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        微小RNA-142-3p靶向Rictor誘導(dǎo)動(dòng)脈粥樣硬化相關(guān)的內(nèi)皮細(xì)胞凋亡*

        2020-12-03 13:42:54肖麗劉萍秦冰
        中國病理生理雜志 2020年11期
        關(guān)鍵詞:檢測(cè)

        肖麗, 劉萍, 秦冰

        微小RNA-142-3p靶向Rictor誘導(dǎo)動(dòng)脈粥樣硬化相關(guān)的內(nèi)皮細(xì)胞凋亡*

        肖麗, 劉萍, 秦冰△

        (中山大學(xué)附屬第三醫(yī)院神經(jīng)內(nèi)科,廣東 廣州 510630)

        探討微小RNA-142-3p (miR-142-3p)對(duì)動(dòng)脈粥樣硬化(atherosclerosis,AS)進(jìn)程中人主動(dòng)脈內(nèi)皮細(xì)胞(human aortic endothelial cells,HAECs)凋亡的影響及作用機(jī)制。HAECs經(jīng)氧化型低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)作用后,采用RT-qPCR檢測(cè)miR-142-3p的表達(dá)水平。Annexin V-FITC/PI雙染法流式細(xì)胞術(shù)(flow cytometry,F(xiàn)CM)和caspase-3活性檢測(cè)試劑盒檢測(cè)細(xì)胞凋亡。使用TargetScan等生物信息學(xué)軟件預(yù)測(cè)miR-142-3p的潛在靶基因,雙螢光素酶報(bào)告基因?qū)嶒?yàn)驗(yàn)證miR-142-3p與Rictor mRNA 3′-UTR直接靶向結(jié)合。在ox-LDL誘導(dǎo)HAECs凋亡過程中miR-142-3p表達(dá)顯著上調(diào)(<0.05,<0.01)。過表達(dá)miR-142-3p促進(jìn)HAECs凋亡,相反,抑制miR-142-3p的表達(dá)水平可以部分程度緩解ox-LDL介導(dǎo)的內(nèi)皮細(xì)胞(endothelial cells,ECs)凋亡。進(jìn)一步研究發(fā)現(xiàn),為miR-142-3p的下游靶基因,沉默基因抑制了miR-142-3p抑制物(miR-142-3p inhibitor)的抗凋亡作用。Akt/eNOS信號(hào)通路參與了miR-142-3p對(duì)ECs凋亡的調(diào)控。抑制miR-142-3p表達(dá)可促進(jìn)靶基因表達(dá)并激活A(yù)kt/eNOS信號(hào)通路從而發(fā)揮抗凋亡的內(nèi)皮保護(hù)作用。

        微小RNA-142-3p;人主動(dòng)脈內(nèi)皮細(xì)胞;細(xì)胞凋亡;Rictor;動(dòng)脈粥樣硬化

        動(dòng)脈粥樣硬化(atherosclerosis,AS)是一種炎癥性疾病,其啟動(dòng)因素為血管內(nèi)皮細(xì)胞(endothelial cells,ECs)損傷[1]。氧化型低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)作為AS的重要危險(xiǎn)因素之一,可以誘導(dǎo)ECs發(fā)生凋亡,繼而導(dǎo)致血管內(nèi)膜增生、炎癥細(xì)胞浸潤、脂質(zhì)沉積和斑塊破裂[2]。因此,抑制ox-LDL誘導(dǎo)的ECs凋亡對(duì)AS的預(yù)防和治療有重要意義。微小RNA(microRNA, miRNA, miR)是真核生物進(jìn)化中,一種高度保守的、新穎的、內(nèi)源性的非編碼小RNA,其通過降解或轉(zhuǎn)錄抑制靶RNA,負(fù)性調(diào)控超過30%的基因表達(dá)[3]。近年來有研究報(bào)道m(xù)iR-142-3p在心肌細(xì)胞凋亡、纖維化和內(nèi)皮屏障破壞等多種心血管疾病中發(fā)揮重要調(diào)控作用[4]。本研究通過制備ox-LDL誘導(dǎo)的人主動(dòng)脈內(nèi)皮細(xì)胞(human aortic endothelial cells, HAECs)凋亡模型,探討miR-142-3p對(duì)ox-LDL誘導(dǎo)的HAECs凋亡的作用及機(jī)制,為AS的防治提供一定的實(shí)驗(yàn)依據(jù)。

        材料和方法

        1 材料

        HAECs、內(nèi)皮細(xì)胞培養(yǎng)基和內(nèi)皮細(xì)胞生長因子購自ScienCell;胎牛血清(fetal bovine serum, FBS)、總RNA提取試劑Trizol、Opti-MEM培養(yǎng)基和Lipofectamine2000購自Invitrogen; ox-LDL購自北京協(xié)生生物有限公司; miRNA陰性對(duì)照模擬物(negative control mimic, NC mimic)、miR-142-3p模擬物(miR-142-3p mimic)、miRNA陰性對(duì)照抑制物(NC inhibitor)、miR-142-3p抑制物(miR-142-3p inhibitor)、siRNA和陰性對(duì)照siRNA (NC siRNA)購自廣州銳博生物科技有限公司; LY294002、L-NAME、caspase-3活性檢測(cè)試劑盒、CCK-8檢測(cè)試劑盒和Annexin V-FITC/PI凋亡檢測(cè)試劑盒購自江蘇碧云天生物科技有限公司; MTT購自AMRESCO; TaqMan MicroRNA Assays購自Applied Biosystems; mRNA實(shí)時(shí)熒光定量PCR試劑盒(SYBR?Premix Ex TaqTMII Kit)購自TaKaRa;引物由上海生工生物工程有限公司合成;Western blot實(shí)驗(yàn)中所用的Ⅰ抗[Rictor (sc-81538)、p-Akt (Ser473)(sc-514032)、Akt (sc-81434)、p-eNOS (Ser1177 )(sc-81510)、eNOS (sc-376751)、GAPDH (sc-32233)和β-actin (sc-130065)]購自Santa Cruz;辣根過氧化物酶標(biāo)記羊抗兔IgG購自KPL;pMIR-REPORT螢光素酶報(bào)告載體購自Ambion;抗CD31抗體(ab28364)購自Abcam; Alexa Fluor 488標(biāo)記的熒光Ⅱ抗購自EarthOx。

        2 方法

        2.1細(xì)胞培養(yǎng)原代HAECs接種在含F(xiàn)BS(50 mg/L)、青/鏈霉素和生長因子的內(nèi)皮細(xì)胞專用培養(yǎng)基中,培養(yǎng)條件為37℃、5% CO2培養(yǎng)箱內(nèi)培養(yǎng),待細(xì)胞長至80%融合度進(jìn)行傳代,穩(wěn)定培養(yǎng)至第3~6代的細(xì)胞用于后續(xù)實(shí)驗(yàn)。

        2.2細(xì)胞表型鑒定將細(xì)胞接種到12孔板,待細(xì)胞貼壁后培養(yǎng)細(xì)胞至融合度達(dá)30%~50%。采用75%乙醇在室溫條件下固定細(xì)胞15 min, PBS洗滌3次后, 1% Triton-100室溫下破膜10 min,5% BSA室溫條件下封閉30 min。加入1∶100稀釋的抗CD31抗體4℃孵育過夜,PBS洗滌3遍,再加入1∶400稀釋的Alexa Fluor 488標(biāo)記的羊抗兔IgG, 37℃避光孵育30 min,DAPI染核,熒光顯微鏡下觀察并拍照。

        取80%~90%融合的細(xì)胞,胰酶消化后,離心去上清,1% Triton X-100室溫破膜10 min, 5% BSA封閉30 min。加入1∶100稀釋的抗CD31抗體4℃孵育過夜, PBS清洗后再經(jīng)Alexa Fluor 488標(biāo)記的羊抗兔IgG, 37℃避光孵育30 min,流式細(xì)胞術(shù)檢測(cè)。

        2.3細(xì)胞轉(zhuǎn)染和細(xì)胞處理按照說明書分別將50 nmol/L的miR-142-3p mimic和miR-142-3p inhibitor轉(zhuǎn)染至HAECs,陰性對(duì)照組分別轉(zhuǎn)染NC mimic和NC inhibitor,之后再經(jīng)ox-LDL(100 mg/L)作用24 h。為檢測(cè)Akt/eNOS信號(hào)通路的作用,HAECs首先經(jīng)過生理鹽水、LY294002(20 μmol/L)或L-NAME(100 μmol/L)處理2 h,之后轉(zhuǎn)染miR-142-3p inhibitor或NC inhibitor,再經(jīng)ox-LDL(100 mg/L)作用24 h。救援實(shí)驗(yàn):HAECs中分別共轉(zhuǎn)染miR-142-3p inhibitor和50 μmol/L Rictor siRNA或NC siRNA,之后再經(jīng)ox-LDL(100 mg/L)作用24 h。

        2.4caspase-3活性檢測(cè)和流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡首先將待檢測(cè)的各組細(xì)胞密度調(diào)整為5×106/L,離心棄掉上清液,使用PBS洗滌2次,加入100 μL裂解緩沖液,冰上放置15 min,渦旋振蕩15 s,離心5 min。取10 μL蛋白上清液,加入90 μL caspase-3活性檢測(cè)液,再加入10 μL Ac-LEHD-pNA,避光反應(yīng)1~2 h后檢測(cè)結(jié)果。

        將HAECs懸液以1×109/L的濃度接種至6孔板,收集經(jīng)不同處理后的細(xì)胞并用Annexin V-FITC/PI凋亡檢測(cè)試劑盒染色,流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡率。Annexin V-FITC+PI-細(xì)胞為早期凋亡細(xì)胞,凋亡率(%)=早期凋亡細(xì)胞數(shù)/所檢測(cè)細(xì)胞總數(shù)×100%。

        2.5CCK-8實(shí)驗(yàn)測(cè)定細(xì)胞活力HAECs接種于96孔板,每孔100 μL,將培養(yǎng)板置于37℃、5% CO2的培養(yǎng)箱中培養(yǎng)1 d后,在不同處理組的每孔加入10 μL CCK-8溶液,孵育2 h后在450 nm波長下于酶標(biāo)儀測(cè)量吸光度()值。每個(gè)樣品重復(fù)5次。

        2.6RT-qPCR檢測(cè)miRNA的水平Trizol提取總RNA,采用TaqMan MicroRNA Assay Kits在7900HT實(shí)時(shí)熒光定量PCR儀上檢測(cè)各組樣本中miR-142-3p的表達(dá),以U6作為內(nèi)參照,相對(duì)表達(dá)量按照2-ΔΔCt法計(jì)算。miR-142-3p的正向引物序列為5'-GGGTGTAGTGTTTCCTACTT-3',反向引物序列為5'-TTTGGCACTAGCACATT-3'; U6的正向引物序列為5'-CTCGCTTCGGCAGCACA-3',反向引物序列為5'-AACGCTTCACGAATTTGCGT-3'。

        2.7Western blot檢測(cè)蛋白水平提取各細(xì)胞樣本總蛋白,定量,與上樣緩沖液按比例混勻,100℃水浴加熱5 min,經(jīng)8% SDS-PAGE后電轉(zhuǎn)移至PVDF膜上, 5%脫脂牛奶室溫封閉1 h,加入Ⅰ抗, 4℃孵育過夜, TBST洗滌3次,每10 min換液1次;加入Ⅱ抗, 37℃孵育45 min, TBST洗滌3次,每15 min換液1次,在暗室中壓片,然后顯影、定影。

        2.8RT-qPCR檢測(cè)mRNA的表達(dá)Trizol提取各組細(xì)胞樣本的總RNA,以β-actin為內(nèi)參照,檢測(cè)各樣本Rictor的mRNA表達(dá)水平。兩步法PCR擴(kuò)增標(biāo)準(zhǔn)程序?yàn)椋?95℃預(yù)變性30 s; 95℃ 5 s, 60℃ 30 s, 40個(gè)循環(huán)。采用2-ΔΔCt法計(jì)算各樣本相對(duì)表達(dá)量。Rictor的正向引物為5'-GGAAGCCTGTTGATGGTGAT-3',反向引物序列為5'-GGCAGCCTGTTTATGGTGT-3';GAPDH的正向引物序列為5'-AAGGTGAAGGTCGGAGTCAAC-3',反向引物序列為5'-GGGGTCATTGATGGCAACAATA-3'。

        2.9雙螢光素酶報(bào)告基因?qū)嶒?yàn)生物信息學(xué)預(yù)測(cè)顯示miR-142-3p與Rictor的3'-UTR存在結(jié)合位點(diǎn),將含有結(jié)合位點(diǎn)與突變位點(diǎn)的Rictor 3'-UTR片段分別插入螢光素酶報(bào)告基因載體構(gòu)建野生型載體pMIR-Rictor-3'-UTR與突變型載體pMIR-Rictor-3'-UTR Mutant,取生長狀態(tài)良好的HAECs,分別將pMIR-Rictor-3'-UTR、pMIR-Rictor-3'-UTR Mutant與NC mimic、miR-142-3p mimic共轉(zhuǎn)染并置于37℃、5% CO2的培養(yǎng)箱內(nèi)繼續(xù)培養(yǎng)48 h,檢測(cè)各組細(xì)胞的螢光素酶活性。

        3 統(tǒng)計(jì)學(xué)處理

        采用SPSS 23.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析。計(jì)量資料用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示,組間比較采用方差分析,以<0.05為差異有統(tǒng)計(jì)學(xué)意義。

        結(jié)果

        1 血管內(nèi)皮細(xì)胞鑒定

        原代HAECs接種在含F(xiàn)BS、青/鏈霉素和生長因子的內(nèi)皮細(xì)胞專用培養(yǎng)基中,培養(yǎng)條件為37℃、5% CO2培養(yǎng)箱內(nèi)培養(yǎng),待細(xì)胞長至80%融合度時(shí)進(jìn)行傳代,穩(wěn)定培養(yǎng)至第3~6代的細(xì)胞于倒置熒光顯微鏡下觀察示,所培養(yǎng)細(xì)胞CD31表達(dá)陽性,見圖1A。流式細(xì)胞術(shù)結(jié)果顯示,培養(yǎng)細(xì)胞CD31陽性表達(dá)率為(98.4±0.9)%,見圖1B。這說明本實(shí)驗(yàn)所用細(xì)胞為血管內(nèi)皮細(xì)胞。

        Figure 1. Identification of vascular endothelial cells. A: immunofluorescence staining of CD31 in the vascular endothelial cells (×400); B: the expression of CD31 in the vascular endothelial cells was measured by flow cytometry.

        2 miR-142-3p在HAECs凋亡過程中表達(dá)上調(diào)

        HAECs中加入ox-LDL (100 mg/L)分別培養(yǎng)不同時(shí)間(0~24 h),caspase-3活性測(cè)定和流式細(xì)胞術(shù)檢測(cè)顯示ox-LDL呈時(shí)間依賴性地誘導(dǎo)HAECs發(fā)生凋亡(<0.05或<0.01),見圖2A、B,且細(xì)胞活力顯著下降(<0.05或<0.01),見圖2C。RT-qPCR檢測(cè)HAECs凋亡過程中miR-142-3p表達(dá)的變化,結(jié)果顯示隨著HAECs凋亡率的升高, miR-142-3p的表達(dá)逐漸上調(diào)(<0.05或<0.01),見圖2D。

        Figure 2. Effects of ox-LDL on the apoptotic and the expression of miR-142-3p in HAECs. A: caspase-3 activity of HAECs was detected; B: flow cytometric analysis of apoptosis and necrosis; C: the viability of HAECs was measured by CCK8 assay; D: relative expression level of miR-142-3p in HAECs was analyzed by RT-qPCR. Mean±SD. n=3. *P<0.05, **P<0.01 vs 0 h group.

        3 Rictor是miR-142-3p在HAECs中的靶基因之一

        利用生物信息學(xué)軟件(TargetScan、miRanda和miRDB)預(yù)測(cè)miR-142-3p可能的靶基因,挑選出作為下一步的研究對(duì)象。如圖3C所示,Rictor的3'-UTR含有miR-142-3p的結(jié)合位點(diǎn),有可能是miR-142-3p的直接靶基因。為研究miR-142-3p對(duì)Rictor蛋白和mRNA表達(dá)的影響,將miR-142-3p mimic和miR-142-3p inhibitor轉(zhuǎn)染入HAECs。ox-LDL處理抑制了Rictor蛋白的表達(dá),與miR-142-3p的表達(dá)呈負(fù)相關(guān); miR-142-3p mimic顯著降低了Rictor蛋白的表達(dá),而miR-142-3p inhibitor上調(diào)了Rictor蛋白的表達(dá)水平(<0.05),見圖3A。此外,miR-142-3p mimic和miR-142-3p inhibitor的轉(zhuǎn)染對(duì)Rictor mRNA的表達(dá)無明顯影響(圖3B),提示miR-142-3p有可能在轉(zhuǎn)錄后水平調(diào)控基因的表達(dá)。

        為進(jìn)一步驗(yàn)證miR-142-3p是否與Rictor mRNA的3’-UTR直接結(jié)合,我們采用雙螢光素酶報(bào)告基因?qū)嶒?yàn)進(jìn)行鑒定。結(jié)果顯示,與NC mimic組相比, miR-142-3p mimic與pMIR-Rictor-3'-UTR共同轉(zhuǎn)染后螢光素酶活性顯著降低(<0.01),而miR-142-3p mimic和pMIR-Rictor-3'-UTR Mutant共轉(zhuǎn)染組的螢光素酶活性則無明顯變化,見圖3D。這說明miR-142-3p可以通過結(jié)合Rictor 3'-UTR上的預(yù)測(cè)靶位點(diǎn)直接抑制Rictor的表達(dá),而將預(yù)測(cè)的靶位點(diǎn)突變后,miR-142-3p對(duì)Rictor的抑制作用基本消除。因此,在HAECs中是miR-142-3p的直接靶基因之一。

        Figure 3. Experimental validation of Rictor as a target gene of miR-142-3p in HAECs. The HAECs were transfected with NC mimic, miR-142-3p mimic, NC inhibitor and miR-142-3p inhibitor, and further exposed to ox-LDL at 100 mg/L for 24 h. A: the protein expression of Rictor was measured by Western blot; B: the mRNA expression of Rictor was measured by RT-qPCR; C: the putative target site of Rictor mRNA 3'-UTR was determined by computational predictions; D: the HAECs were transfected with pMIR-Rictor-3'-UTR or pMIR-Rictor-3'-UTR Mutant and miR-142-3p mimic or NC mimic, and luciferase activity was measured. Mean±SD. n=3. *P<0.05, **P<0.01 vs NC mimic group; #P<0.05 vs NC mimic+ox-LDL group; △P<0.05 vs NC inhibitor group; ▲P<0.05 vs NC inhibitor+ox-LDL group.

        4 下調(diào)miR-142-3p抑制HAECs的凋亡

        與對(duì)照組相比,miR-142-3p過表達(dá)可以促進(jìn)ox-LDL誘導(dǎo)的HAECs凋亡,相反,轉(zhuǎn)染miR-142-3p inhibitor可顯著降低ox-LDL介導(dǎo)的HAECs凋亡(<0.05或<0.01),見圖4A、B。此外,在本實(shí)驗(yàn)中ox-LDL作用后HAECs的活力下降,過表達(dá)miR-142-3p使HAECs的活力進(jìn)一步下降,而下調(diào)miR-142-3p可以增強(qiáng)HAECs的活力(<0.05),見圖4C。

        Figure 4. Influence of miR-142-3p on apoptosis of HAECs induced by ox-LDL. A: the activity of caspase-3 was determined by luminescent substrate assays; B: the apoptosis and necrosis of HAECs were detected by flow cytometry; C: comparison of the viability of HAECs. Mean±SD. n=3. *P<0.05, **P<0.01 vs NC mimic group; #P<0.05, ##P<0.01 vs NC mimic+ox-LDL group; △P<0.05, △△P<0.01 vs NC inhibitor group; ▲P<0.05 vs NC inhibitor+ox-LDL group.

        5 miR-142-3p通過靶向Rictor調(diào)節(jié)HAECs凋亡

        為進(jìn)一步明確基因在miR-142-3p調(diào)控HAECs凋亡中發(fā)揮的作用,我們進(jìn)行了救援實(shí)驗(yàn)。結(jié)果顯示,轉(zhuǎn)染miR-142-3p inhibitor上調(diào)了Rictor蛋白的表達(dá)(<0.05),而miR-142-3p inhibitor與si-Rictor共轉(zhuǎn)染導(dǎo)致Rictor蛋白表達(dá)水平顯著下降(<0.01),見圖5A。caspase-3活性檢測(cè)和流式細(xì)胞術(shù)凋亡檢測(cè)結(jié)果顯示, miR-142-3p inhibitor減輕了ox-LDL介導(dǎo)的HAECs凋亡(<0.05),而當(dāng)轉(zhuǎn)染si-Rictor抑制Rictor蛋白的表達(dá)后,miR-142-3p inhibitor的內(nèi)皮保護(hù)作用也相應(yīng)被消除(<0.05或<0.01),見圖5B、C。

        Figure 5. miR-142-3p modulated the apoptosis of HAECs by targeting Rictor. A: Rictor protein level in HAECs was decreased by transfection with si-Rictor (measured by Western blot); B: down-regulation of Rictor by si-Rictor blocked the suppressive effect of miR-142-3p inhibitor on caspase-3 activity in ox-LDL-treated HAECs; C: the apoptosis and necrosis of HAECs were detected by flow cytometry. Mean±SD. n=3. *P<0.05 vs NCinhibitor+ox-LDL group; #P<0.05, ##P<0.01 vs miR-142-3p inhibitor+NC siRNA+ox-LDL group.

        6 Akt/eNOS信號(hào)通路參與了miR-142-3p對(duì)HAECs凋亡的調(diào)控

        如圖6A、B所示, ox-LDL作用24 h可降低HAECs中磷酸化Akt (Ser473)和eNOS (Ser1177)水平,而抑制miR-142-3p的表達(dá)部分逆轉(zhuǎn)了這種趨勢(shì);相反的,轉(zhuǎn)染miR-142-3p mimic可減弱Akt和eNOS的磷酸化(<0.05)。采用CCK-8法評(píng)估實(shí)驗(yàn)濃度的選擇性Akt阻斷劑LY294002和eNOS抑制劑L-NAME對(duì)HAECs的細(xì)胞毒性,結(jié)果顯示,與對(duì)照組相比, 20 μmol/L LY294002和100 μmol/L L-NAME作用2 h對(duì)HAECs的活力無顯著影響,見圖6C。選擇性Akt阻斷劑LY294002顯著抑制了miR-142-3p inhibitor介導(dǎo)的Akt和eNOS磷酸化; eNOS抑制物L(fēng)-NAME僅減少eNOS的磷酸化而對(duì)Akt磷酸化無明顯影響,見圖6D。當(dāng)Akt/eNOS信號(hào)通路被LY294002或L-NAME阻斷后,miR-142-3p inhibitor保護(hù)HAECs免受ox-LDL誘導(dǎo)凋亡的作用也被相應(yīng)消除(<0.01),見圖6E、F。以上研究表明, miR-142-3p inhibitor對(duì)HAECs的保護(hù)作用是通過激活A(yù)kt/eNOS信號(hào)通路實(shí)現(xiàn)的。

        Figure 6. Down-regulation of miR-142-3p reduced the apoptosis of HAECs via Akt/eNOS signaling pathway activation. A and B: HAECs were exposed to ox-LDL (100 mg/L, 24 h) after miR-142-3p mimic or inhibitor transfection, and the phosphorylation of Akt and eNOS was detected by Western blot; C: HAECs were treated with LY294002 (LY, 20 μmol/L) or L-NAME (100 μmol/L) for 2 h, and the viability was measured by CCK-8 assay; D: the protein levels of Akt, p-Akt, eNOS and p-eNOS were measured by Western blot; E: the caspase-3 activity was detected by enzyme assay; F: the apoptosis and necrosis were detected by flow cytometry. Mean±SD. n=3. *P<0.05 vs NC mimic group; #P<0.05 vs NC mimic+ox-LDL group; △P<0.05, △△P<0.05 vs NC inhibitor group; ▲P<0.05,▲▲P<0.01 vs NC inhibitor+ox-LDL group; @P<0.05, @@P<0.01 vs miR-142-3p inhibitor+ox-LDL group.

        討論

        ECs凋亡在內(nèi)皮功能障礙和AS發(fā)生過程中發(fā)揮重要作用。ox-LDL是AS啟動(dòng)和發(fā)展的重要危險(xiǎn)因素,本研究以ox-LDL誘導(dǎo)的HAECs為模型探討AS進(jìn)程中與ECs凋亡相關(guān)的miRNAs。

        近年來研究表明,miRNAs廣泛參與了血管新生、損傷、炎癥和衰老等眾多病理生理過程[5]。此外,越來越多的研究證實(shí)在AS背景下,存在很多差異表達(dá)的miRNAs調(diào)控ECs的活力和死亡[6]。尋找凋亡相關(guān)的miRNAs及其下游靶基因?yàn)檠芯縀Cs功能障礙和AS治療提供了新的策略。本研究發(fā)現(xiàn)抑制miR-142-3p的表達(dá)可以上調(diào)靶基因并激活A(yù)kt/eNOS信號(hào)通路從而發(fā)揮抗凋亡的內(nèi)皮保護(hù)作用。

        miR-142-3p定位于人染色體17q22,已被證實(shí)在多種腫瘤中表達(dá)下調(diào)。例如, miR-142-3p通過靶向調(diào)控HMGB1在非小細(xì)胞肺癌(non-small-cell lung carcinoma, NSCLC)中抑制腫瘤細(xì)胞增生并誘導(dǎo)凋亡[7]。此外,miR-142-3p與心血管系統(tǒng)疾病也密切相關(guān),在缺氧復(fù)氧誘導(dǎo)心肌細(xì)胞凋亡和纖維化過程中miR-142-3p是重要的調(diào)節(jié)因子[8]。另一項(xiàng)新近研究表明,血小板來源的miR-142-3p通過血小板來源的微粒轉(zhuǎn)移至ECs,作用于下游靶基因從而誘導(dǎo)ECs凋亡[9]。與此相一致,我們研究也發(fā)現(xiàn),miR-142-3p在ox-LDL誘導(dǎo)HAECs凋亡過程中表達(dá)明顯上調(diào)。過表達(dá)miR-142-3p促進(jìn)了HAECs凋亡,而抑制miR-142-3p可以發(fā)揮抗凋亡的內(nèi)皮保護(hù)作用。ox-LDL根據(jù)作用的濃度和時(shí)間不同,可以誘導(dǎo)ECs發(fā)生凋亡、壞死和增生[10]。ox-LDL濃度小于10 mg/L可以促進(jìn)ECs增生;濃度大于50 mg/L促進(jìn)ECs凋亡;濃度大于250 mg/L主要誘導(dǎo)ECs發(fā)生壞死[11]。在本研究的實(shí)驗(yàn)條件下,100 mg/L ox-LDL作用24 h顯著抑制了HAECs的存活和生長,過表達(dá)miR-142-3p加重了這種抑制作用,而下調(diào)miR-142-3p削弱了這種抑制作用。100 mg/L ox-LDL處理(0~24) h對(duì)HAECs壞死率無顯著影響,轉(zhuǎn)染miR-142-3p inhibitor可以輕微降低細(xì)胞壞死率,但差異無統(tǒng)計(jì)學(xué)顯著性。

        mTOR是一種進(jìn)化上高度保守的非典型的絲氨酸/蘇氨酸蛋白激酶,屬于磷脂酰肌醇3-激酶相關(guān)激酶(phosphatidylinositol 3-kinase-related kinase,PIKK)蛋白家族。近年來大量的研究表明,mTOR信號(hào)通路異常與衰老、2型糖尿病、腫瘤和神經(jīng)退行性疾病等多種重大疾病的發(fā)生和發(fā)展密切相關(guān)。mTOR在生物體內(nèi)以mTOR-Raptor復(fù)合物(mTORC1)和mTOR-Rictor復(fù)合物(mTORC2)的形式存在。mTORC2是由mTOR、mLST8/GβL及Rictor(rapamycin-insensitive companion of mTOR)和mSin1(mammalian stress-activated protein kinase- interacting protein 1)組成的多蛋白激酶復(fù)合物,其中Rictor是mTORC2特異性的組成蛋白,并是其發(fā)揮生物學(xué)作用必不可少的組成部分。mTORC2的主要功能是作為蛋白激酶B(PKB/Akt)的激酶磷酸化其473位絲氨酸(Ser473),在PKB/Akt的激活中起重要作用[12]。Akt激活后可通過磷酸化下游靶蛋白eNOS、BAD caspase-9、Fox1、Fox3, YAP和MDM2發(fā)揮促進(jìn)存活和抑制凋亡作用[13]。在這些靶蛋白中,eNOS活性障礙使NO生物活性降低,導(dǎo)致AS發(fā)生。研究表明,Rictor/Akt/eNOS途徑在調(diào)控血管內(nèi)皮細(xì)胞凋亡和功能障礙方面發(fā)揮重要作用。下調(diào)Rictor表達(dá)抑制Akt活化,減少了ECs增生、遷移和血管新生[14],促進(jìn)了ECs凋亡[15]、炎癥[16]和衰老[17]。相反的,成纖維細(xì)胞生長因子(fibroblast growth factor,F(xiàn)GF)、血管內(nèi)皮生長因子(vascular endothelial growth factor,VEGF)[18]和14,15-環(huán)氧二十碳烯酸[17]可促進(jìn)Rictor蛋白表達(dá)及Akt磷酸化,抑制血管內(nèi)皮細(xì)胞凋亡和衰老。ox-LDL誘導(dǎo)的ECs衰老和氧化應(yīng)激通過抑制Akt/eNOS通路實(shí)現(xiàn)[19]。在本研究中,我們發(fā)現(xiàn)過表達(dá)miR-142-3p降低了Rictor的表達(dá)并抑制Akt磷酸化活化,而miR-142-3p表達(dá)抑制可以促進(jìn)Rictor蛋白的表達(dá)水平和Akt活化。進(jìn)一步采用螢光素酶報(bào)告基因?qū)嶒?yàn)證實(shí)是miR-142-3p的直接靶基因之一。沉默之后miR-142-3p inhibitor的內(nèi)皮保護(hù)作用也相應(yīng)消除。ox-LDL抑制了Akt/eNOS活性,HAECs的凋亡率相應(yīng)增加。盡管miR-142-3p inhibitor增強(qiáng)了Akt/eNOS活性,加入LY294002和L-NAME減少了Akt/eNOS磷酸化,同時(shí)也逆轉(zhuǎn)了miR-142-3p inhibitor的抗凋亡作用。

        綜上所述,抑制miR-142-3p可以通過激活Rictor/Akt/eNOS信號(hào)通路而緩解HAECs的凋亡。本研究為防治內(nèi)皮細(xì)胞功能障礙和AS提供新的治療靶點(diǎn)和思路。

        [1] Paone S, Baxter AA, Hulett MD, et al. Endothelial cell apoptosis and the role of endothelial cell-derived extracellular vesicles in the progression of atherosclerosis[J]. Cell Mol Life Sci, 2019, 76(6):1093-1106.

        [2] Kattoor AJ, Pothineni NVK, Palagiri D, et al. Oxidative stress in atherosclerosis[J]. Curr Atheroscler Rep, 2017, 19(11):42.

        [3] Khor ES, Wong PF. The roles of MTOR and miRNAs in endothelial cell senescence[J]. Biogerontology, 2020, 21(5):517-530.

        [4] Zhao Z, Qu F, Liu R, et al. Differential expression of miR-142-3p protects cardiomyocytes from myocardial ischemia-reperfusion via TLR4/NFκB axis[J]. J Cell Biochem, 2020, 121(8/9):3679-3690.

        [5]譚曉勇,方丹,吳劍波,等. miR-21對(duì)血管內(nèi)皮功能及血管生成調(diào)節(jié)作用的研究進(jìn)展[J]. 中國病理生理雜志, 2017, 33(12):2299-2304.

        Tan XY, Fang D, Wu JB, et al. Advances on studies of miR-21 in vascular endothelial function and angiogenesis [J]. Chin J Pathophysiol, 2017, 33(12):2299-2304.

        [6]王玨,趙海蘋. MicroRNA調(diào)控缺血性及出血性腦血管病過程中免疫反應(yīng)的研究進(jìn)展[J]. 中國病理生理雜志, 2017, 33(2):369-374.

        Wang J, Zhao HP.Progress in study of immune response regulated by microRNAs in process of ischemic and hemorrhagic cerebrovascular diseases[J]. Chin J Pathophysiol, 2017, 33(2):369-374.

        [7] Xiao P, Liu WL. MiR-142-3p functions as a potential tumor suppressor directly targeting HMGB1 in non-small-cell lung carcinoma[J]. Int J Clin Exp Pathol, 2015, 8(9):10800-10807.

        [8] Wang Y, Ouyang M, Wang Q, et al. MicroRNA-142-3p inhibits hypoxia/reoxygenationinduced apoptosis and fibrosis of cardiomyocytes by targeting high mobility group box 1 [J]. Int J Mol Med, 2016, 38(5): 1377-1386.

        [9] Bao H, Yao QP, Huang K, et al. Platelet-derived miR-142-3p induces apoptosis of endothelial cells in hypertension [J]. Cell Mol Biol (Noisy-le-grand), 2017, 63(4): 3-9.

        [10] Zhang Y, Xie Y, You S, et al. Autophagy and apoptosis in the response of human vascular endothelial cells to oxidized low-density lipoprotein [J]. Cardiology, 2015, 132(1): 27-33.

        [11] Chen XP, Xun KL, Wu Q, et al. Oxidized low density lipoprotein receptor-1 mediates oxidized low density lipoprotein-induced apoptosis in human umbilical vein endothelial cells: role of reactive oxygen species[J]. Vascul Pharmacol, 2007, 47(1):1-9.

        [12] Zou Z, Chen J, Yang J, et al. Targeted inhibition of Rictor/mTORC2 in cancer treatment: a new era after rapamycin[J]. Curr Cancer Drug Targets, 2016, 16(4): 288-304.

        [13] Abeyrathna P, Su Y. The critical role of Akt in cardiovascular function[J]. Vascul Pharmacol, 2015, 74:38-48.

        [14] Aimi F, Georgiopoulou S, Kalus I, et al. Endothelial Rictor is crucial for midgestational development and sustained and extensive FGF2-induced neovascularization in the adult[J]. Sci Rep, 2015, 5:17705.

        [15] Jin YP, Valenzuela NM, Ziegler ME, et al. Everolimus inhibits anti-HLA I antibody-mediated endothelial cell signaling, migration and proliferation more potently than sirolimus[J]. Am J Transplant, 2014, 14(4):806-819.

        [16] Barilli A, Visigalli R, Sala R, et al. In human endothelial cells rapamycin causes mTORC2 inhibition and impairs cell viability and function[J]. Cardiovasc Res, 2008, 78(3):563-571.

        [17] Yang C, Pan S, Yan S, et al. Inhibitory effect of 14,15-EET on endothelial senescence through activation of mTOR complex 2/Akt signaling pathways[J]. Int J Biochem Cell Biol, 2014, 50(5):93-100.

        [18] Dormond O, Madsen JC, Briscoe DM. The effects of mTOR-Akt interactions on anti-apoptotic signaling in vascular endothelial cells[J]. J Biol Chem, 2007, 282(32):23679-2386.

        [19] Yao Y, Wang Y, Zhang Y, et al. Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways [J]. Lipids Health Dis, 2017, 16(1):77-86.

        MicroRNA-142-3p modulates atherosclerosis-associated endothelial cell apoptosis via targeting Rictor

        XIAO Li, LIU Ping, QIN Bing

        (,,510630,)

        To investigate the role of microRNA-142-3p (miR-142-3p) in endothelial cell apoptosis during atherosclerosis (AS) and the underlying mechanism.Human aortic endothelial cells (HAECs) were treated with oxidized low-density lipoprotein (ox-LDL). The expression level of miR-142-3p was detected by RT-qPCR. Apoptosis was determined via flow cytometry (FCM) and caspase-3 activity assay. Prediction of the binding site between miR-142-3p and 3’-UTR of Rictor mRNA was performed by bioinformatics analysis and confirmed by dual-luciferase reporter assay.The expression of miR-142-3p was substantially up-regulated during the ox-LDL-elicited apoptosis in HAECs (<0.05,<0.01). Forced expression of miR-142-3p exacerbated apoptosis in HAECs whereas inhibition of miR-142-3p partly alleviated apoptotic cell death mediated by ox-LDL. Further analysis identifiedas a direct target gene of miR-142-3p, andknock-down abolished the anti-apoptotic effect of miR-142-3p inhibitor. Moreover, the Akt/endothelial nitric oxide synthase (eNOS) signaling pathway was found to mediate the beneficial effect of miR-142-3p inhibitor on endothelial cells apoptosis.Down-regulation of miR-142-3p inhibits endothelial cell apoptosis and atherosclerotic development by up-regulating the expression of Rictor and activating the Akt/eNOS signaling pathway.

        MicroRNA-142-3p; Human aortic endothelial cells; Apoptosis; Rictor; Atherosclerosis

        R543.5; R363.2

        A

        10.3969/j.issn.1000-4718.2020.11.002

        1000-4718(2020)11-1928-10

        2020-05-25

        2020-10-06

        國家自然科學(xué)基金資助項(xiàng)目(No. 81701167); 廣東省自然科學(xué)基金資助項(xiàng)目(No. 2017A030310360)

        Tel: 020-85252336; E-mail: qinbingsx@aliyun.com

        (責(zé)任編輯:盧萍,宋延君)

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