Expression Of KLF6-SV2 In Colorectal Cancer And Its Effect On Cell Proliferation And Apoptosis

Background Due to the long pathogenic process and individual differences, CRC has become an ideal model of researching malignant tumor etiology and pathogenesis. KLF6, a member of KLF family, which has classic zinc finger structure, is broadly considered to have anticancer activity. The role of its alternative splicing isoform namely SV2 has not yet been definite in the CRC, therefor understanding SV2 isoform’s function has a great theoretical value for uncovering the pathogenesis of CRC.Objective The aim of this study is to examine the SV2 variant’s expression in CRC samples, adjacent intestinal mucosa and CRC cell strains and to analyze the correlation between KLF6-SV2 and CRC, by using lentiviral transfection into cells strains to construct stable cell lines over-expressing KLF6-SV2 isoform and to identify the effect of over-expression of them, and further to investigate its effectiveness on the biological characteristics in CRC cells and reveal its specific mechanism.Methods 1. FQ-RT-PCR was used to determine KLF6-SV2 m RNA expression in 60 pairs of CRC samples and corresponding adjacent intestinal mucosae. Meanwhile five CRC cell lines were measured with the same method. 2. Lentivirus vector stably overexpressing KLF6-SV2 variant was constructed and transfected into SW480 and SW620 which had relatively low amounts of expression.As a result we acquired the cell models with stable expression of SV2 spliceosome. 3. Cell proliferation, apoptosis and DNA content were measured respectively using MTT assay, Annexin V flow cytometry, and DNA ploidy flow detection to analyze biological functions of SV2 isoform in CRC cells. 4. In order to understand the relationship between SV2 and p53 pathway related molecules, their m RNA and protein expression were measured in cell lines with SV2 over-expression.Results 1. The m RNA expression level of KLF6-SV2 in CRC tissues was(0.783±0.409),which was lower than in corresponding adjacent tissues(1.086±0.449)(P=0.002). SV2 expression in SW480 and SW620 were lower than in HCT116, Lo Vo and HT29(P<0.05). 2. Quantitative PCR showed KLF6-SV2 in over-expression groups of SW480 and SW620 rised up to 226 and 266 folds compared with the control groups respectively(P<0.01), which indicated the cell lines overexpressing SV2 spliceosome were successfully constructed. 3. After transfection, SV2 represented high expression. Proliferation of over-expression group cells decreased remarkably in the third to fifth days of cell culture. G0/G1 phase cells and apoptosis ratio were dramatically increased(P <0.05). 4. Enhanced expression of Bax and p21 were found in over-expression groups(P <0.05), which displayed positive correlationship between KLF6-SV2 and Bax or p21. There was no difference about p53 expression(P> 0.05).Conclusion KLF6-SV2 is decreased expression in CRC. KLF6-SV2 over-expression could efficiently restrain cell proliferation and hinder cells transferring from G1 to S phase and accelerate apoptosis in CRC cells. KLF6-SV2 and p21/Bax genes are positively correlated, which is irrelevant to p53 expression.