Expression Of XAF1 In Gastrointestinal Tumors And Its Effects On Colorectal Cancer Cells

BackgroundColorectal cancer is one of the most common malignant tumors in the world,its prevalence in China is steadily increasing in recent years in association with changes in lifestyle and diet,and its mortality rate remains high.Tumors occurred with or subsequently to oncogene/tumor suppressor gene mutation or abnormal expression. Gene mutations including gene deletion mutant,frameshifi mutation and point mutation,and abnormal expression of gene can happen in transcription or post-transcriptional such as Gene shear and translation.XAF1 is a novo found XIAP-associated factor,which directly interacts with XIAP and reduces apoptosis.Recent study demonstrated that XAF1 could promote apoptosis independently interaction with XIAP.XAF1 is not expressed or at a low level in the cancer cells and tissues,therefore considered as tumor suppressor gene. At present,5 transcription(splice) variants of XAF1 had been identificated,XAF1A, XAF1B,XAF1C,XAF1D and XAF1E,and encoded different proteins.There were many studies about XAF1A.The main function of XAF1A is increasing the apoptosis of tumor cell.XAF1B and XAF1C are reported in the literature,may antagonize XAF1A role in apoptosis,but Chung et al reported that XAF1B,XAF1C,XAF1D, XAF1E had the same role of XAF1A,and all promoted apoptosis.All-trans retinoic acid and interferon could induce the expression of XAF1,and all-trans retinoic acid and interferon are inducer of cell differentiation.The role of XAF1 in cell apoptosis was identified,and defined as tumor suppressor genes. Whether XAF1 participates in the differentiation of tumor cells has still not been reported.The development and succession of tumor are closely related to cell differentiation,proliferation and apoptosis.To reveal tumor cell differentiation, proliferation or apoptosis-related genes is beneficial to elucidate the mechanism of tumor,explore tumor's nature,and is of great scientific significance in cancer therapy.ObjectivesTo investigate the levels of XAF1 splice variants1,2,3 mRNA and protein in colorectal cancer and its Precancerous Lesions,and observe the effect of XAF1A on proliferation and differentiation by over expressiving XAF1A in tumor cell lines.To initially investigate the roles of XAF1 transcription variants in tumor development and progression,and provide research data to clarify the mechanism of tumor development and tumor therapy.Materials and Methods1 Materials:cells involved in this study were all keept by our lab;the fresh and paraffin imbedding samples including cancer and normal gastrointestinal samples, adenoma specimens were obtained from the Department of general surgery or Gastroenterology,Nanfang Hospital,Southern Medical University,China between November 2005 and November 2006.pc-DNA 3.1, pcDNA3.1-xafl-sense,pc/DNA 3.1-XAF1-AS,pGL3 promter containing 1430 bp of survivin promoter,pTOP flash expressing 3X catenin/TCF binding element, pFOP is a dominant-negative mutant of pFOP flash.All plasmids were provided by professor Wang jide; 2 The expression of XAF1 splice variants1,2 and 3 in human gastrointestinal cancer cell lines,colorectal primary tumor,precancerous lesion and MGC803 cell line cultivated in medium with IFN-a were detected by RT-PCR;3 Western blotting was used to determine the XAF1 splice variantsl protein in colorectal cancer cell lines and colorectal primary tumor tissues;4 Immunohistochemistry was used to determine the XAF1 expression in colorectal primary tumor and precancerous lesion;5 Transfected XAF1A to SW480 cell line with liposome;6 Luciferase Reporter Assay were used to determine the activity of survivin and catenin promoter;7 Western blotting were used to determine the expression of E-cadherin,Villin and Pan-CEA;8 MTT assay was performed to assess the effect of XAF1 on cell proliferation;9 Statistical analysis:All statistical analyses were performed with SPSS standard version 11.5(SPSS,Chicago,Illinois).The Results of RT-PCR was using the Chi-square test,The Results of western blotting was using one sample T-test,The Results of Immunohistochemistry was using the Chi-square test,The Results of MTT was using two independent samples T-test,A P-value of less than 0.05 was considered to be significant.The Results of Luciferase Reporter Assay was using Two independent samples T-test,A P-value of less than 0.10 was considered to be significant.Results1 RT-PCR indicated that the 3 Splice variants of XAF1 were either absent or at low levels in tumor cell lines and normal transformated cell line,in 2 semisuspension cell lines,all of the 3 Splice variants were at high levels,western blotting indicated that the level of XAF1A proteinum was agree to the level of XAF1A mRNA,all of XAF1A,XAF1B,XAF1C were increased by IFN-ain MGC803 cell line;2 There was no difference expression of XAF1A in tumor and normal tissues(P＞0. 05),the expression of XAF1B and XAF1C in tumor and adenoma tissues were low than the normal tissue,western blotting indicated that,in tumor,the expression of XAF1A was low compared to its corresp normal tissue,but the difference was not significant(P＞0.05);3 Immunohistochemistry Assay indicated that compared to the normal and hyperplasy tissues,in adenoma and tumor tissues,XAF1 nucleus staining was depressed(P＜0.05),cytolymph staining was increased(P＜0.05),the total staining was depressed(P＜0.05);4 MTT assay indicated that over-expression of XAF1 suppressed SW480 cell growth in variant serum concentration(0.2%,4%,10%)(P＜0.05),when transient transfection XAF1A/XAF1-AS into SW480 and SW1116 cells,the level of inhibition/enhancement follow the quantity of vectors used;5 Luciferase Reporter Assay indicated that over-expression of XAF1 suppressed the activity of survivin and catenin promotor(P＜0.10);6 All of XAF1A,XAF1B,XAF1C were increased by IFN-ain MGC803 cell line,over-expression of XAF1 does not influence the expression of E-cadherin, Villin and Pan-CEA.Conclusions1 XAF1 splice variants1,2 and 3 expressions were either absent or at low levels in tumor cell lines and normal transformated cell line;2 There was no difference expression of XAF1A in tumor and normal tissues,the disorderly expression of XAF 1B and XAF1C in tumor may effect the function of XAF1A;3 In normal tissue,XAF1 staining was main in the nucleus,while in tumor tissue,XAF1 staining was main in the cytolymph,and that XAF1 may play different role in tumor cells owing to its location;4 over-expresison of XAF1 suppressed cancer cell growth;5 over-expression of XAF1 suppressed transcription of survivin and catenin;6 All of XAF1A,XAF1B,XAF1C were increased by IFN-ain MGC803 cell line,over-expression of XAF1 does not induce cancer cell differentiation.