title: [CpG methyltransferase induced down-regulation of claudin-7, -8 and its effects on proliferation and apoptosis of human colorectal cancer HT-29 cells].
abstract: OBJECTIVE
  To explore the regulatory effect of CpG methyltransferase (M.SssI) on expression of claudin-7 and claudin-8, promoting apoptosis and inhibiting proliferation of human colorectal cancer HT-29 cells.
  
  
  METHODS
  HT-29 cells were treated with M.SssI (50 U/ml) for 24 hours. The methylation status of claudin-7 and claudin-8 gene promoters was assayed by bisulfite sequencing PCR (BSP). Real-time PCR with SYBR green I technique was used to detect the relative expression of claudin-7 and -8 mRNA, and claudin-7 and claudin-8 proteins were tested by cell immunofluorescence and Western blotting, while the effect on cell apoptosis was assessed by Hoechst 33342 fluorescence and flow cytometry. Inhibition of cell proliferation was measured by MTT assay.
  
  
  RESULTS
  The amounts of methylated claudin-7 and claudin-8 gene CpGs were 25, 10 in the M.SssI group, 9 and 5 in the PBS group, 0 and 3 in the 5-azacytidine group, respectively. Compared with the PBS group, Claudin-7 and -8 were significantly reduced by M.SssI (P < 0.05), but increased by 5-azacytidine (P < 0.05) at both mRNA and protein levels. Hoechst 33342 staining revealed that HT-29 cells treated with PBS and 5-azacytidine were not significantly different, showing even blue fluorescence, round shape and same cell volume. But the M.SssI group presented more apoptotic cells with intensive white fluorescence intensity. Cytometry indicated that early apoptotic index of the M.SssI group was increased by 84.7%, compared with that of the PBS group (P = 0.002). Measurement of MTT optical density demonstrated that cell growth of the M.SssI group was significantly lower than that of the PBS group (P = 0.002), with an inhibition rate of 32.1%, whereas the proliferation of 5-azacytidine group was similar to that of the PBS group (P = 0.084).
  
  
  CONCLUSIONS
  Our findings suggest that M.SssI can down-regulate claudin-7, -8 mRNA and proteins in the human colon cancer HT-29 cells by up-regulating methylation status of claudin-7 and -8 gene promoters, and finally induce apoptosis and inhibit proliferation of the tumor cells.
authors.affiliation: Department of Gastroenterology and Hepatology, School of Medicine, Nanjing University/Nanjing General Hospital of Nanjing Military Command, PLA, Nanjing 210002, China.
authors.forename: Wen-hui
authors.initials: WH
authors.lastname: Wang
authors.forename: Fang-yu
authors.initials: FY
authors.lastname: Wang
authors.forename: Juan
authors.initials: J
authors.lastname: Wei
authors.forename: Yun-zhu
authors.initials: YZ
authors.lastname: Shen
authors.forename: Chang
authors.initials: C
authors.lastname: Liu
authors.forename: Xiao-chuang
authors.initials: XC
authors.lastname: Shu
mesh_terms: D017209:Apoptosis / Q000502:physiology; D049109:Cell Proliferation; D057167:Claudins / Q000378:metabolism*; D003110:Colonic Neoplasms; D015257:DNA-Cytosine Methylases / Q000378:metabolism*; D015536:Down-Regulation / Q000502:physiology; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D019073:HT29 Cells; D006801:Humans; D012333:RNA, Messenger; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D057167:Claudins; D012333:RNA, Messenger; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases