| This topic before conducting the stage,the author collected a large amount of project related documents and materials,including one-horned lotus ethyl acetate extracts parts(EETT)for breast cancer MCF-7 cell proliferation ability has certain inhibitory effect,at the same time for the development of other ability also has inhibitory effect on tumor,however,EETT three negative breast cancer MDA-MB-231 cells on the impact is not obvious.In contrast,MCF-7 cell and breast cancer MDA-MB-231 cells exist obvious differences,the most critical difference in breast cancer ER MCF-7 cell contains 1 kind of estrogen receptor,but MDA-MB231 cells do not have the receptor,which can be deduced,namely EETT in function and under the influence of estrogen receptors(ER inspire effect,under the validation of gene transfection technology,EETT impact on ER receptor was able to prove it.However,with the addition of ER receptor inhibitors,EETT MCF-7 cell proliferation capacity for breast cancer has certain inhibitory effect,and the development of other ability also has inhibitory effect on tumor,embodied in cell migration,adhesion and invasion,despite the weakening trend of the above three kinds of ability,but has not disappeared,suggesting that EETT for MCF-7 cell inhibitory effect of breast cancer is closely related to estrogen receptor GPR30.In order to verify this problem,the author selected breast cancer SK-BR-3 cells expressing GPR30 receptor in the process of research and experiment,so as to further study and analyze the effects of EETT on invasion,migration and adhesion of breast cancer SK-BR-3 cells,and whether there is a correlation between inhibition ability and GPR30 receptor protein.This experiment mainly includes three parts,which are summarized as follows:(1)Preparation and composition analysis of EETT(2)The effects of EETT on the migration,adhesion and invasion of human breast cancer SK-BR-3 cells are mainly reflected in three aspects.First,the effects of EETT on the invasion ability of breast cancer SK-BR-3 cells are verified by erosion experiment.Second,the effect of EETT on the adhesion ability of SK-Br-3 was verified by adhesion experiment.Thirdly,the effect of EETT on the invasion ability of SK-BR-3 was verified by invasion experiment.(3)The effects of EETT on GPR30 protein and pathway were verified by two methods.First,the effects of EETT on the expression level of GPR30 protein in human breast cancer SK-BR-3 cells were analyzed by Western blot.Second,the effects of EETT on the expression levels of GPR30 signaling pathway related proteins in human breast cancer SK-BR-3 cells were analyzed by Western blot.(4)The effects of EETT on the expression of GPR30 pathway-related proteins EGFR,pEGFR,Akt,p-Akt,ERK1/2 and p-ERK1/2 promoted by GPR30 agonist G1 were analyzed by Western blot.Eett extract was obtained by reflux extraction with petroleum ether,chloroform and ethyl acetate.EETT extract was obtained by rotary evaporation.The analysis of active compounds in ethyl acetate fraction of EETT was carried out by UPLC-TOF-MS.A total of 14 compounds of extracts in ethyl acetate fraction were identified.The 14 identified compounds are listed as follows:(1)sucrose,(2)adenosine,(3)coniferin,(4)lariciresinol,(5)piresil-4-O-β-D-glucoside,(6)olivil,(7)piresil-4-O-β-D-glucoside(isomer),(8)pinoresinol,(9)tianshic acid,(10)hexadecanedioic acid,(11)linoleic acid,(12)palmitic acid,(13)linolenic acid,(14)linolic acid.With the aid of cell migration experiment EETT on SK-BR-3 breast cancer cell migration ability coupling effect.Compared with the estradiol plus EETT group and the estradiol group,the migration probability of the estradiol plus EETT group was decreased in the order of 100μg/mL and 130 μg/mL,and the difference between the groups was statistically significant.The cell adhesion test results showed that compared with the estradiol group,the adhesion rate of the EETT group with the concentration of 100 μg/mL and 130 μg/mL was significantly decreased,and the difference was statistically significant between the two groups.Cell invasion assay results showed that compared with the estradiol group,the invasion rates of the estradiol plus EETT at 100 μg/mL and 130 μg/mL were significantly decreased in the estradiol group,and the difference was statistically significant between the estradiol group and the estradiol group.Western blot analysis showed that the effect of EETT on GPR30 protein was relatively small.The effects of EETT on the protein expression of EGFR,p-EGFR,Akt,p-Akt,ERK1/2 and p-ERK1/2 in GPR30 signaling pathway were determined by Western blot.The results showed that EETT had a certain inhibitory effect on the increase of the relative expression levels of p-EGFR/EGFR,p-Akt/Akt,p-ERK/ERK protein induced by estrogen,and the inhibitory effect was statistically significant with the increase of administration dose.The effect of EETT on the expression of EGFR,p-EGFR,Akt,p-Akt,ERK1/2 and p-ERK1/2 protein in GPR30 signaling pathway promoted by estrogen receptor agonist G1 was determined by Western blot.The results showed that EETT could inhibit the expression of pEGFR/EGFR,p-AKT/AKT,p-ERK/ERK protein induced by GPR30 agonist G1,and the difference was statistically significant.According to the results of relevant studies,EETT can inhibit the migration,invasion and adhesion of breast cancer SK-BR-3 cells by regulating the expression levels of GPR30 pathway-related proteins. |
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