The Experimental Study On The Treatment Of Cervical Cancer With Activation Of The Protein Kinase PKR By The Aurora-A SiRNA

Background:Cervical cancer remains as one of the biggest fillers of women worldwide. Despite significant achievements in the treatment of cervical cancer, it is still a deadly disease that readily metastasize in the earlier period, hence newer therapeutical modalities are needed."High risk" genotypes of the human papillomavirus(HPV), most commonly HPV genotype 16/18, are the primary etiologic agents of cervical cancer. Indeed HPV DNA is detected in 99.7% of cervical carcinomas. Thus, cervical cancer and other HPV-associated malignancies might be prevented or treated by the induction of appropriate viral-antigen-specific immune responses. Two HPV oncogenic proteins, E6 and E7, are critical to the induction and maintenance of cellular transformation, therapeutic vaccines targeting E6 and E7 may provide the best opportunity to control HPV associated malignancies. Various candidate therapeutic HPV vaccines are currently being test in clinical trials, some trials are still ongoing. But, the results of the trials are not as desirable as expected.PKR, the protein kinase regulated by double-stranded RNA (dsRNA), is well established as an important component of the host response to viral infection. The N-terminal region of PKR includes a repeated domain that confers dsRNA-binding activity, and the C-terminal region possesses subdomains necessary for kinase catalytic activity. In addition to the autophosphorylation of PKR that occurs during the dsRNA-mediated autoactivation process, the best-characterized PKR substrate is theαsubunit of protein synthesis eukaryotic initiation factor 2 (eIF-2α). PKR catalyzes the phosphorylation of eIF-2αon serine 51, a modification that alters the translation pattern within cells and leads to an inhibition of protein synthesis under stress conditions, including virus infection . PKR also modulates nuclear factorκB (NF-κB)-mediated signal transduction processes in response to dsRNA.Studies have provided evidence that PKR plays an important role in a variety of physiologic processes, including cell proliferation and death, in addition to the role that PKR plays in the antiviral actions of interferons. But the expression level of PKR in cervical caner have not been described.Aurora-A is a centrosome-associated gene,the amplification or high expression of Aurora-A cause centrosomal anomalies,anuploidy formation and cell transformation .The expression level of Aurora-A in most normal tissues remains low,but the overexpression of Aurora-A is a common event in tumor.Recently,the studies on Aurora-A expression in pancreatic tumor,esophageal cancer,gastric cancer and some other tumors were more, and the RNA interference targeting aurora kinase could suppresses tumor growth in some kinds of human cancer cells. But the study in cevical neoplasm were little.Objective:1. To investigate the expression of PKR in different tissues and cells from normal cervix or cervical cancer;2. To observe the expression level of Aurora-A in cervical tissues and cells, and investigate whether Aurora-A expression correlates with clinicopathologic factors and prognosis of cervical carcinoma patients;3. To observe the bionomics effect in Hela cell by inhibition expression of Aurora-A with RNAi targeting;4. To investigate the feasibility of activation PKR by modified siRNA.Methods:1. Differential expression and activition of PKR was examined by RT-PCR, Western blotting and Immunohistochemistry analysis in different cervical tissues and cell lines;2. Differential expression of Aurora-A was examined by RT-PCR, Western blotting and Immunohistochemistry analysis in different cervical tissues and cell lines; to analyze whether Aurora-A expression correlates with clinicopathologic factors and prognosis of cervical carcinoma patients with statistics;3. Inhibition the expression of Aurora-A with RNAi targeting, and the reproduction of Hela cell were tested by MTT method; and the inhibition effect of siRNA was detected by the RT-PCR, Western-blotting;4. To modify the the siRNA oligonucleotide with single-stranded 3'-poly(A) tails at antisense strands, further more, evaluate its effect on downregulation the expression of Aurora-A and activation of PKR.Result:1. The expression and activation of several proteins in PKR-way are in different level and concerned with HPV infection in normal cervix, CIN I, CIN II, CIN III and cervical cancer;2. The expression level of PACT, the important reactivator of PKR, is direct correlated with the progress in cervical pathological changes, and whether the cancer cell be infected by HPV;3. The expression level of p58IPK, the inhibition factor of PKR, is in same condition as PACT;4. PKR over expressed in cancer tissues, and the expression level is associate with HPV;5. The expression level of p-PKR is upregulated in CIN I~II, but downregulated in cancer tissue with HPV infection. High-risk HPV could depress the activation of PKR;6. The expression and activation level of eIF-2α, the most important substrate of PKR, are similar with the level of PKR in cervical tissue;7. Aurora-A is over expressed in human cervical cancer, just like the condition in other cancers;8. The overexpression of Aurora-A is associated with FIGO stage, tumor differentiation, parametrial invasion and lymphnode or hematogenous metastasis, but not other clinicopathological factors.9. Successfully construct the siRNA of Aurora-A, which been proved could suppress Aurora-A overexpression in Hela cell; 10. By MTT analysis, Aurora-A siRNA has been proved could reduce proliferation of Hela cell;11. Although very weak, modified siRNA shows the ability to activate PKR by Western blotting, and the mechanism of the ability needs more explore.Conclusion:1. The expression and activation level of PKR are associated with HPV infection. PKR over expresses in tumor cell lines without HPV infection;2. It is shown that PKR over expressed and phosphorylated in HPV(-) cell line(C33A), just as in other cancers;3. In cervical tissue and HPV(+) cell lines, the expression and activation of PKR are suppressed by HPV in several ways: such as up regulation the level of p58IPK, reducing transcription of PKR mRNA and suppressing the phosphorylation of eIF-2α;4. Aurora-A over expresses in cervical cancer, and the level of expression correlate with the prognosis of cervical cancer patients;5. Aurora-A siRNA could reduce the expression of Aurora-A and the proliferation in Hela cell, which indicate the feasibility of Aurora-A siRNA being used into treating cervical cancer;6. The siRNA, modified with poly A tail, could enhance silencing effect by activating PKR, but the mechanism is still unclear.