Preliminary Study Of MiR-196b-5p And Ednrb In The Progression Of Hepatocellular Carcinoma

Background:Hepatocellular carcinoma?HCC?is one of the most common malignant tumors universally.The major treatment of HCC is still surgical therapy until now and the prognosis of HCC patients is still poor due to the rising incidence,late diagnosis,constant recurrence and persistent drug resistance.Thus,it is necessary to discover novel diagnostic and prognostic biomarkers for HCC.MicroRNA?miRNA?is one of the types of non-coding RNA and regulates gene expression in transcriptional level and post transcriptional modification.Previous studies demonstrated that the abnormal expression of miR-196b-5p was found in various malignances and could affect various biological processes.However,the clinical value and mechanism of miR-196b-5p in HCC have never been reported.Endothelin receptor type B?EDNRB?is one of the members of G protein-coupled receptor family.Previous studies verified the abnormal expression of EDNRB and that EDNRB participate in the progression of various malignances.The low expression of EDNRB and EDNRB ability to suppress the migration and invasion of HCC cells were confirmed in previous study.However,relationship between miR-196b-5p and EDNRB has not been reported,and the molecular mechanism between them in the progression of HCC remains to be elucidated.Objective:To explore the clinical value and mechanism of miR-196b-5p and EDNRB in HCC.Methods:1.We evaluated the expression level and clinical value of miR-196b-5p inHCC from the data in TCGA database and microarray data in GEOdatabase.2.qRT-PCR was used to detect miR-196b-5p expression level in 67 HCCparaffin embedded tissues and corresponding noncancerous tissues.Correlations between miR-196b-5p expression level and clinicopathologicalcharacteristics were analyzed in our study.3.Comprehensive meta-analysis of miR-196b-5p expression based on TCGA,GEO and qRT-PCR was performed in our study.4.We used 12 online tools to predict target genes of miR-196b-5p,and onlygenes that were simultaneously predicted by five or more than five tools were selected.Subsequently,we acquired the potential target genes of miR-196b-5p by overlapping the predicted target genes and the down-regulated mRNAs in HCC based on TCGA database.We performed GO and DO functional annotation,KEGG pathway enrichment analysis,PPI network construction,Spearman rank correlation test and binding siteprediction of the potential target genes of miR-196b-5p,and we confirmedthe final target gene of miR-196b-5p which needs further study via literaturereviewing.5.We evaluated the expression level and clinical value of EDNRB in HCC from TCGA and GEO database.6.Immunohistochemistry was used to detect EDNRB protein expression level in the above 67 HCC paraffin embedded tissues and corresponding noncancerous tissues.Correlations between EDNRB expression level and clinicopathological characteristics were analyzed in our study.7.Dual-luciferase reporter assay was used to detect whether directly targeted relationship between miR-196b-5p and EDNRB.Results:1.The expression level of miR-196b-5p in HCC tissues was higher than normal control liver tissues based on TCGA sequencing data and GEO microarray data(SMD=0.54,95%CI:0.35-0.73,P _{heterogeneity}=0.206,I²=26.7%).The expression level of miR-196b-5p has correlations with gender?P=0.035?,histologic grade?P=0.041?and vascular invasion?P=0.023?in HCC patients.2.The expression level of miR-196b-5p in HCC tissues was higher than the corresponding noncancerous tissues based on qRT-PCR?P=0.0007?.The expression level of miR-196b-5p has correlations with tumor size?P=0.03?,tumor node?P=0.024?,vascular invasion?P=0.029?and capsular invasion?P=0.026?in HCC patients.3.Comprehensive meta-analysis of miR-196b-5p expression based on TCGA,GEO and qRT-PCR verified that the expression level of miR-196b-5p in HCC tissues was higher than normal control liver tissues(SMD=0.56,95%CI:0.39-0.72,P _{heterogeneity}=0.275,I²=18.3%).4.We used 12 online tools to predict target genes of miR-196b-5p,and only genes that were simultaneously predicted by five or more than five tools were selected.Subsequently,we acquired 107 potential target genes of miR-196b-5p by overlapping the predicted target genes and the down-regulated mRNAs in HCC based on TCGA database.We performed GO and DO functional annotation,KEGG pathway enrichment analysis and PPI network construction of the 107 potential target genes of miR-196b-5p,and then focused on the 9 genes in Pathways in Cancer of KEGG pathway.We performed Spearman rank correlation test between the 9 genes and miR-196b-5p based on TCGA,and negative correlations were found between miR-196b-5p and EDNRB,androgen receptor?AR?,forkhead box O1?FOXO1?and insulin like growth factor 1?IGF1?.Bases complementary were found between miR-196b-5p and EDNRB and FOXO1though binding sites prediction.After literature reviewing,EDNRB was chosen as the target gene of miR-196b-5p which needs further study.5.The expression level of EDNRB in HCC tissues was lower than normal control liver tissues based on TCGA and GEO data(SMD=-1.48,95%CI:-1.63-?-1.33?,P _{heterogeneity}=0.116,I²=32.4%).6.LowexpressionlevelofEDNRBinHCCwasverifiedby immunohistochemistry?P<0.0001?.The expression level of EDNRB has potential to be related to AFP level?P=0.055?in HCC patients,more samples are required in the future studies.7.Dual-luciferase reporter assay found that no directly targeted relationship existed between miR-196b-5p and EDNRB.Conclusions:1.The expression level of miR-196b-5p in HCC tissues was higher than normal control liver tissues.Moreover,miR-196b-5p expression level had correlations with the clinicopathological characteristics such as vascular invasion of HCC.2.The expression level of EDNRB in HCC tissues was lower than normal control liver tissues.3.MiR-196b-5p and EDNRB may play important roles in the invasion and migration of HCC cells,however no directly targeted relationship existed between them,the mechanisms within still need further study.