LncRNAuc.134 Represses Hepatocellular Carcinoma Progression By Regulating The Hippo Pathway Via Inhibition Of Ubiquitin Ligase CUL4A

Purpose and Background:Hepatocellular carcinoma(HCC)is the predominant hepatic malignancies which is one of the most common causes of cancer mortality worldwide.The high rate of tumor recurrence and metastasis is a major factor contributing to the poor prognosis of HCC patients.Therefore,there is an urgent need for novel insights into the mechanism of recurrence and metastasis in HCC and to identify novel prognostic molecular markers and potential effective therapeutic targets to improve patient survival.Long noncoding RNAs(IncRNAs)are known to regulate different tumorigenic processes,and a growing body of evidence indicates that Hippo kinase signaling is inactivated in many cancers.However,the upstream IncRNA regulators of Hippo kinase signaling in HCC are poorly understood.Experimental Design:We analyzed IncRNAs expression profiling data by IncRNAs microarray in the highly aggressive cell line HCCLM3 compared with the weekly aggressive cell line MHCC97L.Furthermore,we evaluated IncRNA uc.134 expression in clinical samples using in situ hybridization(ISH)and quantitative real-time polymerase chain reaction(qRT-PCR)analysis.The full-length transcript of uc.134 was confirmed using 5'-and 3'-RACE analyses.To investigate the biological function of uc.134,we performed gain-of-function and loss-of function studies both in vitro and in vivo.The underlying mechanisms of uc.134 in HCC were investigated using RNA pull-down,mass spectrometry and RNA immunoprecipitation.Cycloheximide chase and in vivo ubiquitination assays to identify ubiquitination of LATS1 protein.QRT-PCR,Western bloting and mRNA microarray analyses were used to examine the underlying mechamisms of uc.134 in HCC.Results:Among these incRNAs,the ultraconserved noncoding RNA uc.134 exhibited the greatest downregulation in both tissue samples and HCC cell lines.The in situ hybridization assay revealed that uc.134 expression was significantly decreased in 170 paraffin-embedded samples from patients with HCC compared with adjacent tissues,and uc.134 expression directly correlated with patient prognosis.Furthermore,we defined a 1867-bp full-length transcript of uc.134 using 5' and 3'?RACE analysis.The overexpression of uc.134 inhibited HCC cell proliferation,invasion and metastasis in vitro and in vivo,whereas the knockdown of uc.134 produced the opposite results.Furthermore,we confirmed that uc.134(1408-1867nt)binds to CUL4A(592-759aa region)and inhibits its nuclear export.Moreover,we demonstrated that uc.134 inhibits the CUL4A-mediated ubiquitination of LATSI and increases YAPs127 phosphorylation to silence the target genes of YAP.Finally,a positive correlation between uc.134,LATS1 and pYAPs127 was confirmed in 90 paraffin-embedded samples by in situ hybridization and immunohistochemical staining.Conclusion:Our study identifies that a novel IncRNA,uc.134,represses hepatocellular carcinoma progression by inhibiting the CUL4A-mediated ubiquitination of LATS1 and increasing YAPs127 phosphorylation.The use of this lncRNA may offer a promising treatment approach by inhibiting YAP and activating Hippo kinase signaling.