Long Noncoding RNAs: Novel Risk Prediction And Therapeutic Strategies For HCC

BackgroundHepatocellular carcinoma (HCC) is the fifth most common cancer and the thirdleading cause of cancer mortality around the world. HCC causes thousands of deathsworldwide every year, half of which occur in China. Although the diagnosis and treatmentof HCC have greatly improved as a result of advances in imaging modalities andtherapeutic approaches, the prognosis of patients after surgery for HCC remainsunsatisfactory due to the high rate of metastasis and portal vein invasion (PVTT) in HCCpatients. Moreover, the mechanisms underlying hepatocarcinogenesis still remain unclear.Emerging studies have shown that long noncoding RNAs (lncRNAs), which areectopically expressed in many tumours, are involved in the process of tumourigenesis.PurposeTo understand mechanisms of long noncoding RNAs (lncRNAs) in the process ofhepatocellular carcinoma (HCC) and prognostic values of lncRNAs in HCC patients afterhepatectomy.Methods1. lncRNAs analysis of CSQT-2and determination of target genesThe differentially expressed lncRNAs and mRNAs between CSQT-2and Hep3B celllines were assessed by gene array. Then, analysis from the expression levels of lncRNAsand mRNAs and the association of gene sequences of lncRNAs and mRNAs futherverified the target genes (lncRNA and mRNA).2. Association of target genes and characteristics of HCC cell linesTarget genes levels were measured in five HCC cell lines with different metastaticcapacities by qRT-PCR to reduce the bias during target gene selection. The relationship ofexpression level between lncRNA and mRNA was dentified by RNAi and RPA. Theassociation of target genes and characteristics of HCC cell lines were confirmed viatranswell array.3. Prognosis analysis of the predictive value of tumoural target genes expressionA total of655HCC patients, divided into three independent cohorts (245,372and38),were analysed to test the correlation between lncRNAs and hepatocarcinogenesis using quantitative reverse transcriptase polymerase chain reaction assays (qRT-PCR) and in situhybridisation (ISH). To assess the different metastatic stages of HCC in vivo,245patients(cohort1) with different stages of portal vein tumour thrombus (PVTT) were established asa test cohort to determine the relationship between lncRNAs and HCC metastasis. Incohort2,372HCC patients were included in a validation cohort to analyse the predictivevalue of lncRNAs in the prognosis of HCC patients with or without PVTT. In cohort3,38HCC patients who had received systemic therapy with sorafenib after hepatectomy wereanalysed to evaluate the changes in lncRNA levels in response to treatment with sorafenib.Results1. Gene expression profiling revealed many genes that were differentially expressedbetween the CSQT-2(a highly metastatic HCC cell line) and Hep3B cell lines. BX648637,a long noncoding RNA, was identified as metastasis-related long noncoding RNA (MeRL),attracted our attention due to three factors. First, its expression level in the CSQT-2cellline was higher than that of most abnormal genes. Second, it was associated with ICAM1(intercellular adhesion molecule-1), which is involved in metastasis22-24and is alsooverexpressed in the CSQT-2cell line. Third, it had the longest continuous complementarysequence with ICAM1(812bp) among all of the ICAM1-related lncRNAs identified.2. An RNA duplex was formed between MeRL and ICAM1, which increased thestability and expression of ICAM1. Meanwhile, the metastatic capacities of CSQT-2andHep3B was varies with the variation of MeRL expression level.3. MeRL and ICAM1expression differs between tumour tissues and pairedperitumoural tumour tissues. In245PVTT patients (140PVTT type I patients and105typeIII patients), qRT-PCR revealed that the expression of MeRL and ICAM1was higher inPVTT tissues than in paired tumour (parenchyma tumour, T) tissues. Patients with highMeRL and ICAM1expression were significantly more likely to be in the later stages ofPVTT (P=0.0168and P=0.0322). In372HCC patients, including284patients withoutPVTT and88with PVTT, higher expression levels of MeRL and ICAM1were alsoevident in tumour tissues compared with the paired peritumoural (PT) tissues. Patients withtumours containing high levels of MeRL had a shorter overall survival (P<0.001) anddisease-free survival (P<0.001) than those with tumours with low MeRL. In addition, highMeRL was also associated with larger tumour size (P<0.001), more advanced TNM(P=0.001) and a higher rate of intrahepatic metastasis (P<0.001). Moreover, the analysis of 38HCC patients who had received systemic therapy with sorafenib after hepatectomyindicated that patients with high tumoural MeRL expression had poorer outcomes thanthose with low MeRL expression.Conclusions1. HCC cell lines with different metastatic capacities aslo have diferent expression oflncRNAs and mRNAs; and the expression of lncRNAs differs between tumour tissues andpaired peritumoural tumour tissues.2. LncRNAs could involve in hepatocarcinogenesis by regulating the expression ofmRNAs.3. LncRNAs expression level is correlated with the survival of HCC patients andresponse to adjuvant therapy with sorafenib.Potential applications and noveltyThis study provides a new mechanism of the regulatory actions of lncRNAs.High tumoural MeRL expression was associated with higher rates of metastasis andrecurrence and poor prognosis after hepatectomy. These results strongly suggest thatMeRL may serve as a new diagnostic marker for tumour metastasis or recurrence anda specific target for the inhibition of metastasis-related genes in anti-tumour therapy.