GPR30/STAT3 Signal Pathway Promote Triple-negative Breast Cancer MDA-MA-468 Cells Metastatic Machenism

Objective:Many researchers have found that the G protein-coupled estrogen receptor（GPER1/GPR30）have a high positive rates in ER（-）breast carcinoma.This suggests that estrogen may activate GPR30 to accelerate the tumor progress.In other words,GPR30 may as a new therapeutic target for ER（-）breast cancer,which coupled seven Tran-membrane proteins.It can Trans-activate EGFR signal pathway to play a rapid non-genotype effect of estrogen,that may other than ERα/β.More recently,estrogen is not direct but indirect action on EGFR,and then activates the downstream EGFR-MAPK signaling pathway to promote tumor cells proliferation.Many investigations have linked that high EGFR expression in breast cancer indicates poor prognosis,which has important significance to guide the clinical treatment and prognosis.Recently study found GPR30 were significantly correlated with over expression and breast cancer metastasis.So we investigated GPR30 is whether or not associated with EGFR downstream molecules and the impact on breast cancer metastasis.STAT3 is the point of carcinogenic signal pathways,such as EGFR,IL-6/JAK,Src,etc.So whether there is a connection between GPR30 and STAT3 signaling pathway in Triple-negative breast cancers（TNBC）is the purport in our study.Hence,our research uses vitro experiment to explore the relationship between GPR30 and STAT3 in tumor metastasis.Methods:1 Cell cultureMCF-7,MDA-MB-468,MDA-MA-231and MDA-MB-453 cells were cultured in DMEM medium supplemented with high glucose,10%heat-inactivated fetal bovine serum,penicillin 100 U/mL,streptomycin 100μg/mL at 37℃in an incubator,containing 5%CO₂.2 Western blottingThe GPR30 protein content in four breast cancer cell lines were detected by western blotting.The p-stat3 levels of MDA-MB-468 cells by different concentration and time of estrogen（E2）and GRP30’s special stimulator G1.The p-stat3 levels of MDA-MB-468 cells by different concentration and time of estrogen（E2）and GRP30’s special inhibitor G15.3 Immune-fluorescence testImmune-fluorescence test is applied to detect the location of GPR30 in MDA-MB-468 cells.4 Wound healing assay.Migration rate was measured by wound healing assay.5 Statistic analysesThe results were expressed as mean±standard deviation and evaluated with SPSS 21.0 solftware by analysis of variance（One-Way ANOVA）or Kruscal-Wallis Test.Fisher’s Least Significant Difference（LSD）was used between two groups compared.P<0.05 was considered statistically signifi-cant.Results:1 Protein extraction and Western blotting:The ratios of IOD between GPR30 and GAPDH protein were 1.07±0.05,0.84±0.09,0.15±0.04,0.45±0.12 in MCF-7,MDA-MB-468,MDA-MA-231 and MDA-MB-453 cells respectively;The ratios of IOD between p-STAT3 and GAPDH protein were0.98±0.009,1.13±0.041,1.31±0.015,1.58±0.04 in MDA-MB-468 cells with different concentrations of E2;The ratios of IOD between t-STAT3 and GAPDH protein were 1.10±0.09,1.14±0.06,1.15±0.12,1.12±0.1 in MDA-MB-468 cells with different concentrations of E2;The ratios of IOD between p-STAT3 and GAPDH protein were 0.98±0.009,1.13±0.041,1.31±0.015,1.58±0.04 in MDA-MB-468 cells with different times of E2;The ratios of IOD between t-STAT3 and GAPDH protein were 1.07±0.19,1.03±0.15,1.11±0.04,1.09±0.03 in MDA-MB-468 cells with different times of E2;The ratios of IOD between p-STAT3 and GAPDH protein were 0.98±0.017,1.08±0.072,1.29±0.072,1.56±0.059 in MDA-MB-468 cells with different concentrations of G1;The ratios of IOD between t-STAT3 and GAPDH protein were 1.18±0.09,1.08±0.04,1.17±0.05,1.15±0.07 in MDA-MB-468cells with different concentrations of G1;The ratios of IOD between p-STAT3and GAPDH protein were 0.97±0.022,1.15±0.161,1.59±0.031,0.77±0.061 in MDA-MB-468 cells with different times of G1;The ratios of IOD between t-STAT3 and GAPDH protein were 1.07±0.19,1.10±0.02,1.17±0.06,1.10±0.08 in MDA-MB-468 cells with different times of G1;Above all,the results showed that with the increase of the concentrations and times,the expression of p-STAT3 was up-regulated（P<0.05）,which in a time-dependent or concentration-dependent manner.The ratios of IOD between p-STAT3 and GAPDH protein were 0.97±0.029,1.22±0.033,1.23±0.065,0.67±0.073,0.66±0.084 in control group,E2 group,G1 group,E2+G15 group and G11+G15 group respectively;The ratios of IOD between t-STAT3 and GAPDH protein were 1.15±0.06,1.17±0.05,1.15±0.09,1.10±0.10,1.10±0.08in control group,E2 group,G1 group,E2+G15 group and G11+G15 group respectively;These results indicated that after inhibit GRP30,the expression of p-STAT3 was down-regulated（P<0.05）,which implied there is a correlation between GRP30 and STAT3.2 Immune-fluorescence test:GRP30is located in the cytoplasm mainly.3 Wound healing assay.Migration distance of 24h were（106±15.28）μm、（200±26.46）μm、（186±32.15）μm、（90±26.45）μm、（86±15.27）μm、（47±14.98）μm、（56±16.52）μm in control group,E2 group,G1 group,E2+G15 group,G11+G15,E2+G15+S3I-201 group and G1+G15+S3I-201group respectively;Migration distance of 48h were（234±28.92）μm、（375±18.17）μm、（383±18.52）μm、（149±11.02）μm、（163±20.66）μm、（111±14.15）μm、（103±7.37）μm;These results showed that when activate GRP30,the capability of cell migration has enhanced（P<0.05）;when inhibited GRP30,the capability of cell migration has weakened（P<0.05）.Conclusions:1 The active GRP30 can trans-activate STAT3 signal pathway.2 When inhibited GRP30 or STAT3,the ability of tumor cells were significantly weakened on invasion and migration.