| ObjectiveTo detect MIR22HG expression level in HCC and identify the role of MIR22HG in regulation of hepatocellular carcinoma progression,and find out the molecular mechanism of these roles.Methods1.MIR22HG expression and the correlation with the prognosis of HCC patients.qRT-PCR was used to detect the expression of MIR22HG in 52 paired HCC tumor tissues and corresponding non-tumor tissues(52-patinet cohort).Independent sample t test was used to analyze the differences of MIR22HG between HCC tumor tissues and matched non-tumor tissues.TCGA and GEO gene sets were used to validate to expression of MIR22HG in HCC.Next,we performed in situ hybridization(ISH)to detect the expression of MIR22HG in 145 paraffin-embedded HCC tissues(145-patient cohort).Spearman rank correlation method was used to analyze the correlation between MIR22HG expression and clinical pathological features.Kaplan-Meier survival and log-rank test were used to analyze the correlation between MIR22HG expression and the prognosis of HCC patients.The effects of variables on survival were determined by univariate and multivariate Cox proportional hazards modeling.2.The biological effect of MIR22HG on HCC.qRT-PCR was used to detect the expression of MIR22HG in different HCC cell lines.The human MIR22HG sequence was introduced into SK-Hep-1 and SMMC-7721 cells by lentiviral transduction for its stable expression and short-hairpin RNAs specifically targeting MIR22HG were introduced into HCC-LM3 cell line to silence MIR22HG CCK-8,EdU and mouse subcutaneous xenograft models were applied to detect the effect of MIR22HG on proliferation of HCC.Transwell and nude mice lung metastases model were used to detect the effect of MIR22HG on the invasion and metastasis of HCC.3.The relationship between MIR22HG and miR-22-3p.qRT-PCR was used to detect the expression of miR-22-3p in MIR22HG-overexpressed or-silenced cells and 52 paired HCC tumor and corresponding non-tumor tissues.Moreover,TCGA and GEO gene sets were used to validate to expression of miR-22-3p in HCC.Kaplan-Meier survival and log-rank test were used to analyze the correlation between miR-22-3p expression and the prognosis of HCC patients.Pearson correlation analysis was used to test the correlation between MIR22HG and miR-22-3p.At last,transwell was used to investigate whether MIR22HG plays a key role through miR-22-3p in HCC.4.MIR22HG regulates HMGB1 via miR-22-3p.MiRanda?PicTar softwares were used to predict potential target genes of miR-22-3p.qRT-PCR,western blot and luciferase reporter assays were used to demonstrate that MIR22HG regulates HMGB1 expression by splicing into miR-22-3p.In vitro migration and invasion assays were used to prove MIR22HG inhibited invasion and migration in HCC through regulating HMGB1.5.The relationship between MIR22HG and HuR.Starbase v2.0 online software and PAR-CLIP were used to analyze proteins that bind directly to MIR22HG.RIP and RNA pull down assays were used to demonstrate MIR22HG binds directly to HuR.The effect of HuR on the half-life of MIR22HG was examined after treating with cycloheximide.RIP and qRT-PCR were used to illustrate the effect of MIR22HG on binding capabilities of HuR to its target oncogenes.EdU and transwell assays were used to investigate whether MIR22HG can inhibit the biological function of HuR.Results1.Low expression of MIR22HG was associated with poor prognosis in patients with HCC.We used qRT-PCR to detect the expression of MIR22HG in 52 paired HCC tumor tissues and corresponding non-tumor tissues.The results showed that the expression of MIR22HG in HCC tumor tissues was significantly lower than that in matched non-tumor tissues(P<0.01).According to the microarray data from TCGA and GEO gene sets,MIR22HG was significantly downregulated in HCC tissues(P<0.01).Furthermore,the expression of MIR22HG in 145 paraffin-embedded HCC tissues was detected by in situ hybridization.The results also showed that the expression of MIR22HG in HCC was significantly lower than that in matched non-tumor tissues.Moreover,Spearman's rank correlation analysis revealed that the low expression of MIR22HG was significantly associated with the pathological stage(P = 0.004),portal vein tumor thrombus(P = 0.004)and Barcelona stage(P = 0.005),but not with the gender,age,tumor size,cirrhosis,HBV infection,recurrence and metastasis.In addtion,Kaplan-Meier survival analysis and log-rank test were used to analyze the relationship between MIR22HG expression and prognosis of 145 HCC patients.It was showen that patients with low MIR22HG expression had significantly shorter overall survival(OS)and disease-free survival(DFS)than those with high MIR22HG expression(OS P<0.01,DFS P = 0.042).Furthermore,using Kaplan-Meier survival analysis and log-rank test to examine the relationship between MIR22HG expression and the prognosis of HCC patients in the TCGA liver cancer data set,we also found that the overall survival and disease-free survival of HCC patients with low MIR22HG expression were significantly shortened(OS P = 0.015,DFS P=0.003).2.MIR22HG inhibited the proliferation,invasion,migration and metastasis of hepatocellular carcinoma cells.Firstly,we detected the expression of MIR22HG in hepatocellular carcinoma cell lines by qRT-PCR.MIR22HG was highly expressed in HCC-LM3,but relatively low in MHCC-97H,SK-Hep-1,HepG2 and SMMC-7721.Therefore,to evaluate the biological function of MIR22HG in HCC,the human MIR22HG sequence was introduced into SK-Hep-1 and SMMC-7721 cells by lentiviral transduction for stable expression.Additionally,short-hairpin RNAs specifically targeting MIR22HG were introduced into HCC-LM3 cells to silence MIR22HG.The results showed that MIR22HG overexpression significantly inhibited the proliferation of SK-Hep-1 and SMMC-7721 cells by CCK-8 assays(P<0.05),while the inhibition of MIR22HG significantly promoted the proliferation of HCC-LM3 cells by EdU.Furthermore,the effect of MIR22HG on tumor growth in vivo was examined using mouse subcutaneous xenograft models.Consistently,proliferation of tumor xenografts derived from MIR22HG-overexpressing SK-Hep-1 and SMMC-7721 cells were significantly inhibited,while knockdown of MIR22HG significantly enhanced the proliferation of subcutaneous tumor.Then,it was found that overexpression of MIR22HG could dramatically inhibit the mobility and invasiveness of SK-Hep-1 and SMMC-7721 cells by in vitro migration and invasion assays,whereas MIR22HG knockdown promoted the mobility and invasiveness of HCC-LM3 cells.To confirm these data in vivo,MIR22HG-overexpressing SK-Hep-1 cells and MIR22HG-silenced HCC-LM3 cells were intravenously injected into nude mice via tail vein.It was showed that overexpressing MIR22HG inhibited lung metastasis,while silencing MIR22HG significantly accelerated lung metastasis.3.MIR22HG and miR-22-3p were positively correlated.By analyzing the sequence of MIR22HG,MIR22HG was found to be the host gene of miR-22-3p which was quite conserved,suggesting that MIR22HG may be correlated with miR-22-3p.Then we found out overexpression of MIR22HG markedly increased miR-22-3p expression in SMMC-7721 and SK-Hep-1 cells,whereas silencing MIR22HG significantly reduced miR-22-3p expression in HCC-LM3 cells.qRT-PCR results showed that the expression of miR-22-3p in HCC tumor tissues was significantly lower than that in matched non-tumor tissues(P<0.01).Consistently,according to TCGA and GEO gene sets microarray data,miR-22-3p was significantly downregulated in HCC tissues(P<0.01).In addtion,Kaplan-Meier survival analysis and log-rank test were used to analyze the relationship between miR-22-3p expression and prognosis of HCC patients in TCGA data sets.Results showed that patients with low miR-22-3p expression had significantly shorter overall survival(OS)and disease-free survival(DFS)than those with high miR-22-3p expression(OS P<0.01,DFS P<0.01).Furthermore,using Pearson correlation analysis to examine the relationship between the expression of MIR22HG and miR-22-3p in TCGA cohort and 52-patient cohort,we found that MIR22HG and miR-22-3p was positively correlated(52-patient cohort:r=0.589,P<0.01;TCGA cohort:r=0.585,P<0.01).Finally,using gene set enrichment analysis(GSEA),it was found that the low expression of MIR22HG and miR-22-3p were related to the metastasis gene set,suggesting that miR-22-3p and MIR22HG can both inhibit the invasion and metastasis of HCC cells.Next,we found out miR-22-3p mimic could inhibit the invasion and metastasis of SK-Hep-1 and SMMC-7721 cells through in vitro invasion and migration assays,while miR-22-3p inhibitor promoted invasion,metastatic ability in SK-Hep-1 and SMMC-7721 cells.Subsequently,we repressed miR-22-3p expression in MIR22HG-overexpressing SK-Hep-1 and SMMC-7721 cells and showed that miR-22-3p inhibition rescued the migration and invasion capabilities decreased by MIR22HG overexpression.Conversely,miR-22-3p upregulation suppressed the migration and invasion promoted by MIR22HG knockdown in HCC-LM3 cells.These findings suggested that MIR2FF2HG inhibited the biological function of HCC cells via miR-22-3p.4.MIR22HG regulated HMGB1 via miR-22-3p.We performed bioinformatics analysis using the miRanda and picTar algorithms to investigate the targets of miR-22-3p regulated by MIR22HG and overlapping these genes with those up-regulated in liver cancer(GSE14520 and GSE6762 dataset),which yielded 5 candidate genes,including HMGB1,CD147,TIAM1,SP1,and MYCBP.To verify these 5 candidate genes,we found that MIR22HG and miR-22-3p mimic significantly downregulated the expression of HMGB1 protein by qRT-PCR and western blot,while no effect was found on mRNA and protein expression levels of the other four genes,suggesting that HMGB1 may be the target of miR-22-3p.Subsequently,We detected the expression of HMGB1 in HCC by western blot,and observed the high expression of HMGB1 in HCC.Pearson correlation analysis indicated that miR-22-3p was negatively correlated with HMGB1 protein expression.The 3'-untranslated region(UTR)and the non-specific miR-22-3p-binding site of HMGB1 were cloned into a luciferase reporter vector(psiCHECK-wt-HMGB1 or psiCHECK-mut-HMGB1)to detect the effect of miR-22-3p and MIR22HG on the fluorescence of HMGB1 reporter gene.Luciferase reporter assays showed that miR-22-3p mimic transfection significantly reduced the luciferase activity of psiCHECK-wt-HMGB1 while miR-22-3p inhibitor led to increased luciferase activity.In the meantime,MIR22HG overexpression significantly reduced the luciferase activity of psiCHECK-wt-HMGB1;however,this effect was rescued by applying the miR-22-3p inhibitor;nonetheless,both MIR22HG and miR-22-3p showed little effect on the luciferase activity of psiCHECK-mut-HMGB1,indicating that MIR22HG produced miR-22-3p to bind the 3'UTR of HMGB1.Finally,as found in in vitro invasion and migration assays,HMGB1 promoted the invasion and migration of HCC cells while miR-22-3p and MIR22HG inhibited the invasion and migration of HCC cells,which could be abolished by HMGB1,suggesting the inhibition of MIR22HG on HMGB1 expression via miR-22-3p so as to restrain invasion and migration of HCC cells.5.MIR22HG competedly bound with HuR against HuR target genes.StarBase software v2.0(http://starbase.sysu.edu.cn/rbpLncRNA.php)predicted the potential interaction of MIR22HG with HuR protein.Meanwhile,bioinformatics analysis using HuR photoactivatable-ribonucleotide-enhanced cross-linking,immunoprecipitation(PAR-CLIP;GSE28865)analysis revealed that there were multiple binding sites between MIR22HG and HuR,suggesting the derect binding of MIR22HG with HuR.According to RNA-binding protein immunoprecipitation(RIP)assay,a direct binding relationship between MIR22HG and HuR were confirmed and RNA pull-down assay further validated the interaction of MIR22HG and HuR.Owing to the facts that combination of HuR and its target genes can affect the half-life and thus the stability of the target genes,we detected the half-life of MIR22HG in HuR-silenced cells treated with cycloheximide by qRT-PCR.It showed that HuR knockdown significantly shorten the half-life of MIR22HQ demonstrating that HuR bound and stabilized the MIR22HG RNA.Furthermore,we found that overexpressing HuR in the MIR22HG-overexpressed cells could relieve the inhibition of MIR22HG on proliferation,invasion and migration of HCC cells as shown by the results of EdU and transwell assays,suggesting that HuR might mediated the function of MIR22HG.The RIP assays confirmed that HuR directly bound to its target genes such as CTNNB1,CCNB1,HIF1A,BCL2,COX2,C-FOS,MDM2,VEGFA and knockdown of HuR can downregulated the mRNA or protein level of the target genes by qRT-PCR and western blot.We then investigated the effect of MIR22HG on the interaction between HuR and these oncogenes.RIP assays demonstrated that in the presence of MIR22HQ a marked increase in binding between MIR22HG and HuR was observed,which thereby reduced the binding capabilities of HuR to the oncogenes.Accordingly,both mRNA and protein levels of HuR target genes were dramatically reduced after overexpressing MIR22HG,but highly induced in MIR22HG-silencd cells,indicating that MIR22HG competed with these oncogenes for binding with HuR.Conclusion1.MIR22HG was significantly downregulated in HCC tissues and its low expression was associated with poor prognosis in HCC patients.2.MIR22HG can inhibit proliferation,invasion,migration and metastasis of HCC.3.MIR22HG was positively correlated with miR-22-3p,and MIR22HG was spliced into miR-22-3p to inhibit the invasion and migration of HCC cells.4.MIR22HG inhibited the expression of HMGB1 via miR-22-3p to repress the proliferation,invasion and migration of HCC cells.5.MIR22HG competed with HuR target genes for binding with HuR,resulting in the downregulation of HuR target genes,thereby inhibiting the proliferation,invasion and migration of HCC cells. |
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