| BackgroundHepatocellular carcinoma(HCC)is one of the most common malignant tumors in the worldwide.It is the seventh most common cancer in the world and the third cause of cancer death.In China,the fatality rate is the second highest.Although great progress has been made in the treatment of HCC in recent years,the overall therapeutic effect of HCC is still quite unsatisfactory.Recurrence and metastasis of HCC are the critical factors restricting the comprehensive treatment of HCC.Long-term follow-up found that the recurrence rate of HCC patients after radical resection was even more than 70%in 5 years.Therefore,it has important theoretical and realistic significance to elucidate the molecular mechanism and the initiating factors of the metastasis of HCC,to screen and identify new targets for invasion and metastasis of HCC,and to diagnose and intervene the invasion and metastasis of HCC at the molecular level.In recent years,the functional role of microRNAs as expression of posttranscriptional regulator genes in tumors has attracted much attention.Basic research and clinical observation showed that microRNAs may be involved in the occurrence,development,treatment and prognosis of almost all tumors,including hepatocellular carcinoma.MicroRNAs may be involved in the invasion and metastasis of tumors by affecting the motility of tumor cells,angiogenesis and microenvironment of tumors.Although the role of microRNAs in HCC has been reported,the role and mechanism of microRNAs in HCC metastasis remain unclear.miR-605-3p is a novel microRNA molecule first found in bladder cancer.It has been reported that its expression is significantly down-regulated in bladder cancer and is closely related to the metastasis of bladder cancer.However,the expression pattern,functional role and potential mechanism of miR-605-3p in HCC have not been reported in the literature.Therefore,we will analyze the correlation between the potential anti-oncogene miR-605-3p and the invasion and metastasis of HCC from cell and clinical tissue specimens,and explore the possible molecular mechanism of the involvement of miR-605-3p in the invasion and metastasis of HCC through bioinformatics analysis combined with experimental verification.It provides an important theoretical basis to further explore the molecular signal regulatory network of invasion and metastasis of HCC and to develop new therapeutic strategies targeting at miR-605-3p for invasion and metastasis of HCC.ObjectiveThe current study is to investigate the miR-605-3p expression in HCC tissue specimens and clinical significance of miR-605-3p in HCC.In vitro and vivo assay will be used to explore the effects of mimics and inhibitors of miR-605-3p on the migration,invasion and in vivo metastasis of HCC cell lines.Finally,the mechanism of SNHG16-miRA-605-3p-TRAF6-NF-κB regulatory network based on ceRNA mode in the occurrence and development of HCC was discussed.Methods(1)Quantitative real-time polymerase chain reaction(qRT-PCR),Chi-square test,Fisher,s exact probability method,multiple logistic regression analysis,Kaplan-Meier survival curve and Cox regression model were used to analyze the expression of miR-605-3p in 78 cases of HCC and its correlation with clinicopathological parameters and prognosis.The expression of miR-605-3p in 8 pairs of fresh tissue specimens with intrahepatic metastasis of HCC and 8 pairs of fresh tissue specimens without intrahepatic metastasis of HCC were analyzed by qRT-PCR,and the relationship between miR-605-3p and intrahepatic metastasis of HCC was evaluated.(2)The expression of miR-605-3p in one normal hepatocyte line(L02)and 5 liver cancer cell lines with different metastasis potential(HepG2,Hep3B,MHCC97L,MHCC97H,HCCLM3)was analyzed by qRT-PCR.(3)Mimics and inhibitors of miR-605-3p were constructed.Then,miR-605-3p-mimics and miR-605-3p-inhibitor were transfected into HCC cell lines with high and low expression of miR-605-3p,respectively.The transfection efficiency was tested.(4)Transwell matrix gel chamber test and cell scratch healing test were used to detect the effect of miR-605-3p expression on the migration and invasion of HCC cells and to construct a lung metastasis model of HCC cells in nude mice to detect the effect of miR-605-3p on the metastasis ability of HCC cells in vivo.(5)After transfection of HCC cell lines with high and low expression of miR-605-3p by miR-605-3p-mimics and miR-605-3p-inhibitors,the classical signal pathways related to HCC metastasis were detected to explore the possible mechanism of miR-605-3p inhibiting HCC metastasis.It was found that miR-605-3p was mainly involved in regulating the activation of NF-κB signal pathway and promoting the activation of EMT pathway to play a role in inhibiting HCC metastasis.(6)Bioinformatics predicts target genes that can interact with miR-605-3p,predicts that miR-605-3p can bind to 3’UTR of TRAF6.TRAF6 is an important binding protein of NF-κB signaling pathway by consulting literatures.(7)The correlation between the expression of miR-605-3p and TRAF6 in clinical HCC specimens was analyzed by qRT-PCR.(8)Double luciferase assay confirmed that miR-605-3p could bind directly to the 3’UTR region of TRAF6,and qRT-PCR and Western blot assay confirmed that miR-605-3p could inhibit the expression of target genes at the post-transcriptional level of TRAF6.(9)Rescue test was used to verify whether miR-605-3p could inhibit the metastasis of HCC by targeting the expression of TRAF6 and affecting the activation of NF-κB signaling pathway.(10)Bioinformatics predicts the LncRNAs that may compete with the target gene of miR-605-3p through the endogenous competition of miR-605-3p.qRT-PCR was used to detect the expression of SNHG family in HCC tissues.SNHG16,the most differentially expressed LncRNA,was selected as the LncRNA to further explore the mechanism of ceRNA involved in the regulation of miR-605-3p and HCC metastasis.(11)The correlation between the expression of miR-605-3p and SNHG16 in clinical HCC specimens was analyzed by qRT-PCR.(12)Nuclear plasma separation test was used to identificate the SNHG16 expression and localization.(13)Double luciferase assay confirmed that miR-605-3p could bind to SNHG 16 directly.SNHG 16 and TRAF6 play the role of ceRNA through endogenous competitive miR-605-3p MREs.(14)Transwell matrix gel chamber test and cell scratch healing test were used to detect the effect of SNHG16 expression on the migration and invasion of HCC cells.(15)Western blot was used to detect the effects of important signal proteins in NF-κB signaling pathway.qRT-PCR was used to detect the expression of classical target genes related to downstream and metastasis of NF-κB signaling pathway.The activation of NF-κB signaling pathway was detected by double luciferase reporter gene.The nuclear shuttle of NF-κB protein was detected to analyze the effect of SNHG16 on NF-κB signaling pathway by immunofluorescence.(16)The rescue test confirmed that SNHG16 played these roles through endogenous competition for miR-605-3p.Results(1)The expression of miR-605-3p was negatively correlated with the metastasis potential of HCC.(2)miR-605-3p can inhibit the transfer of HCC cell lines in vitro and in vivo.(3)It was found that miR-605-3p inhibited EMT mediated by NF-κB signaling pathway mainly by inhibiting the nuclear entry of NF-KB/p65,and finally inhibited the metastasis of HCC.(4)It was confirmed that SNHG16 and TRAF6 could protect TRAF6 from the inhibition of these microRNAs through endogenous competition of miR-605-3p,and ultimately increase the expression level of TRAF6 and activate the NF-κB signaling pathway.ConclusionThe regulation network of SNHG16-miR-605-3p-TRAF6-NF-KB promotes the activation of NF-κB signaling pathway.And the EMT process proceeds continuously,which leads to the metastasis of HCC. |
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