| Hepatocellular carcinoma (HCC) with high malignancy and poor prognosis, early diagnosis can improve the prognosis of patients. Low sensitivity of serum AFP could easily lead to missed diagnosis of HCC. With the rapid development of molecular biology, more and more of the HCC-associated markers have been reported. The sensitivity and specificity of single marker are difficult to break through AFP. Diagnosis of multiple markers can improve the diagnostic rate of HCC. Fluorescence quantitative PCR (QRT-PCR) had detected 16 HCC-associated genes expression in our laboratory from which optimized 6 genes and established molecular diagnostic index (MDI), that increased sensitivity and specificity of diagnosis of HCC.AIM:Screening HCC-associated genes which expression levels were specificity and optimizing the combination of genes to establish MDI to verify its value in diagnosis. METHODS:From the cDNA library established by our laboratory and literature to screen 4 tumor suppressor genes and 9 oncogenes. To choose 60 tissue samples from surgical resection at random including 10 normal liver (NL),10 liver cirrhosis (LC), 40 HCC and paracancer, total RNA was extracted then reverse transcripted cDNA. Apply RT-PCR to detect the express level of 13 genes in normal liver, liver cirrhosis, hepatocellular carcinoma. G3PDH as control,2-△△CT method to calculate the relative gene expression levels. When the value of 2-△△CT>3 or<0.33, the gene had a differential expression. HCC-associated genes were picked out whose expression specifically and largely different from the other tissues. Then the MDI was established according to the number of exceptionally express genes. ROC curve was used to preliminary evaluate the MDI diagnostic value.RESULTS:In 40 cases of carcinoma,65.0%,75.0%,67.5% of the specimens expression had a<3-fold normal liver in tumor suppressor genes PRDM2, IGFBP3, DLC1; 87.5%,77.5%,82.5%,85.0%,67.5% of the specimens expression had a >3-fold normal liver in oncogenes GPC3, STMN, CCNA2, BIRC5, AFP, respectively. The expression level was significant difference between carcinoma and liver cirrhosis tissues (0.19 vs 0.57; 0.09 vs 0.53; 0.12 vs 0.42; 191.08 vs 3.30; 9.96 vs 0.96; 20.03 vs 1.58; 13.21 vs 1.45; 24.20 vs 0.83, all P<0.05). Only 30.0% of the specimens had a<3-fold normal liver in WWOX, while 10.0% of the specimens had a>3-fold normal liver in CD133; UBD had 90.0% of the specimens>3-fold normal liver, but also had 70.0% cirrhosis specimens>3-fold normal liver, the specificity was poor; At last PRDM2, IGFBP3, DLC1, GPC3, STMN, CCNA2, BIRC5, AFP were selected as HCC highly associated genes. Based on the relative value of QRT-PCR to draw scatter, the expression of tumor suppressor genes PRDM2, IGFBP3, DLC1 had a significantly decreasing trend from the normal liver, liver cirrhosis to the cancer group, while oncogenes GPC3, STMN, CCNA2, BIRC5, AFP were opposite. MDI was 2.2±1.5,3.0±1.6,2.9±1.5,6.3±1.2 in liver cirrhosis, surgical margin, para-carcerous (N2), and cancer tissues, respectively. Carcinoma group was significantly different from liver cirrhosis group, surgical margin and para-carcerous (N2) groups. Liver Cirrhosis as control group, when MDI was 3.5 or greater diagnosed HCC, receiving operative curves (ROC) sensitivity and specificity were 100% and 90%, accuracy was 98%, area under curves (AUC) was 0.995. para-carcerous and surgical margin as control group, when MDI was 4.5 or greater diagnosed HCC, ROC curve sensitivity were both 90%, specificity was 81% and 79%, accuracy was 91% and 90%, AUC was 0.952 and 0.946, respectively. MDI was from 4 to 8 in tumor group of 40 HCC specimens. If classified MDI of 7,8 as a high while 6,5,4 as a low MDI group, log-rank test was used to survival analysis of these two groups and Kaplan-Meier survival curves was drew. The result displayed that survival curve of low MDI group was higher and more flat than high MDI group (had statistical difference, P=0.04) that suggested a better prognosis of low MDI group.CONCLUSI ON:Expression levels of HCC-associated genes in normal liver, cirrhosis, surgical margin, para-carcerous and cancer tissues were detected by QRT-PCR, indicating that the expression of GPC3 and other genes was obvious difference in each group. Interestingly, the gene expression level was different in cancer tissues, indicating that genetic changes present in HCC and are closely related with tumor characteristics. Optimization of PRDM2, IGFBP3, DLC1, GPC3, STMN, CCNA2, BIRC5, AFP successfully established MDI. Applied ROC curve analysis of 40 HCC and other specimens, results showed that the MDI had a high accuracy in the diagnosis of HCC, that could effectively identify HCC and benign tissues (liver cirrhosis tissue, para-carcerous and surgical margin). Meanwhile, patients with low MDI had good prognosis. This experiment confirmed the value of MDI in the diagnosis of HCC and provided methodology and basis for establishment of MDI in serum in the next step. |
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