Effects Of Estrogen Receptor Subuints On The Biological Behaviors And Thl/Th2-type Cytokine Secretion Of MCF-7

Objective:Human breast cancer cell line MCF-7 was used as parent cell,using RNA interference technology to construct stable cell lines with different expression of ERa/ERβ and investigating the impact of different ER subunits on biological characteristics and Thl/Th2-type cytokine secretion levels of these cells.Methods:MCF-7 with stable ERa and ERβ expression was used as parental cell line.Using pGenesil-1 plasmid as vector,ERa or ERβ gene was silenced by liposome transfection method and the stable cell lines with different ERa/ERp expression were obtained.The three lines were respectively named as:M/HK(ERαhigh/ERβhigh,negative control),M/siα(ERαlow/ERβhigh)and M/siβ(ERαhigh/ERβlow).Then the following experiments were made:1.Western blot was used to analyse the transfection efficiency.2.MTT was used to measure the ability of cell proliferation.3.Flow cytometry technique was used to test the cell cycle distribution.4.Semi-quantitative RT-PCR was poformed to analyse the expression levels of anti-apoptosis gene Bcl-2,Bcl-xl and XIAP.5.Double layer soft-agar colony formation test was used to test the ability of cell tumorigenesis.6.Matrigel adhesion test was used to detect the ability of cancer cell adhesion.7.Sandwich ELISA was used to measure the secretion levels of IFN-γ and IL-4.Results:1.Western blot test results displayed that the highest interference efficiency of ERa or ERβ was 77.7%and 68.3%;the ER expression did not change in M/HK cells.The stable cell lines with different ERα/ERβ expression were constructed successfully.2.MTT analysis results revealed that after 24h and 48h of incubation,compared with the M/HK cells(0.56±0.004,0.63±0.002),the proliferation rate of M/sia cells(0.55±0.008,0.60±0.008)and M/sip cells(0.58±0.008,0.69±0.001)was no significantly different(P>0.05);After 72 h and 96 h of incubation,compared with the M/HK cells,the proliferation rate of M/sia cell was lower(0.71±0.098,0.81±0.087 vs 0.88±0.145,1.07±0.156,P<0.01)and that of M/sip cell was higher(0.71±0.098,0.81±0.087 vs 1.01±0.086,1.23±0.124,P<0.01).3.Flow cytometry results revealed that,compared with M/HK cell,the proportion of GONG,increased significantly in M/sia cells(52.75±3.43%vs 65.25±2.08%,P=0.015),the proportion of S reduced(31.04±1.39%vs 15.90±1.52%;P<0.01),the proportion of G2～M did not change significantly(16.21±2.64%vs 18.86±1.20%,P>0.05).Compared with M/HK cells,the proportion of G0～G1 reduced in M/siβ cells(52.75±3.43%vs 40.56±6.67%,P=0.017),the proportion of S increased(31.04±1.39%vs 43.16±4.67%,P<0.01),the proportion of G2-M did not change significantly(16.21±2.64%vs 16.28±2.00%,P>0.05).4.Semi-quantitive RT-PCR results showed that,compared with M/HK cell,the anti-apoptosis gene XIAP expression was lower in M/siα cells(P<0.01);the anti-apoptosis gene Bcl-2,Bcl-xl and XIAP expression were higher in M/sβcells(P<0.01).5.Double layer soft-agar colony formation expriment results displayed that the number of colony was less in M/sia cells than that in M/HK cells(50.13±3.78 vs 21.25±2.63,P<0.01);the number of colony was more in M/siβ than that in M/HK cells(50.13±3.78 vs 67.85±7.32,P<0.01).6.Cell adhesion experiment results displayed that the absorbance at 570nm of the M/HK cell,M/sia cells and M/siβ cells was 0.70±0.08,0.48±0.09 and 0.65±0.12 respectively.Compared with M/HK cells,the adhesion ability of M/siα cells was significantly lower(P<0.01)and that of M/siβ cells was no different(P>0.05).7.Sandwich ELISA results showed that,compared with M/HK cells,the secretion of IFN-γ was enhanced in M/siα cells(54±2 pg/mL vs 10.2±8 pg/mL,P<0.01),the secretion of IFN-γ was inhibited in M/siβ cells(54±2 pg/mL vs 15±1pg/mL,P<0.01);the secretion of IL-4 was no different(M/HK 38±4pg/mL,M/sia 42±8pg/mL,M/siβ 44±7 pg/mL,P>0.05).Conclusion:1.Different ERcα/ERβ expression can affect biological characteristics of MCF-7.The overexpression of ERa can promote the growth,invasion,metastasis of MCF-7;while the overexpression of ERβ can inhibit the growth of MCF-7.Therefore,regulating ERa/ERβ expression may be a new treatment strategy for breast cancer.2.Different ERa/ERβ expression can modulate the secretion levels’ of Thl-type cytokine IFN-γ in MCF-7,but have no effect on the secretion levels of Th2-type cytokine IL-4.So the different ERa/ERβ expression may influence the Thl/Th2 balance in breast cancer microenvironment and then regulate the local immunity indirectly.