才忠民,趙曉勇
(南方醫(yī)科大學附屬花都醫(yī)院骨科,廣東 廣州 510800)
骨質(zhì)疏松癥(OP)是一種骨代謝性疾病,常見于中、老年人,也是促進其他骨科疾病惡化的誘因[1].Wnt/β-catenin信號轉(zhuǎn)導通路在人體普遍存在,Wnt與存在于細胞膜上的相關蛋白受體結(jié)合,誘導細胞內(nèi)β-catenin蛋白進入細胞核,轉(zhuǎn)錄共活化因子β-catenin是該信號通路的核心部分和最終目標.NPY是氨基酸組成的一種特異性神經(jīng)遞質(zhì),分布于多處的神經(jīng)系統(tǒng)中,在調(diào)節(jié)各種基本生理功能中起著至關重要的作用.Y1R是第一個被發(fā)現(xiàn)為孤兒受體,位于人類染色體4q31.3-q32上[2].本研究中應用低氧處理成骨細胞株,構(gòu)建骨質(zhì)疏松癥體外細胞模型,然后采用Wnt/β-catenin信號轉(zhuǎn)導通路抑制劑DKK1處理,進一步深入分析DKK1對體外骨質(zhì)疏松癥模型的C518、MC3T3-E1成骨細胞株的作用機制和影響結(jié)果,為闡明骨質(zhì)疏松癥的發(fā)病機制和靶向治療策略的研究提供了新的方向.
選擇C518、MC3T3-E1成骨細胞株,C518細胞株購自上海生命科學院細胞資源中心.MC3T3-E1細胞株購自ATCC.C518細胞的培養(yǎng)液是RPMI 1640,MC3T3-E1細胞的培養(yǎng)液是DMEM/F12.
1.2.1 NPY-Y1基因過表達和沉默
取一半各細胞株,分為:空載體組、空白對照組、NPY-Y1過表達組、NPY-Y1基因沉默組,分別加入特異慢病毒(NPY-Y1-cDNA)和空載慢病毒各20 μL,進行孵育過夜處理.
1.2.2 細胞分組
未予任何處理的C518、MC3T3-E1細胞株為空白對照組(Con組)、NPY-Y1基因沉默組及過表達處理的C518、MC3T3-E1細胞株分為NPY-Y1基因沉默組(sh NPY-Y1組)、NPY-Y1過表達組(NPY-Y1OE組).
1.2.3 Wnt/β-catenin信號通路抑制劑 DKK1處理細胞和Western blot驗證
接種4×105各組細胞于六孔板中,加入含5 μM DKK1(Stem RD,USA)的新鮮培養(yǎng)基,72小時后收獲細胞.取提取蛋白的細胞,棄上清,PBS洗2次.孵育后電泳分析結(jié)果.
1.2.4 Real time PCR檢測細胞中的NPY-Y1、Runx-2和Osterix的mRNA
細胞置入1.5 mL EP管內(nèi),加入1.0 mL的Trizol溶液,4℃,12 000 rmp離心15 min.RNA的再溶解,棄去上層懸液,適度干燥RNA沉淀,再在管中加20 μL的DEPC水溶解.引物見表1.
表1 Real time-PCR檢測NPY-Y1、Runx-2和Osterix的引物結(jié)果Tab.1 Primers of NPY-Y1,Runx-2 and Osterix used in Real time-PCR and detection results
1.2.5 Western blot檢測細胞中的NPY-Y1、Runx-2和 Osterix蛋白差異
蛋白樣本進行電泳,NPY-Y1、Runx-2中加入山羊抗兔二抗處理.然后依次進行電泳、轉(zhuǎn)膜、封閉、孵育一抗、孵育二抗.實驗重復3次,取均值進行專業(yè)軟件分析結(jié)果.
1.2.6 雙熒光素酶報告基因?qū)嶒?/p>
細胞用Lipofectamine 2 000轉(zhuǎn)染質(zhì)粒,每組3個復孔數(shù)據(jù)取平均值,而后計算各組的Luciferase熒光值/Renilla熒光值,得到校正后的Luciferase表達強度值.
1.2.7 Rescue實驗
細胞接種于24孔板,分組設置:pcDNA3+pcDNA3(0.6 μg+0.4 μg);pcDNA3+pNPY-Y1(0.6 μg+0.4 μg);p NPY-Y1+p sh Runx-2(0.6 μg+0.4 μg).沉淀孵育,采用溶液置換后進行蛋白半定量驗證.
1.2.8 RNA-seq測序及分析
取1 μL RNA溶液進行濃度及純度測定.構(gòu)建標準文庫,嚴格設定入庫條件,質(zhì)檢后進行用RNA-seq測序,然后運用SOAPaligner/SOAP2將其與參考序列進行比對分析.
應用Prism 9軟件分析,多組間的比較采用單因素方差分析,兩組間的比較采用t檢驗,P<0.05為差異有統(tǒng)計學意義.
在C518、MC3T3-E1細胞中,利用Wnt/β-catenin 抑制劑 DKK1(30 μmol/L)孵育細胞24 h.Western blot檢測,結(jié)果顯示DKK1對NPY-Y1、Runx-2和 Osterix顯著下調(diào),差異明顯,具有統(tǒng)計學意義(P<0.05),見表2和圖1-圖4.
表2 Wnt/β-catenin抑制劑DKK1處理細胞后蛋白變化(±s)Tab.2 Protein changes in cells after being treated with Wnt/β-catenin inhibitor DKK1(± s)
表2 Wnt/β-catenin抑制劑DKK1處理細胞后蛋白變化(±s)Tab.2 Protein changes in cells after being treated with Wnt/β-catenin inhibitor DKK1(± s)
項目NPY-Y1 Runx-2 Osterix t值P值C518MC3T3-E1 DKK1(-)12.20±1.57 10.55±1.47 11.24±1.39 2.779 0.027 DKK1(+)10.14±1.61 6.25±1.02 6.33±1.05 2.971 0.022 DKK1(-)11.96±1.81 10.43±1.35 11.24±1.33 2.995 0.020 DKK1(+)9.13±1.94 6.58±1.59 6.19±1.28 2.977 0.021
圖1 Wnt信號通路抑制劑DKK1作用C518細胞株后蛋白表達結(jié)果Fig.1 Protein expression of C518 cell line after being treated with Wnt signaling pathway inhibitor DKK1
圖2 Western blot驗證信號通路抑制劑DKK1作用C518細胞株后蛋白表達結(jié)果Fig.2 Verification of protein expression of C518 cell line treated with signaling pathway inhibitor DKK1 by Western blot
圖3 Western blot檢測信號通路抑制劑DKK1作用MC3T3-E1細胞株后蛋白表達結(jié)果Fig.3 Detection of protein expression of MC3T3-E1 cell line treated with signaling pathway inhibitor DKK1 by Western blot
圖4 Western blot驗證信號通路抑制劑DKK1作用MC3T3-E1細胞株后蛋白表達結(jié)果Fig.4 Verification of protein expression of MC3T3-E1 cell line treated with signaling pathway inhibitor DKK1 by Western blot
DKK1未處理時sh NPY-Y1組和NPY-Y1OE組Wnt的mRNA明顯高于Con組,DKK1處理后sh NPYY1組和NPY-Y1OE組Wnt的mRNA顯著下調(diào).shNPY-Y1組和NPY-Y1OE組的NPY-Y1 mRNA差異顯著.Runx-2和 Osterix mRNA較DKK1未處理時顯著下調(diào),P<0.05,見表3和圖5.
表3 Real time PCR 檢測C518細胞Wnt、NPY-Y1、Runx-2 和 Osterix mRNA(±s,ng·mL-1)Tab.3 Detection of Wnt,NPY-Y1,Runx-2 and Osterix mRNA in C518 cells by Real time PCR(± s,ng·mL-1)
表3 Real time PCR 檢測C518細胞Wnt、NPY-Y1、Runx-2 和 Osterix mRNA(±s,ng·mL-1)Tab.3 Detection of Wnt,NPY-Y1,Runx-2 and Osterix mRNA in C518 cells by Real time PCR(± s,ng·mL-1)
Wnt 9.27±1.55 11.46±1.38 13.48±1.36 2.730 0.031項目Con sh NPY-Y1 NPY-Y1OE t值P值DKK1(-)DKK1(+)NPY-Y1 5.19±1.32 6.05±1.17 10.31±1.55 2.763 0.030 Runx-2 5.88±1.43 7.04±1.21 14.59±1.33 2.799 0.029 Osterix 6.76±1.21 8.05±1.20 14.58±1.34 2.788 0.028 Wnt 5.26±1.45 6.29±1.43 7.28±1.44 2.872 0.024 NPY-Y1 5.20±1.31 11.88±1.36 13.38±1.57 2.997 0.023 Runx-2 3.89±1.42 4.15±1.68 5.42±1.33 2.996 0.023 Osterix 3.75±1.22 5.17±1.62 6.50±1.19 2.866 0.025
圖5 Real time PCR 檢測C518細胞Wnt、NPY-Y1、Runx-2和 Osterix mRNA差異Fig.5 Differences detection of Wnt,NPY-Y1,Runx-2 and Osterix mRNA in C518 cells by Real time PCR
DKK1未處理時sh NPY-Y1組和NPY-Y1OE組Wnt的蛋白明顯高于Con組,DKK1處理后sh NPY-Y1組和NPY-Y1OE組Wnt的蛋白顯著下調(diào).NPY-Y1組和NPY-Y1OE組的NPY-Y1蛋白與DKK1處理前差異顯著.Runx-2和Osterix mRNA較DKK1未處理時顯著下調(diào),差異顯著,有統(tǒng)計學意義(P<0.05),見表4和圖6.
表4 Western blot檢測C518細胞Wnt、NPY-Y1、Runx-2 和 Osterix 蛋白(±s,μg·mL-1)Tab.4 Detection of Wnt,NPY-Y1,Runx-2 and Osterix proteins in C518 cells by Western blot(± s,μg·mL-1)
表4 Western blot檢測C518細胞Wnt、NPY-Y1、Runx-2 和 Osterix 蛋白(±s,μg·mL-1)Tab.4 Detection of Wnt,NPY-Y1,Runx-2 and Osterix proteins in C518 cells by Western blot(± s,μg·mL-1)
Con sh NPY-Y1 NPY-Y1OE t值P值DKK1(-)DKK1(+)項目Wnt 9.18±1.32 14.79±2.52 15.83±2.48 2.885 0.023 NPY-Y1 8.33±1.47 12.21±1.33 16.78±1.69 2.897 0.022 Runx-2 7.69±1.52 13.74±1.68 15.87±1.05 2.871 0.024 Osterix 8.57±1.30 14.25±1.69 16.97±1.32 2.875 0.024 Wnt 6.17±1.34 10.23±1.65 12.27±1.63 2.963 0.022 NPY-Y1 8.34±1.48 12.30±1.28 16.93±1.97 2.715 0.028 Runx-2 7.71±1.50 8.02±1.46 9.63±1.25 2.693 0.030 Osterix 8.58±1.29 10.17±1.62 12.39±1.20 2.557 0.032
圖6 Western blot檢測C518細胞Wnt、NPY-Y1、Runx-2和 Osterix蛋白差異Fig.6 Differences detection of Wnt,NPY-Y1,Runx-2 and Osterix protein in C518 cells by Western blot注:組間比較,*P<0.05
驗證NPY-Y1直接結(jié)合靶基因Runx-2,根據(jù)載體構(gòu)建的基本步驟得到啟動子報告載體.嚴格按照標準試劑盒操作,組間比較,差異顯著,有統(tǒng)計學意義(P<0.05),見表5和圖7、圖8.
表5 細胞中雙熒光素酶報告系統(tǒng)對質(zhì)粒luciferase熒光強度表達結(jié)果(±s)Tab.5 Fluorescence expression intensity of luciferase plasmid in cells in dual-luciferase reporter system(± s)
表5 細胞中雙熒光素酶報告系統(tǒng)對質(zhì)粒luciferase熒光強度表達結(jié)果(±s)Tab.5 Fluorescence expression intensity of luciferase plasmid in cells in dual-luciferase reporter system(± s)
項目pGL3-Control-luc+pGL3-Basic-luc+pGL3-Runx-2-luc t值P值C518 31.56±3.67 8.64±1.25 22.45±2.23 3.125 0.019 MC3T3-E1 27.59±2.46 6.31±1.34 18.57±2.01 3.330 0.016
圖7 C518細胞中雙熒光素酶報告系統(tǒng)對質(zhì)粒luciferase熒光強度表達Fig.7 Fluorescence expression intensity of luciferase plasmid in C518 cells in dual-luciferase reporter system
圖8 MC3T3-E1細胞中雙熒光素酶報告系統(tǒng)對質(zhì)粒luciferase熒光強度表達Fig.8 Fluorescence expression intensity of luciferase plasmid in MC3T3-E1 cells in dual-luciferase reporter system
NPY-Y1OE組Runx-2的啟動子活性高于Con組,sh NPY-Y1組Runx-2的啟動子活性低于Con組.結(jié)果見表6和圖9、圖10.
表6 雙熒光素酶報告系統(tǒng)驗證轉(zhuǎn)錄因子NPY-Y1調(diào)控Runx-2啟動子活性(±s)Tab.6 Verification of transcription factor NPY-Y1 regulating promoter activity of Runx-2 by dual-luciferase reporter system(± s)
表6 雙熒光素酶報告系統(tǒng)驗證轉(zhuǎn)錄因子NPY-Y1調(diào)控Runx-2啟動子活性(±s)Tab.6 Verification of transcription factor NPY-Y1 regulating promoter activity of Runx-2 by dual-luciferase reporter system(± s)
D K K 1(-)組別C o n s h N P Y-Y 1 N P Y-Y 1 OE t值P值C 5 1 8 6.5 4±1.0 5 3.2 9±0.6 7 9.6 7±1.4 7 2.8 7 1 0.0 2 4 M C 3 T 3-E 1 6.2 1±1.0 1 3.1 4±0.5 3 9.3 2±1.3 6 2.8 7 5 0.0 2 3 D K K 1(+)C 5 1 8 6.5 8±1.0 3 5.3 1±0.6 9 1 3.3 6±1.6 8 2.8 5 3 0.0 2 5 M C 3 T 3-E 1 6.1 8±1.0 2 5.1 9±0.5 2 1 2.2 8±1.5 1 2.8 1 2 0.0 2 6
圖9 C518細胞NPY-Y1調(diào)控Runx-2啟動子活性結(jié)果Fig.9 Regulation of NPY-Y1 on promoter activity of Runx-2 in C518 cells
圖10 MC3T3-E1細胞NPY-Y1調(diào)控Runx-2啟動子活性結(jié)果Fig.10 Regulation of NPY-Y1 on promoter activity of Runx-2 in MC3T3-E1 cells
在外源敲減Runx-2后,能夠被順利的調(diào)整回復原來的表達水平,說明敲減Runx-2能挽救NPY-Y1對Runx-2表達的促進作用.結(jié)果見表7和圖11、圖12.
表7 挽救實驗中Real time PCR檢測C518細胞Runx-2的mRNA水平(±s)Tab.7 Detection of Runx-2 mRNA level in C518 cell by Real time PCR in rescue experiment(± s)
表7 挽救實驗中Real time PCR檢測C518細胞Runx-2的mRNA水平(±s)Tab.7 Detection of Runx-2 mRNA level in C518 cell by Real time PCR in rescue experiment(± s)
DKK1(-)DKK1(+)項目Con sh NPY-Y1 NPY-Y1OE t值P值pcDNA3+pcDNA3 0.78±0.05 0.57±0.06 0.89±0.04 3.250 0.017 pcDNA3+p NPY-Y1 7.32±1.25 5.54±1.36 7.76±1.49 3.365 0.016 p NPY-Y1+p sh Runx-2 0.39±0.06 0.28±0.03 0.45±0.05 3.152 0.018 pcDNA3+pcDNA3 0.79±0.04 0.68±0.05 0.97±0.02 3.412 0.015 pcDNA3+p NPY-Y1 7.33±1.26 7.02±1.33 9.58±1.26 3.553 0.014 p NPY-Y1+p sh Runx-2 0.40±0.06 0.34±0.02 0.48±0.04 3.114 0.019
圖11 挽救實驗中Real time PCR檢測不同組C518細胞Runx-2的mRNA結(jié)果Fig.11 Detection of Runx-2 mRNA in different groups of C518 cells by Real time PCR in rescue experiment
圖12 挽救實驗中Western blot檢測不同組C518細胞Runx-2的蛋白水平Fig.12 Detection of protein level of Runx-2 in different groups of C518 cells by Western blot in rescue experiment
RNA-seq對C518細胞不同組別差異表達基因測序中,NPY-Y1與Runx-2和Osterix差異比較分析、熱圖及分層聚類見表8,圖13.
表8 RNA-seq的NPY-Y1與Runx-2和Osterix差異表達結(jié)果Tab.8 Differential expression of NPY-Y1,Runx-2 and Osterix in RNA-seq
圖13 RNA-seq差異表達mRNA的熱圖及分層聚類Fig.13 Thermal diagram and hierarchical clustering of differential expression mRNA in RNA-seq
骨質(zhì)疏松癥目前發(fā)病率日益增多,已經(jīng)不局限于中老年人,甚至在青年人群中也常有發(fā)病[3].骨質(zhì)疏松癥可以誘發(fā)其他骨代謝性疾病的發(fā)生,目前骨質(zhì)疏松癥的發(fā)病機制尚不明確[4].介導Wnt/β-catenin信號轉(zhuǎn)導通路的調(diào)控與骨質(zhì)疏松癥發(fā)病中的成骨細胞靶向分化過程密切相關,Wnt基因按照其生物學功能差異可以劃分為各自具有不同生物學功能的亞群[5].在促進骨成熟的生成方面,接受胞外信息后與LRP/Fz相結(jié)合,通過系列傳導將信息傳遞給下游的β-catenin,進而發(fā)揮重要作用[6].Runx-2基因和Osterix基因是成骨細胞在細胞分化前期重要基因,是促進骨發(fā)育中的成骨細胞靶向分化成熟的特異性基因,兩者各自在成骨分化不同時期起到調(diào)控作用[7-9].Osterix是Runx-2下游作用靶點,對成骨細胞具有高度特異性,影響多種成骨基因表達[10].本文研究運用Wnt/β-catenin信號轉(zhuǎn)導通路特異性抑制劑DKK1,阻斷信號蛋白的傳遞[11-13].NPY在骨質(zhì)疏松癥中表達增加,作用可能是通過與Y1R結(jié)合而發(fā)揮的,在骨質(zhì)疏松癥模型中,內(nèi)源性和外源性NPY都可以發(fā)揮抗成骨細胞作用[14].
筆者應用Wnt/β-catenin信號通路抑制劑DKK1處理細胞并用Western blot驗證,研究結(jié)果為,DKK1對NPY-Y1、Runx-2和Osterix顯著下調(diào),差異顯著,有統(tǒng)計學意義.通過研究表明DKK1處理使各組中Wnt/β-catenin通路抑制,結(jié)果顯示,NPY-Y1、Runx-2和Osterix mRNA及蛋白表達水平顯著下降.這就明顯說明信號轉(zhuǎn)導通路的阻斷,完全抑制了轉(zhuǎn)錄因子NPY-Y1對靶基因Runx-2和Osterix的調(diào)控,考慮轉(zhuǎn)錄因子NPY-Y1與靶基因Runx-2和Osterix是直接結(jié)合作用.
研究結(jié)果證實,DKK1未處理時sh NPY-Y1組和NPY-Y1OE組Wnt的mRNA和蛋白明顯高于Con組,DKK1處理后sh NPY-Y1組和NPY-Y1OE組Wnt的mRNA和蛋白顯著下調(diào).shNPY-Y1組和NPY-Y1OE組的NPY-Y1 mRNA和蛋白差異顯著.Runx-2和Osterix mRNA和蛋白較DKK1未處理時明顯下調(diào).NPY-Y1OE組Runx-2的啟動子活性高于Con組,sh NPY-Y1組Runx-2的啟動子活性低于Con組.DKK1處理后結(jié)果差異顯著,有統(tǒng)計學意義(P<0.05).在外源敲減Runx-2后,Runx-2蛋白定量上調(diào),說明敲減Runx-2能挽救NPY-Y1對Runx-2表達的促進作用.DKK1處理后結(jié)果差異顯著,有統(tǒng)計學意義(P<0.05).
不同組別的RNA-seq研究中的結(jié)果顯示,C518和MC3T3-E1成骨細胞株測序為顯著差異基因為45個,其中定量上調(diào)的基因為25個,定量下調(diào)的基因為19個,NPY-Y1、Runx-2和Osterix基因為最為明顯的特異性基因.通路分析為Wnt/β-catenin信號轉(zhuǎn)導通路.
綜上所述,通過DKK1處理,抑制 Wnt/β-catenin信號通路活性,抑制NPY-Y1、Runx-2和 Osterix基因表達.揭示了NPY-Y1介導的Wnt/β-catenin信號轉(zhuǎn)導通路調(diào)控可能在骨質(zhì)疏松癥發(fā)病的分子機制中起著重要作用.