Effection Of Overexpression Of SSAT2on Chemosensitivity In Renal Carcinoma And The Mechanisms

Part1Identification of the SSAT2expression plasmid and theexpression of SSAT2plasmid in Caki-1cellsObjective：To identificate the eukaryotic expression plasmid ofpcDNA3.1(D)/V5-His-SSAT2and observe the expression of SSAT2Plasmid in Caki-1cells.Methods： The plasmids pcDNA3.1(D)/V5-His-SSAT2weredigested by KpnⅠ/XbaⅠand sequenced to confirm the insertedsequence．The plasmids were transfected into renal cancer cell line Caki-1by X-treme GENE HP，Western blot analysis were used to detect theV5-SSAT2expression of transfected cells.Results: After KpnⅠ/XbaⅠ digestion,5537bp and577bp were cut,the agarose gel electrophoresis show that the size of cut fragment wasconsistent with the target fragment.The sequencing proved that genesequence of the amplified SSAT2was fully consistent with the sequencewith GeneBank report.Conclusions: The plasmids pcDNA3.1(D)/V5-His-SSAT2had beenconstructed correctly; SSAT2plasmids were successfully transfected into Caki-1cells. Part2The effection of Overexpression of SSAT2on cellproliferation, apoptosis in renal carcinoma Caki-1cell lineObjective：To investigate the effects of overexpression of SSAT2oncell proliferation, apoptosis of renal carcinoma Caki-1cell line underhypoxic conditions, and furthermore analyze the mechanisms.Methods：Plasmid pcDNA3.1(D)/V5-His-SSAT2was transfected intoCaki-1cells, The cells were cultured48h under Hypoxia condition,Inverted phase contrast microscope was used to observe the morphologychange of Caki-1cells, Westernblot analysis were used to detect theexpression of hypoxia-inducible factor-1α(HIF-1α) protein. Apoptoticand necrosis cells were detected by flow cytometry. MTT assay was used toassess cell proliferation.Results: As compared with untransfected group and empty vectorgroup, Protein of HIF-1α in Caki-1cells were downregulated by SSAT2transfection(P<0.01）. the connection between the cells reduced,Tansfectedtumor cell showed lower proliferation rate than untransfected group（0.238±0.027）and empty vector group（0.283±0.017）after24h. thedifference was obvious at72h（P<0.01）.The necrosis and apoptosis rate of empty vector group was6.60±0.53、5.72±1.57, butV5-SSAT2groupnecrosis and apoptosis rate was18.3±1.23、7.39±2.71, SSAT2overexpression could enhance the necrosis and apoptosis rate than the cellsuntransfected (P <0.05）.Conclusions: SSAT2overexpression could inhibit the proliferation，and enhance the necrosis, by downregulating the protein expression ofHIF-1α. Part3The effection of overexpression of SSAT2on thechemosensitivity in renal carcinoma Caki-1cell lineObjective：To investigate the effects of overexpression of SSAT2on chemotherapy sensitivity of renal carcinoma Caki-1cell line underhypoxic conditions.Methods： After Plasmid pcDNA3.1(D)/V5-His-SSAT2wastransfected into Caki-1cells,0.125μg/ml、0.25μg/ml、0.5μg/ml、1μg/ml、2μg/ml、4μg/ml Adriamycin,0.625μg/ml、1.25μg/ml、2.5μg/ml、5.0μg/ml、10.0μg/ml Cisplatin was administrated，The cells were cultured inHypoxia condition at48h, MTT assay was used to assess cell inhibitionrate；The cells were administrated with0.5μg/ml Adriamycin or2.5μg/mlCisplatin，MTT detected the OD490at24h、48h、72h. Results: Significant increase in chmosensitivity of the Caki-1cellswas observed after transfected，P<0.01. when0.5ug／ml Adriamycin wasadministrated,The inhibition rate of V5-SSAT2group was（52.7±9.6）%,while the inhibition rate of two controls were （39.5±8.9）%and（41.0±7.6）%，P<0.01. when2.5ug／ml Cisplatin was administrated,Theinhibition rate of V5-SSAT2group was（40.1±3.4.）%, while the inhibitionrate of two controls were（23.8±5.5）%and（29.3±5.4）%, P <0.01. After0.5ug／ml Adriamycin or2.5ug／ml Cisplatin were administrated，thecell show lower proliferation after24h, P<0.05.Conclusion: SSAT2overexpression could increase sensitivity ofrenal carcinoma cell line Caki-1to Adriamycin and Cisplatin，targetingSSAT2maybe a very promising strategy for renal carcinoma therapy.