ObjectiveTo explore the relationship between aberrant promoter CpG islands methylation status of E-cadherin gene and hepatocarcinogenesis, and to assess its significance in clinical early diagnosis of hepatocellular carcinoma (HCC). MethodsSurgically resected specimens, among which cancerous and corresponding noncancerous liver tissues from 34 HCC patients, 10 liver cirrhosis from patients without HCC and normal liver tissues from 4 accidental deaths, were collected in West China Hospital. Breast cancer cell line MDA-MB-435 with promoter CpG islands hypermethylation of E-cadherin as positive control was gained from the Cell Bank of Chinese Academy of Sciences in Shanghai. The methylation status of promoter CpG island of E-cadherin gene was detected by nested methylationspecific polymerase chain reaction (nested-MSP). ResultsE-cadherin gene promoter CpG islands hypermethylation was found in 61.76% (21/34) of cancerous tissues, in 29.41% (10/34) of noncancereous tissues from the 34 HCC patients and in 50.00% (5/10) liver cirrhosis from patients without HCC. None of the 4 normal liver samples were detected E-cadherin mehylation positive. Moreover, the methylation of E-cadherin gene was significantly more frequent in 34 cancerous than that in corresponding noncancerous liver tissues (Plt;0.05), which had no significant difference between the 10 cirrhotic samples and cancerous or non-cancerous liver tissues (Pgt;0.05). In 34 cancerous samples, with the combination of both biomarkers of E-cadherin methylation and AFP400 (serum AFP level at a cutoff of 400 μg/L), the diagnostic sensitivity of HCC increased to 82.35%. ConclusionsThe aberrant promoter methylation of E-cadherin gene may play a vital role in the development and progression of HCC. Moreover, it might be an early event in hepatocarcinogensis. It is of high value to make further study to confirm the significance of E-cadherin gene methylation in clinical diagnosis and therapy.
ObjectiveTo explore the effect of small interference RNA (siRNA) on the metastasis and invasion of A549 cells after silence the E-cadherin gene (CDH1).MethodsThe CDH1 gene in A549 cells was silenced by siRNA technology, and RT-QPCR and Western blot were used to detect the gene expression and protein expression after silencing. The cells were divided into control group (Control), siRNA negative group (siRNA-NC) and siRNA gene silencing group (siRNA-CDH1). CCK-8 detected cell viability after the gene silencing in 12 h, 24 h, 48 h and 72 h, and cell scratch test to detect cell migration ability. Transwell chamber was used to detect cell invasiveness, and Western blot method was used to detect the expression level of key factors which involved in cell migration, invasion, such as vimentin, matrix metalloproteinases (MMP-2 and MMP-9), and N-cadherin. RT-QPCR was employed to detect the mRNA expression of vimentin, MMP-2, MMP-9, Slug and Snail.ResultsAfter CDH1 silencing, the activity of A549 cells increased significantly and was time-dependent, compared with Control group and siRNA-NC group (P<0.05). The cell scratch test and Transwell chamber test showed that the cell migration and invasion ability increased significantly after CDH1 silencing, compared with Control group and siRNA-NC group (P<0.05). The expression of vimentin, MMP-9, MMP-2 and N-cadherin in siRNA-CDH1 group increased significantly. The mRNA expression level of vimentin, MMP-9, MMP-2 and Slug and Snail also increased. Compared with Control group and siRNA-NC group, the difference was statistically significant (P<0.05).ConclusionCDH1 plays an important role in tumor proliferation and invasion, and its deletion can increase the proliferation and invasion ability of A549 cells.