Expression And Associations Of GRIM-19and STAT3in Human Breast Cancer

Backgroud:GRIM-19(genes associated with retinoid-IFN-induced mortality-19) was originally isolated as a growth suppressive gene using a genetic screen by Kalvakolanu. GRIM-19located on human chromosome19p13.1-13.2, coding for a protein of144amino acids, The GRIM-19protein has a molecular mass16.0kDa. GRIM-19located in nucleus, cytoplasm and mitochondria. Overexpression of GRIM-19enhances cell death, Down-regulation of GRIM-19causes cell proliferation and vicious transformation. GRIM-19, as part of the mitochondrial complex I assembly, plays important role in respiratory. STAT3(Signal transducer and activator of transcription-3) is a cytoplasmic transcription factor that coupled the Tyrosine phosphorylation signal.It is reported that STAT3is over-expressed in most kinds of cancer cells. GRIM-19binds to STAT3gene and inhibits its transcription. The role to up-regulate anti-tumor gene and promote the survival of cancer cell by constitutively activated STAT3can be inhibited by GRIM-19.Objective:To evaluate the expression of GRIM-19and STAT3in patients suffering from breast carcinoma and to determine their correlation with clinicopathological features,Methods:All patients with primary breast carcinoma underwent modified radical mastectomy at Qilu Hospital of Shandong University (Jinan, China) from July2011to September2013. The median age of patients was48.5years (range,39-65years).20of93patients were examined by western bolt and IHC, the others were examined by RT-PCR. Tumor samples and matched non-tumorous tissues were obtained immediately after mastectomy, then snap frozen in liquid nitrogen, and kept at-80℃until further analysis. All molecular markers (ER, PR, HER-2/neu, P53, and Ki-67) of these patients were available. 1. Western blotTotal protein lysates were made from frozen tissues using RIPA buffer, containing protease inhibitor cocktail, Protein concentrations were measured using the BCATM Protein Assay Kit. Protein were separated on10%sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the objective proteins were transferred onto polyvinylidene fluoride (PVDF) membrane. After blocking in TBS-T with5%non-fat dry milk2hours at20℃, membranes were incubated with anti-GRIM-19polyclonal antibody (1:500dilution), anti-STAT3monoclonal antibody (1:5000dilution), and monoclonal anti-β-actin antibody (1:1500dilution) overnight at4℃, The membrane was washed three times with TBS-T for10min each, incubated for2hours in TBS-T with HRP conjugated goat anti-rabbit/mouse secondary antibody (GRIM-191:1500dilution, STAT31:2000dilution, β-actin1:1500dilution), and washed three times with TBS-T for10min each. Antibodies bound to membrane immobilized proteins were visualized by enhanced chemiluminescence using the ECLTM Western blotting detection reagents.2. ImmunohistochemistryGRIM-19and STAT3expressions were determined by immunohistochemical, staining of tissue sections using affinity-purified mouse anti-GRIM19polyclonal antibody (sc-99086,1:100dilution. Santa Cruz, CA) and rabbit anti-STAT3monoclonal antibody (Cell Signaling Technology,1:100dilution. Beverly, MA). Histopathological assessment was performed on paraffin sections of4μm in thickness. Immunohistochemistry was performed on sections after incubating the tissue in3%aqueous hydrogen peroxide for10min, followed by washing in0.01mmol/L phosphate-buffered saline and treatment with1%normal goat serum for10min at room temperature. Antigen retrieval of GRIM-19and STAT3was done by heating sections using a microwave oven for two10-min periods, respectively in0.01mol/L sodium citrate buffer (pH6.0) and EDTA buffer (PH8.0). Sites of primary antibody binding were identified by streptavidin-peroxidase kit followed by reaction with substrate,3,3’diaminobenzidine tetrahydrochloride (DAB)(Santa Cruz, CA). A section of lung squamous carcinoma, previously proven to STAT3positive was used as a positive control. Normal human pancreas was used as positive control for GRIM-19. Omission of the primary antibody was used as negative control.The overall amount of staining was determined by staining intensity (I) and the proportion/extent (E) of stained tumor and matched non-tumorous cells according to a previously reported method. A proportion score was assigned to represent the estimated proportion of positively stained tumor cells (0≤10%,1=11%-25%,2=26%-50%,3>50%). The average estimated intensity of staining in positive cells was assigned as an intensity score (0=none,1=weak,2=intermediate,3=strong). The staining score was calculated by multiplying the score of intensity and extent of staining. Staining scored>2fell into the category of positive, while staining scored≤2were marked as ’negative’ for GRIM-19or STAT3expression.3. Real-time RT-PCRThe RNA of tumor samples and matched non-tumorous tissues were isolated by Trizol, The RNA DNase was treated and reverse transcribed by using the RT reagent Kit (Thermo). The reaction mixtures were subjected to37℃for20min, followed by85℃for5s,4℃forever. For quantitative RT-PCR, the resultant cDNA were performed in triplicate with a Real-Time PCR System (Applied Biosystems), using SYBR green (TaKaRa) within the gene-specific primers GRIM-19, STAT3, GAPDH which served as a normalization reference. Each20μl RT-PCR mix included10μl SYBR Premix Ex TagTM,0.2μl PCR F Primer,0.2μl PCR R Primer,2.0μl cDNA,7.6μl H2O. The reaction mixtures were subjected to an initial denaturation of95℃for10min, followed by36cycles of95℃for5s,55℃for30s, then subjected to a melting curve analysis. Real-time RT-PCR with each sample, using the two primer sets, was conducted in triplicate to ensure consistency in cycle threshold (Ct) values. The amplification was followed by melt curve analysis and the changes in the Ct values were calculated by the equation ΔCt=Ct target-Ct input where target and input refers to GRIM-19, STAT3and GAPDH respectively. The fold differences were calculated as per the formula2-Δ(ΔCt).Results:1. GRIM-19and STAT3expressions were determined by Western blot and IHC in20breast cancer tissues and corresponding nontumorous tissues. Both results have clearly shown that GRIM-19was lost or severely depressed in breast carcinomas with respect to matched nontumorous tissues (P<0.001), and STAT3was inversely over-expressed in breast cancer tissues with respect to matched nontumorous tissues (P<0.001), which were supported by RT-PCR analysis in another73breast samples and matched nontumorous tissues in mRNA level (P<0.001).2. There was negative correlations between the expression of GRIM-19and STAT3in breast cancer tissue in one patients (r=-0.68, P<0.001) in the result of Western blot analysis, which was supported by IHC (r=-0.49, P=0.028) and RT-PCR analysis (r=-0.60, P<0.001).3. The result of Western blot analysis has shown that the expression of STAT3was associated with ER (P=0.030), and there are correlations between the expression of HER-2/neu and GRIM-19(P=0.037), which were both revealed by Western blot analysis.4. The mRNA expression level of GRIM-19and STAT3were associated with histological grade and Ki-67(P<0.05), which were displayed by RT-PCR analysis. Further more, the expression of STAT3was associated with the presence of lymph node metastases (P=0.024) in the result of RT-PCR analysis.Conclusion:GRIM-19was down-regulated and the STAT3was up-regulated in primary breast carcinomas, and there was an inverse correlation between the expression of GRIM-19and STAT3in both protein and mRNA levels. The down-regulation of GRIM-19may reduce the inhibiton of STAT3activity, and plays a role in the carcinogenesis and progress of breast cancer.