Study On The Expression Of ANXA5 In Human Uterine Cervical Carcinoma And Its Function

Annexins are a group of at least 12 structurally related proteins that share a core domain containing four repeats (eight for annexin VI) of about 70 to 80 conserved amino acids. Each of these repeats contains Ca²⁺ and phospholipid-binding sites. The N-terminus, specific for each annexin, contains phosphorylation sites for protein kinase C (PKC) as well as for other kinases. Annexins have been associated with a wide variety of functions including membrane organization, exocytosis, endocytosis, ion channel regulation, ion channel activity, and membrane to cytoskeleton linkage. In contrast with others, Annexin A5, which was more often considered as a major intracellular Ca²⁺-binding protein, has a very short N-terminus which is not phosphorylated. It is known for inducing Ca²⁺ influx by forming Ca²⁺ channels and for inhibiting enzyme activities linked to Ca²⁺ activation such as phospholipase A2 and protein kinase C. Annexin A5 has been thus suggested to mediate Ca²⁺ signalization, cell cycle regulation, signal transduction, membrane trafficking and organization. It also binds to negatively charged phosphatidylserine (PS) with high affinity in the presence of Ca²⁺ ions. This binding of Annexin A5 to PS has been accounted for its anticoagulant effect and apoptosis examing.Cancer of the uterine cervix is currently one of the most common malignancies in women worldwide and is second only to breast cancer in both incidence and mortality. Worldwide, there are more than 370,000 women diagnosed yearly with an overall survival rate of 40%. Of note, this high rate of mortality has remained relatively static. Thus, despite advances in chemoradiation, its primary therapeutic modality, the unacceptable current survival rates argue strongly for the development of early diagnosis and prognosis, the mechanism of tumor formation and accordingly for novel therapies. The development of molecular profiling techniques for genomic and proteomic analysis has introduced a new approach to cancer research aimed at discovering differential gene and protein expression associated with cancer development and progression.Using human uterine cervical squamous cell carcinoma as the tumor model, this study find the change of annexin A5 expression during the development of uterine cervical squamous cell carcinoma and provides the early diagnosis marker for it. To further study the function of annexin A5, the annexin A5 recombinant plasmids were constructed and then transfected into the uterine cervical carcinoma cell lines to overexpress the ANXA5 protein. On the other hand, the ANXA5 expression was suppressed by using RNA interference technology. From these two methods, we observe the change of the cell on proliferation and apoptosis, with which we conclude that ANXA5 protein has the function of facilitating cell apoptosis and supress cell proliferation. Finally, we discuss the possible mechanism through which ANXA5 protein acts on the carcinoma cells.Part I Study on the relationship between annexin A5 and human uterine cervical squamous cell carcinoma.Objective: To study whether the expression of annexin A5 is different between uterine cervical squamous cell carcinoma and normal uterine cervical tissue.Methods1 Specimen collection: Twenty-five fresh tissues of uterine cervical squamous cell carcinoma and fifteen fresh normal uterine cervical tissues were collected from the affiliated hospital of Chengde Medical College, also from which twenty-six carcinoma and fifteen normal paraffin imbedded specimen were obtained.2 Western blotting and immunohistochemistry were employed to examine the annexin A5 expression difference on the protein level between the tumor group and the normal group.3 In situ hybridization was used to detect the annexin A5 expression difference between the tumor group and the normal group on the RNA level.4 Western blotting and immunohistochemistry were used to examine the annexin A5 expression in the uterine cervical carcinoma cell lines.Results1 The annexin A5 expression in the tumor group is much higher than that of the normal group on both the protein level and the RNA level (p<0.05).2 With the differentiation of the carcinoma from well to poor, the annexin A5 expression increased. The difference between well-differentiated and moderated-differentiated group has no difference (p>0.05) while the annexin A5 expression of both the two groups have significant difference when compared with that of the poor-differentiated group(p<0.05).3 Annexin A5 protein expressed in the uterine cervical carcinoma cell lines Hela and SiHa cells, which is in accordance with that of the carcinoma tissue.Conclusion: There exists some relation between annexin A5 and the development of uterine cervical squamous cell carcinoma.Part II Study on the function of annexin A5Objective1 The recombinant plasmids of annexin A5 were constructed and stably transfected into the uterine cervical carcinoma cells to overexpress annexin A5 protein. The function of annexin A5 can be deduced from the biological action change of cells.2 RNAi technology was employed to suppress the annexin A5 expression in HeLa cells. With the biological action change, the annexin A5 function can be concluded.Methods1 Construct two kinds of recombinant plasmids pcDNA3.1-ANXA5 and pEGFP-ANXA5: E. coli including pcDNA3.1 plasmid and E. coli including annexin A5 gene were propagated in a large scale, after extracting plasmids, they were digested with BamHI and XhoI enzyme and annexin A5 gene was obtained by PCR. Then pcDNA3.1 and annexin A5 gene was linked together by T4 linkase. The procedure of obtaining the pEGFP-ANXA5 plasmid was the same as that of the pcDNA3.1-ANXA5 plasmid except that the E. coli were bearing the pEGFP plasmid.2 Transfection: After the sequences of the two recombinant plasmids were proved correct, the two kinds of plasmids were transfected into the uterine cervical carcinoma cell lines SiHa and Hela cells respectively. Screened by G418 for about three weeks, the positive clones were selected.3 Identification of positive cells: To SiHa cells transfected with pcDNA3.1-ANXA5 plasmid, it was concluded to be positive clone if the annexin A5 expression was much higher than that of the untransfected cells by using western blotting. To Hela cells transfected with pEGFP-ANXA5 plasmid, it was concluded to be positive clones if green fluorescence can be seen under the fluorescence microscope.4 Examination of the cell function: The proliferation was examined by using MTT method; Apoptotic ratio of the transfected cells and untransfected cells were examined by using FITC-annexinV-PI.5 Screening siRNA: The three pairs of siRNA synthesized from biology company were transfected into Hela cells respectively. The annexin A5 expression was examined at 48h after transfection so as to screen the siRNA which had the strongest suppression effect.6 RNA interference: The siRNA having the strongest suppression effect was transfected into Hela cells and 48 hours later, the cells were examined.7 Examination of the cell function: Hela cells were planted into the 96-well plate and dealt with siRNA. 48 hours later the proliferation ratio was examined by using MTT method. The apoptotic ratio of the transfected cells and untransfected cells was examined by using the apoptosis examining box.Results1 The sequences of anxA5 gene in the pcDNA3.1-ANXA5 and pEGFP-ANXA5 plasmid were proved correct by using the sequencing apparatus. 2 The proliferation ratio of the Hela cells overexpressing ANXA5 was much lower than that of the untransfected cells(p<0.05); The apoptosis ratio of the Hela cells overexpressing ANXA5 was higher than that of the untransfected cells(p<0.05).3 The proliferation ratio of the Hela cells transfected with siRNA oligo was the same as that of the untransfected cells(p>0.05); The apoptosis ratio of the Hela cells transfected with siRNA oligo was less than that of the untransfected cells (p<0.05).Conclusion: Annexin A5 protein had the function of facilitating apoptosis and suppressing proliferation of the tumor cells.Part III Study on the mechanism of ANXA5 acting on tumor cells Objective: To discuss the possible mechanism of annexin A5 protein on the uterine cervical carcinoma cells.Methods1 PKC expression was explored in both the uterine cervical carcinoma tissues and the normal uterine cervical tissues with western blotting and immunohistochemistry.2 The expression of PKC, bcl-2, bax, P53 and PCNA were examined in both the RNAi Hela cells and the normal Hela cells.Results1 There's no significant difference of PKC expression between uterine cervical carcinoma and normal uterine cervical tissue (p>0.05).2 To compare with the normal Hela cells, the expression of the several kinds of genes of the RNAi Hela cells were as follows:(1) PKC expression is less than that of the normal cells(p<0.05);(2) bcl-2 gene expression is more than that of the normal cells(p<0.05);(3) bax gene expression is less than that of the normal cells(p<0.05);(4) p53 gene expression is less than that of the normal cells(p<0.05);Conclusion(1) Annexin A5 protein exerting its function may be through the route of PKC;(2) Annexin A5 expression is in accordance with that of the apoptosis facilitating genes such as bax and p53 and is contrary to that of the apoptosis suppressing gene such as bcl-2.(3) Annexin A5 is a kind of apoptosis facilitating gene.