Roles And Clinical Diagnostic Values Of Long Non-coding RNA In Hepatocellular Carcinoma

Hepatocellular carcinoma （HCC） is the sixth most common malignant tumor and the third leading cause of cancer-related death globally. HCC is characterized by aggressiveness, invasiveness, especially intrahepatically, and frequent recurrence after resection. Exploring the detailed mechanism contributing to HCC initiation and as well as the early metastasis of HCC is urgent for improving the early diagnosis and prognosis. Previous studies mainly focused on those protein coding genes, particularly in genetics, epigenetics and signal transduction. Along with the development of high-throughput sequencing, non-coding RNA has been identified to account for over 95% in the whole human genome. The complicated interaction between the protein-coding RNA and non-protein-coding RNA has been proved to be crucial in the pathogenesis and development of HCC. As the major component of non-coding RNA, the long non-coding RNA also known as lncRNA, is a new star involving in multiple human disease. The functional effects of lncRNA have been widely recognized, including regulating gene expression through modulation of chromatin remodeling, controlling of gene transcription, posttranscriptional mRNA processing, protein function or localization, and intercellular signaling, and were also reported as a biomarker for predicting survival, metastasis, and in the diagnosis of multiple diseases. Location-associated lncRNA was reported to interact with target protein via a cis-regulatory process especially for the Flank 10kb class lncRNA. In addition, previously research has identified KRT19 as the biomarker for biliary or hepatic progenitor cell; however, HCC patients with a positive stain of KRT19 frequently indicated a poor prognosis. Despite the hypothesis that KRT19 might associate with tumor metastasis, the detailed mechanism still remains unknown. In this study, based on the bioinformatical analysis, we mainly focused on the lncRNA entitled Linc00974 located in the upstream of KRT19 within 25kb away, aiming to demonstrate whether Linc00974 was involved in the pathogenesis or development of HCC via a KRT109 dependent approach. Furthermore, the circulating fragment of KRT19, also known as CYFRA21-1 has been applied as potential biomarker in the diagnosis of lung cancer. Due to the limitation of poor sensitivity and specificity of traditional biomarker, the early diagnosis or the metastasis prediction of HCC is inferior. A new, minimally invasive and more specificity biomarker for the diagnosis or metastasis prediction of HCC were necessary. Therefore, we also screened the potential circulating lncRNA as fingerprint for the early diagnosis of HCC through a high-throughput microarray.Based on the combination of bioinformatics analysis, transcriptome microarray and molecular biological technique, we detected the detailed interaction between Linc00974 and KRT19. Firstly, we found that Linc00974 was highly upregulated in in tumor tissues comparing with the corresponding adjacent tissues. As Linc00974 has not been reported in human disease, we next confirmed non-protein-coding ability and proved the cytoplasmic subcellular location in cell lines according to the results of RT-PCR amplified with separated cytoplasm RNA and nuclear RNA. Secondly, based on previously report, we identified the hypomethylation level of CpG island in promoter region of Linc00974 which might account for the aberrant expression by using the Bisulfite sequencing PCR assay. Besides, we divided the HCC patients into two group including 27 KRT19 positive and 123 KRT19 negative according to the immunohistochemistry and immunofluorescence stain. Pearson analysis revealed that Linc00974 was positive correlated with expression of KRT19 and with tumor size, TNM stage and metastasis of HCC patients. Ectopic expression of Linc00974 could promote the cell proliferation and invasion ability with a suppression of cell apoptosis and cell cycle arrest by upregulation KRT19; however, no difference was observed in KRT19 negative cells. Further in vivo model including subcutaneous tumor xenotransplantation model and tail vein xenograft model demonstrated that Linc00974 could promote the growth and metastasis of HCC through a KRT19 independent pathway. The important role of Linc00974 in tumorigenesis and metastasis helped to delineate an underlying mechanism of the interaction between Linc00974 and KRT19. As reported, lncRNA could regulate the target protein mainly by directing binding to the target protein. Thus, to further validate the association between Linc00974 and KRT19, we performed an RNA immunoprecipitation （RIP） assay; unfortunately, we did not observe a significantly higher enrichment level of Linc00974 with KRT19 antibody compared with nonspecific IgG control antibody. Based on the subcellular location and Flank 10kb theory, we predicated there might be a potential transmitter or’bridge-like factor’ involved in the network through posttranscriptional modification. We conducted the bioinformatics prediction for co-regulation of miRNA while miR-642 was selected as the candidate gene. We finally identified that miR-624 might act as the competing endogenous RNA （ceRNA） in regulating Linc00974 and KRT19, which induced the upregulation of KRT19 through a "sponge" effect, resulting in the activation of the Notch and TGF-β pathways as detected by cDNA microarray.Even though, whether this lncRNA could be applied as potential biomarker for the diagnosis of HCC still remains unclear. Alpha Fetal Protein （AFP） has long been used for the diagnosis or monitoring the recurrence; however, despite the high sensitivity of AFP detection, the specificity of AFP detection have frequently been reported poor in clinical application. A new, novel factor with high sensitivity and specificity is necessary for the monitoring of early diagnosis or early metastasis of HCC. Based on current results, we first detected the circulating expression of Linc00974 to validate its ability for diagnosis of HCC; second, a circulating lncRNA microarray was employed to screening the candidates in matched HCC plasma sample （both pre-operation and post-operation）. The risk score analysis, ROC （Receiver operating characteristic curve） analysis and cluster analysis has been used for estimating the prediction ability of candidates under a strict quality control. Our research has achieved the following results:1. Linc00974 was expressed as fragment in plasma. The specific fragment entitled as Linc00974F-l was upregulated in HCC patients while decreased after the hepatectomy. By merging the Linc00974F-1 with CYFRA21-1, the area under the curve of ROC analysis for HCC diagnosis was 0.764 while for prediction the small HCC and early metastasis of HCC was 0.834 and 0.866, respectively which indicating a novel index for the early diagnosis and prediction early metastasis in HCC patients.2. A remarkable different expression profile of lncRNAs was observed in HCC patients and healthy controls. In order to screen the biomarker predication the tumorigenesis of HCC, we merged the up-regulated lncRNA transcripts in HCC patients with the decreased lncRNA transcripts in HCC patients post-operation and finally obtained 43 lncRNA transcripts. Next, Filtering of all the 43 deregulated transcripts for high signal intensity （≥5） and at least 2-fold deregulation yielded 13 lncRNA candidates. After validating those candidates through a risk score analysis （20 pairs as training set while 147 cases and 180 controls as validation set） and ROC analysis, we finally obtained RP11-160H22.5, XLOC₀14172 and LOC149086 as potential biomarker for HCC which were stable upregulated in HCC patients instead of chronic hepatitis or liver cirrhosis while decreased after operation. ROC analysis found that AUC of combination of three indexes in predicting HCC summed up to 0.896 with the sensitivity and specificity of 91% and 92. Further analysis revealed that the combination of XLOC₀14172 and LOC149086 in predicting the early metastasis of HCC was up to 0.934. A deeper analysis was performed which indicated that most of the patients presented a decreased level of the three levels after operation; however, some patients indicated an increased level after operation. According to the results reported, the abnormal increased factor originate from the primary tumor was highly associated with the metastasis. We further calculated the correlation between the de-regulation and metastasis of the three lncRNAs. The results indicated that mainly of the patients with increased level of lncRNAs after operation share the feature of metastasis, the upregulated lncRNAs might associate with some lncRNAs vectors such as exosomes in patients’ circulation microenvironmentIn conclusion, we studied the roles and molecular mechanisms of functional long non-coding RNAs in HCC initiation and development. Our researches provide new views and landscapes to understand the complex signaling network in HCC, and potential new strategies for the prognosis and therapy of HCC.