Simutaneous Control Of Non-coding RNA On The G2/M Phase Of The Cell Cycle In Hepatocellular Carcinoma

Part OneObjective : To screen for differentially expressed mi RNAs between primary Hepatocellular Carcinoma and adjacent normal tissues.Methods: Affymetrix miRNA microarray technology was used to drtect the changes of miRNA expression between hepatocellular carcinoma tissues and its matched adjacent normal tissues.We did hierarchical clustering analysis to filter differentially expressed miRNA.Then Real-Time PCR was applied to verify microarray data in later experiments.Results: There were 81 differentially expressed miRNAs in 12 pairs of HCC tissues and adjacent normal liver tissues,including 12 up-regulated genes and 69 down-regulated genes.By microarray analysis,it was found that mi RNA-1269 a was the most differentially expressed in up-regulated genes,whereas mi R-199a-3p was the most differentially expressed in down-regulated genes.Conclusion:The differentially expressed mi RNAs screened by microarrays can provide new ideas for targeted therapy of liver cancer-related specific markers,and can also provide a basis for the in-depth study of non-coding RNAs.Part TwoObjective : To identify differentially expressed lncRNAs and mRNAs between primary Hepatocellular Carcinoma and adjacent normal tissues.Methods: LncRNA-mRNA microarray technology was used to detect the changes of lncRNAs and mRNAs expression between hepatocellular carcinoma tissues and its matched adjacent normal tissues.Differentially expressed mRNAs and lncRNAs were screened and subjected to hierarchical cluster analysis.To filter differentially expressed mRNAs,we did GO and Pathway analysis with the help of bioinformatics methods.Then Real-Time PCR was applied to verify microarray data in later experiments.Results: Compared with normal adjacent tissues,there were 2,093 differentially expressed mRNAs in HCC tissues,of which 635 were up-regulated genes and 1458 were down-regulated genes.At the same time there were 1704 differentially expressed lnc RNAs,of which 607 were up-regulated and 1097 were down-regulated.GO and Pathway analysis involved in cell cycle,REDOX,signal transduction ion transport,immune response,cell adhesion,and binding functions.Conclusion : Differentially expressed mRNAs screened by microarrays may be involved in cell cycle,signal transduction,and other important aspects.At the same time,the differentially expressed lncRNAs were screened to establish a basis for the construction of non-coding RNA regulatory networks,providing a theoretical basis for the occurrence and development of HCC at the molecular level.Part ThreeObjective: To constructed lncRNA-miRNA-mRNA regulatory networks Using bioinformatics techniques,and to explore possible regulatory mechanisms among non-coding RNAs.Methods: We used bioinformatics technology to construct miRNA-mRNA,lncRNA-mRNA and miRNA-lncRNA co-expression networks through co-expression correlation analysis.We constructed lncRNA-miRNA-mRNA regulatory networks based on three expression networks to identify possible signaling pathways.It was using Real-time PCR to verified the previously screened differential non-coding RNAs and target genes on the corresponding signaling pathways.Result: Through the mutual verification of the three expression networks,we found that there may be a regulatory network of lncRNAP26302-mi RNA-125b-2-3p-PLK1.The target genes CHEK1 and CCNA2 of mi R-125b-2-3p are in the same gene pathway(The G2/M DNA damage checkpoint)as the target gene PLK1 of lncRNAP26302.There is an upstream and downstream relationship between these target genes in the G2/M DNA damage checkpoint pathway.Real-time PCR validation was performed on these genes.,and the verification results are consistent with the results of the microarray experiment.Conclusion: MiR-125b-2-3p and lncRNAP26302 affect the gene pathway of G2/M over-pathway through the regulation of their respective target genes.This study further explored the occurrence and development of primary hepatocellular carcinoma from the molecular level,providing a basis for the future search for a new diagnosis of hepatocellular carcinoma and new treatment options.