Proteasomes reactivator REG gamma enchances oncogenicity of MDA-MB-231 cell line via promoting cell proliferation and inhibiting apoptosis.
To investigate the effect of proteasomes reactivator REG gamma on cell cycle and apoptosis in vitro and in vivo. In vitro, we first constructed recombinant plasmid of PcDNA3.1-REGgamma and then transfected REGgamma into MDA-MB-231 cell line. We confirmed the transfection efficiency by Western blot. Subsequently, we observed cell growth, cycle and colony formation. Specific proliferative molecule proliferating cell nuclear antigen (PCNA) and apoptosis related signal molecule Caspase-3 was assayed by immmunohistochemistry and absorption spectrometry, respectively. In vivo, we successfully established transplantation tumor nude mice model. We determined REGgamma mRNA level in the transplantation tumor tissue. Then, FCM was used to determine cell cycle, apoptosis and CD16. Finally, we employed immunohistochemistry to determine P21 positive expression. However, the cells transfected with REGgamma grew more rapidly compared with non-transfected ones. Increased cells were observed in S+G2+M phase and S phase in the REGgamma transfected group. PCNA expression level in the transfected cells was higher than that in non-transfected ones. In vivo, we observed the similar phenomenon including more rapid tumor growth, higher REGgamma mRNA expression, decreased cells number in G0/G1 phase and G2/M phase, increased cells in S phase and decreased apoptosis in the transfected group. In the study of related molecules, we also found related molecules P21 and CD16 positive expression rate were obviously lower than non-infected ones. In present study, we found oncogenicity of MDA-MB-231 cell transfected with REGgamma was enhanced, which might be realized via REGgamma promoting cell growth, inhibiting cell apoptosis, degrading P21 and suppressing activation of NK, suggesting REGgamma promoting tumor growth is a process involving multiple factor mechanisms.
Tian
M
M
Chongqing Medical University Department of General Surgery, First Affiliated Hospital, Chongqing, China.
Xiaoyi
W
W
Xiaotao
L
L
Guosheng
R
R
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology / Q000648:ultrastructure; D053148:Caspase 3 / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D008325:Mammary Neoplasms, Experimental / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D046529:Microscopy, Electron, Transmission; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017452:Receptors, IgG / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D001324:Autoantigens; D050759:Cyclin-Dependent Kinase Inhibitor p21; C099558:Ki antigen; D017452:Receptors, IgG; D053148:Caspase 3; D046988:Proteasome Endopeptidase Complex