In recent years, the dysregulation of lncRNA expression profile has been studied widely to uncover the underlying molecular mechanism contributing to the development of human cancers. With the rapid development on the microarray technology and sustaining increase on the coverage of human lncRNA, certain number of lncRNAs have been found to be associated with the carcinogenesis. Microarray technology possesses several advantages, including high-throughput property covering whole human genome, high sensitivity, and capable of providing lncRNA-mRNA correlation analysis. However, due to the expansive price of employing microarray chip, the studies often conducted in a limited sample size. Under such condition, the credibility of lncRNA may affected by random error or outlier. Therefore, it is imperative to conduct qPCR validation on larger sample size to verify the results generated by microarray analysis. In present study, we first investigated the lncRNA and mRNA expression profile in 10 HCC tissue and normal tissue collected from 10 male HCC patients with chronic HBV infection. Compared with the normal tissue, we identified 946 upregulated and 571 downregulated lncRNAs. As for mRNAs, 1,720 upregulated and 1,106 downregulated mRNAs were found by same criteria. Furthermore, qPCR was performed in 30 pairs of tissues to validate the expression level of 22 selected lncRNAs. Overall, the results between different methods showed high consistency except one lncRNA, suggesting the solid performance of microarray analysis.
We conducted the GO enrichment analysis to investigate the potential function and pathway of differentially expressed mRNAs. Surprisingly, the enriched GO terms were mostly involved with the metal ion response, including zinc and cadmium. The results supported evidence from the study conducted by Ebara et al, which observed accumulation of cooper-metallothionein (Cu-MT) in HCC tissue while the zinc metallothionein(Zn-MT) level was significantly higher in surrounding normal liver tissue. It is general acknowledged that Zn has potent anti-oxidative ability, and Zn deficiency leads to enhanced oxidative stress and increased production of inflammatory cytokines. The oxidative stress may trigger harmful inflammatory response, and responsible for cancer. The subjects we included in the present study have undergone chronic HBV infection before the onset of HCC, and HBV is capable of integrating into the host genome during the course of infection, and consequently lead to oncogene activation, tumor-suppressor gene inactivation, or other genome instability. Interestingly, the HBV integration can be found in both HCC tissue and normal tissue. However, the next-generation sequencing revealed that the viral integration occurred more frequently in HCC tissue than in paired non-tumor tissues. We assume that the elevated integration level and chronic inflammation are able to disrupt the zinc/cooper ratio in liver tissue, and consequently produce free radical formation and further induce the damage of hepatocyte. Unlike Cu and Zn, cadmium (Cd) has been considered as a hazardous element to human and classified as a human carcinogen. This toxic element is widely distributed in the human organs after absorption, with the major portion of the body burden located in the liver and kidney. By employing rat liver epithelial cell line, previous study observed the Cd-induced malignant transformation and significantly down-regulated the expression of apolipoprotein E (ApoE) which was recently established as a suppressor of cell invasion. The source of exposure to Cd can be various, one of the most common one is smoking due to the high concentrations of Cd in tobacco plant, and despite the efforts of tobacco control, half of Chinese male were smokers according to the latest survey. Moreover, due to the absence of environmental regulation in early stage of Chinese industry, certain parts of soil were contaminated with the waste water, and the rice which is the staple food of Chinese population would accumulate high concentration of Cd. The pathway analysis also collaborates our hypothesis, as can be seen, the most enriched pathway in differentially expressed mRNA was mineral absorption (hsa04978). Based on the evidences above mentioned, we can conclude that the intake of some specific inorganic elements was involved with the development of HCC in Chinese male population. However, the further investigations such as epidemiological study focusing on inorganic elements exposure should be conducted to verify our assumption.
KIF20A, a member of Kinesin family, plays a critical role in cytokinesis, and more importantly, has been associated with the development and progression of various kinds of human cancers. The in vitro experiments conducted by Gasnereau et al. showed that the product of KIF20A mRNA MKlp2 was highly elevated in human hepatoma cell lines, while it can not be detected in normal human hepatocytes. The essential function of KIF20A involves with the cell adhesion, spreading, migration and proliferation. Other individual studies employing RNA-silencing method yielded conclusive results, and the cell viability and invasion capacity were significantly suppressed in cancer cell lines[25–26]. The findings of previous studies broadly supported the microarray analysis results of present study which demonstrated a 26.44-fold overexpression of KIF20A in HCC tissues when compared with normal tissues. On the other hand, the key molecular for the inhibition of migration and cell growth, HEPACAM was found to be 49.56-fold underexpressed in HCC tissues. The treatment of HEPACAM-overexpressing adenovirus in cancer cell line was able to promote the inhibitory effects on cell proliferation, viability and protein expression. Based on the evidence obtained from in vitro experiment, the underlying mechanism of inhibitory effects caused by HEPACAM was involved with the inhibition of Wnt/β-catenin signaling pathway which was considered as cancer-related pathway if aberrantly activated. The results reflected highly activation of cell migration and proliferation in malignant liver hepatocyte.
In the microarray analysis, we identified a large number of dysregulated lncRNAs which have not yet been reported in similar studies. With the subsequent qPCR validation conducted both in microarray analysis samples and in twice amount of independent samples, we observed high consistency between two methods. Among those differentially expressed lncRNAs, TCONS_00008984 exhibited astonishing elevation in HCC tissue with a 768.94-fold expression. Notably, this extreme high expression was not caused by few outliers, instead, it can be attributed to unanimous increase in qPCR validation. Given the extreme high elevation and absence of outlier, TCONS_00008984 has potential to serve as a diagnostic marker or prognosis indicator, despite its biological function remains clear currently. Epidemiological study focusing on the association between HCC clinical features and the expression level of TCONS_00008984 should be conducted to assess its capability of serving as a biomarker. In vitro experiment essential to reveal its underlying mechanism in the carcinogenesis of HCC should be performed as well.
In summary, the present study detected a great many differentially expressed lncRNAs and mRNAs by comparing HCC tissues and normal tissues with genome-wide microarray analysis, and subsequent qPCR validation showed high consistency. GO enrichment and pathway analysis revealed that the mRNAs related to inorganic ions response were involved with carcinogenesis, especially those associated with Zn and Cd. These findings suggested that the exposure to some specific inorganic elements may possibly involve with the risk of developing HCC, and such exposure should not be neglected. Epidemiological study should be implemented to investigate the synergic effect between inorganic elements exposure and chronic HBV infection, and subsequent interventions should be enhanced to reduce the incidence of HCC.