| Background and ObjectiveHepatocellular carcinoma(HCC)is one of the most common malignancies in our country,with high incidence and mortality rates.Metastasis and recurrence has become one major obstacle for further improving the long-term survival of HCC patients.Therefore,it is critical to elucidate the mechanisms involved in HCC tumorigenesis and metastasis.Long noncoding RNAs(IncRNAs)are recently found a class of non-protein-coding RNAs,which are closely related to tumorigenesis and development of tumors.Long noncoding RNA regulator of reprogramming(linc-ROR)was first discovered in induced pluripotent stem cells(iPSCs),significantly correlated with tumor growth,apoptosis,metastasis,and pluripotency,suggesting the potential value of linc-ROR as a therapeutic target.Recently studies have showed that IncRNAs could act as competing endogenous RNAs(ceRNAs),which can combine with miRNA response elements(MREs)and inhibit miRNAs functions and activities,subsequently modulating the derepression of miRNA targets at the level of post-transcriptional regulation.Linc-ROR is highly expressed in breast cancer,bladder cancer,lung cancer,etc.,and it is closely related with the malignancy of tumors.Nevertheless,there is no relevant research about the function of linc-ROR and its underlying mechanism involving in HCC.In the present study,we comprehensively explore the expression,clinical pathological significance and biological roles of linc-ROR in HCC.Moreover,to further explore the mechanisms of the linc-ROR/miRNA/mRNA pathway regulatory network in the invasion and metastasis of HCC,with the aim of better elucidating the mechanisms of linc-ROR dysregulation and laying a foundation for formulating novel therapeutic target for treatment of HCC.Materials,Methods and ResultsPart I The Analysis of Expression and Clinical Significance of linc-ROR in HCC Materials and Methods1.QRT-PCR was performed to dectect the linc-ROR expression in 20 of 88 primary HCC and corresponding non-tumor liver tissues obtained from HCC patients.Next,expression of linc-ROR was applied to all the 88 HCC tissues and corresponding non-tumor liver tissues.2.The correlations between linc-ROR expression levels and the clinical factors as well as prognosis were determined in the whole 88 HCC tissues.Results1.It was observed that the relative level of linc-ROR was significantly upregulated in 20 cases of HCC tissues in comparison with corresponding non-tumor liver tissues.2.In 88 cases of HCC tissues,linc-ROR expression level was associated with clinicopathological characteristics including lymph node metastasis,vascular invasion,TNM stage and recurrence.The Kaplan-Meier survival analysis indicated that the upregulated expression of linc-ROR was significantly correlated with shorter OS(p<0.001)and DFS(p<0.001)of HCC patients.Part II The Impacts of linc-ROR on Biological Behaviors of HCC Cells Materials and Methods1.Real-time RT-PCR analysis was performed to detect the expression of linc-ROR in a panel of human HCC cell lines with different metastatic potentials(HepG2,SMMC-7721,HCCLM3 and MHCC97-H).2.A linc-ROR overexpression vector was constructed and introduced into low metastatic HCC cell lines(HepG2 and SMMC-7721)through whereas shROR vector was introduced into highly metastatic HCC cell lines(HCCLM3 and MHCC97-H).Wound healing assays and transwell assays with or without Matrigel were performed to detect the in vitro cell migration and invasion abilities.3.The stable HepG2/linc-ROR and HCCLM3/shROR cells were transplanted into the left hepatic lobe of nude mice to establish the orthotopic HCC animal models.After 10 weeks,mice were sacrificed,and their livers and lungs were dissected and prepared for histological analysis.The intrahepatic and lung metastasis status was determined compared with the control groups.4.Western blotting and Immunofluorescence assays were performed to analyze the effect of linc-ROR on the expression of epithelial and mesenchymal molecular makers in HCC cells.Results1.Linc-ROR expression increased progressively from the normal human hepatocyte cell line(HH),to low metastatic HCC cell lines(HepG2 and SMMC-7721),and finally to highly metastatic HCC cell lines(HCCLM3 and MHCC97-H).2.Wound healing and transwell assays confirmed the positive effect of linc-ROR on migration and invasion capacity after linc-ROR overexpression in HepG2(or SMMC-7721)cells,whereas inhibited migration and invasion occurred after knockdown of linc-ROR in HCCLM3(or MHCC97-H)cells.3.The incidence and number of both intrahepatic and lung metastasis in the linc-ROR group was significantly increased,in compared with the control group.Likewise,the incidence and number of both intrahepatic and lung metastasis in the shROR group was significantly decreased,as compared to the control group.Furthermore,the difference was further confirmed by hematoxylin and eosin(HE)staining of liver and lung sections.4.Upregulation of linc-ROR could lead to decreased expression of epithelial markers(E-cadherin and β-catenin)and increased expression of mesenchymal markers(N-cadherin and Vimentin)in HepG2 and SMMC-7721 cells.In contrast,in linc-ROR-downregulated HCCLM3 and MHCC97-H cells,the expression of epithelial markers was markedly increased and the expression of mesenchymal markers was significantly decreased.Linc-ROR could induce EMT phenotype of HCC cells to support migration and invasion.Part III The Analysis of Molecular Mechanisms of linc-ROR/miR-145/ZEB2 pathway in Regulating the Motility and Metastasis of HCC Materials and Methods1.Bioinformatics prediction miRNA which could interact with linc-ROR.RT-PCR was performed to examine the expression level of miR-145 in HCC cells stably transfected with linc-ROR or sh-ROR.And RT-PCR was also performed to examine the expression level of linc-ROR when miR-145 was changed in HCC cells.2.Verification linc-ROR combination with miRNA by dual-luciferase reporter gene assay,RIP assay and RNA abundance measure.3.Wound healing assays,transwell assays with or without Matrigel,western blotting and immunofluorescence assays were performed to detect miR-145 in vitro cell migration and invasion abilities.Analysis whether linc-ROR could function as sponge for miR-145 to promote the biological function of HCC.4.RT-PCR and western blot analyses the effects of linc-ROR and miRNA on the mRNA and protein expression of target gene.RT-PCR analyses the relationship of expression levels among linc-ROR,miRNA and its target gene in HCC tissues.Results1.Bioinformatics prediction showed that miR-132、miR-133、miR-145-5p、miR-181a、miR-19a-5p、miR-200b、miR-205-5p、miR-214、miR-219-5p、miR-320c、miR-34a-5p、miR-454、miR-520c-3p、miR-99b-3p and let-7a-1 had MREs which could be combined with linc-ROR.We then focused on miR-145,which exhibited the greatest change(4.73 fold).The miR-145 expression level was correlated negatively with linc-ROR expression level.There was no difference in linc-ROR levels after knockdown of miR-145 or ectopic expression.2.The luciferase reporter targeting linc-ROR was constructed and co-transfected miR-145 mimics in HCC cells,dual-luciferase reporter gene assay showed that linc-ROR and miR-145 were combined.RIP assay farther confirmed that miR-145 is a bona fide linc-ROR-targeting miRNA.In HCC cells,the abundance of linc-RoR were comparable to or higher than miR-145.3.Inhibition of miR-145 mimics the functions of linc-ROR overexpression in HCC cells.The function of linc-ROR in HCC cells in vitro was partially reversed by miR-145.4.In HepG2 and SMMC-7721 cells,the ectopic expression of linc-ROR upregulated ZEB2 at the transcript and protein levels.Co-transfection of linc-ROR overexpression plasmid and miR-145/mimics could significantly reduce ZEB2 expression level.In HCCLM3 and MHCC97-H cells,shROR decreased ZEB2 at the transcript and protein levels.Similarly,miR-145/inhibitors restored the decrease of ZEB2 expression in the HCC cells transfected with shROR.MiR-145 expression level in HCC tissues was significantly lower than those in the adjacent normal tissues.Additionally,by linear regression analysis,miR-145 level and linc-ROR level were negatively correlated.Meanwhile,the expression of ZEB2 in HCC and corresponding non-tumor tissues was also measured by qRT-PCR.It was showed that ZEB2 had high expression in HCC tissues compared with those in the corresponding normal tissues.Besides,ZEB2 and linc-ROR expression levels were positively correlatedConclusions1.Linc-ROR was significantly upregulated in HCC tissues.The upregulation of linc-ROR was correlated with metastasis and poor survival in HCC patients.2.Linc-ROR could promote invasion and metastasis as well as the EMT process of HCC cell in vitro and in vivo.3.The linc-ROR/miR-145 axis promotes invasion and metastasis in hepatocellular carcinoma via induction of epithelial-mesenchymal transition by targeting ZEB2.4.In conclusion,we discovered that linc-ROR was highly expressed in HCC tissues and played a key role in regulating HCC metastasis.Linc-ROR competitively binds to miR-145,and subsequently up-regulates the expression of its target gene ZEB2 to promote the migration and invasion of HCC cells.Collectively,our research implicates the relevance of linc-ROR/miR-145/ZEB2 regulatory network as a potential therapeutic target for the highly aggressive and malignant HCC cancers. |
