Regulation Of Gene Expression Related To Apoptosis Of ANXA2 Caco2

Background and ObjectiveColorectal caneer can serious damage people’s lives for its high incidence, low early diagnosis rate and easy palindromia, etc. Now, the traditional treatments of malignant tumor include surgical resection, radiotherapy and chemotherapy. Nevertheless, the specificity of these treatments is low and the postoperative recurrence and mortality rate remain high. With the rapid development of molecular biology, gene target therapy has become one of the most promising therapeutic methods for the malignant tumor in the future. The study of colorectal cancer cell malignant transformation related genes and its functions, analysis the relationship of these gens will contribute to clinical therapy. Research shows that ANXA2as cell malignant transformation related gene is overexpression in colorectal cancer tissue or cell and closely related with cancer cell proliferation and matastasis, therefore ANXA2can be a serum biomarker for colorectal cancer. In this study we knockdown ANXA2expression in caco2cell used RNAi technology then analysis the relationship of the ANXA2expression and apoptosis of caco2cell and research how silenced ANXA2regulating its related genes (S100A10、FAK、tPA、β-actin) expression. The aim of this research is to explore the possibility of taking ANXA2as target gene for therapy of colorectal cancer and supply evidence and experimental basis for tumor gene therapy.Methods1. On the basis of the sequences of ANXA2searched from GenBank database, designed interference and control sequences targeted ANXA2according to the principles of primer design, then cloned into the vector pU6H1-GFP to construct recombinant plasmids.2. Recombinant plasmids were transfected into the caco2cells by S-TranG transfection reagent with optimized conditions, at the same time set up negative control; then detected GFP expression at60h post-transfection to value the transfection efficiency and ANXA2inhibition rate.3. Apoptosis of caco2cells treated with MitoView633was further investigated by flow cytometry (FCM).4. Karyon morphology and mitochondrial membrane potential of caco2cells treated with Hoechst33258, MitoView633was respectively observed by inverted microscope and laser scanning confocal microscopy (CLSM).5. The mRNA and protein levels of ANXA2related genes (S100A10, FAK, tPA, β-actin) were analysed by RT-PCR and Western Blot at96h post-transfection in order to discuss the relationship among the five genes.Results1. We designed four different ANXA2-targeting siRNAs and a control siRNA, then the recombinants were constructed successfully; only the best interference ANXA2vector and the control vector were selected to perform this experiment.2. The optimized S-TranG transfection conditions:about70%cell confluence before transfection,450μl complex contains lOμl S-TranQ6μg plasmid and PBS,15min plasmid/S-TranG complex formation time, lml DMEM, then the mixing was added into30mm dish, supplement lml DMEM after6-7h with5%CO2、37℃incubation.3. About80%GFP expression at60h post-transfection suggesting that ANXA2was effectively inhibited.4. The results of FCM showed that the fluorescence density of the cells was significantly reduced in ANXA2interfered groups (p<0.05), suggesting that the normal membrane potential of mitochondria has been changed.5. Moreover, some characteristics of apoptosis cells were also observed in ANXA2silenced cells.6. The results of RT-PCR and Western Blot showed that when ANXA2expression was interfered, the S100A10and tPA expression were significantly reduced, but the FAK and β-actin expression had no obvious changes.ConclusionRNAi can significantly downregulate ANXA2expression in caco2cells, the expression level of ANXA2in caco2cellls could anti-apoptosis in certain degree; at the same time, it also regulate some genes realated with invasion and metastasis of cancer cells by vavious ways. So, ANXA2is hopeful to become a target gene for colorectal cancer gene therapy.