Experimental Study Of The Role Of REGγ In Breast Cancer

PART ONE EXPRESSION AND SIGNIFICANCE OF REGγIN HUMAN BREAST CANCER CELLS AND BREAST EPITHELIAL CELLObjective:To study the expression and significance of REGγin human breast cancer cells and breast epithelial cell.Methods:Western blotting and immunocytochemistry were applied to detect the expression of REGγin human breast cancer cells(MCF-7,MDA-MB-231) and breast epithelial cell(HBL-100).Results:①REGγlevels of the breast cancer cells were obviously higher than those of breast epithelial cell(P＜0.05).②REGγlevels of MDA-MB-231 were obviously higher than that of MCF-7.Conclusion:The expression level of REGγis increased obviously in breast cancer cell lines,and is more important in the cell line with higher potency of malignancy. PART TWO EXPRESSION AND SIGNIFICANCE OF REGγIN HUMAN BREAST CANCER,BENIGN TUMOR AND TUMOR-ADJACENT TISSUESObjective:To study the expression and significance of REGγin human breast cancer,benign tumor and tumor-adjacent tissues.Methods:Immunohistochemistry was applied to detect the expression of REGγin human breast cancer,benign tumor and tumor-adjacent tissues.Results:①REGγlevel of human breast cancer were obviously higher than that of tumor-adjacent tissues(P＜0.05).②REGγlevel in T≤2cm were obviously lower than those in T＞2cm group the pathological gradeⅢgradeⅠandⅡ(P＜0.05).③REGγlevel in axillary lymph node negative were obviously lower than those in axillary lymph node positive.④REGγlevel of CerBb-2(+) group were obviously higher than those of c-erBb-2(-) group(P＜0.05).⑤REGγlevel of ER(-) group were obviously higher than those of ER(+) group and(P＜0.05).⑥There were no significant differences of REGγlevels between with gradeⅢgroup and gradeⅠandⅡgroup(P＞0.05).Conclusion:REGγlevel increased in T＞2cm,axillary lymph node positive,c-erBb-2(+) and ER(-) tumors,but the expression level had no relation with pathlogical grade PART THREE CONSTRUCTION AND IDENDIFICATION OF RECOMBINED REGγshRNA PLASMID IN BREAST CARCINOMA CELLSObjective:To construct and idendify the recombined small hairpin RNAs(shRNA) plasmid targeted to REGγgene,which can knock down the REGγgene of breast carcinoma cells.Methods:Small hairpin RNAs(shRNA) targeted to REGγcoding gene were designed and synthesized according to the principle mentioned by Elbashir and Reynolds.It was cloned into pGenesil-1 which worked as a transcription vector to construct recombined plasmid named pshRNAREGγ-1,pshRNAREGγ-2 and pshRNAREGγ-HK(negative control plasmid).The recombined plasmid was extracted in middle quantity and transferred into breast carcinoma cells by Lipofectamine^TM 2000.Realtime-quantitive-RT-PCT and western blotting were used to detect REGγmRNA and protein respectively.Results:Three recombined plasmids:pshRNAREGγ-1, pshRNAREGγ-2 and pshRNAREGγ-HK were constructed successfully. Stable transfected cell lines were gained by G418 screen after transfection by Lipofetamine^TM 2000.REGγmRNA was suppressed to 36%,48%和38%,46%and in contrast to untransfected cell lines detected by realtime-quantitive-RT-PCR,and REGγprotein was knocked down to 38.23%,45.12%and 36.43%,48.46%measured by western blotting in cells transfected by pshRNA REGγ-1 and pshRNAREGγ-2 plasmid in MDA-MB-231 cell and MCF-7 cell respectively,pshRNAREGγ-2 is better than pshRNA REGγ-1.Conclusions:REGγprotein in breast carcinoma cells can be knocked down effectively by recombined plasmid pshRNAREGγ-1 and pshRNA REGγ-2.The effect of pshRNAREGγ-2 is better than that of pshRNA REGγ-1. PART FOUR STUDY OF THE SIGNIFICANCE AND ALTERATION OF RELATIVE PROTEINS IN pshRNAREGγPLASMID TRANSFECTED BREAST CANCER CELL LINESObjective:To investigate the influence on the cell growth,cell cycle, apoptosis and the change of levels for relative proteins by treated with shRNAREGγ-2 in different breast cancer cell lines.Methods:The variations of mRNA and protein level of REGγ,SRC-3,PCNA and p21 in different pshRNAREGγ-2 plasmid transfected breast cancer cell lines were tested by real-time-quantitative-RT-PCR and western blotting as well as immunocytochemistry respectively;The cell proliferation rate and apoptosis were measured by MTT,flow cytometer and transmission electron microscope,dTdT mediated dUTP nick end labeling(TUNEL) method.Results:The REGγmRNA and protein levels were significantly decreased in both transfected groups,but there were no change of the other three proteins' mRNA levels:SRC-3,PCNA and p21,and the variations of these proteins levels were:PCNA level was significantly decreased,and SRC-3 level was significantly increased in both cell lines (P＜0.05);p21 level was increased in MDA-MB-231cell(P＜0.05) but was no significant change in MCF-7cell.There were more apoptotic bodies were detected in MDA-MB-231 cell(P＜0.05) than those in MCF-7cell;the G1 phase cells percentage was increased with the decrease of S phase cells in MDA-MB-231 cell(P＜0.05) and the transfected cell growth was stepped down,but on the contrary,the S phase cells was increased in MCF-7cell(P＜0.05) with an accelarated cell growth.Conclusions:REGγhas different roles in MDA-MB-231cell and MCF-7cell.REGγstimulates cell proliferation by degradation of p21 and increases the PCNA in MDA-MB-231cell,and inhibits the proliferation of MCF-7 cell by degradation of SRC-3.