The Effect Of SURVIVIN And KSP Expression On The Apoptosis Of Breast Cancer Cells MDA-MB-231 Was Studied

Background & Objective : Experimental and clinical studies have confirmed that growth and reproduction of tumor cells relate to the abnormal expression of some oncogenes and tumor suppressor gene, in which survivin gene and kinesin spindle protein(kinesin spindle protein, KSP) are particularly important. Survivin gene is a new member of inhibiting apoptosis protein family(IAP) found in recent years. Survivin is a tumor specific protein. Survivin does not or less express in normal tissues(thymus, genitalexcept) cell, but specially and highly expresses in embryonic tissues and malignant tumors(such as breast cancer, gastric cancer, colorectal cancer, lung cancer etc.), and is closely related to the differentiation of the tumor cells proliferation and invasion metastasis. Due to the powerful ability to regulate cell proliferation and anti- apoptosis, and thus survivin becomes the new target of a hot tumor chemotherapy in recent years; Kinesin spindle protein(KSP or Eg5), is a member of the kinesin subfamily5(kinesin-5). KSP plays a key role in formation of bipolar spindle, replication, chromosome separation of cell mitosis early stage and centrosome splitting of the mitotic process. In addition to chromosome segregation process during mitosis, KSP obviously involves the occurrence and development of tumor. KSP is highly expressed in many tumor cell lines, such as breast cancer, lung cancer. Therefore KSP has also become a very important target of cancer chemotherapy. In theory, it is possible to inhibit cancer cell reproduction or promote its apoptosis by inhibitting expression of these oncogenes.There are the advantages of high specificity, good effect, easy preparation, convenient operation for small interfering RNA(siRNA) technology. siRNA has become the first choice of inhibiting gene expression. Cancer cell growth was inhibited by down-regulating expression of a single oncogene in the previous studies. In order to enhance the effect of siRNAs, we co-transfected survivin and KSP siRNA and down-regulating expressions of the two genes in breast cancer MDA-MB-231 cells to study the effect on cell growth and invasion ability and to provide a new strategy for gene therapy of breast cancer.Methods : The siRNAs targeting survivin and KSP were chemically synthesized and modified, then transfected into breast cancer cells MDA-MB-0231 respectively by protoplasm nano-technology synthesis transfection reagent Entranster?-R. The proliferation and apoptosis of the MDA-MB-0231 were investigated by MTT assay and Flow cytometry(FCM). The relative expression of mRNAs and proteins were detected by q RT-PCR and Western Blot.Results:The effects on inhibiting proliferation of breast cancer MDA-MB-231 cells were obviously for single or double-transfecting two genes siRNAs, but the double-transfection was better. Expression levels of survivin and KSP mRNA and protein were significantly lowered(P<0.01). 100 nmol/L is the best concentration of siRNA, 48 h after transfection reached the best effect. The cell proliferation rates of survivin, KSP and survivin + KSP groups were respectively 0.66 ± 0.01, 0.67 ± 0.03, 0.54 ± 0.01. The rates of apoptosis were(11.61 + 0.89)%,(16.33 + 0.94)%,(45.73+ 1.11)%, which was significant difference(P<0.01) compared with the control group. In each group, the expression of survivin and KSP mRNA was significantly inhibited(P<0.01), Bax mRNA expression was increased significantly(P<0.01), Bcl-2 mRNA expression decreased significantly(P<0.01). Double co-interferencing group among the groups, cell proliferation rate was the lowest, apoptosis rate was the highest, increase of Bax mRNA and protein expression and decrease of Bcl-2 mRNA and protein expression were most significantly.Conclusion:These results suggest that double down-regulating expressions of survivin and KSP genes can effectively inhibit breast cancer cell proliferation and promote apoptosis by siRNAs. The effect of double down-regulating survivin and KSP is better than down-regulating single gene. Therefore, co-interferencing survivin and KSP gene is a new potential method for tumor therapy. Moreover, SURVIVIN and KSP could be as effective targets of anticancer.