SOST基因3'-UTR双荧光素酶报告载体构建及调控mRNA的鉴定
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摘要
为构建SOST基因3'-UTR双荧光素酶基因报告载体,并通过检测荧光素酶活性,初步分析可能调控SOST基因表达的MicroRNA并鉴定,本研究利用PCR方法,根据SOST基因3'-UTR序列信息设计其扩增引物,以293T细胞基因组DNA为模板PCR扩增SOST基因的3'-UTR序列,将其克隆到Psi-CHECK2载体中形成双荧光素酶基因报告载体;运用在线miRNA靶预测数据库确定靶向SOST的miRNA;阳离子脂质体法将miRNA miminc、miRNA inhibitor与SOST3'-UTR Psi-CHECK2、mutSOST3'-UTR Psi-CHECK2载体分别共转染于常规培养的293T细胞中,然后检测荧光素酶活性,并与空白、阴性对照(NC)比较。结果表明:经酶切及基因测序验证,成功构建SOST3'-UTR Psi-CHECK2、mutSOST3'-UTR Psi-CHECK2载体;在线miRNA靶预测数据库预测到SOST基因可能是miR-218-5p的作用靶点;双荧光检测结果显示miR-218-5p miminc(0.09±0.03)较空白组(0.19±0.05)、NC组(0.19±0.02)荧光酶素活性下降(p<0.01),miR-218-5p inhibitor (0.50±0.03)荧光酶素活性明显升高(p<0.01),mutSOST3'-UTR Psi-CHECK2载体各组间均无差异(p>0.01)。我们的研究表明,本研究成功构建SOST基因3'-UTR段Psi-CHECK2载体,初步证实miR-218-5p对SOST基因有调控作用。
In order to construct the dual luciferase gene reporter vector of 3'-UTR of SOST gene(Psi-CHECK2)and initially analyze and identify microRNAs that may regulate the expression of SOST gene by detecting luciferase activity, in this study, PCR amplification method was used to design the amplification primers based on the3'-UTR sequence information of SOST gene, and the 3'-UTR sequence of SOST gene was amplified by PCR using293 T cell genomic DNA as a template, and then cloned into Psi-CHECK2 vector to form a dual luciferase gene reporter vector. The online miRNA target prediction database was used to determine the miRNAs targeting SOST.Cationic liposome assay was used to co-transfection miRNA miminc, miRNA inhibitor and SOST3'-UTR Psi-CHECK2 and mutSOST3'-UTR Psi-CHECK2 vectors into conventional 293 T cells. Luciferase activity was detected and compared with blank and negative control(NC). The result showed that the SOST3'-UTR Psi-CHECK2 and mutSOST3'-UTR Psi-CHECK2 vectors were successfully built that identified by enzyme digestion and gene sequencing. The online miRNA target prediction database predicted that the SOST gene might be the target of miR-218-5 p; double fluorescence detection results showed luciferase activity of miR-218-5 p miminc(0.09±0.03)was lower than that of the blank group(0.19±0.05), luciferase activity of NC group(0.19±0.02) was decreased(p<0.01),Fluorescein activity of miR-218-5 p inhibitor(0.50±0.03) was significantly increased(p<0.01), and there was no difference between the groups of mut SOST3'-UTR Psi-CHECK2 vector(p>0.01). Our study indicated that this study successfully constructed the Psi-CHECK2 vector of the 3'-UTR segment of SOST gene, and preliminarily confirmed that mir-218-5 p might have regulatory effect on SOST gene.
In order to construct the dual luciferase gene reporter vector of 3'-UTR of SOST gene(Psi-CHECK2)and initially analyze and identify microRNAs that may regulate the expression of SOST gene by detecting luciferase activity, in this study, PCR amplification method was used to design the amplification primers based on the3'-UTR sequence information of SOST gene, and the 3'-UTR sequence of SOST gene was amplified by PCR using293 T cell genomic DNA as a template, and then cloned into Psi-CHECK2 vector to form a dual luciferase gene reporter vector. The online miRNA target prediction database was used to determine the miRNAs targeting SOST.Cationic liposome assay was used to co-transfection miRNA miminc, miRNA inhibitor and SOST3'-UTR Psi-CHECK2 and mutSOST3'-UTR Psi-CHECK2 vectors into conventional 293 T cells. Luciferase activity was detected and compared with blank and negative control(NC). The result showed that the SOST3'-UTR Psi-CHECK2 and mutSOST3'-UTR Psi-CHECK2 vectors were successfully built that identified by enzyme digestion and gene sequencing. The online miRNA target prediction database predicted that the SOST gene might be the target of miR-218-5 p; double fluorescence detection results showed luciferase activity of miR-218-5 p miminc(0.09±0.03)was lower than that of the blank group(0.19±0.05), luciferase activity of NC group(0.19±0.02) was decreased(p<0.01),Fluorescein activity of miR-218-5 p inhibitor(0.50±0.03) was significantly increased(p<0.01), and there was no difference between the groups of mut SOST3'-UTR Psi-CHECK2 vector(p>0.01). Our study indicated that this study successfully constructed the Psi-CHECK2 vector of the 3'-UTR segment of SOST gene, and preliminarily confirmed that mir-218-5 p might have regulatory effect on SOST gene.
引文
Cha X.Y.,and Hu Y.,2014,Research progress in osteoporosis related signaling pathways,Zhongguo Guzhishusong Zazh(Chinese Journal of Osteoporosis),20(2):205-209(查小云胡予,2014,骨质疏松相关信号通路研究进展,中国骨质疏松杂志,20(2):205-209)
Chang J.C.,Christiansen B.A.,Murugesh D.K.,Sebastian A.,Hum N.R.,Collette N.M.,Hatsell S.,Economides A.N.,Blanchette C.D.,and Loots G.G.,2018,SOST/sclerostin improves posttraumatic osteoarthritis and inhibits MMP2/3 expression after injury,J.Bone Miner.Res.,33(6):1105-1113
Cui X.B.,Li S.,Li T.T.,Peng H.,Jin T.T.,Zhang S.M.,Liu CX.,Yang L.,Shen Y.Y.,Li S.G.,Li N.,Li Y.,Hu J.M.Jiang J.F.,Suo J.,Qi Y.,Liang W.H.,Wang L.H.,Dang HW.,Li L.,Cao W.W.,Wei Y.T.,Yin L.B.,Wu C.Y.,Yuan X.L.,Zhou H.,Zheng Y.,Chen Y.Z.,and Li F.,2016,Targeting oncogenic PLCE1 by mi R-145 impairs tumor proliferation and metastasis of esophageal squamous cell carcinoma,Oncotarget,7(2):1777
Leupin O.,Piters E.,Halleux C.,Halleux C.,Hu S.,Kramer T.Morvan F.,Bouwmeester T.,Schirle M.,Bueno-Lozano M.Fuentes R.F.J.,Itin P.H.,Boudin E.,Freitas F.D.,Jennes K.Brannetti B.,Charara N.,Ebersbach H.,Geisse S.,Lu C.X.Bauer A.,Hul W.V.,and Kneissel M.,2011,Bone overgrowth-associated mutations in the LRP4 gene impair sclerostin facilitator,J.Biol.Chem.,268(22):19489-19500
Li Z.,Wong S.H.,Shen J.,Chan M.T.,and Wu W.K.,2016,The The role of microRNAS in ankylosing spondylitis,Medicine(Baltimore),95(14):3325-3327
Taipaleenm?ki H.,Farina N.H.,van Wijnen A.J.,Stein J.L.,Hesse E.,Stein G.S.,and Lian J.B.,2016,Antagonizing mi R-218-5p attenuates Wnt signaling and reduces metastatic bone disease of triple negative breast cancer cells,Oncotarget,7(48):79032-79046
van Bezooijen R.L.,ten Dijke P.,Papapoulos S.E.,and L?wik C.W.,2005,SOST/sclerostin,an osteocyte-derived negative regulator of bone formation,Cytokine and Growth Factor Reviews,16(3):319-327
Wang X.,Cui Y.,Zhao Y.L.,and Cui M.J.,2017,Advances in the study of osteosclerotic proteins in chronic kidney dis ease,Guangdong Yixue(Guangdong Medical),38(2):322-324(王雪,崔燕,赵永利,崔明姬,2017,骨硬化蛋白在慢性肾脏病的研究进展,广东医学,(2):322-324)
Wu J.,Wu L.,and Jin Q.H.,2016,Expression of SOST andβ-catenin in cartilage and subchondral bone in patients with knee osteoarthritis at different stages,Zhongguo Guzhishusong Zazhi(Chinese Journal of Osteoporosis),22(6):689-694(吴疆,吴龙,金群华,2016,SOST和β-catenin在不同分期膝骨关节炎患者软骨及软骨下骨表达的研究,中国骨质疏松杂志,22(6):689-694)
Zhou L.T.,Feng Y.T.,Dai J.Y.,and Ou Y.J.,2017,Advances in the regulation of mi RNA in adipose stem cell differentiation,Zhongguo Xiufu Chongjian Waike Zazhi(Chinese Journal of Reconstructive Surgery),(12):1506-1511(周兰庭,冯雁婷,戴景兴,欧阳钧,2017,mi RNA调控脂肪干细胞分化的研究进展,中国修复重建外科杂志,(12):1506-1511)
Chang J.C.,Christiansen B.A.,Murugesh D.K.,Sebastian A.,Hum N.R.,Collette N.M.,Hatsell S.,Economides A.N.,Blanchette C.D.,and Loots G.G.,2018,SOST/sclerostin improves posttraumatic osteoarthritis and inhibits MMP2/3 expression after injury,J.Bone Miner.Res.,33(6):1105-1113
Cui X.B.,Li S.,Li T.T.,Peng H.,Jin T.T.,Zhang S.M.,Liu CX.,Yang L.,Shen Y.Y.,Li S.G.,Li N.,Li Y.,Hu J.M.Jiang J.F.,Suo J.,Qi Y.,Liang W.H.,Wang L.H.,Dang HW.,Li L.,Cao W.W.,Wei Y.T.,Yin L.B.,Wu C.Y.,Yuan X.L.,Zhou H.,Zheng Y.,Chen Y.Z.,and Li F.,2016,Targeting oncogenic PLCE1 by mi R-145 impairs tumor proliferation and metastasis of esophageal squamous cell carcinoma,Oncotarget,7(2):1777
Leupin O.,Piters E.,Halleux C.,Halleux C.,Hu S.,Kramer T.Morvan F.,Bouwmeester T.,Schirle M.,Bueno-Lozano M.Fuentes R.F.J.,Itin P.H.,Boudin E.,Freitas F.D.,Jennes K.Brannetti B.,Charara N.,Ebersbach H.,Geisse S.,Lu C.X.Bauer A.,Hul W.V.,and Kneissel M.,2011,Bone overgrowth-associated mutations in the LRP4 gene impair sclerostin facilitator,J.Biol.Chem.,268(22):19489-19500
Li Z.,Wong S.H.,Shen J.,Chan M.T.,and Wu W.K.,2016,The The role of microRNAS in ankylosing spondylitis,Medicine(Baltimore),95(14):3325-3327
Taipaleenm?ki H.,Farina N.H.,van Wijnen A.J.,Stein J.L.,Hesse E.,Stein G.S.,and Lian J.B.,2016,Antagonizing mi R-218-5p attenuates Wnt signaling and reduces metastatic bone disease of triple negative breast cancer cells,Oncotarget,7(48):79032-79046
van Bezooijen R.L.,ten Dijke P.,Papapoulos S.E.,and L?wik C.W.,2005,SOST/sclerostin,an osteocyte-derived negative regulator of bone formation,Cytokine and Growth Factor Reviews,16(3):319-327
Wang X.,Cui Y.,Zhao Y.L.,and Cui M.J.,2017,Advances in the study of osteosclerotic proteins in chronic kidney dis ease,Guangdong Yixue(Guangdong Medical),38(2):322-324(王雪,崔燕,赵永利,崔明姬,2017,骨硬化蛋白在慢性肾脏病的研究进展,广东医学,(2):322-324)
Wu J.,Wu L.,and Jin Q.H.,2016,Expression of SOST andβ-catenin in cartilage and subchondral bone in patients with knee osteoarthritis at different stages,Zhongguo Guzhishusong Zazhi(Chinese Journal of Osteoporosis),22(6):689-694(吴疆,吴龙,金群华,2016,SOST和β-catenin在不同分期膝骨关节炎患者软骨及软骨下骨表达的研究,中国骨质疏松杂志,22(6):689-694)
Zhou L.T.,Feng Y.T.,Dai J.Y.,and Ou Y.J.,2017,Advances in the regulation of mi RNA in adipose stem cell differentiation,Zhongguo Xiufu Chongjian Waike Zazhi(Chinese Journal of Reconstructive Surgery),(12):1506-1511(周兰庭,冯雁婷,戴景兴,欧阳钧,2017,mi RNA调控脂肪干细胞分化的研究进展,中国修复重建外科杂志,(12):1506-1511)