The Regulatary Networks Construction Of LncRNA-miRNA-mRNA In HepG2 Cell Lines By Using Strainded Specific RNA Sequencing

There is still a lack of a systematic understanding for mechanism of the development of HCC especially little knowledge of different pathogenic factors and their interaction. Unidentified key gene or molecular abnormalities can improve the diagnostic rate of HCC and targeted nterventional therapy. Recent discoveries show non-coding RNA plays an important role in the occurrence, development and metastasis of HCC. Further study of the molecular mechanisms of non-coding RNA in the process of liver cancer to find new diagnostic markers and therapeutic targets has become a hot topic in cancer research. Tumor necrosis factor a(TNF-a), a cytokine produced primarily by the LPS stimulated macrophages, has a variety of inflammatory cytokine biological activities through interacted with other cytokines to activate immune responses, mediate systemic inflammation, and induce tumors necrosis. In this study, Liver (cancer) cell lines HL-7702/HepG2 were used as the research materials and divided into two experimental groups:one was the HL-7702/HepG2 group; another group was HepG2 cell lines, one of them was treated with TNF-α for 3 hours. Total RNA was isolated and then sequenced by strand-specific RNA sequencing technology with rRNA depletions. mRNA/IncRNA expression profiling data were obtained using bioinformatic method. For differentially expressed mRNAs our research conducted function annotation. Relationships of miRNA-mRNA and miRNA-lncRNA were got using DIANA-TarBase v7.0 and IncBase software respectively, we finally constructed the regulatory network of IncRNA-miRNA-mRNA. This research attempts to find related miRNAs, mRNAs and IncRNAs in the process of liver cancer and TNF-a induced apoptosis and build IncRNA-miRNA-mRNA three-molecular regulatory network. The goal of our study is to filter out specific mRNA/IncRNA as a tumor biomarker for early diagnosis, evaluation and prognosis of malignant indicators and also provide a basis theory for molecular biology in HCC. In this thesis, our study was divided into two main parts:1. HL-7702 and HepG2 cell lines were cultured and sequenced by strand-specific RNA-seq after ribosomal RNA depletion. And then bioinformatics methods were applied for the sequencing data quality control, reads mapping and transcripts assembling. Through the expression data of mRNA and IncRNA by FPKM normalization, we selected differentially expressed mRNAs and IncRNAs. We obtained 3035 differentially expressed mRNAs totally, of which 1384 upregulated and 1651 downregulated, and 57 differentially expressed IncRNAs, of which 23 upregulated and 34 downregulated (statistically standard |log2 ratio|>1, FDR<0.01). According to the GO annotation and KEGG pathway analysis, we found at least 23 clusters were connected with cell proliferation and cell apoptosis-related deaths. We also found that four tumor metabolic pathways associated with:ErbB, JAK-STAT, mTOR and WNT, which indicated that the associated genes involved in those biological processes and might promote the proceeding of liver cancer. Combined with the previous miRNA profiling data from our group, we used DIANA TOOL a bioinformatics analysis software tool to build the IncRNA-miRNA-mRNA regulatory network to explore the post-transcriptionally regulatory functions of these non-coding RNAs in the process of liver cancer.2. HepG2 cell lines were cultured, divided into two groups and treated with TNF-a for 3 hours choosing one group. Both groups were sequenced and analyzed using the same method as the group one applied. We totally picked up 114 differentially expressed mRNAs with 102 upregulated and 12 downregulated, and 46 differentially expressed IncRNAs with 38 upregulated and 8 downregulated. Through GO and KEGG pathway analysis, we found that some inflammatory cytokines such as IL6, IL1B and IL8 participated in TNF-a-mediated apoptosis pathway and cancer. Furthermore, we also noticed those dysregulated genes NFKIBA, NFκB1, IKBKE in NF-κB pathway involved in TNF-a-mediated tumor cell apoptosis. Combined with the previous miRNA profiling data from our group, we used DIANA TOOL a bioinformatics analysis software tool to build the IncRNA-miRNA-mRNA regulatory network to explore the post-transcriptionally regulatory functions of these non-coding RNAs in the process of liver cancer.This study selected HL-7702 and HepG2 cell line as the research object, of which a group of HepG2 cell lines was treated by TNF alpha. We hope to find the molecular mechanism in the process of tumor cell proliferation and TNF alpha mediated tumor cell apoptosis and get mRNA and non coding RNA regulatory network diagram in tumor cell. However, our research results need more experimental support.