| Objective:A growing body of evidences have shown that long noncoding RNAs(lnc RNAs)play important roles in hepatocellular carcinoma(HCC).This study aimed to explore the expression level,clinical significance and biological function of a new lnc RNA,LINC01146 in hepatocellular carcinoma.At the same time,bioinformatic methods were used to predict the potential molecular mechanism of LINC01146 in HCC.Methods:1.The lnc RNA microarray,the Cancer Genome Atlas(TCGA)database and the Gene Expression Omnibus(GEO)database were used to explore the expression level of LINC01146 in HCC tissues in the first part.The edge R package was utilized to analysis the results of lnc RNA microarray in R language.The t-test was used to explore the expression level of LINC01146 in HCC tissues.The Kaplan-Meier analysis was used to explore the association between LINC01146 and overall survival of HCC patients.The random-effects model was used for Meta-analysis of 13 GEO datasets included in the study.Afterwards,the q RT-PCR(real-time fluorescence quantitative polymerase chain reaction)was utilized to verify the expression level of LINC01146 in human HCC tissue samples.The paired t test was performed to compare the expression level of LINC01146 in HCC tissues and adjacent normal tissues.TheX~2 test was performed to explore the relationship between the expression of LINC01146 and the clinical characteristics of HCC patients.The Kaplan-Meier analysis was used to investigate the association between LINC01146 and overall survival of HCC patients.The Cox proportional risk model was used to explore independent influencing factors for prognosis in HCC patients.2.We carried out cell experiments in vitro and animal experiments in vivo in the second part.MHCC97H and Huh7 cells were used to establish stably overexpression LINC01146 HCC cell lines,while Huh7 and Hep3B cells were used to establish stably knockdown LINC01146 HCC cell lines.The CCK-8 and plate clone formation assays were used to assess the effects of overexpression and knockdown of LINC01146 on the proliferation ability of HCC cells in vitro.The Transwell chambers were used to evaluate the effects of overexpression and knockdown LINC01146 on migration and invasion of HCC cells in vitro.The flow cell cycle and apoptosis experiments were conducted to investigate the effects of overexpression and knockdown LINC01146 on the cell cycle and apoptosis of HCC cells in vitro.Afterwards,the tumor forming model in nude mice was performed to explore the effect of LINC01146 on the proliferation ability of HCC cells in vivo.HCC cells with overexpression and knockdown LINC01146 were injected into nude mice in order to explore the influence of overexpression and knockdown LINC01146 on the proliferation of subcutaneous tumor in vivo.The Hematoxylin-eosin staining(HE)was performed to observe the differences in tumor histomorphology between the knockdown LINC01146 group and the control group.The immunohistochemical(IHC)staining were used to compare the expression level of Ki-67 between the knockdown LINC01146 group and the control group.3.The bioinformatic methods were performed to explore the potential molecular mechanism of LINC01146 in HCC.The GEPIA,MEM and TANRIC websites were used to predict the co-expression genes of LINC01146 online.The Web Gestalt online site was used to conduct the Gene Ontology(GO)function annotation and the KOBAS 3.0 online site was used to construct the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment.The STRING database was used to construct a PPI network.The t test was used to explore the expression levels of core genes in HCC and the Pearson correlation analysis was used to explore the correlation between core genes and LINC01146based on TCGA database.Results:1.LINC01146 was down-regulated in HCC tissues through lnc RNA microarray analysis(FC=3.92,P=6.93E-4,FDR=0.027).The t test results showed that the expression level of LINC01146 was downregulated in HCC tissues based on TCGA database(3.08±1.55 vs.3.81±0.51,t=6.76,P<0.001).The Kaplan-Meier analysis showed that the expression of LINC01146 was associated with 5-year overall survival in HCC patients(X~2=4.70,P=0.030,N=330).The results of Meta-analysis showed that the expression level of LINC01146 was lower in HCC tissues than that in normal tissues(SMD=-0.97,95%CI[-1.33,-0.60],Z=-5.15,P<0.001).In addition,there was no significant publication bias in this Meta-analysis(P=0.656).Furthermore,the q RT-PCR results showed that the expression level of LINC01146 was significantly decreased in HCC tissues compared with adjacent normal tissues(t=-7.44,P<0.001).In addition,the expression level of LINC01146 was correlated with tumor size(X~2=8.02,P=0.005),tumor number(X~2=5.44,P=0.020),microvascular invasion(X~2=4.55,P=0.033),satellite nodules(X~2=7.31,P=0.007),HBV DNA content(X~2=5.57,P=0.018),and BCLC grade(X~2=9.19,P=0.010).The result of Kaplan-Meier analysis showed that low expression of LINC01146 was associated with poor prognosis of HCC patients(X~2=16.58,P<0.001,N=85).Moreover,the result of Cox proportional risk model indicated that high expression level of LINC01146 may be an independent protective factor for prognosis of HCC patients(HR=0.38,95%CI[0.16,0.92],P=0.033).2.We successfully constructed overexpression LINC01146 in MHCC97H cells(t=109.80,P<0.001)and Huh7 cells(t=21.93,P<0.001).The CCK-8assays showed that overexpression of LINC01146 inhibited the proliferation of HCC cells(MHCC97H:F=65.79,P<0.001;Huh7:F=532.10,P<0.001).The results of plane clone formation experiment showed that overexpression of LINC01146 inhibited the proliferation of HCC cells(MHCC97H:t=18.80,P=0.003;Huh7:t=29.26,P=0.001).The results of flow cell cycle test showed that overexpression LINC01146 retarded the cycle progression of Huh7 cells(G1 phase:t=16.47,P<0.001;S phase:t=14.02,P<0.001).The results of flow cell apoptosis test showed that overexpression LINC01146 promoted the apoptosis of HCC cells(Huh7:t=20.01,P<0.001).The Transwell assays showed that overexpression LINC01146 inhibited the migration(MHCC97H:t=15.65,P<0.001;Huh7:t=18.32,P<0.001)and invasion ability(MHCC97H:t=20.64,P<0.001;Huh7:t=26.75,P<0.001)of HCC cells.on the contrast,knockdown of LINC01146 was successfully constructed in HCC cell lines(Huh7:t=22.28,P<0.001;Hep3B:t=22.98,P<0.001).The CCK-8 results showed that knockdown of LINC01146 promoted the proliferation of HCC cells(Huh7:F=80.86,P<0.001;Hep3B:F=155.60,P<0.001).The results of plane clone formation experiment showed that knockdown of LINC01146 promoted the proliferation of HCC cells(Huh7:t=51.40,P<0.001;Hep3B:t=36.89,P=0.001).The results of flow cell cycle test showed that knockdown of LINC01146 accelerated the cell cycle of Huh7 cells(G1 phase:t=25.16,P<0.001;S phase:t=14.63,P<0.001).The results of flow cell apoptosis test showed that knockdown of LINC01146inhibited the apoptosis of HCC cells(Huh7:t=29.74,P<0.001).The Transwell assays showed that knockdown of LINC01146 promoted the migration of HCC cells(Huh7:t=18.35,P<0.001;Hep3B:t=13.27,P=0.006)and invasion(Huh7:t=9.33,P=0.001;Hep3B:t=13.33,P<0.001).3.The results of tumor formation in nude mice showed that overexpression of LINC01146 inhibited the volume(F=63.11,P<0.001)and mass(t=4.60,P=0.001)of the tumors.In contrast,knockdown of LINC01146 increased the volume(F=89.06,P<0.001)and mass(t=4.82,P=0.001)of tumors.Moreover,the HE staining results showed that tumor cells in the knockdown LINC01146group were irregular in morphology,with different nuclear sizes and shapes,obvious atypia,active mitosis,and pathological mitosis.The IHC results showed that the positive expression of Ki-67 protein was significantly increased in the knockdown LINC01146 group compared to the control group(t=17.05,P<0.001).4.The results of pathway enrichment analysis showed that the co-expressed genes of LINC01146 were mainly enriched in“metabolic pathway”,“complement and coagulation cascade”and other pathways.At the same time,a total of 12 core genes(FETUB、TTR、LPA、ALDH8A1、SERPINA4、AGXT、APOH、SERPINF2、F13B、ORM2、KNG1、F11)that interacted with LINC01146were found in PPI network.The t test results indicated that the expression levels of these core genes were all significantly lower in HCC tissues(P<0.05).Furthermore,the results of Pearson correlation analysis indicated that the expression levels of these core genes were all correlated with LINC01146(P<0.05).Conclusion:LINC01146 was downregulated in HCC tissues,and low expression level of LINC01146 was associated with poor prognosis of HCC patients.Overexpression of LINC01146 could inhibit the proliferation,migration and invasion of HCC cells,and promote the apoptosis of HCC cells in vitro.On the contrary,knockdown of LINC01146 could promote the proliferation,migration and invasion of HCC cells,and inhibit the apoptosis of HCC cells in vitro.In addition,overexpression of LINC01146 inhibited tumorigenesis of HCC cells in vivo,while knockdown of LINC01146 promoted tumorigenesis of HCC cells in vivo.Bioinformatics analysis showed that LINC01146 may affect the process of HCC by regulating the“metabolic pathway”and“complement and coagulation cascade”pathways. |
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