Investigation Of The Expression Of ErbB3in Renal Cell Carcinoma And Its Clinical Significance

Part one: the ErbB3mRNA and protein expression in renal cell carcinoma(RCC) and normal tissuesObjective:1, To study the ErbB3mRNA and protein expression in renal cellcarcinoma (RCC) and normal tissues；2. To study the relationship between the ErbB3mRNA expression and the TNM stage of RCC.Method:1. Regular RT-PCR and real-time quantitative RT-PCR (qRT-PCR) werecarried out to detect the mRNA expression levels of ErbB3in38cases of renal cellcarcinoma tissues and their corresponding normal renal tissues. The proteinexpression levels of ErbB3were also investigated by Western Blotting analysis,2.The clinical significance of ErbB3expression in RCC, as well as the relationshipbetween ErbB3expression and TMN staging was statistically analyzed.Results: Our regular RT-PCR and qRT-PCR results showed that among38patientswith RCC,17(44.74%,17/38) of which had significantly higher expression levels ofErbB3in RCC as compared with their corresponding normal adjacent tissues. On thecontrary, the mRNA levels of ErbB3in RCC of6cases (15.79%,6/38) were lowerthan that of their corresponding normal adjacent tissues. Still, the mRNA expressionlevels of ErbB3in RCC in15(39.47%,15/38) cases remained pretty much the sameas their self-paired controls. Meanwhile，the higher protein expression levels of ErbB3in RCC were further confirmed by Western Blotting analysis. Interestingly, maybedue to the small size of samples, our statistical analysis showed that there was nosignificant relationship between the expression levels of ErbB3and TNM staging ofRCC (P=0.379>0.05, r=0.184).Conclusion: The expression levels of ErbB3mRNA and protein in renal cellcarcinoma tissues were significantly higher than that of the self-paired normal tissues,which indicating that ErbB3may play a role in tumorigenesis of RCC. While, thelacking of relationship between the expression levels of ErbB3and TNM staging of RCC may hint that the ErbB3does not paly important role in aggressive RCCprogression. Part two: effects of lentivirus mediated-ErbB3gene overexpression on hallmarksof A498cells.Objective: To investigate the effects of lentivirus mediated-ErbB3geneoverexpression on hallmarks of A498cells.Method:1. HEK293T and A498cells were maintained in DMEM/F12andRPMI1640supplemented with10%fetal bovine serum, respectively. All cell lineswere cultured in a37oC humidified atmosphere containing95%air and5%CO2andwere split twice a week.2. Lentiviral expression control vector pLEX-MCS orpLEX-ErbB3and lentivirus packaging plasmids psPAX2and pMD2.G wereco-transfected into virus packaging cell line HEK293T using PEI following thestandard procedure. After24h, the culture media were changed with freshDMEM/F12medium (10%fetal bovine serum). The virus in conditioned mediumwere collected in the following two consecutive days, and then filtered with lowprotein-binding filters before they were aliquoted and stored at-80°C.3. A498cellswere infected with either control or ErbB3-expressing lentivirus mediated bypolybrene with a final concentration of8ug/ml and the overexpression of ErbB3wereanalyzed with RT-PCR, qRT-PCR and Western Blotting.4. The effects ofoverexpression of ErbB3on growth of A498cells were analyzed by MTS assay inconsecutive6days post-infection.5. The effects of overexpression of ErbB3onsensitivity of A498to cisplatin were also analyzed by MTS assay.6. Statisticalanalyses of the experimental data were performed using a two sided student’s t-test.Significance was set at P <0.05.Results: As shown by RT-PCR and qRT-PCR, the mRNA level of ErbB3wassignificantly higher in ErbB3-expressing lentivirus infected A498as compared withcontrol lentivirus infected A498. The overexpression of ErbB3protein was alsoconfirmed by Western Blotting analysis. Our further MTS results showed that the growth and proliferation rate was significantly increased in ErbB3-overexpressingA498. Besides, the survival rate of ErbB3-overexpressing A498after exposure tocisplatin was dramatically increased as compared.Conclusion: The lentivirus expression system can mediate the overexpression of bothmRNA and protein of ErbB3in A498successfully. The overexpression of ErbB3notonly promoted the growth and proliferation of A498, but also resulted in its resistanceto cisplatin. Thus, we presumed that overexpression of ErbB3may be associated withdrug resistance in RCC.