Effect Of SiRNA Against ObR On The Proliferation And Apoptosis Of SK-BR-3 Cell Line And Mechanism Exploration

Background and Objective:Obesity is a risk factor for breast cancer and isassociated with poor prognosis and drug resistance.Leptin,the protein product of theobese gene, which controlling food intake and energy balance.In cellular models,leptin has been shown to activate proliferation, angiogenesis,and invasion.Leptinaction is mediated through the transmembrane leptin receptor ObR. The signalingpathways known to be activated by ObR include JAK/STAT,MAPK andPI3K,et,al.Recent studie suggested that in SK-BR-3 breast cancer cell line,leptin cantransactivate HER2 through both the epidermal growth factor receptor HER1 andJAK2 pathways.Here,we first tested whether the ObR and HER2 can be coexpressedin breast cancer biopsies and the relationship of OB/ObR expression with theclinicopathlogical features and prognosis;then we studied the effect of siRNA againstObR on the proliferation and apoptosis of SK-BR-3 cell line and explored themechanisms.Methods:(1) The expression of OB and ObR in breast cancer tissues wasevaluated with immunohistochemistry;(2) A specific ObR siRNA oligonucleotidewere constructed. The ObR siRNA was transient transfected into SK-BR-3 cell line that have coexpression of ObR and HER2;(3) The results of transfections wereconfirmed by qreal-time PCR and Western blotting;(4) The effect of siRNA on theproliferation of SK-BR-3 cell lines was evaluated using MTT assay;(5) The effect ofsiRNA on the apoptosis of SK-BR-3 cell line was evaluated by Annexin V andTUNEL;(6) Change of HER2 protein expression was detected by western blot assay.Results:(1) Positive expressions of OB and ObR were noted in 79.8% and 85.1%of the samples. The expression of OB and ObR in breast cancer tissue was notsignificantly related to the clinicopathological characteristics,including ages,menopause, tumor size,pathological type,lymph node metastasis and distantmetastasis,( P>0.05). The expression of OB and ObR was not significantly related tothe ER,PR and P53 status,(P>0.05).The expression of ObR showed a significantcorrelation with the HER2 status ,(P=0.04);(2) The expression of OB wassignificantly related to the overall survival(OS),(p=0.008). For the tamoxifen-treatedpatients, postmenopause patients, triple negative patients and lymph node metastasispatents ,the OS of the OB-positive group was significantly shorter than that of thenegative group, (p=0.05, p=0.036, p=0.005, p=0.005);(3)ObR-targeted siRNA transfectionremarkably decreased the ObR expression at the mRNA and proteinlevels;(4)ObR-targeted siRNA transfection remarkably decreased the growth of SKBR-3 cells;(5)ObR-targeted siRNA transfection remarkably increased the apoptosis ofSK-BR-3 cells;(6) ObR-targeted siRNA transfection remarkably decreased HER2expression at the protein level.Conclusion:ObR and HER2 coespression in breast cancer tissues,ï¼›downregulationof ObR expression with ObR-targeted siRNA remarkably decreased thegrowth and increased the apoptosis of SK-BR-3 cells;and the inhibition of HER2expression might be related to the mechanisms.