Screening Differently Expressed Circrna In Colorectal Cancer And Studying Its Function

Colon cancer is one of the most common gastrointestinal tumor all over the world and seriously harm to human health.Recent years,with the improvement of the living standard and the transformation of dietary structure,the incidence and mortality of colon cancer stay at a high level in our nation.Early diagnosis and treatment can effectively improve the prognosis of patients,but for the advanced group,there is little we can do.The primary reason for the dilemma is that the pathogenesis of colon cancer is still unclear.Gene mutation,tumor stem cell and inflammatory response are mainly research direction in the pathogenesis of colon cancer.Many researchs have proved that the abnormal expression of genes play an important role in the oncome and advance in colon cancer.The expression of genes are influenced by many factors.With the developemment of gene sequencing and analyzing,scientists gradually realize that circRNA is a novel non-coding RNA which have conserved sequence and can stably express in mammals.CircRNAs can regulate the expression of gene at transcriptional or posttranscriptional.Many researchs show that the abnormal expression and regulation of circRNAs can affect the occurrence and course of cancers,include hepatoma,gastric carcinoma and colorectal cancer.CircRNAs are abundant and multiple in human being.Now only a little of them were studied,so search circRNAs which are differently expressed between the tumor tissue and adjacent normal tissue and have important biological function in colorectal cancer can help clarify the pathogenesis of colorectal cancer.Aims:Screen and identify one circRNA which participates in regulation of the progress in colon cancer.Methods:1)Download RNA sequencing data of twelve paired colorectal cancer and its adjacent normal tissue from sequence read archive?SRA?.Then select differently expressed circRNAs by the FASTQ,TopHat2 and find circ software combine with UCSC database according to the setting standard.Predict the downstream targets of these circRNAs by targetscan,miRanda,starbase database.Structure circRNA-miRNA-mRNA co-expression regulatory networks for the circRNAs which can get good prediction from all of the three softwares.At last,analyze the GO and pathway of these circRNAs by DAVID,Pathway Studio software combine with KEEG database.2)We preliminarily verifid these circRNAs by qRT-PCR.Then analyzed the circRNA-miRNA-mRNA regulatory networks.Ultimately elected one for further study.Agarose gel electrophoresis and ligation point sequencing were used to certify the circRNA have cyclic structure.RNase R test is used to confirm the stability of this circRNA.3)Collect surgical specimens and clinicopathologic data of 40 patients with colorectal cancer,then detecte the expression level of circHIPK3 in colorectal cancer and its adjacent normal tissues by qRT-PCR,analyze whether there is significant difference between the two groups.Find out the clinical factors which related to the expression of circHIPK3.4)Cultivate HT29,SW480,LoVo,CAC02 and FHC cell,test the expression level of circHIPK3 in these cell lines,select appropriate cell line for functional experiment.Structure and transfect overexpressed plasmid,measure transfection efficiency,complete proliferation,apoptosis and migration assays.Speculate the regulatory mechanism of circHIPK3 by bioinformatics analysis and previous study,then analyze the correlation between circHIPK3 and its possible targets miRNA by qPCR.Results:1)Twelve differently expressed circRNA were found by bioinformatics software,six of them were up-regulation,the rest were down-regulation,four of them could get good prediction from all of the three softwares.GO and pathway analyze show that these circRNAs may take part in the biological process of apoptotic cycle regulation,cell proliferation,drug resistance and tissue remodeling,their signal path are concentration in Foxo and P53.2)Hsa_circ₀000400 and hsa_circ₀000284 were demonstrated have different expression in five paired tissues?p<0.05?,hub analysis suggest that hsa_circ₀000284?circHIPK3?have more node.Agarose gel electrophoresis and ligation point sequencing certify circHIPK3 have cyclic structure,RNase R test show it have a good stability.3)The expression of circHIPK3 are significantly down-regulated in the cancer tissues than in the adjacent normal tissues?p<0.05?,the different expression of circHIPK3 is only associated with the metastasis of tumor.4)The expression of circHIPK3 is also significantly down-regulated in the tumor cell line than in FHC cell?p<0.05?.LoVo and SW480 cell line are used for functional experiment.We found that overexpress circHIPK3 can weaken the proliferation and migration ability of LoVo and SW480 cell,meanwhile promote their apoptosis.The analysis result suggest that circHIPK3 may participate in process regulation of colorectal cancer by circHIPK3-miR-20a-5p-P53 axis.The ?CT of circHIPK3 is negative correlated with miR-20a-5p?r =-0.7378,p = 0.0149?.Conclusion:The expression of circHIPK3 is significantly down-regulated in the colorectal cancer tissues and tumor cell lines.the expression of circHIPK3 are related to the tumor metastasis in patient.CircHIPK3 can weaken the growth and migration ability in tumor cell.CircHIPK3 maybe a tumor suppressor regulator in colorectal cancer.