The Interaction Of ERα And ERβ In The Regulation Of Differentiation And Proliferation Of Vertebral Growth Plate Chondrocyte

Objective:To explore the interaction of the ERa and ERβ in regulating mice adolescence vertebral epiphyseal plate chondrocyte differentiation and proliferation,and the role of smad4signaling transduction in ERa and ER(3regulating the Runx2and BMP2expression.Methods:genbank database was used to retrieve the mouse ERa, ERβ and Smad4cDNA sequences and their transcripts.The siRNA sequences targeting and silencing ERa, ERβ and Smad4mRNA were designed which were contructed into gene silencing lentiviral vector and packaged as lentiviral particles. Mice vertebral growth plate chondrocytes are extracted, grouped and were transfected target gene ERa-shRNA-PLL3.7, ERβ-shRNA-PLL3.7, and negative control vectors, respectively, to research the role of ERa and ERβ regulating mice chondrocyte differentiation and proliferation. Then, the cells were grouped with hierarchical design and were transfected target gene RNAi vectors to observe the smad4signaling in ERa and ERβ regulating Runx2and BMP2gene expression in chondrocytes.The cells cycle was analyzed, the cells growth curves were plotted and the Runx2and BMP2expression was detected in each group.SPSS16.0software was used to analyze the differences between the groups, P<0.05was considered statistically significant difference.Results:(1)After the chondrocyte cells were transfected with ERa-shRNA-PLL3.7, ERP-shRNA-PLL3.7and Smad4-shRNA-PLL3.7, respectively, the expression of corresponding target gene was decreased, there was significant difference, P<0.05.(2) After the cells were transfected, the OD value (A490) were measured by MTT assay, respectively, and the cells growth curve were plotted.the result showed that descending order of the OD values in four groups were ER(3RNAi group> control group> ERaRNAi group> United silent group, there were significant differences between the groups, P<0.05.The cells cycle was analyzed by flow cytometry at3th day after transfected, the result showed that the descending order of the cell percentages in S and G2phase: ERβRNAi group> control group> ERaRNAi group> United silent groups, there were significant differences between the groups, P<0.05.the Runx2,BMP2gene expression were detected by Realtime-PCR at10th day in four groups in descending orde:ERβRNAi group> control group> ERaRNAi group> United silent group,there were significant differences between the groups,P<0.05.(3)hierarchical design was used to further detected OD value in10subgroups at3th,5th,7th and9th day after transfected,the result show the descending order:ERβRNAi-ERa excited-Smad4normal group> ERβRNAi-ERa excited-Smad4RNAi group> ERβRNAi-ERa normal-Smad4normal group>ERβRNAi ERa normal-Smad4RNAi group>ERs normal-Smad4normal group> ERs normal-Smad4RNAi group> ERaRNAi-ERβ excited-Smad4normal group>ERaRNAi-ERβnormal-Smad4normal group> ERaRNAi-ERβ excited-Smad4RNAi group>ERaRNAi-ERβ normal-Smad4RNAi group.Comparison between ERβRNAi-ERa excited-Smad4RNAi group and ERβRNAi-ERa excited-Smad4normal group, as well as ERβRNAi-ERa normal-Smad4RNAi group and ERβRNAi-ERa normal-Smad4normal group, there was no significant difference, P>0.05;However, there were significantly different among other groups, P<0.05. Descending order of the Runx2gene expression in each subgroup was basically the same as OD value. The descending order of BMP2gene expression was that ERβRNAi-ERa excited-Smad4normal> ERβRNAi-ERa excited-Smad4RNAi group> ERβRNAi ERa normal-Smad4normal> ERpRNAi-ERa normal-Smad4RNAi group> ERs normal-Smad4normal groups> ERs normal-Smad4RNAi group> ERaRNAi-ERβ excited-Smad4normal> ERaRNAi-ERβ excited-Smad4RNAi group> ERaRNAi-ERβ normal-Smad4normal> ERaRNAi-ERβ normal-Smad4RNAi group. Whether in ERβRNAi group or in ERaRNAi group, Comparison between Smad4RNAi subgroup and Smad4normal subgroup, there were no significant differences,P>0.05. Conclusion:Both ERa and ERβ could regulate vertebral growth plate cartilage cell differentiation and proliferation. The role of regulation of ERa was greater than ERβ.When the expression of ERa and ERβ were normal, ERβ has partially inhibit ERa function.However, when the expression of ERa significantly reduced, ERβ can partially compensate for ERa function ERP also enhanced the chondrocyte differentiation and proliferation by partly reinforced Runx2gene expression by Smad4signaling, but it may also by non-Smad4signaling pathway.However, the regulation of ERa and ERβ on BMP2gene expression by non-Smad4signaling.