CpG Methyltransferase/RNA Interference Induced Down-regulation Of Claudin-7and Its Effects On Proliferation And Apoptosis Of Human Colorectal Cancer Cells

Background:Colorectal cancer (CRC) is the fourth most common malignant cancer in the world with400,000males and380,000females suffering from this disease every year. Due to early detection by screening programs and removal of precancerous adenomatous polyps, mortality resulted from CRC has been highly prevented. However, there are still almost390,000deaths resulting from CRC occurring annually and the relative survival rate is less than40%. Metastasis of tumor is the primary cause of fatality in colorectal cancer patients.Claudin-7is a tight tight junction protein. In relation to CRC, a number of reports suggest that overexpression of claudin-7may aid metastasis of CRC and dissemination of cancer cells which are the primary causes of fatality in colorectal cancer patients. Hence, in this research, we picked claudin-7to demonstrate if epigenetic knockdown effect by claudin-7specific siRNA and CpG Methyltransferase (M.SssI) had any influence on colorectal cancer cells proliferation and apoptosis, which would offer distinctive prospects for alternative cancer treatments.Objective:The effects of CpG Methyltransferase (M.SssI) and RNA interference (RNAi) on expression of tight junction-related transmembrane protein claudin-7, apoptosis and proliferation was investigated in human colorectal cancer cells. Methods:Bisulfite sequencing PCR (BSP) was conducted to analyze the methylation status of claudin-7promoters region in HT-29cells after treatments of M.SssI, Phosphate Buffered Saline (PBS) and methyltransferase inhibitor5-Azacytidine (5-Aza) as control. Real-time PCR with SYBR green I technique was used to detect the relative expression of claudin-7mRNA in HT-29and HCT-116cells, and claudin-7protein was tested by cell immunofluorescence and Western blotting. Besides, the effect on cell apoptosis was assessed by Hoechst33342fluorescence and flow cytometry. Inhibition of cell proliferation was measured by Cell CountingKit-8(CCK-8).Results:The amounts of methylated claudin-7gene CpGs were significantly increased by M.SssI group than in PBS while5-Aza down-regulated the methylated CpGs. Claudin-7in HT-29and HCT-116cells was greatly reduced after treatments of M.SssI and RNAi, at both mRNA and protein levels. Hoechst33342staining revealed HCT-116cells treated with M.SssI presented more apoptotic cells with intensive white fluorescence intensity comparing with PBS and5-Aza cells showing even blue fluorescence, round shape and same cell volume. Similarly, early apoptotic indexes in M.SssI and RNAi group were increased. In contrast, measurement of CCK-8demonstrated that cell growth of the M.SssI and RNAi group was significantly lower than separate controls.Conclusions:These results suggest that M.SssI and RNAi could reduce expression of claudin-7at different levels, induce apoptosis and inhibit proliferation of colorectal cells.