沉默微小RNA-218表達(dá)保護(hù)STZ誘導(dǎo)的糖尿病大鼠腎組織

楊海波， 王清俊， 李蘇童， 陳小琳， 慕 婷

(1西安市中心醫(yī)院腎臟內(nèi)科， 陜西 西安 710003； 2銅川礦務(wù)局中心醫(yī)院腎臟內(nèi)科， 陜西 銅川 727000)

沉默微小RNA-218表達(dá)保護(hù)STZ誘導(dǎo)的糖尿病大鼠腎組織

楊海波△， 王清俊2， 李蘇童1， 陳小琳1， 慕 婷1

(1西安市中心醫(yī)院腎臟內(nèi)科， 陜西 西安 710003；2銅川礦務(wù)局中心醫(yī)院腎臟內(nèi)科， 陜西 銅川 727000)

目的： 探討沉默微小RNA-218(microRNA-218， miR-218)表達(dá)對(duì)鏈脲佐菌素(streptozotocin，STZ)誘導(dǎo)的糖尿病腎病大鼠腎臟組織的保護(hù)作用及其可能機(jī)制。方法: 采用單次腹腔注射STZ (50 mg/kg)方法制備糖尿病大鼠模型并構(gòu)建miR-218短發(fā)夾RNA(short hairpin RNA，shRNA)慢病毒載體。SD大鼠被隨機(jī)分為健康對(duì)照組、糖尿病模型組、空載慢病毒組及miR-218-shRNA組。于自動(dòng)生化儀上檢測不同時(shí)點(diǎn)(4、8和12周)大鼠血糖、24 h尿蛋白量、血清肌酐(serum creatinine，SCr)及血尿素氮(blood urea nitrogen，BUN)含量。實(shí)時(shí)熒光定量PCR(RT-qPCR)檢測腎臟組織miR-218的表達(dá)。RT-qPCR和Western blot檢測血紅素氧合酶1(heme oxygenase-1，HO-1)、腎病蛋白(nephrin)和p38絲裂原激活的蛋白激酶(p38 mitogen-activated protein kinase, p38 MAPK)的mRNA及蛋白表達(dá)水平。Caspase-3活性檢測試劑盒檢測caspase-3活性。末端脫氧核苷酸轉(zhuǎn)移酶介導(dǎo)的dUTP缺口末端標(biāo)記(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling，TUNEL)法檢測腎臟組織細(xì)胞凋亡。結(jié)果: 與健康對(duì)照組相比，STZ處理后大鼠miR-218表達(dá)水平顯著升高。同時(shí)模型大鼠的血糖、24 h尿蛋白量、SCr及BUN含量顯著升高(P<0.05)；模型大鼠腎臟組織中HO-1和nephrin的mRNA和蛋白表達(dá)水平顯著降低，而p38 MAPK蛋白的磷酸化水平顯著升高；另外，模型大鼠腎臟組織中的caspase-3活性也顯著升高。模型大鼠感染miR-218-shRNA后，miR-218表達(dá)水平顯著下降并可以顯著逆轉(zhuǎn)上述效應(yīng)。miR-218-shRNA組腎臟組織細(xì)胞的凋亡水平顯著低于糖尿病模型組及空載慢病毒組。結(jié)論: miR-218參與了糖尿病大鼠的腎臟損傷，慢病毒載體沉默其表達(dá)能有效抑制腎臟組織細(xì)胞的凋亡，提示miR-218可以作為糖尿病腎病的基因治療靶點(diǎn)。

微小RNA-218； 慢病毒載體； 糖尿??； 腎臟

糖尿病腎病(diabetic nephropathy，DN)是糖尿病主要的微血管并發(fā)癥，也是導(dǎo)致終末期腎病的主要原因[1]。微小RNA(microRNA， miRNA)是一類具有調(diào)控功能的非編碼RNA分子，廣泛參與生長發(fā)育、增殖及凋亡等生物學(xué)過程。已知有多種miRNA、分子及信號(hào)通路參與了DN的病理過程[2-4]。我們前期的體外研究發(fā)現(xiàn)[5]，microRNA-218(miR-218)在高糖培養(yǎng)的腎小球足細(xì)胞中過表達(dá)，并且通過調(diào)節(jié)血紅素氧合酶1(heme oxygenase-1，HO-1)促進(jìn)足細(xì)胞凋亡。而miR-218在體內(nèi)是否發(fā)揮作用還有待進(jìn)一步驗(yàn)證。因而，本研究擬通過建立miR-218短發(fā)夾RNA(short hairpin RNA，shRNA)慢病毒載體模型，在體內(nèi)探討其對(duì)鏈脲佐菌素(streptozotocin，STZ)誘導(dǎo)的糖尿病大鼠的腎臟保護(hù)作用。

材 料 和 方 法

1 動(dòng)物及主要試劑

清潔級(jí)雄性SD大鼠，6周齡，體重160～200 g，購自第四軍醫(yī)大學(xué)實(shí)驗(yàn)動(dòng)物中心；STZ購自Sigma；抗HO-1、腎病蛋白(nephrin)、p38絲裂原激活的蛋白激酶(p38 mitogen-activated protein kinase, p38 MAPK)、磷酸化p38 MAPK(phosphorylated p38 MAPK，p-p38)、caspase-3抗體及辣根過氧化物酶標(biāo)記的 II 抗購自Santa Cruz Biotechnology；TRIzol試劑購自Invitrogen；One Step PrimeScript miRNA cDNA Synthesis Kit購自TaKaRa；M-MLV Reverse Transcriptase購自Promega；SYBR Green qPCR Master Mix購自Thermo Fisher；末端脫氧核苷酸轉(zhuǎn)移酶介導(dǎo)的dUTP缺口末端標(biāo)記(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling，TUNEL)試劑盒購自Roche；慢病毒表達(dá)載體和包裝質(zhì)粒購自上海吉瑪制藥技術(shù)有限公司；T4 DNA連接酶購自NEB；caspase-3活性檢測試劑盒購自碧云天生物技術(shù)有限公司。

2 主要方法

2.1 構(gòu)建miR-128 shRNA慢病毒載體以抑制miR-218的表達(dá) 參考miRBase數(shù)據(jù)庫(http://www.mirbase.org/)小鼠miR-218基因成熟序列(MIMAT0000663)：5’-UUGUGCUUGAUCUAACCAUGU-3’，設(shè)計(jì)分別帶有BamH I和XhoI酶切位點(diǎn)的miR-218 shRNA序列(5’-GGATCCCACATGGTTAGATCAAGCACAATTCAAG AGATTGTGCTTGATC-TAACCATGTTTTTTTCCTCGAG-GG-3’)和陰性對(duì)照序列，并送由上海生工生物有限公司合成；將miR-218 shRNA單鏈退火并通過T4DNA連接酶連接到pLenti-H1載體上，4 ℃過夜；轉(zhuǎn)化DH5α感受態(tài)細(xì)胞后，均勻涂布于含氨芐青霉素的LB平板上并挑取菌落。PCR鑒定陽性菌落(PCR反應(yīng)條件： 95 ℃ 5 min； 95 ℃ 20 s， 62 ℃ 20 s， 72 ℃ 30 s， 35個(gè)循環(huán)； 72 ℃ 5 min)。培養(yǎng)陽性克隆并測序驗(yàn)證。將10 μg構(gòu)建好的pLenti-H1-miR-218-shRNA與6 μg Δ8.9及6 μg VSVG共轉(zhuǎn)染293T細(xì)胞。72 h后，高速離心并收集細(xì)胞上清，用0.45 μm濾器過濾上清液以除去細(xì)胞碎片。采用逐孔稀釋滴度測定法測定病毒滴度。

2.2 動(dòng)物模型的制備及分組 大鼠飼養(yǎng)于(23± 2)℃，12 h晝夜交替。大鼠適應(yīng)性飼養(yǎng)1周后，按體重隨機(jī)分組。造模前禁食12 h, 然后將1% STZ以50 mg/kg腹腔注射入模型組大鼠體內(nèi)，健康對(duì)照組注射等量的檸檬酸緩沖液(0.01 mmol/L，pH=4.5)。72 h后測全血血糖，血糖≥16.7 mmol/L視為造模成功。繼續(xù)飼養(yǎng)1周后，將模型組進(jìn)一步細(xì)分為糖尿病模型組(注射生理鹽水，每只100 μL)，空載慢病毒組[注射空載慢病毒液，感染復(fù)數(shù)(MOI)=100]及miR-218-shRNA組(注射miR-218慢病毒液，MOI=100)，均經(jīng)大鼠尾靜脈注射。

2.3 24 h尿蛋白、血清肌酐(serum creatinine，SCr)及血尿素氮(blood urea nitrogen，BUN)含量的檢測 大鼠造模成功后繼續(xù)飼養(yǎng)1周，然后進(jìn)行慢病毒注射。注射時(shí)點(diǎn)為分別為第1周、第5周和第9周剛開始時(shí)；并在第4周、第8周及第12周留取24 h尿蛋白、SCr和BUN并將其稀釋至合適的工作濃度，于全自動(dòng)生化分析儀(HITACHI)檢測大鼠24 h尿蛋白、SCr和BUN含量。

2.4 RT-qPCR方法檢測miRNA及mRNA的表達(dá)水平[5]腎臟組織研磨后裂解組織，用TRIzol試劑抽提總RNA并用分光光度發(fā)測定其含量和濃度。miR-218檢測方法參照miRNA逆轉(zhuǎn)錄試劑盒說明書反轉(zhuǎn)錄合成cDNA，并參照SYBR Green qPCR Master Mix試劑盒操作方法檢測miR-218表達(dá)水平(以U6為內(nèi)參照)。miR-218的上游引物為5’-GCGCTTGTGCTTGATCTAA-3’，下游引物為5’-GTGCAGGG-TCCGAGGT-3’； U6的上游引物為5’-CTCGCTTCGGCAGCACA-3’，下游引物為5’-AACGCTTCACGAA-TTTGCGT-3’。目標(biāo)基因mRNA的檢測方法為：參照mRNA逆轉(zhuǎn)錄試劑盒操作說明反轉(zhuǎn)錄合成cDNA，并參照SYBR Green qPCR Master Mix試劑盒操作方法檢測目標(biāo)基因mRNA表達(dá)水平(以β-actin為內(nèi)參照)。HO-1的上游引物為5’-CCATAGGCTCCTTCCTCCTTTC-3’，下游引物為5’-GGCCTTCTTTCTA-GAGAGGGAATT-3’； nephrin的上游引物為5’-AGCTCGTGTCTCCCAGAGT-3’，下游引物為5’-CGTTCACGTTTGCAGAGATGT-3’； β-actin的上游引物為5’-TTCCTTCTTGGGTATGGAAT-3’，下游引物為5’-GAGCAATGATCTTGATCTTC-3’)。均采用2-ΔΔCt方法計(jì)算相對(duì)表達(dá)量。

2.5 Western blot檢測蛋白表達(dá) 腎臟組織研磨后裂解組織并測定蛋白的濃度。取30 μg樣品，經(jīng)十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳分離后將其轉(zhuǎn)移到硝酸纖維素膜上；然后用含3% 脫脂奶粉和0.1% Tween-20的PBS緩沖液對(duì)膜進(jìn)行封閉，37 ℃, 封閉1 h后，用含0.1% Tween-20的PBS清洗膜2次；加入抗HO-1 (1∶1 000)、抗nephrin (1∶1 500)、抗p38 (1∶1 000)、抗p-p38(1∶1 000)及抗β-actin(1∶2 000)抗體，4 ℃孵育過夜；加入辣根過氧化物酶標(biāo)記的Ⅱ抗(1∶ 1 000)， 37 ℃孵育閉1 h；測定蛋白條帶。

2.6 Caspase-3活性的檢測 腎臟組織經(jīng)裂解勻漿后，按caspase-3檢測試劑盒說明書進(jìn)行操作，于405 nm處檢測吸光度。

2.7 TUNEL試劑盒檢測細(xì)胞凋亡率 將腎臟組織做成石蠟切片(5 μm)。按照Roche TUNEL試劑盒說明進(jìn)行操作。組織常規(guī)脫蠟水化，然后加入100 μL蛋白酶K室溫孵育15 min；加入50 μL TUNEL反應(yīng)混合液，37 ℃避光孵育1 h；浸入0.3% H2O2中20 min再加入HRP標(biāo)記的鏈親合素，37 ℃避光孵育30 min，DAB染色；經(jīng)蘇木素復(fù)染，每個(gè)腎臟組織標(biāo)本選取6張切片，并用Image-Pro Plus 6.0圖像分析軟件計(jì)算凋亡指數(shù)。凋亡指數(shù)(%)=凋亡細(xì)胞數(shù)/總細(xì)胞數(shù)×100%。

3 統(tǒng)計(jì)學(xué)處理

實(shí)驗(yàn)數(shù)據(jù)采用統(tǒng)計(jì)學(xué)軟件SPSS 22.0進(jìn)行分析。數(shù)據(jù)采用平均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示。多組間比較采用單因素方差分析，用Bonfferoni校正的t檢驗(yàn)進(jìn)行各組均數(shù)間的兩兩比較。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 腎臟組織miR-218表達(dá)

RT-qPCR實(shí)驗(yàn)表明，隨著STZ注射后大鼠飼養(yǎng)時(shí)間的延長，腎臟組織中糖尿病模型組miR-218水平顯著高于健康對(duì)照組(P<0.05)。注射miR-218-shRNA慢病毒載體可顯著降低miR-218水平，說明了miR-218-shRNA慢病毒載體構(gòu)建成功，見圖1。

Figure 1.The expression of miR-218 in the kidney tissues at different time points. W: weeks. Mean±SD.n=4.*P<0.05vshealthy control group;#P<0.05vsempty vector group.

圖1 各組不同時(shí)點(diǎn)(4周、8周和12周)腎臟組織中miR-218的表達(dá)水平

2 血尿生化指標(biāo)檢測結(jié)果

與健康對(duì)照組相比，糖尿病模型組血糖、24 h尿蛋白、SCr及BUN含量顯著升高(P<0.05)。與糖尿病模型組及空載病毒組相比，沉默miR-218后miR-218-shRNA組血糖、24 h尿蛋白、SCr及BUN含量顯著下降，差異具有統(tǒng)計(jì)學(xué)意義(P< 0.05)，見表1～3。

表1 4周時(shí)大鼠血尿生化指標(biāo)的檢測結(jié)果

*P<0.05vshealthy control group;#P<0.05vsempty vector group.

表2 8周時(shí)大鼠血尿生化指標(biāo)的檢測結(jié)果

*P<0.05vshealthy control group;#P<0.05vsempty vector group.

表3 12周時(shí)大鼠血尿生化指標(biāo)的檢測結(jié)果

*P<0.05vshealthy control group;#P<0.05vsempty vector group.

3 HO-1、nephrin和p38 MAPK表達(dá)及caspase-3活性檢測

體外實(shí)驗(yàn)表明，miR-218可以靶向作用于HO-1并激活p38 MAPK的表達(dá)。因而我們從體內(nèi)實(shí)驗(yàn)去驗(yàn)證這一結(jié)果。如圖2所示，miR-218-shRNA慢病毒轉(zhuǎn)染大鼠后繼續(xù)飼養(yǎng)8周，與健康對(duì)照組相比，糖尿病模型組HO-1和nephrin的mRNA及蛋白水平顯著降低，總p38 MAPK蛋白水平不變，但p38 MAPK蛋白磷酸化水平升高。與此同時(shí)，沉默miR-218表達(dá)后，HO-1水平顯著升高，總p38 MAPK蛋白水平不變，p38 MAPK蛋白磷酸化水平降低。Caspase-3活性檢測結(jié)果表明，與健康對(duì)照組相比，糖尿病模型組的caspase-3活性顯著升高(P<0.05)，而沉默miR-218表達(dá)會(huì)使caspase-3活性降低。

Figure 2. The expression of HO-1， nephrin and p38 MAPK, and the caspase-3 activity in the kidney tissues of rats. A: the caspase-3 activity; B: the mRNA expression of HO-1 and nephrin; C: the protein expression of HO-1, nephrin and p38 MAPK (p38). Mean±SD.n=4.*P<0.05vshealthy control group;#P<0.05vsempty vector group.

圖2 各組大鼠腎組織HO-1、nephrin和p38 MAPK表達(dá)及caspase-3活性的變化

4 TUNEL法檢測腎臟組織細(xì)胞凋亡

TUNEL實(shí)驗(yàn)結(jié)果表明，4周、8周和12周時(shí)健康對(duì)照組大鼠腎組織的TUNEL陽性細(xì)胞數(shù)分別為(3.16±0.53)%、(5.13±0.57)%和(7.05±0.56)%，糖尿病模型組凋亡指數(shù)分別為(6.59±0.74)%、(10.43±1.76)%和(15.65±1.03)%，與同一時(shí)點(diǎn)健康對(duì)照組相比顯著升高(P<0.05)。而miR-218-shRNA組4周、8周和12周時(shí)TUNEL陽性細(xì)胞數(shù)為(5.25±0.86)%、(7.75±0.97)%和(9.26±1.12)%，顯著低于同一時(shí)點(diǎn)糖尿病模型組，說明miR-218的沉默可以有效減少腎臟組織的細(xì)胞凋亡，見圖3。

Figure 3.The numbers of TUNEL positive cells in the kidney tissues of the rats at different time points (×200). W: weeks. Mean±SD.n=4.*P<0.05vshealthy control group;#P<0.05vsempty vector group.

圖3 不同時(shí)點(diǎn)各組大鼠腎組織TUNEL陽性細(xì)胞數(shù)的比較

討 論

研究表明在糖尿病腎病中miRNA發(fā)揮著重要的調(diào)節(jié)因子和藥物靶點(diǎn)作用[6]。足細(xì)胞特異性敲除Dicer (一種在miRNA合成過程中起重要作用的核酸內(nèi)切酶)會(huì)導(dǎo)致蛋白尿的增多及腎小球硬化[7- 8]。miR-29c在db/db糖尿病鼠腎小球中高表達(dá)且沉默其表達(dá)可以顯著降低蛋白尿和腎小球系膜基質(zhì)的聚集[9]。本實(shí)驗(yàn)組前期的研究也表明，miR-218在高糖培養(yǎng)的腎小球足細(xì)胞中過表達(dá)，且可以通過調(diào)節(jié)HO-1和p38 MAPK的活性誘導(dǎo)足細(xì)胞凋亡[5]。但miR-218在體內(nèi)是否參與腎臟保護(hù)作用尚不清楚。研究表明miR-218在癌癥的發(fā)生發(fā)展中發(fā)揮著重要作用。在髓母細(xì)胞瘤病人中，miR-218下調(diào)并扮能夠抑制腫瘤的發(fā)生[10]。在口腔鱗狀細(xì)胞癌中，DNA甲基化使miR-218沉默并抑制蛋白激酶B(protein kinase B, Akt)的磷酸化[11]。在宮頸癌中，過表達(dá)miR-218可通過靶向結(jié)合LIM和SH3蛋白1(LIM and SH3 protein 1，LASP1)抑制HeLa細(xì)胞生長[12]。本文以STZ誘導(dǎo)的大鼠為動(dòng)物模型，驗(yàn)證了miR-218在糖尿病腎病中的作用。首先，研究將50 mg/kg STZ腹腔注射入大鼠體內(nèi)，72 h后全血血糖結(jié)果表明注射STZ的大鼠血糖≥16.7 mmol/L，表明動(dòng)物模型構(gòu)建成功。RT-qPCR結(jié)果表明在STZ誘導(dǎo)的大鼠腎臟組織內(nèi)，miR-218表達(dá)水平顯著高于健康對(duì)照組，與體外實(shí)驗(yàn)結(jié)果一致。與此同時(shí)，Kanki等[13]也表明miR-218在高糖誘導(dǎo)的腎細(xì)胞中高表達(dá)，說明miR-218可能參與了糖尿病引起的腎病的發(fā)展進(jìn)程。本文研究也發(fā)現(xiàn)抑制miR-218的表達(dá)可顯著改善由STZ引起的大鼠24 h尿蛋白量、SCr及BUN水平升高。同時(shí)，抑制miR-218表達(dá)也會(huì)影響大鼠血糖水平。研究表明在胰島細(xì)胞MIN6中，過表達(dá)miR-218可顯著降低胰島素分泌，并降低葡萄糖敏感性，而抑制miR-218可顯著促進(jìn)胰島素分泌[14]。Chang等[15]發(fā)現(xiàn)在3T3-L1脂肪細(xì)胞中，胰島素可通過PI3K和蛋白激酶C依賴的信號(hào)通路上調(diào)HO-1蛋白表達(dá)。因而我們推測，抑制miR-218升高可促進(jìn)細(xì)胞內(nèi)胰島素分泌，從而降低動(dòng)物血糖水平。體外實(shí)驗(yàn)表明，miR-218是通過靶向作用于HO-1，進(jìn)而調(diào)節(jié)p38 MAPK的活性發(fā)揮促進(jìn)足細(xì)胞凋亡的作用，抑制p38 MAPK的活性可減弱足細(xì)胞的凋亡。研究表明在糖尿病腎病患者及小鼠體內(nèi)，p38 MAPK的磷酸化水平升高[16-17]。HO-1是一個(gè)血紅素分解代謝的限速酶，可降解血紅素為等摩爾一氧化碳、膽綠素和鐵離子。抑制HO-1會(huì)導(dǎo)致胞內(nèi)血紅素的堆積[18]，而血紅素可通過活化Toll樣受體4活化p38 MAPK[19]。近期的研究也表明，在骨髓來源的不成熟樹突細(xì)胞中，抑制HO-1表達(dá)后，血紅素表達(dá)升高，從而使磷酸化p38 MAPK的表達(dá)水平顯著升高，達(dá)到調(diào)節(jié)樹突細(xì)胞的功能和表型的作用[20]。Liu等[21]發(fā)現(xiàn)，在急性腎損傷骨髓來源的間充質(zhì)干細(xì)胞，HO-1可負(fù)調(diào)節(jié)p38 MAPK磷酸化。上述研究表明HO-1可通過調(diào)節(jié)血紅素的表達(dá)水平負(fù)調(diào)節(jié)p38 MAPK磷酸化。本研究進(jìn)一步驗(yàn)證了miR-218作用的分子機(jī)制，實(shí)驗(yàn)結(jié)果表明miR-218能夠下調(diào)HO-1的表達(dá)，上調(diào)p38 MAPK的活性。大量研究表明，磷酸化p38 MAPK水平在糖尿病腎病患者中升高，且激活p-p38 MAPK會(huì)促進(jìn)腎小球系膜細(xì)胞增殖及系膜外基質(zhì)大量合成和積聚[22]。同時(shí)HO-1可通過上調(diào)系膜細(xì)胞p21蛋白(一種與細(xì)胞凋亡相關(guān)的細(xì)胞周期蛋白)表達(dá)抑制腎小球系膜細(xì)胞增殖[23]。本文研究提示，HO-1可能是通過抑制p-p38 MAPK活化，從而延緩腎小球系膜增殖達(dá)到保護(hù)腎臟的作用。

慢病毒載體是一種新型的病毒載體系統(tǒng)，可有效地將目的基因?qū)氲絼?dòng)物或人的腦、腎臟等組織及原代細(xì)胞和穩(wěn)定細(xì)胞系中，具有感染效率高，安全和持續(xù)穩(wěn)定的表達(dá)目的基因特點(diǎn)，具有潛在的生物基因治療價(jià)值[24]。Espana-Agusti等[25]通過超聲引導(dǎo)注射慢病毒載體至腎小管，建立了長效的體內(nèi)調(diào)節(jié)基因表達(dá)的方法。本文通過構(gòu)建慢病毒介導(dǎo)的miR-218-shRNA載體并轉(zhuǎn)染大鼠腎臟，發(fā)現(xiàn)miR-218-shRNA可有效沉默miR-218基因的表達(dá)，為糖尿病腎病的治療提供了有效的靶點(diǎn)。

綜上所述，本實(shí)驗(yàn)結(jié)果表明miR-218在STZ誘導(dǎo)的大鼠腎臟組織中高表達(dá)，慢病毒載體沉默miR-218基因表達(dá)可以顯著抑制腎臟組織的凋亡，提示miR-218有望成為治療糖尿病腎病的治療靶點(diǎn)。

[1] Fineberg D, Jandeleit-Dahm KA, Cooper ME. Diabetic nephropathy: diagnosis and treatment[J]. Nat Rev Endocrinol, 2013, 9(12):713-723.

[2] Trionfini P, Benigni A, Remuzzi G. MicroRNAs in kidney physiology and disease[J]. Nat Rev Nephrol, 2015, 11(1): 23-33.

[3] Fernandez-Fernandez B, Ortiz A, Gomez-Guerrero C, et al. Therapeutic approaches to diabetic nephropathy-beyond the RAS[J]. Nat Rev Nephrol, 2014, 10(6):325-346.

[4] 趙樂萍， 金雷鋼， 施立華， 等.CXCL16缺失緩解STZ誘導(dǎo)的糖尿病小鼠的腎臟病變[J]. 中國病理生理雜志, 2016, 32(2):327-332.

[5] Yang H, Wang Q, Li S. MicroRNA-218 promotes high glucose-induced apoptosis in podocytes by targeting heme oxygenase-1[J]. Biochem Biophys Res Commun, 2016, 471(4):582-588.

[6] Kato M, Putta S, Wang M, et al. TGF-β activates Akt kinase through a microRNA-dependent amplifying circuit targeting PTEN[J]. Nat Cell Biol, 2009, 11(7):881-889.

[7] Harvey SJ, Jarad G, Cunningham J, et al. Podocyte-specific deletion of dicer alters cytoskeletal dynamics and causes glomerular disease[J]. J Am Soc Nephrol, 2008, 19(11):2150-2158.

[8] Shi S, Yu L, Chiu C, et al. Podocyte-selective deletion of dicer induces proteinuria andglomerulosclerosis[J]. J Am Soc Nephrol, 2008, 19(11): 2159-2169.

[9] Long J, Wang Y, Wang W, et al. MicroRNA-29c is a signature microRNA under high glucose conditions that targets Sprouty homolog 1, and itsinvivoknockdown prevents progression of diabetic nephropathy[J]. J Biol Chem, 2011, 286(13):11837-11848.

[10]Venkataraman S, Birks DK, Balakrishnan I, et al. MicroRNA 218 acts as a tumor suppressor by targeting multiple cancer phenotype-associated genes in medulloblastoma[J]. J Biol Chem, 2013, 288(3):1918-1928.

[11]Uesugi A, Kozaki K, Tsuruta T, et al. The tumor suppressive microRNA miR-218 targets the mTOR component Rictor and inhibits AKT phosphorylation in oral cancer[J]. Cancer Res, 2011， 71(17)：5765-5778.

[12]邱 瑜， 黃建平， 周勤仙， 等. Hsa-miR-218 靶向調(diào)控LASP1對(duì)宮頸癌HeLa細(xì)胞生長的影響[J]. 中國病理生理雜志, 2015, 31(9):1572-1577.

[13]Kanki M, Moriguchi A, Sasaki D, et al. Identification of urinary miRNA biomarkers for detecting cisplatin-induced proximal tubular injury in rats[J]. Toxicology, 2014, 324:158-168.

[14]Lang H, Ai Z, You Z, et al. Characterization of miR-218/322-Stxbp1 pathway in the process of insulin secretion[J]. J Mol Endocrinol, 2015, 54(1):65-73.

[15]Chang CL, Au LC, Huang SW, et al. Insulin up-regulates heme oxygenase-1 expression in 3T3-L1 adipocytes via PI3-kinase- and PKC-dependent pathways and heme oxygenase-1-associated microRNA downregulation[J]. Endocrinology, 2011, 152(2):384-393.

[16]Maestroni A, Tentori F, Meregalli G, et al. Inhibition of MAP-kinase cascade normalizes the proliferation rate of fibroblasts from patients with type 1 diabetes and nephropathy[J]. J Diabetes Complications, 2005, 19(5):291-296.

[17]Lim AK, Nikolic-Paterson DJ, Ma FY, et al. Role of MKK3-p38 MAPK signalling in the development of type 2 diabetes and renal injury in obese db/db mice[J]. Diabetologia, 2009, 52(2):347-358.

[18]Wagener FA, Volk HD, Willis D, et al. Different faces of the heme-heme oxygenase system in inflammation[J]. Pharmacol Rev, 2003, 55(3):551-571.

[19]Figueiredo RT, Fernandez PL, Mourao-Sa DS, et al. Characterization of heme as activator of Toll-like receptor 4[J]. J Biol Chem, 2007, 282(28): 20221-20229.

[20]Al-Huseini LM, Aw Yeang HX, Hamdam JM, et al. Heme oxygenase-1 regulates dendritic cell function through modulation of p38 MAPK-CREB/ATF1 signaling[J]. J Biol Chem, 2014, 289(23):16442-16451.

[21]Liu N, Wang H, Han G, et al. Alleviation of apoptosis of bone marrow-derived mesenchymal stem cells in the acute injured kidney by heme oxygenase-1 gene modification[J]. Int J Biochem Cell Biol, 2015, 69:85-94.

[22]Adhikary L, Chow F, Nikolic-Paterson DJ, et al. Abnormal p38 mitogen-activated protein kinase signalling in human and experimental diabetic nephropathy[J]. Diabetologia, 2004, 47(7):1210-1222.

[23]Kumar D, Bhaskaran M, Alagappan L, et al. Heme oxygenase-1 modulates mesangial cell proliferation by p21waf1upregulation[J]. Ren Fail, 2010, 32(2): 254-258.

[24]Naldini L, Trono D, Verma IM. Lentiviral vectors, two decades later[J]. Science, 2016, 353(6304):1101-1102.

[25]Espana-Agusti J, Tuveson DA, Adams DJ, et al. A minimally invasive, lentiviral based method for the rapid and sustained genetic manipulation of renal tubules[J]. Sci Rep, 2015, 5:11061.

(責(zé)任編輯： 林白霜， 羅 森)

Protective effect of microRNA-218 silencing on kidney tissue in STZ-induced diabetic rats

YANG Hai-bo1, WANG Qing-jun2, LI Su-tong1, CHEN Xiao-lin1, MU Ting1

(1DepartmentofNephrology,Xi’anCentralHospital,Xi’an710003,China;2DepartmentofNephrology,TongchuanMiningCentralHospital,Tongchuan727000,China.E-mail:haiboyangsea@163.com)

AIM: To investigate the protective effect of microRNA-218 (miR-218) silencing on kidney tissue of streptozotocin (STZ)-induced diabetic nephropathy rats and the potential mechanism. METHODS: The diabetic rat model was established by a single intraperitoneal injection of STZ (50 mg/kg). Meanwhile, the miR-218 short hairpin RNA (shRNA) lentiviral vector was constructed. The Sprague-Dawley rats were randomly divided into 4 groups： healthy control group, diabetes group, empty vector group and miR-218-shRNA group. The blood glucose, 24 h urinary protein, serum creatinine (SCr) and blood urea nitrogen (BUN) in the rats at different time points (4, 8 and 12 weeks) were measured by an automated analyzer. The expression of miR-218 was detected by RT-qPCR, while the expression of heme oxygenase-1 (HO-1), nephrin and p38 mitogen-activated protein kinase (p38 MAPK) at mRNA and protein levels in the kidney tissues was determined by RT-qPCR and Western blot. The caspase-3 activity was detected by caspase-3 activity assay kit, and the cell apoptosis of the kidney tissues was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). RESULTS: Compared with healthy control group, the expression of miR-218 was significantly increased in STZ-treated rats. Meanwhile, the concentrations of blood glucose, 24 h urinary protein, SCr and BUN were significantly increased in STZ-treated rats (P<0.05). The mRNA and protein expression of HO-1 and nephrin was significantly decreased， while the level of phosphorylated p38 MAPK was significantly increased in STZ-treated rats. In addition, the activity of caspase-3 was also significantly increased in STZ-treated rats. When the model rats were infected with miR-218-shRNA, the expression of miR-218 was significantly decreased and the above effects were markedly reversed. Furthermore, TUNEL results showed that compared with diabetic group and empty vector group, miR-218 silencing significantly attenuated the cell apoptosis in the kidney tissues in miR-218-shRNA group. CONCLUSION: miR-218 is involved in the kidney injury in diabetic rats, and silencing of miR-218 by lentiviral vector-mediated miR-218-shRNA transfection effectively inhibits kidney cell apoptosis, suggesting that miR-218 is a potential target for the treatment of diabetic nephropathy.

MicroRNA-218; Lentiviral vector; Diabetes; Kidney

1000- 4718(2017)07- 1251- 07

2016- 09- 19

2017- 04- 10

R587.2； R363.2

A

10.3969/j.issn.1000- 4718.2017.07.016

雜志網(wǎng)址： http://www.cjpp.net

△通訊作者 Tel: 029-87218916; E-mail: haiboyangsea@163.com