Knockdown of survivin gene suppresses the tumorigenicity and enhances chemosensitivity in hepatocellular carcinoma (HCC)

Survivin, the inhibitor of apoptosis (IAP) family member, was suggested to control cell division and inhibit apoptosis. Expression of this protein was only found in transformed cell lines and human tumor tissues, such as colorectal cancer, but not in terminally differentiated adult tissue. Survivin mRNA was frequently expressed in HCC and its protein expression was shown to be highly correlated to the proliferation index (Ki-67). The present study aimed at studying the effect of survivin on the tumorigenicity and chemosensitivity in HCC via the establishment of an HCC cell line (PLC) with the stably knockdown of survivin gene (PLC-k). The viability of PLC-k showed 13.3% and 33.3% lower than that of the cell line transfected with vector control (PLC-v) at 24- and 48-hour time points, respectively. Addition of cisplatin reduced the cell viability of PLC-k cells to 50% and 53% at 24- and 48-hour time points, respectively. Whereas PLC-v cells showed no significant change and 21% decrease in cell viability at 24- and 48-hour time points, respectively. To elucidate the cellular activity and the responses to chemotherapeutic drug treatment in PLC-k and PLC-v cells, apoptotic and proliferation assays were performed. By Annexin V staining, early apoptosis in the two cell lines were similar at 24 hour after cell seeding (PLC-v: 7.15% and PLC-k: 8.67%). After cisplatin treatment, the changes in the percentage of apoptotic cells were similar in the two cell lines, with the increase of around 1.4-fold and 2.2-fold at 24 and 48 hour time point, respectively. In the proliferation analysis, cells were seeded and cell numbers were counted at 24, 48 and 72 hour time points. Numbers of PLC-k cells were significantly lower than PLC-v cells in all the 3 time points, implying the proliferation rate of PLC-k was significantly lower than PLC-v. After 24-hour cisplatin treatment on the two cell lines, PLC-k showed significant decrease (40.4%) in cell number comparing to untreated cells, while there was no significant decrease in PLC-v cells. At 48-hour time point, more dramatic decrease in cell number was detected in PLC-k (63%) comparing to PLC-v (31.4%). In in vivo study, the cell lines were subcutaneously injected into nude mice for tumorigenicity assay. PLC-v induced tumor growth in all 12 mice, whereas PLC-k can only induce tumor growth in 2 out of 12 mice. Besides, the PLC-k-induced tumors grew dramatically slower than PLC-v-induced tumors. The above results demonstrated the role of survivin on tumor growth and chemosensitivity in HCC. The significant decrease in the cell viability of PLC-k and its responses to drug treatment may be due to the inhibition of cell proliferation rather than induction of apoptosis. To further investigate the function of survivin genes on cell proliferation and chemosensitivity in HCC, cell cycle analysis by flow cytometry and studying cell cycle protein expressions will be performed