| Objective:To investigate the expression of long non-coding RNAs (IncRNAs) in human liver cancer, we constructed differentially expressed lncRNAs profiling. Then we screened and identified some lncRNAs which are closely associated with the pathogenesis and progression of liver cancer and analyzed the feasibility of differentially expressed lncRNAs as molecular markers for early diagnosis, invasion, metastasis and prognosis for liver cancer. This will provide new strategies for the diagnosis and treatment of liver cancer.Methods:In this study, we screened raw microarray experimental datas of liver cancer in the NCBI GEO DateSets database and analyzed the expression of differentially expressed long noncoding RNAs in liver cancer using the significance analysis of microarray (SAM) software to construct the differentially expressed lncRNAs profiling. And the function of these differentially expressed lncRNAs was assessed by DAVID online software. Then we selected the actin filament-associated protein1-antisense RNA1(AFAP1-AS1) to verify its function. The expression of AFAP1-AS1was preliminarily detected in fresh liver cancer tissues and their corresponding adjacent normal tissues from30pairs by real-time fluorescence quantitative PCR (q-PCR). Then the expression of AFAP1-AS1was detected in82cases digestive system tumor tissues and their corresponding adjacent normal tissues using multi-tumor tissue microarrays (TMA)(including11cases esophageal carcinoma,11cases gastric carcinoma,26cases liver cancer and34cases colorectal cancer). The expression of AFAP1-AS1was significant lower in liver cancer tissues compared to non-cancerous tissues, which was further verified by in situ hybridization (ISH) in other70cases including liver cancer and non-cancerous tissues. We analyzed the relationship between the expression of AFAP1-AS1and their clinicopathological features, and assessed its role in tumor lymph node metastasis. Results:There was46differentially expressed lncRNAs obtained from microarray datas of liver cancer analyzed by SAM, including19up-regulated lncRNAs and27down-regulated lncRNAs. RPL23AP64、 TPT1-AS1、C3P1、AFAP1-AS1、WDFY3-AS2、FAM99B、TTTY6may have important function in liver cancer by the functional analysis using DAVID online software. Preliminary functional verification found that the expression of AFAP1-AS1in30pairs of fresh liver cancer tissues was significantly lower than that in their corresponding adjacent normal liver tissues (P<0.05). Multi-tumor tissue microarray showed that the expression of AFAP1-AS1in esophageal cancer was higher than that in their corresponding adjacent normal esophageal tissues (P<0.05), whereas it was lower in liver cancer tissues than that in their corresponding adjacent normal liver tissues (P<0.05). There was no significant difference between the expression of AFAP1-AS1in gastric cancer and their corresponding adjacent normal tissues. This was also seen in colorectal carcinoma (P>0.05). Further tests also showed that the expression of AFAP1-AS1in other70pairs of liver cancer tissues was significantly lower than that in their corresponding adjacent normal liver tissues, and it was closely associated with clinical stage and lymph node metastasis (P<0.05). The sensitivity, specificity, percent agreement, positive predictive value and negative predictive value of AFAP1-AS1as the metastasis molecular marker was91.23%、28.21%、65.62%、68.75%and65.00%, respectively.Conclusion:The differentially expressed lncRNAs profiling of liver cancer was constructed successfully, including19up-regulated lncRNAs and27down-regulated lncRNAs. The expression of AFAP1-AS1was down-regulated and was closely associated with clinical stage and lymph node metastasis. AFAP1-AS1may be a useful molecular marker for clinical diagnosis of liver cancer. |
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