Using Rnai To Study The Role Of Estrogen Receptor Gene ERβ On Vertebral Linear Growth During Adolescence

Objective:To construct the targeting ERβ-specific RNAi vector then, it was packaged into virus particles.Methods:Two transcript variants of ER(3cDNA sequences can be retrieved in genbank website."SiRNA Target Finder" software from Ambion’s was used to design the Sense Strand Sequence of the siRNA targeting and silencing ER(3gene. construct ERβ-specific targeting gene silencing vector. The most effective recombinant plasmid screened for ERβ gene knockdown was packaged into retroviruses. The mouse osteoblastic cell lines MC3T3-E1was divided into3groups, of which two groups were transfected with recombinant plasmid ER(3RNAi and negative control plasmid ERLRNAi, Group3was the control group, the same amount of PBS Instead of plasmids.3groups of cells cultured under the same conditions. In each group, the Cell growth curve was drawn; the cell cycle were analyzed; alkaline phosphatase (ALP) activity and the formation of calcium nodules were detected; the expression of the OPG and RANKL were analyzed.36mice, Of which18female mice,18male mice, were randomly assigned and divided into3groups, respectively, ERβRNAi group, blank control group and negative control group. Prepare gene silencing animal models. The length and weight were measured respectively, in1day,2weeks,4weeks and6weeks after animal models were made. The vertebral tissue sections were stained with the HE and Masson trichrome stain for observing the changes in vertebral epiphyseal plate cell layer and differences in trabecular bone density. TUNEL was used to analyze the differences of apoptosis under the epiphyseal plate; Immunohistochemical method for detection of the expression of the cytokines of OPG and RANKL SPSS16.0software was used to analyze whether the differences was significantin.Results:the two RNAi targeting sequences designed, which homolog-ous sequences can be retrieved in the mouse ERβ gene transcripts but in the other mouse genome, can simultaneously target the two ERβ gene transcripts. Plasmid DNA sequencing proved that the recombinant plasmid containing shRNA sequences. Detection of transfected cells showed that the gray ratio of the two bands of amplified fragments of the two ER(3RNAi group were significantly more decreased than the other groups, and the gray ratio of the bands of the ERβRNAi-1was more decreased than the ERβRNAi-2. there was significant difference, P<0.05. The virus titer T of the packaged retroviral pasmids ERβRNAi was106.67±0.25IU/ml. The growth curve showed that the cell proliferation in the ERβRNAi group was more significantly enhanced than the other two groups. Flow cytometry showed that the percentage of cells G1in the ERβRNAi group was significantly less, while the S phase and G2phase cells was significantly higher than the other groups; The ALP activity in the RNAi group was more than in the other groups; the bone nodules formed in RNAi group were a larger number and larger volume than the other groups; The OPG’s Ct value in RNAi group was relatively lower; the Ct value of the RANKL is higher than the other groups, a significant difference, P <0.05. In female mice, the mice’s length added value in the ERβRNAi group were higher, while the gained weight less in the ERβRNAi group than in the other two groups, a significant difference, P<0.05. Vertebral tissue sections produced showed that the vertebral trabecular bone under the growth plate were more intensive, but the proliferation of epiphyseal cartilage layers were thinner in the ERβRNAi group than that in the other groups. And the top growth plate cartilage shown structural disorder in ERβRNAi group.The number of apoptosis cells in the vertebral epiphyseal plate was more, and the the expression of OPG under the vertebral cartilage layer was more increased, but the expression of RANKL decreased in ERβRNAi group than that in the other groups, P<0.05. However, in male mice group, the above test results were not significantly different.Conclusion:The highly efficient ERβ-specific targeting retrovirus RNAi vector were able to effectively silence the expression of ERβ gene. In vivo and in vitro, the knockdown of the ERβ expression can enhance osteoblast proliferation, increase the OPG expression and the ALP activity, decrease RANKL expression, therefore, the bone formation was increased. According to test results ERβ silence, we can reversely infer that The expression of the ERβ may directly and/or indirectly inhibit the cartilage cells’ transformation into osteoblasts and osteoblasts’ proliferation, maturation and mineralization. Activation of cytokines in the OPG/RANK/RANKL signaling pathway, including OPG and RANKL, etc., indirectly inhibit the apoptosis of osteoclasts and promote bone resorption, Subsequently, the spinal vertebrae linear growth was reduced.