for significant microRNAs,analyze the function of these target genes and their key signaling pathways.Methods:1.To collect the same patient first-diagnosed and drug-resistance plasma of non-surgical and first-diagnosed small cell lung cancer patients diagnosed by histopathology from July 2017 to December 2018 in Yunnan Cancer Hospital,and screen the differential expression microRNAs of chemoresistance in plasma of small cell lung cancer patients by small RNA sequencing;2.The total RNA of the small cell lung cancer parental cell line H69 and its multi drug-resistant strain H69AR cells were extracted,and the RNA was reverse transcribed into cDNA using the miRNA First-Strand cDNA Synthesis Kit,and U6 was used as an internal reference,real-time quantitative PCR experiments were performed to verify the expression levels of differentially expressed microRNAs;3.TargetScanHuman 7.2,miRDB,miRTarbase and other databases were used to predict the target genes of differentially expressed microRNAs,and the functions of these target genes and their key signaling pathways were analyzed by GO and KEGG databases.Results:1.A total of 12 plasma samples from 6 SCLC patients with every patient's first-diagnosed and drug-resistance plasma samples were sent for small RNA sequencing.Six patients were treated with first-line chemotherapy with etoposide(VP-16)combined with platinum drugs.The results of screening for differentially expressed microRNAs of chemotherapy resistance were as follows:628 samples of A,645 samples of B,1200 samples of C,2875 samples of D,1089 samples of E and 3702 samples of F.There are 20 differentially expressed microRNAs in 6 samples.2.Real-time quantitative PCR confirmed that miR-369-5p,miR-484,miR-584-5p and miR-1247-5p were up-regulated in the multi drug-resistant cell line H69AR compared with the small cell lung cancer parental cell line H69;miR-485-5p,miR-192-5p,miR-150-5p,miR-3529-3p,miR-4446-3p,miR-363-3p,miR-320e,miR-3184-3p,miR-378a-3p,miR-625-5p,miR-375-3p,miR-4488,miR-615-3p and miR-92a-5p were down-regulated in the multi drug-resistant cell line H69AR;miR-6842-3p and miR-423-5p were no significant change in the multi drug-resistant cell line H69AR.3.Pick out the significantly differentially expressed miR-369-5p,mR-584-5p,miR-485-5p,miR-192-5p,miR-150-5p,miR-3529-3p,miR-4446-3p,miR-3184-3p,miR-378-3p,miR-615-3p and miR-484,using TargetScanHuman 7.2 software to predict their target genes,and then analyzing the function of these target genes and their key signaling pathways through GO and KEGG databases,and found that these target genes are mainly involved in gene transcription,cell cycle,cell growth,cell death and intercellular signal transduction,and play biological functions such as catalytic activity,protein-binding transcription factor activation,and nucleic acid-binding transcription factor activity.Significant enrichment of KEGG PATHWAY resulted in two signaling pathways that are more interesting in small cell lung cancer:PI3K-AKt signaling pathway and Notch signaling pathway.Conclusions:1.By performing small RNA sequencing on the six small cell lung cancer patients with every patient first-diagnosed and drug-resistance plasma samples,20 microRNAs with differential expression of six patients' chemotherapy resistance were obtained.2.Real-time quantitative PCR was performed in small cell lung cancer cell lines.Compared with the parental cell H69,4 microRNAs were up-regulated in their multidrug resistant cell line H69AR;14 microRNAs were down-regulated in their multidrug resistant cell line H69AR;and 2 microRNAs were no significant change in the multi drug-resistant cell line H69AR.3.The target genes of differentially significant microRNAs were analyzed by GO database,and these target genes were mainly involved in important biological processes such as gene transcription,cell cycle,cell growth,cell death and intercellular signal transduction,and exerted catalytic activity and protein-binding transcription.Biological functions such as factor activation and nucleic acid-binding transcription factor activity;two significant signaling pathways in small cell lung cancer were obtained by KEGG PATHWAY significant enrichment analysis:PI3K-AKt signaling pathway and Notch signaling pathway. |
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