| Objective: Triple-negative breast cancer(TNBC)is a kind of refractory breast cancer that neither express estrogen receptor,progesterone receptor nor Her-2 receptor.With the characteristics of young onset,high recurrence rate,poor hormone therapeutic efficacy and grave prognosis,TNBC has always been a research focus for domestic and foreign scholars[1].In recently years[2-3],some researchers have found that G protein-coupled receptor(GPR30)had a higher positive expression rate in some of breast cancer tissues with negative estrogen receptors and estrogen could continue to function through the combination with GPR30.Therefore,it is conceivable that GPR30 may be a new target for TNBC treatment.Compare with classical estrogen receptor,GPR30 has a different mechanism of action which it plays a quick genotype non-estrogenic effects by trans-activation of epidermal growth factor receptor(EGFR)and its downstream signaling pathway.Many studies[4-5] suggest that high EGFR expression is important to guide breast cancer treatment and also predicts poor prognosis.According to another studies[6],GPR30 indirectly activate EGFR-MAPK/Erk signaling pathway to promote tumor cells proliferation.So the question is whether there are interactions between GPR30 and other EGFR downstream signaling pathways impacting TNBC transfer.Phosphatidylinositol 3-kinase(PI3K)is an important signal molecule in EGFR downstream signaling pathway.In many malignant tumor cases,PI3K/Akt signal pathway has abnormal activation and over expression[7-9],which suggests this signal pathway may also play an important role in tumor metastasis process.This paper discusses whether there are interactions between GPR30 and PI3K/Akt signaling pathway,and working together to impact breast cancer metastasis.Method: 1 Cell Culture: in the incubator with 5% CO2 at 37℃,culturing breast cancer cell line by high sugar DMEM containing 15% fetal bovine serum.2 Using Western-blot experiments to detect 4 kinds GPR30 protein expression situation in breast cancer cell lines from different pathological types.3 Detecting GPR30 position in TNBC cell MDA-MB-468 by immunofluorescence staining test.4 Stimulated by estradiol(E2)and GPR30 specific agonist(G1),detect p-MDA-MB-468 cells PI3 K protein expression levels by Western-blot test with concentrationt and time gradient stimulation increasing.5 Using Western-blot to detect p-PI3 K protein expression levels in MDA-MB-468 cell after GPR30 specific blocker(G15)stop E2 and G1 activating GPR30.6 By Wound healing and Transwell invasion test detecting the influence of GPR30-PI3K/Akt signaling pathway on the ability of tumor cells migration and invasion.7 Statistical method: data processing by SPSS 21.0,measurement data displayed by M±QR or±s,the groups were compared by ANOVA or KW H rank sum test,pairwise comparison between groups using LSD method,with P <0.05 the difference was statistically significant.Results: 1 Four kinds of breast cancer cell lines are MDA-MB-468,MCF-7,MDA-MA-231 and MDA-MB-453.Detect the protein expression of GPR30 by Western blot,the result are 1.07+/-0.05,0.84,0.15+/-0.04,0.04 0.09 mm+/-0.12.The results suggest that GPR30 high expression in triple negative cells MDA-MB-468 and ER positive cells MCF-7,while in MDA-MA-231,another TNBC cell line,GPR30 almost do not express(P <0.05).2 As a result of the immunofluorescence test,GPR30 mainly located in cell cytoplasm.3 E2 concentration gradient are 0ng 5ng 10 ng 20ng.After the stimulation of MDA-MB-468 cells,p-PI3 K protein expression levels are: 0.98±0.009,1.13±0.041,1.31±0.015,1.58±0.04.E2 time gradient as 0min,5min,10 min,20min.p-PI3 K protein expression levels are: 0.98±0.018,1.17±0.103,1.54±0.035,0.73±0.068,G1 concentration gradient are 0nM,1nM,10 nM,100n M.After stimulation of MDA-MB-468 cells,p-PI3 K protein expression levels are: 0.98±0.017,1.08±0.072,1.29±0.072,1.56±0.059;G1 gradient time as 0min,5min,10 min,20min,p-PI3 K protein expression levels are: 0.97±0.022,1.15±0.161,1.59±0.031,0.77±0.061.Statistical analysis,with the increasing of GPR30 agonist concentration and time,p-PI3 K protein expression levels also increase gradually,and in a concentration and timedependent manner(P <0.05).The experiment results indicate that after GPR30 agonist activation,it may trans-activated EGFR downstream PI3K/Akt signaling pathway and participate in promoting tumor progression by increasing p-PI3 K protein expression.4 Testing by Western blot,p-PI3 K protein semi-quantitative levels in Control group,E2 treatment group,G1 treatment group,E2+G15 treatment group,G1 + G15 treatment group are 0.97 ± 0.029,1.22 ± 0.033,1.23 ± 0.065,0.67 ± 0.073,0.66 ± 0.084.The result of the experiment shows that p-PI3 K protein expression increase in E2 and G1 treatment group,but in G15 treatment group which specific GPR30 blocker added,p-PI3 K protein expression level decreased(P<0.05).This result provides further evidence that there may be correlations between GPR30 and PI3 K / Akt signaling pathway.5 Scratch test is did for these groups below: control group,E2 treatment group,G1 treatment group,E2+G15 treatment group,G1+ G15 treatment group and G1 + PI3 K inhibitor treatment group.Cell migration of each group is(76.67±15.28)um、(256.67±20.82)um、(260±36.05)um 、(65±22.91)um 、(61.67±10.41)um 、(58.33±27.54)um 、(63.33±32.15)um.The result shows that compare with control group,cellular migration ability significantly increases in E2 and G1 treatment groups(P<0.05).And cellular migration ability significantly decreases when GPR30 specific blocker used to block the activation effect of GPR30 or PI3 K specific blocker to the phosphorylation of PI3K(P<0.05).The results suggest that activation of GPR30-PI3K/Akt signaling pathway may affect breast cancer cells migration.6 Grouping same with scratch test,transwell test is did.The membrane cell number in each groups are(17±2.65),(48±5.57),(47.33±4.04),(14.33±3.06),(13.67±2.08),(12.67±3.05)and(13.66±3.06).Similar with scratch,this result clarifies GPR30-PI3K/Akt signaling pathway has important effects on breast cancer invasion and metastasis further more.Conclusion:1 There may be some kind of interaction between GPR30 and PI3K/Akt signaling pathway for the dependence of p-PI3 K protein expression levels on concentration and time of E2 and G1.2 GPR30 can trans-activate the PI3K/Akt signaling pathway and make a great influence on tumor invasion and migration after it activated by GPR30 agonist.But the tumor invasion and migration ability decrease when the cut down the ignaling pathway.3 Based on the above results confirm our preliminary,there may be correlations between GPR30 and PI3K/Akt signaling pathway and make a further influence on cancer cells invasion and metastasis. |
PDF Full Text Request