| BackgroundHepatocellular carcinoma(HCC)is a common malignant tumor,due to its high morbidity and mortality,poses a serious threat to human health worldwide.Surgical resection has been considered the best strategy for HCC therapy.However,the prognosis of patients after resection remains poor,mainly because of the high recurrence and metastasis rates.In clinical studies,surgical resection of various types of tumours with propofol-based total intravenous anaesthesia(TIVA)can reduce the risk of recurrence and metastasis;however,the mechanism underlying these effects of propofol is still elusive.Propofol is an extensively used,intravenous anaesthetic agent that plays complex roles in the inhibition of tumourigenesis and tumour metastasis in human malignancies.Meanwhile,in vitro and in vivo experiments have indicated that propofol affects the biological behaviour of hepatocellular carcinoma.However,the underlying mechanisms have not been fully understood.The aberrant expression of miRNA has been detected in a variety of human malignancies,suggesting that they might play essential roles in tumourigenesis and tumour development.In this study,we found that propofol inhibits the proliferation,migration,invasion and EMT of hepatocellular carcinoma in vivo and in vitro through upregulation of the potentially associated miR-219-5p.Subsequently,the functional assayswill be further explored.Objectives1.To verify that Propofol suppressed HCC cells proliferation and migration in vitro and induced expression of the tumour suppressor candidate miR-219-5p.2.To explore the mechanism of propofol-induced miR-219-5pexpression inhibited EMT of HCC cells through suppression of Wnt/?-catenin signalling;and figure out RAB18 transcripts are direct targets of miR-219-5p.3.To evaluate the expression levels of RAB18 in HCC cell lines,tissues and matched adjacent non-tumor liver tissues.And to better understand the clinical relevance of RAB18 expression in HCC.To explore the role of RAB 18 in hepatocarcinogenesisand HCC progression,and provide a new theory for metastasis of hepatocellular carcinoma.Contents and methods1.Propofol suppressed HCC cells proliferation and migration in vitro and induced expression of the tumour suppressor candidate miR-219-5p.(1)We used MTT assay,colony formation,wound-healing and transwell assays to address the effect of propofol on the proliferation and invasion of HCC in vitro.Using miRNA array to detect differentially expressed miRNAs in cells with propofol treatment and detecting the expression of miR-219-5p by RT-qPCR.(2)We explore the influence of propofol-induced miR-219-5p expression on cellproliferation and migration in vitro.To verify the findings in vivo,we establishedsubcutaneousxenografttumor and orthotopic xenograft tumor models in nude mice.2.Propofol-induced miR-219-5p inhibits EMT of HCC cells through suppression of Wnt/?-catenin signalling,miR-219-5p directly targets RAB18.(1)Western blot,RT-qPCR and immunohistochemistry were performed to detect the expressions EMT-related markers.The activation status of Wnt/?-catenin pathway was detected by western blot.(2)Through TargetScan and miRwalk algorithms,RAB 18 was predicted to be a direct target of miR-219-5p,RAB 18 expression was detected by western blot and RT-qPCR in miR-219-5p overexpressing HCC cells.Luciferase reporter assay was used to determine miR-219-5p direct targeting the RAB 18.3.RAB 18 promotes proliferation and metastasis in HCC andthe molecular mechanism.(1)RAB18 is associated with pathoclinical features and clinical associations of RABI18 in HCC;RAB18 expression in tumour and matched adjacent non-tuIllorous tissues was assayed by immunohistochemistry(IHC).Statistical analysis to determine the prognostic value of RAB18 in HCC patients.(2)Western blot and RT-qPCR were used to detect the expression of RAB18 in HCC cells lines.(3)Establishment and identification of HCC cells clones with stable knockdown RAB18 expression.We explore the influence of RAB18 on cell proliferation and migration in vitro.Then we established subcutalleous xenograft tumor and metastatic lung tumor models in nude mice.Xenografts model innude mice was conducted to detect the tumor growth capability in vivo.(4)Western blot,RT-qPCR and immunohistochemistry were performed to detect the expression of cell cycle-regulation related genes and EMT-related markers,theexpression of Ki-67 in xenograft tumor was examined by immunohistochemistry.Results1.Propofol suppressed HCC cells proliferation and migration in vitro and induced expression of the tumour suppressor candidate miR-219-5p.Propofol attenuated tumour initiation and its metastatic potential in vivo and in vitrothrough upregulation of the tumour suppressor miR-219-5p.2.Propofol-induced miR-219-5p inhibited EMT of HCC cells through suppression of Wnt/?-catenin signaling,miR-219-5p directly targets RAB18.3.Expression level of RAB 18 was significantly increased in HCC tissues and associated with poor prognosis.The expression of RAB 18 is positively correlated with invasiveness in HCC cell lines;RAB 18 promotes HCC cells proliferation,migration and invasion in vitro.The subcutaneous xenograft tumor and metastatic lung tumor models in nude mice showed that RAB 18 could promote HCC growth and metastasis in vivo.We examined levels of EMT-related makers and cyclin,the results showed that RAB 18 promotes EMT and cell cycle.ConclusionsPropofol attenuated proliferation,invasion and migration of HCC in vitro and in vivo.The inhibitory effects may be achieved by upregulation of miR-219-5p expression,which suppresses Wnt/?-catenin signalling and subsequently regulates progression,metastasis and EMT of HCC,miR-219-5p directly targets RAB18.Abnormal RAB18 expression promoted the proliferation and metastasis ofHCC cells,thus contributing to poor prognosis.Identification of the detailed molecular mechanisms of RAB18 involvement in hepatocarcinogenesis and HCC progression is a future research direction.Nevertheless,RAB18 could serve as a predictive marker of HCC prognosis and a therapeutic target for the treatment of HCC. |
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