The Role Of Long Noncoding RNA HClnc1 In Hepatocellular Carcinoma Patients

Background and Aim: Primary liver cancer(PLC)is the fourth most frequently diagnosed cancer worldwide and the second most frequent cause of cancer death and with the highest incidence in Asia,particularly China.Hepatocellular carcinoma(HCC)is the main type of primary liver cancer Partial hepatectomy remains the most commonly used curative therapy modality for HCC,but the prognosis of these HCC patients remains poor because of the high frequencies of metastasis andheterogeneity.In HCC,the lncRNAs promote carcinogenesis and metastasis through multiple mechanisms,including miRNA sponging,epigenetic modification,and transcriptional regulation.Long noncoding RNAs(lncRNAs)are emerging molecules that mediate various types of tumorigenesis,but their functions and mechanisms in human hepatocellular carcinoma(HCC)remain poorly understood.Hence,there is an urgent need to investigate the molecular mechanism involved in HCC progression in order to identify biomarkers for early detection and perform novel rational targeted therapeutic strategies.METHODS: Differentially expressed lncRNAs in HCC tissues were identified by expression microarray analysis and quantitative real-time PCR and in situ hybridization.Overall survival was evaluated by the Kaplan–Meier survival curve and the Log-rank test.The Cox proportional hazards model was used to determine the independent factors,which were based on the variables selected by a univariate analysis.To generate the ROC curves,patients were classified as surviving either longer or shorter than the median OS.To compare the pathologic and clinical value of the six overexpressed lncRNA candidates with that of the tumour-node-metastasis(TNM)stage for prognosis,we calculated their accuracy using prediction error curves.To validate whether HClnc1 is an authentic lncRNA,we examined its protein-coding potential by using independent predictive algorithms,including Coding Potential Calculator(CPC),PhyloCSF,Kozak sequences(score:-0.012,ribosomal binding profiling,ENCODE regulation and conservation of HClnc1(https://genome.ucsc.edu/ENCODE/),RNAfold(https://rna.tbi.univie.ac.at/).Furthermore,the in vitro translation assay did not show any protein/peptide products from HClnc1.In vitro experiments include cell proliferation,cell cycle,apoptosis,invasion and drug-resistence.The role of HClnc1 in vivo was identified by mice bearing HCC cells model.The functional roles of HClnc1 in HCC were demonstrated by a series of in vitro and in vivo experiments.Comprehensive identification of RNA-binding proteins(ChIRP),RNA immunoprecipitation and luciferase analyses were used to demonstrate the potential mechanisms of HClnc1.RESULTS: The present investigation identified that lncRNA HClnc1 played a critical role in the progression and metastasis of HCC using high throughput screening with microarray and proteomics combined with bioinformatics analysis and molecular biological techniques.Its biological function and regulation mechanisms are concluded as follows.First,lncRNA HClnc1 was closely associated to the prognosis and survival rate(OS and RFS)of HCC patients.To investigate the lncRNA expression profiles in HCC tissues,we analysed five pairs of HCC tissue samples using lncRNA microarray.Through expression intensity sorting within the HCC tumour and nontumour groups,the ten most increased lncRNAs in HCC tumour tissues compared to nontumour tissues were identified.These changes were further verified in a larger group containing HCC and matched nontumour tissue samples from two independent groups using qRT-PCR and ISH.High HClnc1 expression was more frequently observed in HCC patients with microvascular invasion and an advanced TNM stage.High HClnc1 expression was significant independent factors affecting the OS and RFS of HCC patients after hepatectomy.ROC analysis indicated that the combination of HClnc1 and TNM stage was more precise in predicting clinical outcome than the TNM stage alone.To validate whether HClnc1 is an authentic lncRNA,we examined its protein-coding potential by using independent predictive algorithms.We further examined its short peptide coding potential by using PhyloCSF.HClnc1 showed a very low codon substitution frequency score.HClnc1 also did not contain any valid Kozak sequences.These predictions were further verified by ribosome profiling,which revealed no/minimal ribosome binding of HClnc1;the ENCODE regulation and conservation of HClnc1 are clearly illustrated.Furthermore,the in vitro translation assay did not show any protein/peptide products from HClnc1.These data strongly support that HClnc1 is a lncRNA.Our study documents a novel lncRNA,HClnc1,one of the top upregulated lncRNAs in HCC,which functions as an oncogene.Hence,we select the HClnc1 as the object of the study.Second,HClnc1 directly regulates biological behaviour of HCC.By using gain of function and lose of function in vitro and in vivo,we found that cells were arrested at the G2/M checkpoint by HClnc1 knockdown,accompanied by induced cell apoptosis.HClnc1 promote cell proliferation and inhibit apoptosis.In addition,we found that HClnc1 can facilitate migration and invasion of HCC cells.Likewise,tumour growth and weight in the xenograft mouse tumour models were dramatically increased owing to HClnc1 overexpressing.Moreover,HClnc1 knockdown also sensitized LM3 and Huh7 cells to 5-fluorouracil and oxaliplatin,which are standard TACE treatment regimens for HCC.These data indicate that HClnc1 facilitates HCC progression by regulating HCC cell proliferation and metastasis and increasing chemotherapy resistance.Third,HClnc1 promotes HCC tumourigenesis by interacting with the pyruvate kinase 2(PKM2)protein.To screen potential protein targets of HClnc1 in HCC cells,HClnc1-interacting proteins in Huh7 cells were concentrated by biotinylated antisense HClnc1 RNA and identified by mass spectrometry analysis.Among all five candidate proteins identified by mass spectrometry,only PKM2 interacted with HClnc1.HClnc1-overexpressing cells developed more capillary-like structures than the negative control cells,which was abrogated by PKM2 knockdown.Ectopic HClnc1 expression could bind and increase the stability of PKM2 by reducing its ubiquitous modification.STAT3 signalling was activated in HClnc1-or PKM2-overexpressing HCC cells,and PKM2 knockdown abrogated HClnc1-induced STAT3 activation.By contrast,HClnc1 knockdown inhibited STAT3 activation,which was rescued by PKM2 overexpression.The Western blot results further showed that ectopic HClnc1 expression increased the phosphorylation of STAT3(Tyr705),which was blocked by PKM2 knockdown.By contrast,HClnc1 knockdown inhibited STAT3 phosphorylation,which was rescued by overexpression of PKM2.Consistent with these results,downstream targets of the STAT3 pathway(BCL2L1,CDH1,CCND1,MCL1,BIRC5,MMP2,and MMP9)were also significantly upregulated in HClnc1-overexpressing HCC cells,and their expression was partially inhibited by PKM2 knockdown.High HClnc1 expression and a high PKM2 level in HCC tissues were associated with reduced OS and RFS.CONCLUSION: 1.Our study identified a novel lncRNA,HClnc1,which was one of the top upregulated lncRNAs in HCC.2.High expression of the HClnc1 was significantly associated with a poor prognosis in HCC patients.Univariate and multivariate regression analyses demonstrated that HClnc1 expression was an independent predictor of HCC aggressiveness with significant HRs for predicting clinical outcome.Combined with PKM2 expression profiling,HClnc1 provides a more precise clinical outcome prediction than conventional TNM stage classification alone.3.HClnc1 could function as an oncogene by facilitating HCC cell proliferation and metastasis.HClnc1 could bind and increase the stability of the PKM2 protein by reducing its ubiquitous modification to facilitate STAT3 transcriptional activity and aerobic glycolysis.In addition,PKM2 was upregulated in HCC tissues and correlated with HClnc1 expression and patient survival.