邵維斌, 路 萍, 夏春英, 李 忻, 荀 康, 馮松濤, 趙 倩

(江蘇省鎮(zhèn)江市第一人民醫(yī)院 腎臟內(nèi)科, 江蘇 鎮(zhèn)江, 212002)

microRNA-192在晚期糖基化終產(chǎn)物誘導(dǎo)人腹膜間皮細(xì)胞上皮-間葉轉(zhuǎn)化中的作用

邵維斌, 路 萍, 夏春英, 李 忻, 荀 康, 馮松濤, 趙 倩

(江蘇省鎮(zhèn)江市第一人民醫(yī)院 腎臟內(nèi)科, 江蘇 鎮(zhèn)江, 212002)

目的探討microRNA-192(miR-192)對(duì)晚期糖基化終產(chǎn)物(AGEs)誘導(dǎo)人腹膜間皮細(xì)胞上皮-間葉轉(zhuǎn)化(EMT)的調(diào)控作用。方法人腹膜間皮細(xì)胞、轉(zhuǎn)染miR-192抑制物后人腹膜間皮細(xì)胞和轉(zhuǎn)染miR-192抑制物陰性對(duì)照的人腹膜間皮細(xì)胞在含AGEs的培養(yǎng)基培養(yǎng)72 h， 以M199培養(yǎng)基和含80 mM BSA的M199培養(yǎng)基為對(duì)照，然后運(yùn)用實(shí)時(shí)熒光定量PCR法檢測(cè)miR-192和mRNA的表達(dá)情況，運(yùn)用蛋白質(zhì)印跡法檢測(cè)蛋白的表達(dá)情況。結(jié)果在AGEs刺激后，人腹膜間皮細(xì)胞miR-192、膠原蛋白I(CollagenI)mRNA、α-平滑肌肌動(dòng)蛋白(α-SMA)mRNA和蛋白表達(dá)水平顯著上升 (P<0.05)，而E-鈣黏蛋白(E-cadherin) mRNA和蛋白表達(dá)水平顯著下降(P<0.05)。與轉(zhuǎn)染miR-192抑制物陰性對(duì)照的人腹膜間皮細(xì)胞相比，轉(zhuǎn)染miR-192抑制物的人腹膜間皮細(xì)胞miR-192、Collagen I mRNA、α-SMAmRNA和蛋白表達(dá)水平顯著下降 (P<0.05)，而E-cadherin mRNA和蛋白表達(dá)水平顯著上升(P<0.05)。結(jié)論AGEs可能通過上調(diào)miR-192表達(dá)誘導(dǎo)人腹膜間皮細(xì)胞EMT。miR-192抑物可能通過下調(diào)miR-192表達(dá)阻止AGEs誘導(dǎo)人腹膜間皮細(xì)胞EMT。miR-192在AGEs誘導(dǎo)人腹膜間皮細(xì)胞EMT中起重要調(diào)控作用。

晚期糖基化終產(chǎn)物; 間皮細(xì)胞; 上皮-間葉轉(zhuǎn)化; microRNA-192

隨著腹膜透析的進(jìn)行，腹膜會(huì)出現(xiàn)新生血管和纖維化等結(jié)構(gòu)改變，引起腹膜功能衰竭[1-2]。透析液中高濃度葡萄糖、AGEs等可引起腹膜間皮細(xì)胞E-鈣黏蛋白等表達(dá)下降, α-平滑肌肌動(dòng)蛋白等表達(dá)上升，發(fā)生EMT[3-4], 腹膜間皮細(xì)胞EMT可能在腹膜發(fā)生新生血管形成和纖維化等結(jié)構(gòu)改變的過程中起主導(dǎo)作用[4-5]。MicroRNA是一類非編碼小RNA, 參與多種疾病調(diào)控。研究[6-7]發(fā)現(xiàn), microRNA-192在糖尿病腎組織細(xì)胞轉(zhuǎn)分化和纖維化過程中起重要調(diào)控作用。本研究探討microRNA-192在AGEs誘導(dǎo)人腹膜間皮細(xì)胞EMT中的調(diào)控作用，現(xiàn)報(bào)告如下。

1 材料與方法

1.1 原代人腹膜間皮細(xì)胞的培養(yǎng)

人腹膜間皮細(xì)胞分離、培養(yǎng)及鑒定參照文獻(xiàn)[8]的方法進(jìn)行。

1.2 分組

① 對(duì)照組, M199培養(yǎng)基; ② AGEs, 含80 mM AGEs-BSA的M199培養(yǎng)基; ③ 牛血清白蛋白(BSA)組，含80 mM BSA的M199培養(yǎng)基。第3代人腹膜間皮細(xì)胞在上述各組培養(yǎng)基中培養(yǎng)72 h; ④ miR-192抑制物組，轉(zhuǎn)染miR-192抑制物的腹膜間皮細(xì)胞，在含80 mM AGEs-BSA的M199培養(yǎng)基培養(yǎng)72 h; ⑤ miR-192抑制物對(duì)照組，轉(zhuǎn)染陰性對(duì)照的人腹膜間皮細(xì)胞在含80 mM AGEs-BSA的M199培養(yǎng)基72 h。

1.3 實(shí)時(shí)熒光定量PCR法檢測(cè)miRNA-192和

E-cadherin mRNA、α-SMA mRNA、Collagen

I mRNA的表達(dá)情況

細(xì)胞總RNA抽提按Trizol試劑盒的方法進(jìn)行，轉(zhuǎn)錄按照cDNA逆轉(zhuǎn)錄試劑盒說明書進(jìn)行，運(yùn)用實(shí)時(shí)定量PCR試劑盒檢測(cè)，按公式2-△△Ct求出基因表達(dá)量[9]。E-cadherin、-SMA、CollagenI、miRNA-192引物序列及相關(guān)條件見文獻(xiàn)[7, 10]。

1.4 蛋白質(zhì)印跡法檢測(cè)E-cadherin、α-SMA蛋白

的表達(dá)[10]

人腹膜間皮細(xì)胞裂解蛋白經(jīng)SDS-PAGE凝膠電泳后電轉(zhuǎn)移至PVDF膜，用5%脫脂奶粉封閉2 h后，分別加入小鼠抗E-cadherin抗體(美國(guó)BD)、小鼠抗α-SMA抗體和小鼠抗β-actin抗體4 ℃過夜，洗膜后加辣根過氧化酶標(biāo)記的人抗小鼠IgG抗體, 37 ℃1.5 h, 洗膜后加ECL試劑, X線自顯影顯示結(jié)果。

1.5 抑制物的合成與轉(zhuǎn)染

miR-192抑制物，序列為5-GGCUGUCAAUUCAUAGGUCAG-3(購(gòu)自上海吉瑪生物公司)，抑制陰性對(duì)照寡核苷酸，序列為5″-CAGUACUUUUGUGUAGUACAA-3(購(gòu)自上海吉瑪生物公司)。按照試劑說明書進(jìn)行轉(zhuǎn)染操作。

1.6 統(tǒng)計(jì)學(xué)方法

各組間資料比較用方差分析，兩兩比較用Student-Newman-Keals 檢驗(yàn)(運(yùn)用SAS軟件進(jìn)行統(tǒng)計(jì)分析)。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

2.1 AGEs對(duì)人腹膜間皮細(xì)胞microRNA-192、α-SMA、E-cadherin、和Collagen I mRNA表達(dá)的影響

實(shí)時(shí)定量PCR檢測(cè)結(jié)果顯示，與對(duì)照組及BSA組相比, AGEs刺激72 h后, microRNA-192、Collagen I和α-SMA mRNA的表達(dá)水平顯著上升(P<0.05), 而E-cadherin mRNA表達(dá)水平顯著下降(P<0.05)。見圖1。

與對(duì)照組及BSA組相比，#P<0．05。圖1 AGEs刺激后間皮細(xì)胞E?cadherin、α?SMA、CollagenImRNA和microRNA?192的相對(duì)表達(dá)情況

2.2 AGEs對(duì)人腹膜間皮細(xì)胞α-SMA、E-cadherin蛋白表達(dá)的影響

蛋白質(zhì)印跡法結(jié)果顯示，與對(duì)照組及BSA組相比, AGEs刺激72 h后, α-SMA蛋白表達(dá)水平顯著增加(P<0.05), 而E-cadherin 蛋白表達(dá)水平顯著下降(P<0.05)。見圖2。

與對(duì)照組及BSA組相比，#P<0．05。圖2 AGEs刺激后間皮細(xì)胞組α?SMA、E?cadherin蛋白的相對(duì)表達(dá)情況

2.3 microRNA-192抑制物對(duì)在AGEs刺激后人腹膜間皮細(xì)胞E-cadherin、α-SMA、CollagenI mRNA、microRNA-192表達(dá)的影響

實(shí)時(shí)定量PCR檢測(cè)結(jié)果顯示，與miR-192抑制物對(duì)照組相比, microRNA-192抑制物轉(zhuǎn)染后人腹膜間皮細(xì)胞在AGEs刺激72 h后, microRNA-192、Collagen I mRNA、α-SMA表達(dá)水平顯著下降(P<0.05)，而E-cadherin mRNA表達(dá)水平顯著上升(P<0.05)。見圖3。

與miR?192抑制物對(duì)照組及AGEs組相比，#P<0．05。圖3 miR?192抑制物作用后間皮細(xì)胞E-cadherin、α?SMA、CollagenImRNA和microRNA?192的表達(dá)情況

2.4 microRNA-192抑制物對(duì)在AGEs刺激后人

腹膜間皮細(xì)胞α-SMA、E-cadherin蛋白表達(dá)的影響

Western 印跡結(jié)果顯示，與miR-192抑制物對(duì)照組相比, microRNA-192抑制物轉(zhuǎn)染后人腹膜間皮細(xì)胞在AGEs刺激72 h后, α-SMA蛋白表達(dá)水平顯著下降(P<0.05), 而E-cadherin 蛋白表達(dá)水平顯著增加(P<0.05)。見圖4。

3 討 論

腹膜透析已成為終末期腎衰患者主要的治療措施之一。目前，越來越多終末期腎衰患者首選腹膜透析療法。隨著腹膜透析進(jìn)行，透析液中高濃度葡萄糖、AGEs、葡萄糖降解產(chǎn)物等可引起腹膜結(jié)構(gòu)和功能改變，導(dǎo)致腹膜纖維化和功能衰竭，妨礙了腹膜透析的正常進(jìn)行。

與miR?192抑制物對(duì)照組及AGEs組相比，#P<0．05。圖4 miR?192抑制物作用后間皮細(xì)胞組α?SMA、E?cadherin蛋白的表達(dá)情況

長(zhǎng)期腹膜透析患者腹膜出現(xiàn)表層間皮細(xì)胞消失、纖維化、透明樣血管病變及大量新生血管形成等病理改變。腹膜明顯新生血管形成和纖維化是引起腹膜功能衰竭的主要原因。作者先前的研究發(fā)現(xiàn)，高濃度葡萄糖或AGEs等能刺激腹膜間皮細(xì)胞轉(zhuǎn)分化[10-11], 發(fā)生EMT的腹膜間皮細(xì)胞可能是腹膜透析患者腹膜組織中肌纖維母細(xì)胞的主要來源之一[4, 12]。這些轉(zhuǎn)分化的腹膜間皮細(xì)胞能產(chǎn)生多種細(xì)胞因子如血管內(nèi)皮生長(zhǎng)因子、轉(zhuǎn)化生長(zhǎng)因子β等細(xì)胞因子具有促進(jìn)血管內(nèi)皮細(xì)胞增殖，刺激體內(nèi)新血管形成、調(diào)節(jié)組織分化和參與纖維化等作用，腹膜間皮細(xì)胞EMT可能在腹膜新生血管形成和纖維化的過程中起關(guān)鍵作用。這些證據(jù)提示腹膜間皮細(xì)胞損傷在腹膜透析時(shí)腹膜結(jié)構(gòu)和功能改變中起主導(dǎo)作用。因而研究AGEs損傷腹膜間皮細(xì)胞的機(jī)制對(duì)防治腹膜透析時(shí)腹膜結(jié)構(gòu)改變和功能衰竭具有重要意義。

MicroRNA是一類內(nèi)生的、長(zhǎng)度約為20～24個(gè)核苷酸的功能性非編碼小RNA，廣泛參與生長(zhǎng)發(fā)育、細(xì)胞分化、細(xì)胞凋亡、腫瘤發(fā)生等過程[13], 與多種疾病相關(guān)[14-16]。最近有研究發(fā)現(xiàn), MicroRNA-192在糖尿病腎組織細(xì)胞轉(zhuǎn)分化和纖維化過程中起重要調(diào)控作用，但MicroRNA-192對(duì)于AGEs引起腹膜間皮細(xì)胞EMT過程中是否同樣具有調(diào)控作用，目前尚不清楚。

本研究發(fā)現(xiàn), AGEs刺激后，人腹膜間皮細(xì)胞miR-192、Collagen I mRNA、α-SMA mRNA和α-SMA蛋白表達(dá)水平明顯上升，而E-cadherin mRNA和E-cadherin蛋白表達(dá)水平顯著下降，表明AGEs可能通過上調(diào)miR-192表達(dá)誘導(dǎo)人腹膜間皮細(xì)胞EMT。本研究還發(fā)現(xiàn)，轉(zhuǎn)染miR-192抑制物的人腹膜間皮細(xì)胞經(jīng)AGEs刺激后miR-192、Collagen I mRNA、α-SMA mRNA和α-SMA蛋白表達(dá)水平顯著下降，而E-cadherin mRNA和E-cadherin蛋白表達(dá)水平顯著上升，表明miR-192抑制物可能通過下調(diào)miR-192表達(dá)阻止AGEs誘導(dǎo)人腹膜間皮細(xì)胞EMT。因此miR-192可能在AGEs誘導(dǎo)人腹膜間皮細(xì)胞上皮-間葉轉(zhuǎn)化中起重要調(diào)控作用。

[1] Mehrotra R, Devuyst O, Davies S J, et al. The Current State of Peritoneal Dialysis[J]. J Am Soc Nephrol, 2016, 27: 3238-3252.

[2] Devuyst O, Margetts P J, Topley N. The pathophysiology of the peritoneal membrane[J]. J Am Soc Nephrol, 2010, 21: 1077-1085.

[3] Vargha R, Endemann M, Kratochwill K. Ex vivo reversal of in vivo transdifferentiation in mesothelial cells grown from peritoneal dialysate effluents[J]. Nephrol Dial Transplant, 2006, 21: 2943-2947.

[4] Aroeira L S, Aguilera A, Sánchez-Tomero J A, et al. Epithelial to Mesenchymal Transition and Peritoneal Membrane Failure in Peritoneal Dialysis Patients: Pathologic Significance and Potential Therapeutic Interventions[J]. J Am Soc Nephrol, 2007, 18: 2004-2013.

[5] Yanez-Mo M, Lara-Pezzi E, Selgas R, et al. Peritoneal dialysis and epithelial-to-mesenchymal transition of mesothelial cells[J]. N Engl J Med, 2003, 348: 403-413.

[6] Putta S, Lanting L, Sun G, et al. Inhibiting microRNA-192 ameliorates renal fibrosis in diabetic nephropathy[J]. J Am Soc Nephrol, 2012, 23: 458-69.

[7] Krupa A, Jenkins R, Luo D D, et al. Loss of MicroRNA-192 promotes fibrogenesis in diabetic nephropathy[J]. J Am Soc Nephrol, 2010, 21: 438-47.

[8] 邵維斌, 錢家麒. 從網(wǎng)膜組織法培養(yǎng)人腹膜間皮細(xì)胞[J]. 腎臟病與透析腎移植雜志, 2001, 10: 92-94.

[9] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[J]. Methods, 2001, 25: 402-408.

[10] 邵維斌, 荀康, 李忻, 等. 高濃度葡萄糖誘導(dǎo)大鼠腹膜間皮細(xì)胞上皮-間葉轉(zhuǎn)化[J]. 實(shí)用臨床醫(yī)藥雜志, 2010, 17: 4-7.

[11] 邵維斌, 彭淑娟, 夏春英, 等. 晚期糖基化終產(chǎn)物誘導(dǎo)大鼠腹膜間皮細(xì)胞上皮-間葉轉(zhuǎn)化[J]. 實(shí)用臨床醫(yī)藥雜志, 2011, 19: 6-9.

[12] Liu Y, Dong Z, Liu H, et al. Transition of mesothelial cell to fibroblast in peritoneal dialysis: EMT, stem cell or bystander[J]. Perit Dial Int, 2015, 35: 14-25.

[13] Adams B D, Parsons C, Walker L, et al. Targeting noncoding RNAs in disease[J]. J Clin Invest, 2017, 127: 761-771.

[14] Ameis D, Khoshgoo N, Iwasiow B M, et al. MicroRNAs in Lung Development and Disease[J]. Paediatr Respir Rev, 2017, 22: 38-43.

[15] Laffont B, Rayner K J. MicroRNAs in the Pathobiology and Therapy of Atherosclerosis[J]. Can J Cardiol, 2017, 33: 313-324.

[16] Oliveto S, Mancino M, Manfrini N, et al. Role of microRNAs in translation regulation and cancer[J]. World J Biol Chem, 2017, 8: 45-56.

TheroleofmicroRNA-192inprogressionofadvancedglycationendproducts-inducedepithelial-to-mesenchymaltransitionofhumanperitonealmesothelialcells

SHAOWeibin,LUPing,XIAChunying,LIXin,XUNKang,FENGSongtao,ZHAOQian

(DepartmentofNephrology,ZhenjiangFirstPeople′sHospital,Zhenjiang,Jiangsu, 212002)

ObjectiveTo investigate the role of microRNA-192 (miR-192) in the regulation of epithelial mesenchymal transition induced by advanced glycation end products (AGEs) in human peritoneal mesothelial cells.MethodsThe human peritoneal mesothelial cells, the human peritoneal mesothelial cells transfected by miR-192 inhibitor and the human peritoneal mesothelial cells transfected by miR-192 inhibitor negative control were cultured for 72 hours in culture medium containing AGEs (80mM); M199 medium and medium with M199 medium containing 80mM BSA as negative control, the expression of miR-192 and mRNA was detected by real-time quantitative PCR, and the expression of miR-192 and mRNA was detected by Western blotting.ResultsAGEs significantly upregulated the expression of miR-192, Collagen I mRNA, α-smooth muscle actin(α-SMA) mRNA and protein(P<0.05), while significantly downregulated the E-cadherin mRNA and protein expression(P<0.05). Compared with human peritoneal mesothelial cells transfected by miR-192 inhibitor, the expression of miR-192 and Collagen I mRNA and α-SMA mRNA and protein was decreased (P<0.05), while E-cadherin mRNA and protein expression was significantly increased (P<0.05).ConclusionAGEs may induce EMT of human peritoneal mesothelial cells by upregulation of miR-192 expression. MiR-192 inhibitor may prevent AGEs-inducing EMT of human peritoneal mesothelial cells by down-regulation of miR-192 expression, and may play an important regulatory role in EMT of human peritoneal mesothelial cells induced by AGEs.

peritoneal mesothelial cells; epithelial-to-mesenchymal transition; advanced glycation end products; microRNA-192

R 459.5

A

1672-2353(2017)19-095-04

10.7619/jcmp.201719027

2017-05-21

江蘇省鎮(zhèn)江市科技項(xiàng)目(SH2013051)