The Relationship Between The Expression Of RASSF1A And The Sensitivity To Cisplatin Among A549Cell

Objective: Acquired chemo-resistance is an important reason offailure in lung cancer treatment. Previous reports indicated that thealteration of RASSF1A expression might be a key factor for regulating thechemo sensitivity in cancer. Thus, the present study intends to investigatethe relationship between RASSF1A and the sensitivity to cisplatin in A549cell using cell transfect technology for the purpose of finding a biomarkerpredicting chemo sensitivity of A549cell to cisplatin and providing theexperimental support to the individualized chemotherapy for the patientsof lung cancer.Methods: A549cell and its homologous cisplatin-resistant cell lineA549-DDP cell were cultured.A549cell with high RASSF1A levels was transfected with smallinterference RNA specific to RASSF1A to low RASSF1A expression.Semi-quantitative RT-PCR and western-blot analysis were performed toconfirm the expression of RASSF1A mRNA or protein before and aftercell transfect. Exposing A549cells to different concentrations(0-20μmol/l) of cisplatin, MTT assays were used to evaluate cell viability and the IC50value to cisplatin before and after transfect was calculated respectively;Exposing A549cells to the same concentration(10μmol/l) of cisplatin,Flow cytometric analysis was used to assess apoptosis before and aftercell transfect.A549-DDP cell with low RASSF1A levels was transfected withPCMV5-RASSF1A plasmid to enhance RASSF1A expression;Semi-quantitative RT-PCR and western-blot analysis were performed toconfirm the expression of RASSF1A mRNA or protein before and aftercell transfect. Exposing A549-DDP cells to differentconcentrations(0-70μmol/l) of cisplatin, MTT assays were used toevaluate cell viability and the IC50value to cisplatin before and aftertransfect was calculated respectively; Clone forming tests were used toevaluate the clone forming ability before and after cell transfect.Exposing A549-DDP cells to the same concentration(20μmol/l) ofcisplatin, Flow cytometric analysis was used to assess apoptosis beforeand after cell transfect.Results: RT-PCR and western-blot analysis indicated that RASSF1AmRNA and protein levels decreased greatly in A549cell transfected withsiRNA-RASSF1A. Exposing A549cell to cisplatin for24h, IC50value tocisplatin increased greatly in A549cell with lower levels of RASSF1Athan that in A549cell （[19.4±2.3）μmol/l vs（11.4±1.8）μmol/l, P=0.009]. With the same concentration of cisplatin, the apoptosis percentagedecreased greatly in A549cell with lower levels of RASSF1A than that ofA549cell [（14.4±0.91）%vs（31.9±0.31）%, P=0.01].RT-PCR and western-blot analysis indicated that RASSF1A mRNAand protein levels were increased greatly in A549-DDP cell transfectedwith pCMV5-RASSF1A. Exposing A549-DDP cell to cisplatin for24h,IC50value to cisplatin decreased greatly in A549-DDP cell withexogenous expression of RASSF1A than that in A549-DDP cell[(39.9±6.3) μmol/l vs (53.0±5.8) μmol/l, P=0.036]; After exposingA549-DDP cell to cisplatin for5days, the clone number of A549-DDPcells with exogenous expression of RASSF1A was significantly reducedthan that of A549-DDP cells; with the same concentration of cisplatin, theapoptosis percentage increased greatly in A549-DDP cell with exogenousexpression of RASSF1A than that of A549-DDP cell[(7.1±0.01)%vs(3.8±0.18)%, P=0.002].Conclusion: The alteration of RASSF1A expression may be a criticalfactor for regulating chemo-sensitivity for cancer and it needs deeplystudy about whether RASSF1A expression acts as biomarker forpredicting chemo-sensitivity of lung cancer patients in clinic.