miR-675調(diào)控低氧誘導(dǎo)肺動(dòng)脈平滑肌細(xì)胞的增殖

王貝諾 劉雪萍 張君國(guó) 賀斌峰

·論著·

miR-675調(diào)控低氧誘導(dǎo)肺動(dòng)脈平滑肌細(xì)胞的增殖

王貝諾1劉雪萍2張君國(guó)3賀斌峰1

目的探討miR-675對(duì)低氧誘導(dǎo)大鼠肺動(dòng)脈平滑肌細(xì)胞(PASMCs)增殖的調(diào)控作用及相關(guān)機(jī)制。方法低氧(1% O2)處理PASMCs 48 h，利用Real time-PCR檢測(cè)0、6、12、24和48 h時(shí)各個(gè)時(shí)相點(diǎn)miR-675的表達(dá)水平，應(yīng)用Western blot檢測(cè)低氧處理PASMCs 48 h 后靶基因REPS2蛋白的表達(dá)情況。先合成miR-675抑制劑(inhibitor)再將其和陰性對(duì)照(NC)轉(zhuǎn)染PASMCs細(xì)胞，24 h后再進(jìn)行低氧處理從0h至48 h，應(yīng)用MTT法檢測(cè)0、12、24、48 h時(shí)細(xì)胞的活力，并檢測(cè)REPS2蛋白的表達(dá)狀況。構(gòu)建野生型和突變型REPS2 3′UTR 插入pMIR-REPORTTMluciferase vector載體，并將其與pRL-TK質(zhì)粒共轉(zhuǎn)染PASMCs細(xì)胞，之后將miR-675 inhibitor和NC分別轉(zhuǎn)染PASMCs細(xì)胞，利用雙熒光素酶報(bào)告基因檢測(cè)活性。在常氧下先分別轉(zhuǎn)染miR-675 inhibitor或模擬物(minic)48 h，再檢測(cè)PASMCs中REPS2的表達(dá)水平。結(jié)果隨著低氧處理時(shí)間的延長(zhǎng)，miR-675 在PASMCs細(xì)胞中的表達(dá)水平逐漸增高(P<0.05)。而與對(duì)照組比較，REPS2蛋白在低氧處理的PASMCs細(xì)胞中表達(dá)顯著下調(diào)(P<0.05)。miR-675 inhibitor組在24 h和48 h的細(xì)胞活力顯著低于對(duì)照組和miR-NC組(P<0.01)。熒光素酶報(bào)告基因結(jié)果說明REPS2是miR-675的靶基因。在常氧環(huán)境上調(diào)miR-675可顯著下調(diào)REPS2蛋白表達(dá)，而在常氧和低氧環(huán)境下抑制miR-675的水平可明顯升高REPS2蛋白表達(dá)。結(jié)論miR-675通過下游靶基因REPS2調(diào)控低氧誘導(dǎo)的PASMCs的異常增殖，可能是低氧肺動(dòng)脈高壓、肺源性心臟病等疾病治療的潛在靶點(diǎn)。

miR-675； 低氧； 肺動(dòng)脈平滑?。?肺血管重構(gòu)； REPS2； 增殖

低氧肺血管重構(gòu)(hypoxia pulmonary vascular remodeling, HPR)及肺動(dòng)脈高壓是慢性阻塞性肺疾病(chronic obstructive pulmonary diseases, COPD)發(fā)展為肺源性心臟病和呼吸衰竭的關(guān)鍵病理學(xué)基礎(chǔ)[1]。在我國(guó)COPD、慢性肺源性心臟病、高原肺水腫、高原心臟病等多種與肺血管重構(gòu)關(guān)系密切的重要疾病已成為社會(huì)的主要負(fù)擔(dān)和臨床診治的難點(diǎn)。目前認(rèn)為肺血管重構(gòu)的主要特征之一是在各種刺激因素下(包括低氧)肺動(dòng)脈平滑肌異常增生導(dǎo)致的血管結(jié)構(gòu)重塑[2]。因此，探索阻止和逆轉(zhuǎn)肺動(dòng)脈平滑肌細(xì)胞的異常增殖依然是防治肺血管重構(gòu)的重點(diǎn)。

普遍存在于各種細(xì)胞內(nèi)的microRNAs (miRNA)是一種非編碼RNA，一般長(zhǎng)度僅有18-25bp。研究表明miRNA或miRNA與其他非編碼RNA協(xié)同參與了各種生理、病理生命現(xiàn)象的調(diào)控，其主要的調(diào)控機(jī)制是通過與靶基因mRNA的3′UTR完全/不完全互補(bǔ)結(jié)合，起到抑制mRNA的翻譯或介導(dǎo)mRNA的降解，從而抑制靶蛋白的表達(dá)及其功能[3]。最近的miRNA的研究表明，miRNA在低氧誘導(dǎo)肺動(dòng)脈高壓、慢阻肺中發(fā)揮著重要的作用[4-5]。既往研究表明，miR-675在低肌力和低非脂肪量指數(shù)(Fat-free mass idex, FFMI) 的COPD患者血漿中呈高表達(dá)[6]，而低肌力和低FFMI意味著高死亡率[7]，提示miR-675的高表達(dá)與COPD患者預(yù)后較差關(guān)系密切。其他研究還表明，上調(diào)miR-675可促進(jìn)多種腫瘤的增殖、轉(zhuǎn)移和侵襲[8-10]。但目前miR-675在低氧肺血管重構(gòu)中的表達(dá)及其作用尚不清楚。本研究主要檢測(cè)miR-675在低氧誘導(dǎo)的肺動(dòng)脈平滑肌細(xì)胞中的表達(dá)，并探索其在增殖中的作用及其可能的機(jī)制，為低氧肺血管重構(gòu)、慢阻肺及肺心病的治療提供新的靶點(diǎn)。

材料與方法

一、主要材料

DMEM培養(yǎng)基GIBCO公司，胎牛血清購(gòu)自PAN公司，TRIzol regent 購(gòu)自sigma公司，lipofectamine 2000購(gòu)自Invirogen公司；陰性對(duì)照(Negative control, NC)、miR-675 minic和miR-675 inhibitor均購(gòu)自上海生工；逆轉(zhuǎn)錄試劑盒、T4連接酶、HindⅢ和Spe I限制性內(nèi)切酶購(gòu)自Fermentas 公司；實(shí)時(shí)定量試劑盒購(gòu)自Roche公司；REPS2抗體購(gòu)自abcam公司，蛋白裂解液和GAPDH單克隆抗體購(gòu)自上海夢(mèng)至生物科技有限公司；CKK-8試劑盒購(gòu)自江蘇碧云天；pMIR-REPORTTMluciferase和pRL-TK 質(zhì)粒載體購(gòu)自clonetech生物技術(shù)公司；熒光素酶報(bào)告基因檢測(cè)試劑盒購(gòu)自peromega公司。

二、研究方法

1. 大鼠肺動(dòng)脈平滑肌細(xì)胞(pulmonary artery smooth muscle cells, PASMCs)的培養(yǎng)： PASMCs細(xì)胞用含有10%胎牛血清的DMEM培養(yǎng)基進(jìn)行培養(yǎng)，取對(duì)數(shù)生長(zhǎng)期的細(xì)胞進(jìn)行實(shí)驗(yàn)。

2. PASMCs的低氧處理：將PASMCs原有培養(yǎng)基棄去，加入1%胎牛血清的DMEM培養(yǎng)基中培養(yǎng)12 h進(jìn)行同步化，然后棄去培養(yǎng)基加入10%胎牛血清的DMEM培養(yǎng)基，將細(xì)胞放入低氧培養(yǎng)箱中以1% O2下進(jìn)行低氧處理。

3. miR-675表達(dá)的檢測(cè)： 按照TRIzol Regent說明書提取細(xì)胞的總RNA。應(yīng)用頸環(huán)法檢測(cè)miR-675的表達(dá)。miR-675的特異逆轉(zhuǎn)錄引物為5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAG

ACTGAGCG-3′，內(nèi)參U6的逆轉(zhuǎn)錄引物為5′-CAAAATATGGAACGCTTC-3′，逆轉(zhuǎn)錄條件為42 ℃ 60 min，72 ℃ 5 min。 逆轉(zhuǎn)錄獲得的cDNA進(jìn)行實(shí)時(shí)熒光定量PCR檢測(cè)miR-675的表達(dá)，以U6作為內(nèi)參，序列見表1。每個(gè)待測(cè)基因設(shè)2個(gè)復(fù)孔。數(shù)據(jù)通過BIO-RAD CFX96系統(tǒng)進(jìn)行處理，按照公式RQ=2-△△CT計(jì)算各組間的關(guān)系。

表1 基因引物序列

4. miR-675 模擬物(minic)、miR-675抑制物(inhibitor)及NC轉(zhuǎn)染PASMCs ： 將miR-675 minic、miR-675 inhibitor及NC干粉用125 μl RNase-free H2O 配制成20μmol/L的儲(chǔ)存液。miR-675 minic轉(zhuǎn)染PASMCs細(xì)胞的步驟如下：① 將PASMCs消化后，在24孔板中接種約2×105個(gè)/孔的細(xì)胞；②過夜培養(yǎng)后，棄去培養(yǎng)基，每孔加入40 μl的opti-MEM培養(yǎng)基；③將2.5 μl濃度為20μM 的miR-675 minic加入到47.5l opti-MEM中，輕輕混勻，室溫孵育5 min，將另外一支裝有48μl opti-MEM中EP管中加入2 μl轉(zhuǎn)染試劑lipofectamine 2000，輕輕吹打混勻，室溫孵育5 min；④將lipofectamine 2000吸入裝有miR-675 minic的EP管中，輕輕混勻，室溫孵育20 min；⑤將100μl混合試劑加入24孔板中，充分混勻；⑥細(xì)胞培養(yǎng)48 h后進(jìn)行PCR和Western blot等檢測(cè)。miR-675 inhibitor及NC的轉(zhuǎn)染同miR-675 minic轉(zhuǎn)染步驟。

5. MTT檢測(cè)細(xì)胞增殖：取對(duì)數(shù)生長(zhǎng)期的PASMCs細(xì)胞消化后制成細(xì)胞懸液，將細(xì)胞濃度調(diào)整到1×104/孔，接種于96孔板。過夜培養(yǎng)后進(jìn)行低氧或轉(zhuǎn)染處理，處理完成后繼續(xù)培養(yǎng)48 h后換液，每孔加入10 μl(5 mg/ml)的MTT溶液。37 ℃孵育4 h后，每孔加入150 μl Formanzan溶液，繼續(xù)孵育15 min可觀察到Formanzan全部溶解。用酶標(biāo)儀在570 nm處檢測(cè)各孔吸光度(OD值)，空白孔調(diào)零。

6. 熒光素酶報(bào)告基因檢測(cè)：構(gòu)建野生型和突變型REPS2 3′UTR pMIR-REPORTTMluciferase vector載體；并將上述載體分別與pRL-TK質(zhì)粒共轉(zhuǎn)染PASMCs細(xì)胞。培養(yǎng)24 h后分別將等量的miR-675 minic和NC轉(zhuǎn)染PASMCs；轉(zhuǎn)染48 h后，檢測(cè)螢火蟲和海腎熒光素酶活性。

7. Western blot檢測(cè)REPS2蛋白的表達(dá)：在培養(yǎng)皿中加入200 μl蛋白裂解液，置于冰上裂解10 min，然后將其收集，于4 ℃、以離心半徑8 cm、12 000 r/min離心20 min，取上清液。蛋白變性后進(jìn)行SDS-PAGE電泳。蛋白轉(zhuǎn)印后，將PVDF膜室溫封閉1 h，然后將膜放入含有REPS2抗體 (1︰1 000)及GAPDH抗體(1︰20 000)的抗體稀釋液中，4 ℃過夜。TBST洗3次后孵育二抗，用ECL化學(xué)發(fā)光法檢測(cè)REPS2和GAPDH蛋白的表達(dá)水平， GAPDH作為內(nèi)參來校正各自目的蛋白。

三、統(tǒng)計(jì)學(xué)方法

結(jié) 果

一、低氧干預(yù)PASMCs后miR-675的表達(dá)水平

利用qPCR檢測(cè)了miR-675在低氧處理后PASMCs 中的表達(dá)。發(fā)現(xiàn)在低氧處理PASMCs的6、12、24和48 h時(shí)相點(diǎn)miR-675的表達(dá)分別是0 h的1.21±0.03、1.81±0.11、2.59±0.14和3.13±0.07倍(P<0.05)，miR-675在低氧處理后PASMCs 中表達(dá)呈時(shí)間依賴性，見圖1。

圖1 qPCR檢測(cè)miR-675在低氧處理的PASMCs細(xì)胞中的表達(dá)；注：*與0 h，P<0.05；**與0 h，P<0.05

二、轉(zhuǎn)染miR-675 inhibitor對(duì) 低氧處理下PASMCs細(xì)胞活力的影響

分別將miR-675 inhibitor和NC 轉(zhuǎn)染PASMCs細(xì)胞24 h后，將細(xì)胞置于1% O2的低氧環(huán)境下處理48 h，利用qPCR檢測(cè)PASMCs細(xì)胞中的miR-675表達(dá)水平，發(fā)現(xiàn)miR-675 inhibitor和NC組中miR-675的相對(duì)表達(dá)量分別是對(duì)照組的(50.00±6.01)%和(99.67±2.00)%，miR-675 inhibitor組中miR-675的表達(dá)顯著低于NC組和單純低氧組(P<0.05) ，見圖2A。我們發(fā)現(xiàn)低氧處理24 h、48 h后，Hypoxia組、Hypoxia+NC組和Hypoxia+miR-675 inhibitor組細(xì)活力均顯著高于低氧處理0 h和12 h這三組的細(xì)胞活力(P<0.05) ，并且在低氧處理24 h、48 h時(shí)Hypoxia+miR-675 inhibitor組細(xì)胞活力顯著低于Hypoxia組和Hypoxia+NC組(P<0.05)。在各個(gè)時(shí)相點(diǎn)Hypoxia組和Hypoxia+NC組細(xì)胞活力無顯著差異(P<0.05)，見圖2B。上述研究說明下調(diào)miR-675的表達(dá)可顯著抑制低氧誘導(dǎo)PASMCs異常增殖。

圖2 下調(diào)miR-675對(duì)PASMCs細(xì)胞活力的影響；注：A：qPCR檢測(cè)PASMCs轉(zhuǎn)染miR-675 inhibitor和NC后的miR-675的表達(dá)；*與Hypoxia組比較，P<0.01；^與Hypoxia+NC組比較，P<0.01；B：MTT檢測(cè)PASMCs細(xì)胞轉(zhuǎn)染miR-675 inhibitor和NC后，其對(duì)低氧誘導(dǎo)PASMCs的細(xì)胞活力影響。*與0 h比較，P<0.01；^與12 h組比較，P<0.01；#與Hypoxia組比較，P<0.01；&與Hypoxia+NC組比較，P<0.01

三、REPS2是miR-675作用的下游直接靶點(diǎn)

通過生物信息學(xué)分析，認(rèn)為REPS2可能是為miR-675的靶基因。分別構(gòu)建REPS2野生型和突變型3′UTR質(zhì)粒熒光素酶報(bào)告載體，見圖3A。上述載體與pRL-TK質(zhì)粒、miR-675 minic或NC一起共轉(zhuǎn)染PASMCs細(xì)胞，然后檢測(cè)熒光素酶活性。結(jié)果顯示野生型REPS2 3′UTR質(zhì)粒和miR-675 minic共轉(zhuǎn)染組的熒光素酶活性低于野生型REPS2 3′UTR質(zhì)粒和NC共轉(zhuǎn)染組(P<0.01)。miR-675 minic和突變型REPS2 3′UTR質(zhì)粒共轉(zhuǎn)染組與突變型REPS2 3′UTR質(zhì)粒和NC共轉(zhuǎn)染組相比較，兩組熒光素酶活性無顯著差異(P>0.05)。上述研究結(jié)果證實(shí)REPS2是miR-675的作用靶點(diǎn)，見圖3B。此外，我們還發(fā)現(xiàn)miR-675 minic轉(zhuǎn)染PASMCs細(xì)胞后，其REPS2蛋白的表達(dá)較NC組可顯著降低(P<0.05)，見圖3C。而用miR-675 inhibitor抑制PASMCs中miR-675的表達(dá)，REPS2蛋白的表達(dá)水平較NC組明顯升高(P<0.05)，見圖3D。進(jìn)一步說明REPS2是miR-675的直接靶點(diǎn)。

圖3 REPS2是miR-675的靶點(diǎn)；注：A：miR-675與野生型和突變型REPS2 3′UTR端的示意圖；B：熒光素酶報(bào)告基因檢測(cè)miR-675與REPS2 3′UTR端的結(jié)合情況；*與野生型REPS2 3′UTR質(zhì)粒和miR-675 minic共轉(zhuǎn)染組，P<0.01；C和D：轉(zhuǎn)染miR-675 minic/miR-675 inhibitor與NC 48 h后，western blot檢測(cè)PASMCs細(xì)胞中REPS2蛋白的表達(dá)情況

四、下調(diào)miR-675促進(jìn)低氧處理PASMCs中REPS2的表達(dá)

前面的研究發(fā)現(xiàn)低氧處理后PASMCs中miR-675的表達(dá)顯著升高，但其靶點(diǎn)REPS2的表達(dá)并不清楚。因此，我們檢測(cè)低氧處理PASMCs 48 h后REPS2蛋白的表達(dá)情況，見圖4A。發(fā)現(xiàn)低氧處理后REPS2較常氧組顯著下調(diào)。轉(zhuǎn)染miR-675 inhibitor、NC后繼續(xù)低氧處理48 h后，見圖4B。檢測(cè)發(fā)現(xiàn)miR-675 inhibitor組和NC組REPS2的表達(dá)顯著高于低氧處理組，并且REPS2蛋白在miR-675 inhibitor組的表達(dá)明顯高于NC組。

圖4 下調(diào)miR-675促進(jìn)低氧處理PASMCs中REPS2的表達(dá)；注：A：REPS2蛋白在低氧和常氧下PASMCs中的表達(dá)；B：轉(zhuǎn)染miR-675 inhibitor和NC后，PASMCs繼續(xù)低氧處理后REPS2蛋白的表達(dá)水平

討 論

本研究發(fā)現(xiàn)miR-675在PASMCs中隨低氧處理時(shí)間的延長(zhǎng)而表達(dá)逐漸升高。利用miR-675 inhibitor可有效抑制低氧誘導(dǎo)的PASMCs異常增殖。此外，還發(fā)現(xiàn)增殖相關(guān)負(fù)性調(diào)節(jié)因子REPS2是miR-675的直接靶點(diǎn)。因此，我們推測(cè)miR-675通過調(diào)控REPS2，進(jìn)而發(fā)揮調(diào)控PASMCs增殖的作用。

最近研究的表明，miRNA的紊亂在低氧肺血管重構(gòu)的發(fā)生和發(fā)展中發(fā)揮著重要作用[11]。血清中循環(huán)的miRNA，如miR-20, miR-28-3p, miR-34c-5p and miR-100 and miR-7可作為評(píng)估、監(jiān)測(cè)COPD進(jìn)展的生物標(biāo)志物[12]。Courboulin 等[13-15]指出miR-204在肺動(dòng)脈平滑肌中的異常上調(diào)激活NFAT，下調(diào)BMPR2，促進(jìn)IL-6的生產(chǎn)，導(dǎo)致肺動(dòng)脈平滑肌細(xì)胞的異常增殖和凋亡的抵抗。同樣，miR-21在低氧暴露的人PASMCs中也顯著升高[16]，其可能通過下調(diào)PDCD4、SPRY2和PPARα，進(jìn)而發(fā)揮促進(jìn)PASMCs增殖、抵抗凋亡的作用[17]。本實(shí)驗(yàn)發(fā)現(xiàn)，低氧上調(diào)PASMCs細(xì)胞中miR-675的表達(dá)。本研究應(yīng)用miR-675 inhibitor下調(diào)PASMCs細(xì)胞中miR-675的表達(dá)，結(jié)果顯示下調(diào)miR-675能抑制低氧誘導(dǎo)的PASMCs的增殖。此外，其他研究表明下調(diào)miR-675也可抑制肝癌、食管癌的增殖[18-19]，但具體的作用機(jī)制尚不清楚。

本研究在PASMCs中驗(yàn)證了REPS2 (RALBP1 associated Eps domain containing 2)是miR-675的靶基因。REPS2也被叫做POB1,其可編碼蛋白復(fù)合物的一部分，從而發(fā)揮調(diào)節(jié)生長(zhǎng)因子內(nèi)吞的作用。進(jìn)一步研究發(fā)現(xiàn)REPS2通過作用于小G蛋白R(shí)al的GTPase活性蛋白，進(jìn)而負(fù)性調(diào)節(jié)生長(zhǎng)因子受體的內(nèi)化和抑制生長(zhǎng)因子相關(guān)信號(hào)通路[20]。最近的研究發(fā)現(xiàn)REPS2在前列腺癌中的表達(dá)顯著降低，并且其可作為評(píng)估乳腺癌和前列腺癌進(jìn)展的潛在分子標(biāo)志物[21]。本研究發(fā)現(xiàn)REPS2是miR-675的靶點(diǎn)，并且調(diào)節(jié)miR-675的表達(dá)可影響REPS2蛋白的表達(dá)[19]。本研究還發(fā)現(xiàn)低氧下REPS2顯著下調(diào)，而此時(shí)PASMCs細(xì)胞由于處于合成期，增殖能力顯著升高[22]。在PASMCs處于低氧情況下時(shí)，miR-675表達(dá)受到抑制可上調(diào)REPS2的表達(dá)，并且下調(diào)的miR-675可抑制低氧誘導(dǎo)的PASMCs異常增殖。上述研究提示miR-675通過REPS2進(jìn)而調(diào)控 PASMCs的增殖。但是REPS2調(diào)控細(xì)胞增殖的具體機(jī)制仍不清楚。Tomassi 等[23]的研究指出REPS2蛋白含有中心脯氨酸富集域(central proline rich domain)，其負(fù)性調(diào)節(jié)激活的EGFR受體的內(nèi)化過程，進(jìn)而影響細(xì)胞的增殖。因此，我們推測(cè)在PASMCs中REPS2也可能通過上述機(jī)制調(diào)控細(xì)胞的增殖。

本研究尚存在一些不足：①研究在體外證實(shí)了miR-675通過REPS2調(diào)控PASMCs細(xì)胞增殖，但在體內(nèi)miR-675是否依然具有抑制PASMCs增殖和阻止低氧肺血管重構(gòu)的作用尚不清楚；②盡管我們發(fā)現(xiàn)REPS2可以調(diào)控低氧誘導(dǎo)的PASMCs的增殖，但是具體機(jī)制依然不明確。

綜上所述，下調(diào)miR-675通過促進(jìn)靶基因REPS2的表達(dá)，進(jìn)而發(fā)揮抑制低氧誘導(dǎo)的PASMCs細(xì)胞異常增殖的作用。上述研究提示miR-675可成為治療低氧肺動(dòng)脈高壓、慢阻肺、肺心病的潛在靶點(diǎn)。

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王貝諾，劉雪萍，張君國(guó)，等. miR-675調(diào)控低氧誘導(dǎo)肺動(dòng)脈平滑肌細(xì)胞的增殖[J/CD]. 中華肺部疾病雜志： 電子版， 2016， 9(6)： 625-630.

miR-675 modulates hypoxia-induced proliferation of pulmonary artery smooth muscle cell by targeting REPS2

WangBeinuo1,LiuXueping2,ZhangJunguo3,HeBinfeng1.

1DepartmentofRespiratoryDiseases,theSecondAffiliatedHospitaloftheThirdMilitaryMedicalUniversity,Chongqing400037,PRChina.2DepartmentofThoracicSurgery,theSecondAffiliatedHospitaloftheThirdMilitaryMedicalUniversity,Chongqing400037,PRChina;3EmergencyDepartment,Fengducountypeople′shospital,Chongqing400037,PRChina

HeBinfeng,Email:ldhbf@tmmu.edu.cn

Objective To explore the mechanism and effect of miR-675 regulation hypoxia induced proliferation of pulmonary artery smooth muscle cells (PASMCs). Method The relative expression of miR-675 have been detected by real time-PCR while PASMCs were under 1% O2hypoxia for 0, 6, 12, 24 and 48 h. The protein expression of REPS2 was measured by western blot when PASMC have been exposed at hypoxia for 48 h. The sequence of miR-675 inhibitor and NC were synthesized. They were transfected into PASMCs for 24 h, and then PASMCs were treated by hypoxia for 48 h. The cell viability and REPS2 protein levels of PASMCs was evaluated at 12, 24 and 48 h by MTT and western blot. The wild-type and mutation of REPS2 3′UTR was inserted into the plasmid of pMIR-REPORTTMluciferase vector, which was co-transfected into PASMCs with pRL-TK plasmid. miR-675 minic and NC was transfected into these cells, which had been transfected pMIR-REPORTTMluciferase vector and pRL-TK plasmid. Firefly and Renilla reniformis luciferase activities were measured 48 h later. Additional, the protein expression of REPS2 was detected when PASMCs was treated with miR-675 inhibitor or minic for 48 h. Result The miR-675 levels was time-depended manner while PASMCs were under hypoxia exposed (P<0.05). The expression of REPS2 protein was significant lower in hypoxia group, compared to normoxia group (P<0.05). The cell viability of PASMCs were makeable decreased under hypoxia 24 and 48 h after PASMCs had been transfected miR-675 inhibitor, compared to NC group(P<0.01). Luciferase assay showed REPS2 was a direct target gene of miR-675. Up-regulation of miR-675 was significant decreased the expression of REPS2 while miR-675 minic was transfected into PASMCs in normoxia. On the contrary, down-regulation miR-675 by miR-675 inhibitor was remarkable elevated REPS2 protein level both normoxia and hypoxia. Conclusion miR-675 regulated the hypoxia induced PASMCs proliferation by target gene REPS2, and it maybe consider as a novel treatment target for hypoxia PAH and cor pulmonary.

miR-675; Hypoxia; Pulmonary vascular remodeling; REPS2; Proliferation

10.3877/cma.j.issn.1674-6902.2016.06.010

國(guó)家自然科學(xué)基金面上項(xiàng)目(81670047)

400037 重慶，第三軍醫(yī)大學(xué)新橋醫(yī)院呼吸內(nèi)科1、胸外科2408200 重慶市豐都縣人民醫(yī)院急診科3

賀斌峰，Email: ldhbf@tmmu.edu.cn

R563

A

2016-11-02)