| Background : Hepatocellular carcinoma(HCC)is one of the most common and aggressive malignant tumors in the world,while the molecular mechanisms underlying HCC tumorigenesis and progression are not completely clear.Despite the major advances achieved in the diagnostic tools of HCC,only one third of the newly diagnosed patients are presently eligible for curative treatments.To reduce metastasis and raise survival rate,the deepgoing exploration of molecular mechanism underlying HCC tumorigenesis and the further search for new molecular targets affecting tumor growth and metastasis are required in both clinical diagnosis and treatment for patients.Non-coding RNAs contain micro RNA(mi RNA)and long non-coding RNA(lnc RNA),which have been demonstrated to participate in the progression of many cancers including HCC.As highly conserved small non-coding RNAs with sizes of 20–25 nucleotides,mi RNAs could negatively regulate gene expression at post-transcriptional level by directly binding to the 3?-untranslated region(3?-UTR)of target messenger RNA(m RNA)to induce m RNA deregulation or translational repression.Lnc RNAs with a size of longer than 200 nucleotides have been reported to participate in the pathogenesis of tumors by affecting different biological processes,including cell proliferation,apoptosis,differentiation and migration.Lnc RNA could bind to DNA,RNA or protein to affect the expression and function of targetgenes.The relationship between lnc RNA and mi RNA was also a hot topic.The aim of our study was to investigate the roles and related mechanisms of mi RNA and lnc RNA in HCC tumorigenesis.Methods:Tissue samples were collected from patients with HCC who underwent partial liver resection at Department of Hepatobiliary and Pancreatic Surgery,the Affiliated Hospital of Qingdao University,and blood samples were collected from HVs and patients with CHB or cirrhosis at Department of Infectious Diseases,the Affiliated Hospital of Qingdao University.HCC cell lines were obtained from the Cell Resource Center of Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences.Human HCC cell lines and normal liver cell line were cultured.Total RNAs of tissues or plasma were extracted and subjected to reverse transcription,high-throughput sequencing for small RNAs and real time quantitative PCR were used to show expression of mi R-92 b in tumor tissues and plasma.q RT-PCR was used for lnc RNA DANCR.The correlations of mi R-92 b and DANCR expression with clinicopathologic characteristics of HCC were analyzed.As circulating ?-fetoprotein(AFP)is frequently used for the diagnosis of HCC,we also examined the relationship between AFP and mi R-92 b in the plasma.In functional analysis,Cell Counting Kit-8(CCK-8)assay was used to evaluate cell proliferation,wound-healing assay was used to evaluate cell metastesis and transwell invasion assay was used to evaluate cell invasion.Mi R-92 b overexpression lentivirus,mi R-92 b knockdown lentivirus,DANCR knockdown lentivirus and the NC lentivirus were purchased.HCC cells were infected with the corresponding lentivirus were injected subcutaneously to the BALB/c nude mice purchased from the Shanghai Institute of Materia Medica and constructed HCC xenograft mouse model.The tumor volumes were calculated to evaluated HCC cell proliferation in vivo.The lungs of mice were fixed with formaldehyde and the fixed tissues were serial sectioned and stained with hematoxylin-eosin,Then lung metastatic nodules were observed and calculated to evaluated HCC cell metastasis in vivo.In mechanic analysis,q RT-PCR and Western blot were used to evaluate the expression of vimentin,CCND1 and MYC in tissues and cell lines.Immunofluorescence analysis was used to evaluate location of ?-catenin in HCC with mi R-92 b mimics transfection.Luciferase reporter assay was used to evaluate the trans-activity of ?-catenin.Statistical analysis was performed using the SPSS program.Statistical significance was calculated by Student's t-test,?2 test or one-way ANOVA.Pearson analysis was used in correlation analysis and the statistical significance of differences between two AUCs was calculated and compared by using Delong's algorithm.Results:Firstly,we showed that mi R-92 b was significantly upregulated in tumor tissues and plasma(p<0.05)of HCC patients.Correlation analysis showed that mi R-92 b expression level was highly correlated with gender and microvascular invasion of HCC patients.Female HCC patients(p =0.041)and HCC patients with microvascular invasion(p =0.026)exhibited higher mi R-92 b expression.The expression of mi R-92 b in the plasma of HCC patients was positively associated with the corresponding AFP level(p<0.001).The correlation indicated that mi R-92 b might be a potential biomarker in the clinical diagnosis of HCC.Functional analysis showed that mi R-92 b could promote cell proliferation(p<0.001),migration and invasion(p<0.05)of HCC cells with mi R-92 b overexpression,while mi R-92 b knockdown could inhibit cell proliferation(p<0.01),migration and invasion(p<0.05)of HCC cells.Our results showed that mi R-92 b overexpression could increase the tumor sizes(p<0.05)and lung metastasis rates.On the other hand,mi R-92 b inhibitor could decrease the tumor sizes(p<0.01)and lung metastasis rates compared with control group.Mechanistic investigations suggested that mi R-92 b could significantly promote the ?-catenin signaling pathway and the expressions of downstream oncogenes by promoting the translocation of ?-catenin protein from cytoplasm to nuclear.Our study suggested the potential value of mi R-92 b as a biomarker in the clinical diagnosis and treatment of HCC patients.Secondly,we explored the roles of lnc RNA DANCR in the diagnosis and treatment of HCC patients.DANCR is considered to act as an oncogene in many cancer types.However,the roles of DANCR in diagnosis and progression of HCC are still unknown.In this study,we showed that DANCR was upregulated in tumor tissues and plasma of patients with HCC(p<0.001).There was a significant positive relationship between plasma DANCR and the corresponding tumor tissue DANCR levels(p<0.001).Plasma DANCR was significantly lower after surgery than that before surgery(p<0.01),which suggested that plasma DANCR might derive from tumor tissues in patients with HCC.Correlation analysis showed that the expression of DANCR in HCC tissues was highly correlated with differentiation(p=0.018),microvascular(p=0.003)and liver capsule invasion(p=0.023)of HCC patients.It was correlated with microvascular(p=0.017)and liver capsule invasion(p=0.004)in plasma.So HCC patients with microvascular and liver capsule invasion exhibited higher expression of DANCR in plasma and tumor tissues.Plasma DANCR exhibited significantly increased discriminatory power for differentiating patients with HCC from HVs and non-HCC patients compared with AFP(p=0.026),which has been used as a biomarker for HCC diagnosis.And further analysis showed that plasma DANCR exhibited significantly increased discriminatory power for differentiating patients with HCC from patients with chronic hepatitis B and cirrhosis compared with AFP(p=0.001).So DANCR can act as a potential biomarker for HCC diagnosis.Furthermore,knockdown of DANCR downregulated the expression of ?-catenin,repressed the ?-catenin pathway and inhibited the expression of ?-catenin downstream genes.CCK-8 and transwell invasion assay showed that downregulation of DANCR in HCC cells could inhibit cell proliferation(p<0.01)and metastasis(p<0.05)of HCC cells.HCC xenograft mouse model showed that HCC cells with DANCR koockdown used to generated tumors could decrease the tumor sizes(p<0.01)and lung meatastasis rates compared with the control group.In conclusion,this study revealed for the first time the importance of DANCR in clinical diagnosis and disease progression of HCC patients.Dissussion and conclusion:MiR-92 b and DANCR were significantly upregulated in tumor tissues and plasma of HCC patients.Mi R-92 b could promote HCC cell proliferation and metastasis in vitro and in vivo.Mi R-92 b activated the ?-catenin signaling in HCC cells.DANCR acted as a more efficient biomarker in HCC diagnosis compared with AFP.Knockdown of DANCR could inhibit cell proliferation and metastasis in HCC cells.In summary,the important roles of mi RNAs and lnc RNAs in HCC are attracting more and more researchers.Although the progress involving the roles of mi RNAs and lnc RNAs in HCC have been impressive,only a small part of mi RNAs and lnc RNAs have been well characterized and a large portion remain to be further defined and explored.Thus,more efforts are urgently needed to better elucidate the function and mechanisms of liver-specific mi RNAs and lnc RNAs in tumorigenesis of HCC.We believe that the growing researches on the roles of mi RNAs and lnc RNAs in hepatocarcinogenesis could pave the way for designing novel therapeutic approaches for HCC in the future. |
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