Experimental Study Of The Role Of Livin In Breast Cancer

PART ONEEXPRESSION AND SIGNIFICANCE OF THE APOPTOTIC INHIBITOR LIVIN IN HUMAN BREAST CANCERObjective:To study the expression and significance of the apoptotic inhibitor Livin in human breast cancer.Methods:Immunocytochemistry, Real-time-quantitive-RT-PCR and Western blot were applied to detect the expression of Livin in human breast cancer cells(MCF-7,ZR-75-30, MDA-MB-231, MDA-MB-435s)and breast epithelial cell(HBL-100). Immunohistochemistry was applied detect the expression of Livin in human breast cancer, benign tumor and tumor-adjacent tissues.Results:①Livin expression of the breast cancer cells were obviously higher than those of breast epithelial cell(P<0.05).②There was no significant difference of Livin expression level between the four breast cancer cells.③Livin expression level of human breast cancer was obviously higher than that of tumor-adjacent tissues and benign tumor.④Livin expression level in T≤2cm group of human breast cancer was obviously lower than that in T>2cm group (P<0.05).⑤Livin expression level in axillary lymph node negative group was obviously lower than that in axillary lymph node positive group (P<0.05).⑥Livin expression level in grade III group of human breast cancer was obviously higher than that in grade I and II group (P<0.05).⑦There were no significant differences of Livin levels between in CerBb-2(+)group and C-erBb-2(-)group (P>0.05), ER(-)group and ER(+) group (P>0.05) .Conclusion:The expression level of Livin is increased obviously in breast cancer cell lines. Livin expression level increased in T>2cm, axillary lymph node positive, grade III tumors, but it had no relation with the CerBb-2 and ER status.PART TWOCONSTRUCTION AND IDENTIFICATION OF LIVIN EUKARYOTIC EXPRESSION VECTOR IN BREAST CANCER CELLSObjective: To construct and idendify the siRNA eukaryotic expression vectors targeting to Livin gene and to study the suppressing effect of Livin-siRNA on the Livin gene expression in MCF-7 , MDA-MB-231 cells.Methods: Three siRNA eukaryotic expression vectors targeting Livin(si-Livin1,si-Livin2,si-Livin3) and negative control vector (si-HK)were constructed. The recombined plasmid was extracted in middle quantity and transfected into MCF-7 and MDA-MB-231 cells by LipofectamineTM 2000. Real-time-quantitive-RT-PCR and Western blot were used to detect Livin mRNA and protein respectively.Results: Three specific siRNA eukaryotic expression vectors targeting Livin and negative control vector were successfully constructed. The Livin siRNA could inhibit the expression of Livin mRNA and protein to varying degree, and si-Livin2 resulted in the highest inhibiting rate.The Livin mRNA and protein expression were decreased significantly in MCF-7 and MDA-MB-231 breast cancer cells by si-Livin2(P<0.05).Conclusion: Three specific siRNA eukaryotic expression vectors targeting Livin (si-Livin1,si-Livin2,si-Livin3)could inhibit the expression of Livin mRNA and protein to varying degree .The effect of si-Livin2 is better than that of si-Livin1 and si-Livin3.PART THREE THE EFFECT OF LIVIN-TARGETED siRNA ON BIOLOGICAL BEHAVIOR OF HUMAN BREAST CANCER CELLS AND POSSIBLE MECHANISMObjective:To investigate the effect of Livin-targeted siRNA on the biological behaviour of breast cancer cell lines MCF-7 and MDA-MB-231 and to study its possible mechanism.Methods: si-Livin2 was transfected into MCF-7 and MDA- MB- 231 cells by LipofectamineTM 2000.The changes of Caspase-3,7,9 and Smac/ DIABLO expressions were detected by Real-Time-quantitative-PCR and Western blot.The effect of suppressing proliferation of MCF-7 and MDA- MB-231 cells was detected by MTT assay. Cell cycles were determined by flow cytometry. Cells ultramicrostructure changes were observed by electron microscope.The effect of inducing MCF-7 and MDA- MB-231 cells apoptosis and inhanced chemotherapy sensitivity of breast cancer cells treated to adriamycin was detected by TUNEL assay.Results: Forty-eight hours after si-Livin2 transfection, compared with the blank and si-HK group, the proliferation of MCF-7 and MDA-MB-231 cells was strongly inhibited(P<0.05). The cells percentage of the G0/Gl phase was increased and the S phase was decreased significantly ( P < 0.05 ) .Rate of apoptosis increased significantly(P<0.05). Typical morphological characteristic of apoptotic cells was observed by electron microscope. Chemotherapy sensitivity of breast cancer cells to adriamycin was enhanced. The mRNA and protein expressions of Caspase-3,7,9 and Smac/DIABLO were all increased in both si-Livin2 transfected groups(P<0.05), but there were no change of the other control groups.Conclusion:Through upregulating the expressions of Caspase-3,7,9 and Smac/DIABLO,Livin-targeted siRNA can inhibit proliferation and induce apoptosis in MCF-7 and MDA-MB-231 cells,and enhance cells chemotherapy sensitivity to adriamycin significantly.PART FOURINHIBITION EFFECT OF LIVIN SIRNA ON THE GROWTH OF HUMAN BREAST CANCER CELL XENOGRAFT IN NUDE MOUSEObjective:To study the inhibitory effect of Livin siRNA eukaryotic expression vector on human breast cancer MDA-MB-231 cell xenograft in nude mice and its mechanism.Methods: MDA-MB-231 cells were transplanted into subcutaneous tissue of nude mice to establish tumor xenograft. Si-Livin2 vectors were injected into tumor xenograft. The tumor growth was monitored. Tumor morphology was observed with HE staining.Ultramicrostructure changes of tumor tissue were observed by electron microscope. Cell apoptosis was detected by TUNEL assay. Immunohistochemistry was applied to detect the expression of Livin in tumor xenograft. The expressions of Livin, Caspase-3,9 protein in tumor tissues were detected by Western blot.Results:The tumor volume was significantly smaller in si-Livin2 group than in blank and control group(P<0.05).Necrosis and cell apoptosis were found in treated group. Typical morphological characteristic of apoptotic cells was observed in treated group by electron microscope. The apoptosis rate of tumor cells was significantly higher in treated group than in blank and control group. The expression of Livin protein was significantly lower in treated group than in blank and control group (P<0.05). Caspase-3 and Caspase-9 expressions were upregulated in treated group compared with other control groups(P<0.05).Conclusion: Livin siRNA can silence the Livin expression in human breast cancer cell xenograft and inhibite the growth of MDA-MB-231 cell xenograft in nude mice.