The Expression And Clinical Significance Of MiR-1180-3p In Hepatocellular Carcinoma

Objective:Hepatocellular carcinoma（HCC）is one of the most common malignant tumors,with a high degree of malignancy and a poor prognosis.Mounting evidence indicates that miRNAs play a critical role in tumorigenesis by participating in a series of biological processes,involving apoptosis,migration and cell proliferation.Indeed,the dysregulation of various miRNAs has been identified in malignant tumors and may serve as prognostic biomarkers and therapeutic targets.This study intends to screen prognosis-related miRNA in HCC patients based on public databases such as GEO and TCGA.Then,the expression levels of miRNA and its effort on clinical features,overall survival and cumulative survival rate were verified in an independent HCC cohort.Methods:1.The expression and functional prediction of miR-1180-3p in HCC based on bioinformatics analysis:The GSE36915 dataset,which contains the miRNA expression profile from the Gene Expression Omnibus（GEO）,was used to select the abnormally expressed miRNAs in HCC,and three significantly overexpressed miRNAs were identified in HCC tissues.We further analyzed the relationship between three upregulation miRNA and the prognosis of HCC using the TCGA database,and found high expression levels of miR-1180-3p were correlated closely with poor outcomes of HCC patients.To further understand the potential mechanism of miR-1180-3p in HCC,the target genes of the selected miRNAs were obtained using 3 miRNA target gene prediction databases,and their functions were analyzed using the online tool Web Gestalt.In addition,to screened the key target genes of mir-1180-3p in HCC,we analyzed the correlation between the expression level of target mRNA in the key KEGG pathway and the expression level of miR-1180-3p based on RNA-seq data and miRNA-seq data from TCGA database.2.The clinical significance miR-1180-3p in Hepatocellular Carcinoma:A total of 114 patients with HCC in our study underwent liver cancer resection at Guangxi Medical University Cancer Hospital from January 2017 to June 2018.All HCC patients were reviewed to analyze clinical features,including age,gender,nation,smoking status,drinking status,HBV infection,Edmonson grade,tumor number,largest tumor size,microvascular invasion（MVI）,tumor necrosis,tumor capsule,tumor thrombus,cirrhosis,steatosis and satellite nodule presence.The patients were followed up regularly by outpatient service and telephone.We analyzed miR-1180-3p expression levels in 114 paired HCC tissue and non-tumor liver tissue by RT-qPCR.To analysis the relationship between the expression of miR-1180-3p and clinical features,the cutoff values of miR-1180-3p was used to classify patients with HCC into two groups（high expression or low expression）.To analysis the relationship between the expression of miR-1180-3p and overall survival and 1-year cumulative survival rate in HCC,the Kaplan-Meier method was applied for univariate survival analysis and Cox proportional hazards regression was conducted for multivariate survival analysis.To evaluate the prognosis prediction efficacy of miR-1180-3p expression in HCC,the receiver operating characteristic（ROC）curve was plotted and the area under the curve（AUC）was calculated.Results:1.The expression and functional prediction of miR-1180-3p in HCC based on bioinformatics analysis:（1）The abnormally expressed miRNAs in HCC:Based on analysis of the miRNA-seq from the GSE36915 dataset,miR-183-5p（log₂FC=2.36,P<0.001）,miR-452-3p（log₂FC=1.73,P<0.001）,and miR-1180-3p（log₂FC=1.21,P<0.001）were significantly overexpressed in HCC compared with adjacent normal tissues（ANTs）.（2）The relation between miRNA expression and the overall survival of HCC patients:Subsequently,we used TCGA datasets to correlate the expression of 3 upregulated miRNAs with overall survival in HCC patients.miR-1180-3p overexpression was found to be related to the short-term survival of HCC patients by Kaplan-Meier survival.miR-183-5p and miR-1180-3p were found to be related to the survival of HCC patients by univariate Cox analysis,and further multivariate Cox analysis showed that the expression of miR-1180-3p（HR=1.25,95%CI=1.07-1.45,P=0.004）was identified as an independent prognostic factor for HCC.（3）miR-1180-3p target prediction and functional analysis:A total of733 target genes of miR-1180-3p were found from three prediction databases for functional enrichment analysis.The GO analyses demonstrated that the target genes were closely related to the proliferation and malignancy of tumors.The KEGG analysis showed that 54 miR-1180-3p target genes were enriched in Ten key signaling pathways,including Thyroid cancer,Thyroid hormone signaling pathway,Ras signaling pathway,Chronic myeloid leukemia,Non-small cell lung cancer,Rap1 signaling pathway,MAPK signaling pathway,Fc gamma R-mediated phagocytosis,VEGF signaling pathway and Pathways in cancer.We further analyzed the correlation between the expression of 54 mRNAs and miR-1180-3p in the key signaling pathway.The results showed that the mRNA expression levels of AKT1,ANGPT4,CASP9,ETS1,GLI2,MYO10,RAPGEF2,RPS6KA1,RXRA,TRAF1 and WNT9B genes were negatively correlated with the expression levels of miR-1180-3p,in which RAPGEF2（r=-0.41,P<0.001）and ETS1（r=-0.39,P<0.001）were significantly correlated with the expression levels of mir-1180-3p.Moreover,RAPGEF2 is mainly involved in MAPK signaling pathway and Rap1 signaling pathway,and ETS1 is mainly involved in the regulation of Ras signaling pathway and cancer pathway.2.The relationship between miR-1180-3p expression and clinical features and prognostic of HCC:（1）The expression of miR-1180-3p in HCC samples:The results of RT-qPCR revealed that miR-1180-3p expression was significantly upregulated in 114 HCC tissues compared with non-tumor tissues（t=-2.099,P=0.038）.（2）The correlation of miR-1180-3p with HCC clinical features:The expression of miR-1180-3p showed a significant positive correlation with the tumor number（χ²=6.818,P<0.05）,miR-1180-3p was overexpressed in HCC samples with multiple tumors compared with single tumor.but there was no significant difference between other clinical features and miR-1180-3p expression.（3）The correlation of miR-1180-3p with prognostic of HCC:In this study,114 patients with HCC were followed up regularly through outpatient visits and telephone calls.The overall survival time of patients was calculated from the operation time as the starting point to the time of death outcome or follow-up deadline（June 2019）.We analyzed the influence of the expression level of miR-1180-3p on the overall survival time and the 1-year cumulative survival rate of patients.The results of Kaplan-Meier analysis and Log-rank test showed that,patients with Age≤54,multiple tumors,MVI,satellite nodule and high miR-1180 expression had a shorter overall survival than those with Age>54,single tumor,none satellite nodule and low miR-1180-3p expression（P<0.05）.Moreover,the 1-year cumulative survival rate of patients with Age≤54 year was lower than those with Age>54 year（84%vs 96%,P=0.029）;the 1-year cumulative survival rate of patients with multiple tumors was lower than those with single tumor（74%vs 94%,P=0.006）;the 1-year cumulative survival rate of patients with MVI was lower than those with non-MVI（74%vs 94%,P=0.006）;the 1-year cumulative survival rate of patients with satellite nodule was lower than those with non-satellite nodule（92%vs 75%,P=0.006）;the1-year cumulative survival rate of patients with high miR-1180 expression was lower than those with low miR-1180-3p expression（93%vs 61%,P=0.011）.Univariate Cox analyses showed that,Age≤54-year,multiple tumors,patients with MVI,satellite nodule and high miR-1180 expression were risk prognostic factor for overall survival of HCC.Furthermore,multivariate Cox analyses indicated that miR-1180-3p expression was an independent prognostic factor for overall survival（HR=3.10,95%CI=1.02-9.47,P=0.047）.（4）The combine effort of miR-1180-3p expression and clinical features on the prognosis of HCC patients:Kaplan-Meier analysis showed that,patients with high miR-1180 expression、Age≤54-year、multiple tumors and MVI had a shorter overall survival than other patients（P<0.001）,and the 1-year cumulative survival rate of them was only 15%.Cox analyses showed that the combine indexes of high miR-1180 expression,Age≤54-year,multiple tumors and MVI was an independent prognostic factor for overall survival（HR=22.2,95%CI=3.71-132.8,P=0.001）.ROC curves showed that,compared with miR-1180-3p expression（AUC=69.03,95%CI=53.97-84.09）,Age（AUC=67.18,95%CI=54.80-79.55）,tumor number（AUC=67.19,95%CI=51.76-82.62）,MVI（AUC=82.62,95%CI=73.03-92.21）,the combine of miR-1180-3p expression and Age（AUC=76.84,95%CI=63.14-90.54）,the combine of miR-1180-3p expression（AUC=72.63,95%CI=55.95-89.31）and tumor number and the combine of miR-1180-3p expression and MVI（AUC=89.30,95%CI=82.93-95.66）,the combine indexes of miR-1180-3p expression,Age,tumor number and MVI（AUC=89.74,95%CI:83.00-96.48）had the highest AUC in predicting poor prognosis at follow-up of 1 year.Conclusions:1.miR-1180-3p may be promoted the development and poor prognosis of HCC through influenced the signaling pathways involved in Ras signaling pathway and MAPK signaling pathway by regulated target mRNA such as RAPGEF2 and ETS1.2.miR-1180-3p is upregulated in HCC and is correlated with tumor number.Up-regulated miR-1180-3p is associated with a poor prognosis.Thus,miR-1180-3p might be useful as an independent prognostic marker for HCC.Moreover,the combined of miR-1180-3p,Age,tumor number and MVI has a higher value in prognosis prediction of HCC.