Analysis Of Differentially Expressed Genes In Hepatocellular Carcinoma And Preliminary Validation Of Long Non-coding RNAs

Hepatocellular carcinoma?HCC?is a prevalence malignant tumor in the world.The mortality of HCC is the third in the malignant tumors.In China,HCC has high rate of mortality and morbidity in patients,which mainly caused by HBV infection.In Guangxi area,HCC is the top one in tumor mortality.Because of the hidden symptoms,fast growth and metastasis,the patients of HCC is late for treatment when they are at the middle-late stage.Although surgical resection and liver transplantation could improve the situation,the quality of patients' life affects by HCC recurrence and metastasis,which is the main problem that we faced.So it is necessnary to uncover the mechanisms in the initiation and development of HCC and seek specific candidate diagnostic biomakers and therepeutic targets,which would porvide powerful evidence for HCC.HCC has been traditionally considered as a high heterogeneity and genetic cancer.Cancers' genes not only result in cell canceration,but also could cause epigenetic regulation,such as DNA methylation,acetylation,ubiquitination,non-coding RNA regulation and so on.In the non-coding RNAs' regulation,rencent studies found that long noncoding RNAs plays a key role in the initation and development of cancer,like PCA3,HOTAIR and H19.Long non-coding RNA?lnc RNA?is a kind of more than 200 nucleotide RNAs in the nucleus or cytoplasm,which could regulate target genes in the transcriptional or post-transcriptional levels and affect the function of tumor cells.With the development of lnc RNAs,numerous studies verified that lnc RNAs have differential expression in special tumor tissues.Because of that reason,lnc RNAs could be useful for early digonosis of cancers and therapeutics.Those lnc RNAs' function provided bright prospects in diagnosis and treatment of HCC.With the completion of Human Genome Project?HGP?and the increasingly development of bioinformatics,genomics and transcriptomics emerge at a historic moment.At the same time,in the post-genomic era,next generation gene sequencing technology is rapidly developing,and this development deeply changed the thinking structure of transcriptomics.RNA microarray,as a common tool in deeply testing transcriptomics,could entirely detect sepecial species' transcriptomics in the mononucleotide level with accurate positions and high sensitivity,which provided entire landscape for cancers researchers and a powerful tool for choosing differentially expressing lnc RNAs and cancers' mechanisms.At present,we do not completely understand differential expressing lnc RNAs in early and middle stage of HCC and related mechanisms.Based on human microarray chips,we entirely analyzed and validated the differential expressing lnc RNAs and m RNA,then significant lnc RNAs and m RNAs were found in the development of HCC.Those lnc RNAs were analyzed for its relationship with m RNAs,and we further found lnc RNAs and related m RNAs' pathways in HCC and its mechanism for the HCC diagnosis and therepy.Methods In the part one,high throughput microarray chip was adopted for differential lnc RNAs and m RNAs.The subgroup of lnc RNAs was analysized for relationship between lnc RNAs and m RNAs.Gene ontology?GO?and KEGG was used for investigating HCC pathways,and GSEA analysized related cancer pathways from the entire transcriptomics genes.In the part two,based on the part one,we validated ten differentital lnc RNAs with RT-PCR in HCC patients.And we further used two independtent populations and ROC analysis to find diagnosis biomarkers from target lnc RNAs in HCC patients.What's more,TCGA data was used for investigating wether target lnc RNAs could be used as prognostic biomarkers.We further found the significant lnc RNAs and m RNAs in co-expressionn network and IPA,which played significant roles in initation and development of HCC and prediced how those lnc RNAs regulated the development of HCC.Results1.The analysis of differentially expressed genes in HCC?1?Using the high-throughput microarray chips,our results showed the differential lnc RNAs 4433 and m RNAs 5210.Among them,there are 4lnc RNAs reported in other studies.?2?Using GO and KEGG,resluts further showed key pathways included cell cycle,splicing pathway in HCC,.?3?Using GSEA,we inverstigated the key related cancer pathways in HCC,including P53 signaling,Toll-like receptor signaling.2.The preliminary validation of long non-coding RNAs?1?We used RT-PCR to validate 9 differential lnc RNAs in the HCC population,including 4 up-regulated lnc RNAs and 5 down-reglated lnc RNAs,which is consistent with microarray chip's results.?2?Using two HCC populations,3 lnc RNAs,which included ENST00000524045,ENST0000042149 and NR₀02763,were validated as diagnosis biomarkers of HCC,especially in the early stage of HCC.ENST00000524045 could use as a biomarker at the early stage of HCC.Furthermore,combined model provided a more powerful biomarker for HCC.?3?Using the TCGA database,our results showed that 3 lnc RNAs included ENST00000443523,ENST00000524045 and ENST00000508147 were related with survival time in HCC prognosis.?4?Using co-expression network,we investigared the interaction between lnc RNAs and m RNAs and found key pathways in the development of HCC,including cell cycle,PPAR signaling pathway and DNA replication.?5?We used IPA to validate the significant genes,such as CCND1,CDK1 in the cell nuclear,which could take part in cell cycle to regulate the development of HCC.Conclusion We found the differential expressing lnc RNAs in the initation and development of HCC,and validated the significant lnc RNAs and related m RNAs,which were related with cell cycle.Target lnc RNAs were validated in HCC patients' population and were associated with the diagnosis and prognosis of HCC.ENST00000524045 and its target gene ZFPM2 could play a key role in HCC mechanism.