肖 洋，杜瑤瑤，高 成，孔 煒

(北京大學(xué)基礎(chǔ)醫(yī)學(xué)院生理學(xué)與病理生理學(xué)系，教育部分子心血管學(xué)重點(diǎn)實(shí)驗(yàn)室, 北京 100191)

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·論著·

microRNA在高磷誘導(dǎo)血管平滑肌細(xì)胞鈣化早期的動(dòng)態(tài)變化

肖 洋，杜瑤瑤，高 成，孔 煒△

(北京大學(xué)基礎(chǔ)醫(yī)學(xué)院生理學(xué)與病理生理學(xué)系，教育部分子心血管學(xué)重點(diǎn)實(shí)驗(yàn)室, 北京 100191)

目的：觀察高磷誘導(dǎo)的血管平滑肌細(xì)胞鈣化早期microRNA表達(dá)變化，分析其可能參與的信號(hào)通路途徑。方法：采用高磷(無(wú)機(jī)磷 2.6 mmol/L)刺激大鼠血管平滑肌細(xì)胞系A(chǔ)7r5鈣化7 d，鄰甲酚酞絡(luò)合酮比色法和考馬斯亮藍(lán)法檢測(cè)細(xì)胞內(nèi)鈣含量，RT-PCR法檢測(cè)平滑肌細(xì)胞表型和鈣化相關(guān)基因的表達(dá)變化，茜素紅染色觀察鈣結(jié)節(jié)。microRNA microarray 法檢測(cè)高磷刺激后0、3、12 h，680種microRNA表達(dá)變化。采用TAM軟件分析不同時(shí)間點(diǎn)間信號(hào)激活情況。結(jié)果：高磷誘導(dǎo)的A7r5細(xì)胞鈣鹽含量升高9.6倍(P<0.05)，平滑肌細(xì)胞表型mRNA(SM-α actin，SM22)下調(diào)(P<0.05)， 鈣化相關(guān)mRNA(BMP2、MSX2、Runx2)上調(diào)(P<0.05)，茜素紅染色后可見(jiàn)高磷刺激組有明顯鈣結(jié)節(jié)。680種microRNA在3個(gè)時(shí)間點(diǎn)的表達(dá)各不相同，只有6種microRNA分別逐級(jí)上調(diào)或漸漸下調(diào)。26種信號(hào)通路被明顯激活，其中包括細(xì)胞凋亡、分化增殖等已知與鈣化相關(guān)的信號(hào)通路。結(jié)論：microRNA參與調(diào)節(jié)血管鈣化是一種動(dòng)態(tài)微調(diào)的過(guò)程，為血管鈣化研究帶來(lái)新的思路。

血管鈣化；肌，平滑，血管；微RNA；信號(hào)轉(zhuǎn)導(dǎo)

血管鈣化是動(dòng)脈粥樣硬化、慢性腎病以及衰老等常見(jiàn)的臨床病理表現(xiàn)，在其發(fā)生時(shí)血管彈性降低、僵硬化，易形成血栓和出現(xiàn)斑塊破裂，血管鈣化是心腦血管疾病高發(fā)病率和高死亡率的重要因素之一。血管鈣化以往被認(rèn)為是一種被動(dòng)過(guò)程，但近年來(lái)研究表明血管鈣化是一種復(fù)雜并且高度調(diào)控的過(guò)程[1]。血管平滑肌細(xì)胞(vascular smooth muscle cell，VSMC)在血管鈣化中起關(guān)鍵作用，其通過(guò)分化成成骨樣細(xì)胞，生成基質(zhì)小泡進(jìn)而在血管壁形成鈣磷沉積病灶，目前對(duì)此過(guò)程了解較少。

microRNA是一種內(nèi)源性表達(dá)的非編碼小RNA，其大小長(zhǎng)約19～23個(gè)核苷酸,剪切加工成熟的microRNA與蛋白質(zhì)復(fù)合物結(jié)合形成RNA沉默復(fù)合體，與靶mRNA進(jìn)行不同程度的堿基互補(bǔ)配對(duì)，降解靶mRNA或抑制靶mRNA的翻譯。研究證實(shí) microRNA在機(jī)體發(fā)育、細(xì)胞分化、心血管疾病、腫瘤、病毒感染等各種生理或病理過(guò)程發(fā)揮重要作用[2]。目前對(duì)于 microRNA與血管鈣化之間的關(guān)系已有初步認(rèn)識(shí)，例如，miR-125b是最早于鈣化人冠狀動(dòng)脈平滑肌細(xì)胞上被發(fā)現(xiàn)顯著下調(diào)的micro-RNA[3]，Wang等[4]觀察到終末期腎病患者相比健康人群循環(huán)系統(tǒng)中miR-15b明顯降低，該研究發(fā)現(xiàn)其可能參與了磷代謝過(guò)程。本課題組于2012年報(bào)道[5]，miR-29a/b能夠抑制血管平滑肌細(xì)胞鈣化。以往對(duì)于microRNA參與鈣化機(jī)制的研究，多數(shù)學(xué)者只關(guān)注鈣化刺激后某一時(shí)間點(diǎn)microRNA的變化,但microRNA參與機(jī)體信號(hào)調(diào)節(jié)是一種動(dòng)態(tài)微調(diào)的過(guò)程，為了能夠?qū)︹}化機(jī)制有更深入的理解，本研究檢測(cè)給予血管平滑肌細(xì)胞鈣化刺激(高磷)后0、3、12 h 3個(gè)時(shí)間點(diǎn)不同microRNA的表達(dá)，以探討在鈣化發(fā)生早期microRNA的動(dòng)態(tài)變化，并分析其可能參與激活的信號(hào)通路。

1 材料與方法

1.1 細(xì)胞培養(yǎng)

大鼠胚胎胸主動(dòng)脈平滑肌細(xì)胞系A(chǔ)7r5購(gòu)自美國(guó) Type Culture Collection (Manassas, VA)公司。A7r5細(xì)胞采用含有10%(體積分?jǐn)?shù))胎牛血清Dulbecco modified Eagle medium (DMEM)培養(yǎng)基，在37 ℃并含有5%(體積分?jǐn)?shù)) CO2的濕潤(rùn)環(huán)境中培養(yǎng)。鈣化血管平滑肌細(xì)胞模型采取給予2.6 mmol/L無(wú)機(jī)磷刺激7 d的方法，期間每2天換液一次。

1.2 細(xì)胞鈣沉積定量實(shí)驗(yàn)

A7r5細(xì)胞給予鈣化刺激7 d后，棄掉培養(yǎng)基，用PBS清洗2遍，加入含有6%(體積分?jǐn)?shù))HCl溶液消化收集細(xì)胞，超聲破碎3次，每次3 s，間隔3 s。12 000 r/min，離心15 min，取上清。采用鄰甲酚酞絡(luò)合酮比色法測(cè)定Ca離子濃度，考馬斯亮藍(lán)法測(cè)定蛋白質(zhì)濃度進(jìn)行標(biāo)準(zhǔn)化，得出細(xì)胞內(nèi)鈣含量。

1.3 細(xì)胞茜素紅染色

A7r5細(xì)胞給予鈣化刺激7 d后，棄掉培養(yǎng)基，用PBS清洗3遍。加入4%(質(zhì)量分?jǐn)?shù))多聚甲醛固定10 min，棄掉多聚甲醛后，PBS清洗3遍，之后加入1%(質(zhì)量分?jǐn)?shù))茜素紅溶液1 mL，浸染30 min。光鏡下采集圖像，可見(jiàn)鈣化組平滑肌細(xì)胞染成紅色的鈣結(jié)節(jié)。

1.4 real-time-PCR定量實(shí)驗(yàn)

A7r5細(xì)胞給予鈣化刺激48 h后，Trizol法(Invitrogen)提取RNA，再采用逆轉(zhuǎn)錄試劑盒(Promega)逆轉(zhuǎn)錄成cDNA，-20℃保存。real-time PCR反應(yīng)采用美國(guó)Mx3000 Multiplex Quantitative PCR System (Stratagene公司, La Jolla, CA)完成。mRNA水平與β-actin進(jìn)行標(biāo)準(zhǔn)化，引物序列見(jiàn)表1。

表1 大鼠相關(guān)mRNA引物序列Table 1 Rats related mRNA primer sequence

1.5 microRNA microarray實(shí)驗(yàn)

A7r5細(xì)胞給予2.6 mmol/L無(wú)機(jī)磷刺激0、3和12 h，用Trizol法(Invitrogen)或miRNeasy minikit (QIAGEN)收集總RNA。采用miRCURYTMHy3TM/Hy5TMPower labeling kit標(biāo)記，與miRCURYTMLNA Array (v.16.0) (Exiqon, Denmark)雜交，清洗后使用Axon GenePix 4000B microarray scanner掃描芯片。得出原始數(shù)據(jù)上傳到NCBI-Pubmed-GEO dataset數(shù)據(jù)庫(kù)，GEO accession: GSE39700。microRNA在3個(gè)時(shí)間點(diǎn)的倍數(shù)變化通過(guò)不同的熒光強(qiáng)度來(lái)顯示，1.5倍以上變化被認(rèn)為差異有統(tǒng)計(jì)學(xué)意義。

1.6 microRNA信號(hào)通路分析

將上述顯著變化的microRNA數(shù)據(jù)，帶入TAM軟件進(jìn)行數(shù)據(jù)庫(kù)分析(www.cuilab.cn/tam)，得出激活的信號(hào)通路數(shù)據(jù)，P<0.05認(rèn)為差異有統(tǒng)計(jì)學(xué)意義，再將P值進(jìn)行負(fù)的常用對(duì)數(shù)換算(-lgP)，作圖得出分析結(jié)果。

1.7 統(tǒng)計(jì)學(xué)分析

采用GraphPad Prism 4軟件進(jìn)行分析，正態(tài)分布的連續(xù)隨機(jī)變量采用平均值±標(biāo)準(zhǔn)誤表示。兩組間比較采用t檢驗(yàn)，成對(duì)數(shù)據(jù)均采用配對(duì)t檢驗(yàn)(雙側(cè)檢驗(yàn))。

2 結(jié)果

2.1 鈣化血管平滑肌細(xì)胞模型的建立

首先在A7r5細(xì)胞上驗(yàn)證鈣化模型是否成立。在給予2.6 mmol/L無(wú)機(jī)磷刺激7 d后，高磷組(Pi)鈣沉積相比對(duì)照組(Ctrl)顯著上調(diào)9.6倍[Ctrl (0.23±0.03) mmol/gvs. Pi (2.44±0.34) mmol/g，n=3，P<0.05，圖1A]，提示鈣化模型上鈣鹽的沉積。采用茜素紅染色法觀察給予高磷刺激后A7r5細(xì)胞鈣化情況，鏡下可見(jiàn)高磷組(Pi)出現(xiàn)明顯紅色鈣結(jié)節(jié)(圖1B)，從組織學(xué)角度證實(shí)鈣鹽在平滑肌細(xì)胞的沉積。進(jìn)一步研究該模型上平滑肌細(xì)胞表型與鈣化相關(guān)mRNA的表達(dá)變化，通過(guò)real-time PCR實(shí)驗(yàn)檢測(cè)發(fā)現(xiàn)給予高磷刺激48 h后，平滑肌細(xì)胞表型分子骨骼肌-α肌動(dòng)蛋白 (skeletal muscle-α actin, SM-α actin)、平滑肌22 (smooth muscle 22， SM22)均下調(diào)(P<0.05)，鈣調(diào)理蛋白(Calponin)有下降趨勢(shì)但差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.131 5, 圖 1C～E)。相反骨形態(tài)發(fā)生蛋白2(bone morphogenetic protein-2， BMP2)、骨細(xì)胞的同源 2異型蛋白(msh homeobox 2，MSX2)、Runt相關(guān)轉(zhuǎn)錄因子2(runt-related transcription factor 2，Runx2)3種鈣化相關(guān)因子均明顯上調(diào)(P<0.05，圖 1F～H)，以上結(jié)果證實(shí)在給予高磷刺激后，A7r5細(xì)胞向成骨樣細(xì)胞轉(zhuǎn)化，同時(shí)證明鈣化模型成立。

*P<0.05， compared with the control (Ctrl) group．

圖1 高磷刺激7 d后，A7r5細(xì)胞內(nèi)鈣含量(A)、茜素紅染色情況(B)和平滑肌細(xì)胞表型mRNA[SM-α actin(C)、Calponin(D)和SM22(E)]，以及鈣化相關(guān)mRNA[BMP2(F)、MSX2(G)和Runx2(H)]表達(dá)變化
Figure 1 A7r5 cell were stimulated by high phosphate for 7d. The cell calcium content (A)， alizarin red staining (B)， smooth muscle cell type mRNA expression of SM-α actin (C)，Calponin (D) and SM22 (E)，Calcified related mRNA expression of BMP2 (F), MSX2 (G) and Runx2 (H)

2.2 microRNA microarray實(shí)驗(yàn)

采用上述模型，通過(guò)microRNA microarray實(shí)驗(yàn)研究給予高磷刺激后0、3和12 h這3個(gè)時(shí)間點(diǎn)microRNA表達(dá)譜變化(圖 2)。原始數(shù)據(jù)已上傳至NCBI-Pubmed-GEO dataset數(shù)據(jù)庫(kù)，GEO accession: GSE39700(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE39700)。

A, scatterplot of miR expression profiles, left panel, 3 h vs. 0 h, right panel, 12 h vs. 3 h； B, hierarchical clustering for microRNA expression profiles in rat VSMCs with high phosphate for 0, 3 or 12 h. Red indicates high relative expression, and green indicates low relative expression.

圖2 給予A7r5細(xì)胞高磷刺激后0、3、12 h 3個(gè)時(shí)間點(diǎn)microRNA表達(dá)變化
Figure 2 Microarray profile of time-series change of microRNA in rat VSMCs stimulated with high phosphate for 0, 3, 12 h

實(shí)驗(yàn)共檢測(cè)680種microRNA，并分別比較不同microRNA在3 h與0 h點(diǎn)間以及12 h與3 h點(diǎn)間的變化。以變化超過(guò)1.5倍認(rèn)為差異具有統(tǒng)計(jì)學(xué)意義，統(tǒng)計(jì)分析結(jié)果見(jiàn)表 2～5。

2.3 給予高磷刺激后0 h與3 h點(diǎn)間所激活的信號(hào)通路

有25種microRNA上調(diào)以及14種microRNA下調(diào)超過(guò)33%(表2～3)。為了探尋0 h與3 h點(diǎn)間不同的信號(hào)通路活動(dòng)情況，對(duì)上述39種microRNA進(jìn)行信號(hào)通路數(shù)據(jù)庫(kù)分析(圖 3)。下調(diào)的microRNA所激活的通路包括細(xì)胞運(yùn)動(dòng)(miR-128、miR-9)、上皮-間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition，EMT,包括miR-542、miR-203、miR-29a)和細(xì)胞死亡(miR-128、miR-203、let-7f)等；上調(diào)的microRNA所激活的通路包括腫瘤抑制基因(let-7b/e、miR-195、miR-29c 等)、脂肪細(xì)胞分化(let-7b/e、miR-378、miR-448)、EMT(let-7b/e、miR-448、miR-29c 等)等。

2.4 給予高磷刺激后3 h與12 h點(diǎn)間所激活的信號(hào)通路

3 h與12 h點(diǎn)間分別有48個(gè) microRNA上調(diào)超過(guò)1.5倍和116個(gè)microRNA下調(diào)超過(guò)33%(表4～5)，數(shù)據(jù)庫(kù)分析后結(jié)果見(jiàn)圖 4。下調(diào)的microRNA除在前面0 h與3 h兩時(shí)點(diǎn)間已激活的通路外，還包括細(xì)胞凋亡(miR-15b、miR-221/222、miR-29a 等)、免疫炎癥反應(yīng)(miR-25、miR-21、miR-126 等)和骨再生(miR-221/222、let-7b/d、miR-24 等)等共計(jì)24條信號(hào)通路被激活，令人意外的是在此時(shí)間段唯一被上調(diào)的microRNA明顯激活的信號(hào)通路只有腦發(fā)育(miR-323、miR-326、miR-9 等)。

2.5 6種逐級(jí)變化的microRNA

第2.3和2.4小節(jié)展示了在A7r5細(xì)胞上給予鈣化刺激后信號(hào)通路的活動(dòng)情況，與預(yù)期相符，模擬血管鈣化發(fā)生早期進(jìn)程，能夠幫助理解microRNA參與鈣化機(jī)制調(diào)節(jié)的動(dòng)態(tài)微調(diào)作用。將上述結(jié)果綜合分析發(fā)現(xiàn)，在3個(gè)時(shí)間點(diǎn)上，只有3種microRNA[miR-183、 miR-664、 miR-9*(*RNA fragment paired with mature microRNA)]逐級(jí)上調(diào)以及另外3種microRNA(miR-542-5P、let-7f、miR-29a)漸漸下調(diào)(圖 5)。

3 討論

本研究首先在大鼠血管平滑肌細(xì)胞系A(chǔ)7r5上驗(yàn)證給予高磷刺激能夠促進(jìn)血管平滑肌細(xì)胞向成骨樣細(xì)胞的轉(zhuǎn)化，確認(rèn)鈣化模型成立，并進(jìn)一步在該模型上觀察680種microRNA在0、3、12 h 3個(gè)時(shí)間點(diǎn)的變化情況，進(jìn)行信號(hào)通路數(shù)據(jù)庫(kù)分析，結(jié)果顯示，microRNA在此過(guò)程中參與多種信號(hào)通路途徑，包括已知與鈣化相關(guān)的細(xì)胞死亡/凋亡、分化遷移、免疫炎癥反應(yīng)和骨再生等[6]，與預(yù)期相符。尤為矚目的是，在這680種microRNA中只有6種是分別逐級(jí)上調(diào)或下調(diào)的，而其他大部分microRNA在3個(gè)時(shí)間點(diǎn)都呈現(xiàn)各種不同的變化方式，再次證實(shí)microRNA參與機(jī)體活動(dòng)是一種動(dòng)態(tài)精細(xì)調(diào)控的過(guò)程。

表2 高磷刺激A7r5細(xì)胞 0 h與3 h點(diǎn)間下調(diào)的microRNATable 2 List of down-regulated microRNA in high-phosphate stimulated A7r5 cell at 3 h vs. 0 h

* RNA fragment paired with mature microRNA. ID, identification.

表3 高磷刺激A7r5細(xì)胞 0 h與3 h點(diǎn)間上調(diào)的microRNATable 3 List of up-regulated microRNA in high-phosphate stimulated A7r5 cell at 3 h vs. 0 h

* RNA fragment paired with mature microRNA. ID, identification.

表4 高磷刺激A7r5細(xì)胞 3 h與12 h點(diǎn)間下調(diào)的microRNATable 4 List of down-regulated microRNA in high-phosphate stimulated A7r5 cell at 12 h vs. 3 h

續(xù)表microRNAnameIDFoldchange(12hvs.3h)Normalized3hNormalized12hrno-miR-338*178250.6156236280.5345622120.329089128rno-miR-503*1481290.5572391540.1827956990.101860921rno-miR-433428530.6325896330.1950844850.123408423rno-miR-107109230.6506696722.5499231951.659157689rno-miR-25426820.2932730832.8387096770.83251714rno-miR-19b109980.4391193630.8832565280.387855044rno-let-7d1459680.4033811534.0937019971.651322233rno-miR-28*1457140.5045587380.5668202760.285994123rno-miR-34b291530.51415416417.921658999.214495593rno-miR-434*112470.585771960.3778801840.221351616rno-miR-99b111840.6277302275.5514592933.484818805rno-miR-495426760.5395163110.5192012290.280117532rno-miR-379110930.5197124541.2211981570.63467189rno-miR-497428470.2928283820.6221198160.182174339rno-miR-195*427230.655321580.2211981570.144955926rno-miR-19a109970.3658138871.7188940090.628795299rno-miR-24-2*429500.524840421.3809523810.724779628rno-miR-32110530.2176143980.4500768050.097943193rno-miR-107*1475360.4567953570.2058371740.094025465rno-miR-1291486450.5435365520.5622119820.305582762rno-miR-871*1481600.5616621580.554531490.311459354rno-miR-743a*1480170.5877558920.3732718890.219392752rno-miR-384-5p428440.27544760.1920122890.052889324rno-miR-142-3p109470.5208202020.6205837170.323212537rno-miR-222*1481330.3126346720.2380952380.074436827rno-miR-3580-3p1485190.5763015140.1597542240.092066601rno-miR-136*425120.595102840.5299539170.315377081rno-miR-99a427080.5480769972.3410138251.283055828rno-let-7i99380.39186082610.832565284.244857982rno-miR-493*111250.5031950660.2841781870.142997062rno-miR-195131480.6225485282.536098311.57884427rno-let-7c1458200.6171944387.0522273434.352595495rno-miR-741-3p1484550.4215341281.8448540710.777668952rno-let-7f177520.2833823050.5529953920.156709109rno-miR-15b172800.42638007915.491551466.605288932rno-miR-16109670.5284715089.781874045.169441724rno-miR-324-3p1457080.5499091570.4132104450.227228208rno-miR-2901482870.2071657640.4254992320.088148874rno-miR-199a-5p295620.56598004326.909370215.2301665rno-miR-376b-3p143040.6560916410.4239631340.278158668rno-miR-34a272170.3898001333.2764976961.277179236rno-let-7a1471620.4462532472.6513056841.183153771rno-miR-211475060.63486876936.0844854122.90891283rno-miR-466d1485940.386560091.3732718890.530852106rno-miR-181b109720.3480601490.6528417820.227228208rno-miR-145426410.5701424423.672811062.094025465rno-miR-93306870.48155174911.861751155.712047013rno-miR-362*424740.6227820420.2642089090.164544564rno-miR-1001459430.4488085240.9078341010.407443683rno-miR-181d1456360.5529614393.3901689711.874632713rno-miR-4941475140.1879272130.4377880180.082272282rno-miR-4961481130.5672115850.4869431640.276199804rno-miR-301a131430.5060331254.6374807992.346718903rno-let-7a-1*/rno-let-7c-2*178880.0535806880.1827956990.009794319rno-miR-674-3p310530.5256396170.6298003070.331047992rno-miR-411174820.6117879670.4930875580.301665034rno-miR-295*1484380.6376101861.8402457761.173359452

續(xù)表microRNAnameIDFoldchange(12hvs.3h)Normalized3hNormalized12hrno-miR-322131500.3639925211.5714285710.571988247rno-miR-214110140.4563069866.5852534563.00489716rno-miR-365110780.6592933326.2334869434.109696376rno-miR-380*112380.5498656650.1674347160.092066601

*RNA fragment paired with mature microRNA. ID, identification.

表5 高磷刺激A7r5細(xì)胞 3h與12 h點(diǎn)間上調(diào)的microRNATable 5 List of up-regulated microRNA in high-phosphate stimulated A7r5 cell at 12 h vs. 3 h

*RNA fragment paired with mature microRNA. ID, identification.

血管鈣化已知是一種高度復(fù)雜、多種信號(hào)通路參與調(diào)控的主動(dòng)過(guò)程，病理性的血管鈣化與動(dòng)脈粥樣硬化、糖尿病、心力衰竭這些心血管疾病的預(yù)后密切相關(guān)。Pasquinelli等[7]最早于2002年在線蟲(chóng)體內(nèi)發(fā)現(xiàn)microRNA(lin-4和let-7)。目前的研究表明microRNA與眾多調(diào)節(jié)途徑相關(guān)聯(lián)，包括生物個(gè)體發(fā)育、病毒防御、組織分化、細(xì)胞增殖和凋亡、脂肪代謝、參與原癌基因作用等。近年來(lái)發(fā)現(xiàn)，microRNA同樣參與血管鈣化的調(diào)控過(guò)程，學(xué)者們?cè)诓煌碾x體、在體鈣化模型上發(fā)現(xiàn)miR-125b、miR-204/205、miR-221/222和miR-29a/b等的表達(dá)下調(diào)[3,5,8-10]， 以及miR-223和miR-135a等的上調(diào)[11-12]。后續(xù)研究發(fā)現(xiàn)過(guò)表達(dá)或敲低miR-204、miR-205、miR-133a和miR-30b/c會(huì)分別抑制或促進(jìn)鈣化，其機(jī)制是通過(guò)直接作用并抑制鈣化相關(guān)轉(zhuǎn)錄因子Runx2的表達(dá)而完成[8-9,13-14]。miR-125b是目前被研究較多與血管鈣化相關(guān)的microRNA，多種鈣化模型上均發(fā)現(xiàn)miR-125b表達(dá)量明顯降低，研究證實(shí)其可能是通過(guò)調(diào)控鈣化相關(guān)信號(hào)通路因子SP7(osterix)以及Ets1起到抑制鈣化的作用[3,15]。miR-135a*、miR-762、miR-714以及 miR-712*被發(fā)現(xiàn)隨著鈣化發(fā)生而表達(dá)上調(diào)，其可能是以抑制鈣離子外排的方式促進(jìn)血管鈣化[12]。miR-223亦有促進(jìn)鈣化的作用，過(guò)表達(dá)miR-223可以抑制Mef2c和RhoB的表達(dá)，提示可能是其作用機(jī)制[11]。

A, down-regulated microRNA； B, up-regulated microRNA.

圖3 0 h與3 h點(diǎn)間被顯著激活的信號(hào)通路
Figure 3 Signal pathways were significantly activated between 0 and 3 h

本研究觀察到microRNA在給予高磷刺激12 h內(nèi)，3個(gè)時(shí)間點(diǎn)上共有26種信號(hào)通路被明顯激活。部分信號(hào)通路早已被報(bào)道參與了鈣化調(diào)控過(guò)程：(1)miR-15b、miR-221/222、miR-29a等19種microRNA參與了細(xì)胞凋亡過(guò)程，Liu等[16]在脊髓損傷模型上觀察到miR-15b與促凋亡基因(PTEN、PDCD4、RAS)和抑制凋亡基因Bcl2的表達(dá)相關(guān)聯(lián)，F(xiàn)u等[17]在腫瘤細(xì)胞系上敲低miR-221可以上調(diào)Bax并下調(diào)Bcl2等凋亡調(diào)控基因，并且miR-15b、miR-221/222、miR-29a均存在與鈣化相關(guān)的報(bào)道；(2)miR-25、miR-21、miR-126等12種microRNA參與了炎癥反應(yīng)：文獻(xiàn)證實(shí)miR-25參與了炎癥因子TNFα誘導(dǎo)的血管平滑肌細(xì)胞增殖作用[18]，miR-21亦被發(fā)現(xiàn)與TGF-β信號(hào)通路及纖維化過(guò)程相關(guān)聯(lián)[19]；(3)miR-19a、let-7b/d、miR-24等11種microRNA參與了骨再生：Palmieri等[20]在研究P-15促進(jìn)骨生成的實(shí)驗(yàn)中發(fā)現(xiàn)有11種microRNA上調(diào)，6種下調(diào)，其中包括本研究同樣發(fā)現(xiàn)的miR-19a、let-7d、miR-221，但其變化方式不同。本研究還發(fā)現(xiàn)在0 h與3 h點(diǎn)間下調(diào)和3 h與12 h點(diǎn)間上調(diào)的microRNA，包括miR-323、miR-326、miR-133b等前后共8種，均激活了腦發(fā)育信號(hào)通路，這一似乎與血管鈣化毫無(wú)關(guān)系的機(jī)制。McCartney等[21]發(fā)現(xiàn)多種腦發(fā)育過(guò)程疾病存在血管鈣化，其誘因包括營(yíng)養(yǎng)不良和血管自身主動(dòng)鈣化，提示血管鈣化與腦發(fā)育存在某種聯(lián)系，同時(shí)驗(yàn)證本研究中的信號(hào)通路分析結(jié)果，但對(duì)此機(jī)制的理解需要更深入地研究。

A, down-regulated microRNA； B, up-regulated microRNA.

圖4 3 h與12 h點(diǎn)間被顯著激活的信號(hào)通路
Figure 4 Signal pathways were significantly activated between 3 and 12 h

本研究發(fā)現(xiàn)的6種逐級(jí)變化的microRNA中，以往工作已證實(shí)miR-29a/b可以通過(guò)抑制高磷誘導(dǎo)的ADAMTS-7表達(dá)的增加，調(diào)控血管鈣化[5]，ADAMTS-7(a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs-7)是一種含Ⅰ型血小板反應(yīng)蛋白基序和解聚素的金屬蛋白酶，其在離體或在體的血管鈣化模型明顯上調(diào)。過(guò)表達(dá)或敲低ADAMTS-7能夠分別加重或減輕鈣化，人類(lèi)GWAS研究同樣證實(shí)ADAMTS-7是血管鈣化和冠心病的致病基因。ADAMTS-7參與鈣化調(diào)控機(jī)制是通過(guò)調(diào)節(jié)一種叫做軟骨寡聚基質(zhì)蛋白(cartilage oligomeric matrix protein，COMP)的降解來(lái)完成的，過(guò)表達(dá)ADAMTS-7可以促進(jìn)COMP降解。COMP是一種內(nèi)源性的血管平滑肌細(xì)胞鈣化抑制因子，其減少或缺失將導(dǎo)致血管平滑肌細(xì)胞失去穩(wěn)態(tài)，造成病理性結(jié)果。本課題組曾證實(shí)miR-29a/b可以直接作用于ADAMTS-7的3′UTR，抑制其表達(dá)，與本研究microRNA microarray結(jié)果相一致，驗(yàn)證了結(jié)果的可信性，同時(shí)提示其他5種microRNA是否同樣在鈣化調(diào)控中起作用。

A, miR-183; B, miR-664; C, miR-9; D, miR-542-5P; E, let-7f; F, miR-29a.

圖5 芯片結(jié)果中6種在3個(gè)時(shí)間點(diǎn)逐級(jí)上調(diào)或下調(diào)的microRNA
Figure 5 6 miRs level in A7r5 cell stimulated by high phosphate for 0,3 and 12 h by miR chip screening

[1]唐朝樞，齊永芬. 關(guān)注血管鈣化發(fā)病新機(jī)制的研究[J]. 中國(guó)醫(yī)學(xué)前沿雜志, 2010, 2(3): 5-8.

[2]朱明燕, 莫中成, 曾高峰. 血管平滑肌細(xì)胞增殖相關(guān) micro-RNA 的研究進(jìn)展[J]. 實(shí)用醫(yī)學(xué)雜志, 2013, 29(21): 3610-3612.

[3]Goettsch C, Rauner M, Pacyna N, et al. miR-125b regulates calcification of vascular smooth muscle cells[J]. Am J Pathol, 2011, 179(4): 1594-1600.

[4]Wang H, Peng W, Ouyang X, et al. Reduced circulating mi-R-15b is correlated with phosphate metabolism in patients with end-stage renal disease on maintenance hemodialysis[J]. Ren Fail, 2012, 34(6): 685-690.

[5]Du Y, Gao C, Liu Z, et al. Upregulation of a disintegrin and metalloproteinase with thrombospondin motifs-7 by miR-29 repression mediates vascular smooth muscle calcification[J]. Arterioscler Thromb Vasc Biol, 2012, 32(11): 2580-2588.

[6]Johnson RC, Leopold JA, Loscalzo J. Vascular calcification: pathobiological mechanisms and clinical implications[J]. Circ Res, 2006, 99(10): 1044-1159.

[7]Pasquinelli AE, Ruvkun G. Control of developmental timing by micrornas and their targets[J]. Annu Rev Cell Dev Biol, 2002, 18: 495-513.

[8]Cui RR, Li SJ, Liu LJ, et al. MicroRNA-204 regulates vascular smooth muscle cell calcificationinvitroandinvivo[J]. Cardiovasc Res, 2012, 96(2): 320-329.

[9]Qiao W, Chen L, Zhang M. MicroRNA-205 regulates the calcification and osteoblastic differentiation of vascular smooth muscle cells[J]. Cell Physiol Biochem, 2014, 33(6): 1945-1953.

[10]Mackenzie NC, Staines KA, Zhu D, et al. miRNA-221 and mi-RNA-222 synergistically function to promote vascular calcification[J].Cell Biochem Funct, 2014, 32(2): 209-216.

[11]Rangrez AY, M’Baya-Moutoula E, Metzinger-Le Meuth V, et al. Inorganic phosphate accelerates the migration of vascular smooth muscle cells: evidence for the involvement of miR-223[J]. PLoS One, 2012, 7(10): e47807.

[12]Gui T, Zhou G, Sun Y, et al. MicroRNAs that target Ca(2+) transporters are involved in vascular smooth muscle cell calcification[J]. Lab Invest, 2012, 92(9): 1250-1259.

[13]Liao XB, Zhang ZY, Yuan K, et al. MiR-133a modulates osteogenic differentiation of vascular smooth muscle cells[J]. Endocrinology, 2013, 154(9): 3344-3352.

[14]Balderman JA, Lee HY, Mahoney CE, et al. Bone morphogenetic protein-2 decreases microRNA-30b and microRNA-30c to promote vascular smooth muscle cell calcification[J]. J Am Heart Assoc, 2012, 1(6): e003905.

[15]Wen P, Cao H, Fang L, et al. miR-125b/Ets1 axis regulates transdifferentiation and calcification of vascularsmooth muscle cells in a high-phosphate environment[J]. Exp Cell Res, 2014, 322(2): 302-312.

[16]Liu G, Keeler BE, Zhukareva V, et al. Cycling exercise affects the expression of apoptosis-associated microRNAs after spinal cord injury in rats[J]. Exp Neurol, 2010, 226(1): 200-206.

[17]Fu B, Wang Y, Zhang X, et al. MiR-221-induced PUMA silencing mediates immune evasion of bladder cancer cells[J]. Int J Oncol, 2015, 46(3): 1169-1180.

[18]Qi L, Zhi J, Zhang T, et al. Inhibition of microRNA-25 by tumor necrosis factor α is critical in the modulation of vascular smooth muscle cell proliferation[J]. Mol Med Rep, 2015, 11(6): 4353-4358.

[19]Dattaroy D, Pourhoseini S, Das S, et al. Micro-RNA 21 inhibition of SMAD7 enhances fibrogenesis via leptin-mediated NADPH oxidase in experimental and human nonalcoholic steatohepatitis[J]. Am J Physiol Gastrointest Liver Physiol, 2015, 308(4): G298-312.

[20]Palmieri A, Pezzetti F, Brunelli G, et al. Peptide-15 changes miRNA expression in osteoblast-like cells[J]. Implant Dent, 2008, 17(1): 100-108.

[21]McCartney E, Squier W. Patterns and pathways of calcification in the developing brain[J]. Dev Med Child Neurol, 2014, 56(10): 1009-1015.

(2015-03-17收稿)

(本文編輯：王 蕾)

Dynamic alteration of microRNA in high phosphorus induced calcification of vascular smooth muscle cell

XIAO Yang, DU Yao-yao, GAO Cheng, KONG Wei△

(Department of Physiology and Pathophysiology, Peking University School of Basic Medical Sciences； China and Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing 100191, China)

Objective：To study the change of microRNA during the early stage of high phosphorus induced vascular smooth muscle cell (VSMC) calcification and its related mechanism.Methods:Theinvitrocalcification model was created through stimulating VSMC cell line A7r5 with high Pi (2.6 mmol/L) for 7 d. The calcification was validated through ocresolphthalein complexone colorimetry to detect the cellular calcium content, real-time PCR to measure the calcification-related gene expression and alizarin red staining to observe the formation of calcium nodules. Based on the cell calcification model, micro-RNA microarray array was applied to screen the profiles of microRNA expression in VSMC following high Pi stimulation for different periods (0, 3 and 12 h). The array data were analyzed by TAM tool to explore the activated signaling pathway.Results: The calcium content of A7r5 cells induced by high Pi was increased 9.6 times high as cells without Pi treatment (P<0.05). VSMC contractile phenotype genes (SM-α actin, SM22) were down-regulated (P<0.05), while calcification-related genes (BMP2, MSX2, Runx2) were up-regulated (P<0.05) in VSMC stimulated by high Pi. The calcium nodules were obviously formed in cells after 7 d high Pi treatment. In microarray experiment, 680 individual microRNAs were detected in high Pi-treated VSMCs at different time points (0, 3 and 12 h). Among these genes, miR-183, miR-664 and miR-9*were increased whereas miR-542-5P, let-7f and miR-29a were decreased in time-dependent manners. Twenty-six kinds of signaling pathways, including cell apoptosis, differentiation and proliferation, were significantly activated. All these activated pathways were associated with calcification. Conclusion:This study implies that microRNA changed in high Pi-induced VSMCs may involve in the process of calcification.

Vascular calcification； Muscle, smooth, vascular； microRNA； Signal transduction

國(guó)家基金委重大國(guó)際合作項(xiàng)目 (81220108004)、國(guó)家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃(973計(jì)劃，2012CB518002)、 國(guó)家自然科學(xué)基金 (81070243、81121061、91339000)和國(guó)家杰出青年基金 (81225002)資助Supported by the Foundation of Major International Cooperation (81220108004)，the National Basic Research Program of China (973 Program， 2012CB518002)， the National Natural Science Foundation of China(81070243, 81121061, 91339000) and the National Science Fund for Distinguished Young Scholars (81225002)

時(shí)間：2016-5-15 13：25：04

http://www.cnki.net/kcms/detail/11.4691.R.20160515.1325.006.html

R331.3

A

1671-167X(2016)05-0756-10

10.3969/j.issn.1671-167X.2016.05.002

△ Corresponding auther’s e-mail, kongw@bjmu.edu.cn