丁林, 李萌陽, 王夢宇, 法鴻鴿, 房芯羽, 王建勛
環(huán)狀RNA Lrp6調(diào)控過氧化氫誘導(dǎo)的H9c2心肌細(xì)胞凋亡*
丁林, 李萌陽, 王夢宇, 法鴻鴿, 房芯羽, 王建勛△
(青島大學(xué)基礎(chǔ)醫(yī)學(xué)院,山東 青島 266021)
探究環(huán)狀RNA Lrp6(circLrp6)對過氧化氫(H2O2)誘導(dǎo)的H9c2心肌細(xì)胞凋亡的影響。通過Sanger測序和RNase R酶切驗(yàn)證circLrp6的環(huán)狀結(jié)構(gòu);細(xì)胞熒光原位雜交分析circLrp6的亞細(xì)胞定位;通過RT-qPCR檢測H2O2處理的H9c2細(xì)胞和缺血再灌注損傷的小鼠心臟組織中circLrp6的差異表達(dá)水平;采用TUNEL染色檢測circLrp6是否影響H2O2誘導(dǎo)的心肌細(xì)胞凋亡水平;通過生物信息學(xué)的方法預(yù)測與circLrp6相互結(jié)合的下游靶點(diǎn)及其結(jié)合位點(diǎn)。circLrp6具有環(huán)狀結(jié)構(gòu),定位于細(xì)胞核,它在H2O2處理后的H9c2心肌細(xì)胞和缺血再灌注的心臟組織中表達(dá)水平下調(diào)(<0.05),過表達(dá)circLrp6對H2O2處理的心肌細(xì)胞具有保護(hù)作用,表現(xiàn)為TUNEL染色陽性率下降(<0.05)。另外,circLrp6具有結(jié)合miRNA和蛋白質(zhì)的潛能。circLrp6對H2O2誘導(dǎo)的心肌細(xì)胞凋亡具有抑制作用。
環(huán)狀RNA Lrp6;心肌細(xì)胞;過氧化氫;細(xì)胞凋亡
近年來,以冠心病為代表的缺血性心臟病已成為全球主要的死亡原因之一,嚴(yán)重威脅著人類的生命健康。研究表明,由于機(jī)體內(nèi)異常的脂質(zhì)代謝而引起的冠狀動脈粥樣硬化是誘發(fā)冠心病的主要原因。血液中的脂質(zhì)附著在動脈壁上會引起血管閉塞,血流受阻,嚴(yán)重時(shí)會引起心肌缺血,危及生命[1]。盡管臨床上經(jīng)皮冠狀動脈介入聯(lián)合藥物恢復(fù)血流灌注的治療方法已逐漸成熟,但急性冠狀動脈綜合征的死亡率仍然很高。這是由于心肌缺血后再灌注會產(chǎn)生大量的活性氧(reactive oxygen species, ROS),造成缺血再灌注損傷,最終導(dǎo)致心律失常、心功能障礙和梗死后心臟重構(gòu)[2-3]。細(xì)胞凋亡是心肌缺血再灌注損傷過程中心肌細(xì)胞的死亡方式之一[4]。目前,調(diào)控該過程的分子機(jī)制尚未完全闡明。因此,深入研究調(diào)控心肌細(xì)胞凋亡的分子機(jī)制將對治療缺血再灌注損傷和冠心病具有重要的意義。
環(huán)狀RNA(circular RNAs, circRNAs)是一類由前體mRNA(pre-mRNA)通過反向剪接而形成的共價(jià)閉合的單鏈環(huán)狀非編碼RNA,它在真核生物中含量豐富、結(jié)構(gòu)穩(wěn)定、進(jìn)化保守,通常以組織特異性的方式表達(dá),在心血管疾病、神經(jīng)退行性疾病和腫瘤等多種疾病中發(fā)揮重要的調(diào)控作用[5-6]。近年來,研究者利用新一代高通量測序技術(shù)在心臟中鑒定出大量的circRNA,其中一部分在心肌細(xì)胞中高表達(dá)的circRNA已被證明在極大程度上能決定心肌細(xì)胞的命運(yùn),調(diào)控心肌細(xì)胞的增殖、衰老和死亡等過程[7]。例如心肌梗死的成年小鼠在缺失由超級增強(qiáng)子調(diào)控的circNfix后,可促進(jìn)心肌細(xì)胞增殖和血管生成,抑制心肌細(xì)胞凋亡,減輕預(yù)后不良[8]。在老年人和老年鼠中高表達(dá)的circFoxo3與細(xì)胞衰老有關(guān),沉默內(nèi)源性的能抑制細(xì)胞衰老,緩解阿霉素誘導(dǎo)的心肌?。?]。抑制小鼠心臟中高表達(dá)的circNCX1(又稱circSLC8A1)可減弱缺血性心肌損傷、心肌肥厚和阿霉素誘導(dǎo)的心肌毒性[10-12]。circRNA_001131通過結(jié)合微小RNA-25-3p(microRNA-25-3p, miR-25-3p)抑制心肌成纖維細(xì)胞中纖維化相關(guān)基因的表達(dá)[13]。然而,還有很多高表達(dá)于心臟的circRNA,其功能和作用機(jī)制尚不清楚,值得我們進(jìn)一步探索。
根據(jù)Werfel等[14]的測序結(jié)果,我們發(fā)現(xiàn)一個(gè)在人類、小鼠和大鼠心臟中都高度保守且高表達(dá)的circRNA,命名為circLrp6。circLrp6可在多種癌癥發(fā)生中發(fā)揮致癌作用,促進(jìn)細(xì)胞增殖和侵襲,抑制細(xì)胞凋亡[15-18],但其在心臟中是否發(fā)揮功能尚不清楚。因此,本研究旨在探討circLrp6對過氧化氫(hydrogen peroxide, H2O2)誘導(dǎo)的心肌細(xì)胞凋亡的影響。
將8周齡的C57BL/6J雄性小鼠[許可證號為SCXK(魯)2019-0003]隨機(jī)分為兩組:實(shí)驗(yàn)組小鼠結(jié)扎冠狀動脈左前降支(left anterior descending, LAD)30 min,再灌注3 h;假手術(shù)組小鼠只穿線不結(jié)扎。所有動物實(shí)驗(yàn)均得到青島大學(xué)醫(yī)學(xué)部倫理委員會的批準(zhǔn)。
大鼠心肌細(xì)胞系H9c2購于上海生命科學(xué)研究院。DMEM培養(yǎng)基(Gibco);胎牛血清(北京全式金);0.25%胰蛋白酶(武漢賽維爾);30% H2O2(國藥);Trizol試劑(Invitrogen);反轉(zhuǎn)錄和實(shí)時(shí)熒光定量PCR試劑盒(南京諾唯贊);轉(zhuǎn)染試劑Lipofectamine 3000(Invitrogen);RNase R(Invitrogen);TUNEL細(xì)胞凋亡檢測試劑盒(上海翌圣);熒光原位雜交(fluorescencehybridization, FISH)試劑盒(上海吉瑪);引物和熒光探針由深圳華大基因和北京擎科生物公司合成;circLrp6的siRNA和過表達(dá)質(zhì)粒由上海吉瑪制藥技術(shù)有限公司合成。倒置熒光顯微鏡(Olympus);倒置雙光子激光共聚焦掃描顯微鏡(Leica);實(shí)時(shí)熒光定量PCR儀(Bio-Rad);NanoDrop分光光度計(jì)(Thermo Fisher)。
3.1細(xì)胞培養(yǎng)和處理H9c2細(xì)胞用含10%胎牛血清、1×105U/L青霉素和100 mg/L鏈霉素的DMEM培養(yǎng)基,并置于37 ℃、5% CO2的恒溫培養(yǎng)箱中培養(yǎng)。當(dāng)細(xì)胞密度達(dá)到80%~90%時(shí),用0.25%胰蛋白酶消化再傳代,使用100 μmol/L H2O2對細(xì)胞進(jìn)行處理。
3.2細(xì)胞轉(zhuǎn)染委托上海吉瑪制藥技術(shù)有限公司構(gòu)建circLrp6過表達(dá)質(zhì)粒并合成用于特異性沉默的小干擾RNA(small interfering RNA, siRNA)和陰性對照(negative control, NC)序列。siRNA序列為5'-AUCAAGUGGCGGCCCCUUUTT-3'和5'-AAAGGGGCCGCCACUUGAUTT-3';NC序列為5'-UUCUCCGAACGUGUCACGUTT-3'和5'-ACGUGACACGUUCGGAGAATT-3'。然后,按照Lipofectamine 3000說明書的操作步驟轉(zhuǎn)染質(zhì)?;騭iRNA。
3.3RT-qPCR按照Trizol試劑說明書的步驟提取心肌細(xì)胞和心臟組織中的總RNA;利用NanoDrop分光光度計(jì)測定RNA的濃度與純度;根據(jù)反轉(zhuǎn)錄試劑盒的說明書將RNA反轉(zhuǎn)錄為cDNA,再進(jìn)行qPCR;使用2-ΔΔCt公式計(jì)算相對表達(dá)量,相應(yīng)引物序列見表1。
表1 RT-qPCR引物序列
F: forward; R: reverse.
3.4circRNA成環(huán)驗(yàn)證(1)反向PCR擴(kuò)增:分別提取H9c2細(xì)胞中的基因組DNA(gDNA)和總RNA,并將總RNA反轉(zhuǎn)錄為cDNA,設(shè)計(jì)并合成用于擴(kuò)增circLrp6的收斂引物和發(fā)散引物,以gDNA和cDNA為模板,用兩種不同的引物進(jìn)行PCR擴(kuò)增。收斂引物的正向序列為5'-TGTAGTTGGAGGCTTGGAGGAT-3',反向序列為5'-CACTTGATGGATCTAAGGCAATAGC-3';發(fā)散引物的正向序列為5'-GGTCATGGCTTGATATACTGGAGTG-3',反向序列為5'-CCAACTACAATCGTCGCATTCTCT-3'。對PCR擴(kuò)增產(chǎn)物進(jìn)行瓊脂糖凝膠電泳檢測,并將反向擴(kuò)增產(chǎn)物進(jìn)行Sanger測序。(2)RNase R耐受實(shí)驗(yàn):每組5 μg總RNA,對照組不加RNase R,實(shí)驗(yàn)組加15 U RNase R,37 ℃金屬浴孵育10~15 min。之后用Trizol試劑按照說明書從反應(yīng)體系中純化RNA,再進(jìn)行RT-qPCR。
3.5FISH檢測在24孔板中,將H9c2細(xì)胞接種在1 cm×1 cm的玻片上,當(dāng)細(xì)胞密度為70%左右,吸棄培養(yǎng)基,用PBS清洗兩遍,用4%的多聚甲醛固定。然后,按照FISH試劑盒的說明書進(jìn)行實(shí)驗(yàn),最后用含DAPI染液的封片劑固定。用倒置雙光子激光共聚焦掃描顯微鏡對染好的玻片進(jìn)行拍照。熒光探針序列為5'-FAM-AGCAACAAAGGGGCCGCCACTTGATGGATCT-3'。
3.6TUNEL細(xì)胞凋亡檢測在24孔板中,將H9c2細(xì)胞接種在1 cm×1 cm的玻片上。然后,吸棄培養(yǎng)基,用PBS清洗兩遍,用4%的多聚甲醛對已處理的細(xì)胞室溫固定15 min。按照TUNEL細(xì)胞凋亡檢測試劑盒的說明書進(jìn)行實(shí)驗(yàn),最后用含DAPI染液的封片劑封片固定。使用倒置熒光顯微鏡對染好的玻片進(jìn)行觀察拍照,計(jì)算TUNEL陽性細(xì)胞核占總細(xì)胞核的比例,得出細(xì)胞凋亡率。
所有數(shù)據(jù)都用GraphPad Prism 8進(jìn)行統(tǒng)計(jì)學(xué)分析,以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示。兩組間比較采用檢驗(yàn),多組間比較采用單因素方差分析,組間的兩兩比較采用Tukey檢驗(yàn)。以<0.05為差異有統(tǒng)計(jì)學(xué)意義。
首先,分析Werfel等[14]的測序結(jié)果,我們發(fā)現(xiàn)了一個(gè)來源于基因2號外顯子、剪接后成熟序列長度為394 nt的circRNA,它在人類、大鼠和小鼠的心臟中都高表達(dá)且保守性高,將其命名為circLrp6(圖1A)。使用circLrp6的收斂引物和發(fā)散引物,以H9c2的cDNA和gDNA為模板進(jìn)行擴(kuò)增,收斂引物可將cDNA和gDNA擴(kuò)增出條帶,而發(fā)散引物只能將cDNA擴(kuò)增出條帶(圖1B)。Sanger測序結(jié)果顯示,反向擴(kuò)增產(chǎn)物的序列與公開的circLrp6序列相一致且5'和3'末端相連(圖1C)。結(jié)合PCR結(jié)果可知,circLrp6序列末端相連來源于反向剪切,并非基因組重排。由于circRNA沒有末端,所以circLrp6相比于線性的GAPDH和Lrp6更耐受核酸外切酶RNase R的消化,結(jié)構(gòu)更加穩(wěn)定(<0.05),見圖1D。我們還檢測了新生大鼠心肌細(xì)胞和成纖維細(xì)胞中circLrp6的表達(dá)豐度。與成纖維細(xì)胞相比,circLrp6在心肌細(xì)胞中表達(dá)豐度更高(<0.05),見圖1E。FISH檢測結(jié)果證明,circLrp6主要定位于細(xì)胞核中(圖1F)。因此,circLrp6是一個(gè)在心肌細(xì)胞中高表達(dá)且進(jìn)化保守的circRNA。
Figure 1.circLrp6 is a conserved cardiac circRNA. A: sequence analysis of phyloP showed that circLrp6 is conserved. B: circLrp6 was a back-splicing formed loop. It was amplified from the cDNA of H9c2 cells by divergent primers, while it could not be amplified from gDNA. C: circLrp6 was generated from the 2nd exon of the Lrp6 gene, and the back-splice junction of circLrp6 was identified by Sanger sequencing. D: total RNA extracted from H9c2 cells was incubated with or without RNase R. RT-qPCR showed the disappearance of GAPDH and linear Lrp6 by RNase R treatment, whereas circLrp6 was not affected. E: relative expression level of circLrp6 in cardiomyocytes and cardiac fibroblasts. F: subcellular localization of circLrp6 was identified by fluorescence in situ hybridization (FISH). Green: circLrp6 probes were labeled with FITC; blue: nuclei were stained with DAPI. Scale bar=20 μm. Mean±SD. n=3. *P<0.05 vs mock; #P<0.05 vs cardiomyocyte.
心臟缺血再灌注后,過度釋放的ROS會造成心肌細(xì)胞損傷和死亡。有研究報(bào)道,100 μmol/L H2O2處理H9c2心肌細(xì)胞可體外模擬缺血再灌注損傷[19]。為了探究circLrp6是否與心肌細(xì)胞凋亡相關(guān),我們用100 μmol/L H2O2處理H9c2心肌細(xì)胞0、1、3、6和12 h后,檢測circLrp6的表達(dá)量變化,結(jié)果顯示H9c2細(xì)胞中circLrp6的表達(dá)量在H2O2處理后持續(xù)下降(<0.05),見圖2A。我們進(jìn)一步檢測了circLrp6在小鼠心肌缺血再灌模型中的表達(dá),與體外處理結(jié)果一致,較于對照組來說,小鼠心肌缺血再灌后circLrp6表達(dá)量減少(<0.05),見圖2B。綜上所述,circLrp6在凋亡的心肌細(xì)胞和缺血再灌注損傷的心肌中表達(dá)水平降低,提示circLrp6可能在心肌細(xì)胞凋亡過程中發(fā)揮重要的作用。
Figure 2.The expression level of circLrp6 decreased in apoptotic cardiomyocytes. A: the expression level of circLrp6 in H9c2 cells with 100 μmol/L H2O2 treatment for indicated times; B: RT-qPCR assay of cardiac circLrp6 in the ischemic zone adjacent to the infarction region of rat hearts; C and D: the expression levels of circLrp6 (C) and Lrp6 mRNA (D) in H9c2 cells transfected with circLrp6 siRNA (si-circLrp6) were detected; E and F: the expression levels of circLrp6 (E) and Lrp6 mRNA (F) in H9c2 cells transfected with circLrp6 overexpression vector (OE-circLrp6) were detected. Mean±SD. n=3. *P<0.05 vs 0 h group; #P<0.05 vs sham group; ▲P<0.05 vs negative control (NC) or empty vector (EV).
為了檢測circLrp6在心肌細(xì)胞凋亡過程中發(fā)揮的功能,我們合成了針對的特異性siRNA和過表達(dá)質(zhì)粒。首先,我們證明了siRNA和過表達(dá)載體分別能在H9c2細(xì)胞中顯著敲減和過表達(dá)(<0.05),見圖2C、D,并對線性Lrp6 mRNA的表達(dá)量沒有影響(圖2E、F)。在此基礎(chǔ)上,我們用50 μmol/L H2O2誘導(dǎo)細(xì)胞凋亡,通過TUNEL染色檢測心肌細(xì)胞的凋亡水平。結(jié)果顯示,敲減可促進(jìn)H2O2誘導(dǎo)的心肌細(xì)胞凋亡,表現(xiàn)為TUNEL染色陽性率升高(<0.05),見圖3A。而過表達(dá)可抑制100 μmol/L H2O2誘導(dǎo)的心肌細(xì)胞凋亡,表現(xiàn)為TUNEL染色陽性率降低(<0.05),見圖3B。綜上所述,circLrp6可顯著抑制H2O2誘導(dǎo)的心肌細(xì)胞凋亡。
Figure 3.circLrp6 inhibited hydrogen peroxide (H2O2)-induced cardiomyocyte apoptosis in vitro. A: H9c2 cells transfected with circLrp6 siRNA (si-circLrp6) were treated with 50 μmol/L H2O2 for 12 h; B: H9c2 cells transfected with circLrp6 overexpression vector (OE-circLrp6) were treated with 100 μmol/L H2O2 for 12 h. Scale bar=50 μm. Mean±SD. n=3. *P<0.05 vs negative control (NC) or empty vector (EV).
通過生物信息學(xué)分析,我們發(fā)現(xiàn)miR-330-5p和miR-326具有與circLrp6相互作用的潛能。CircInteractome數(shù)據(jù)庫[20]預(yù)測結(jié)果顯示,miR-330-5p與circLrp6有1個(gè)潛在的結(jié)合位點(diǎn),miR-326與circLrp6有2個(gè)潛在的結(jié)合位點(diǎn)(圖4A)。另外,我們在CircInteractome、catRAPID[21]和RBPsuite[22]數(shù)據(jù)庫中分別預(yù)測可以與circLrp6相互結(jié)合的蛋白質(zhì),發(fā)現(xiàn)EIF4A3和LIN28A在3個(gè)數(shù)據(jù)庫中都被預(yù)測為能與circLrp6結(jié)合的蛋白(圖4B),結(jié)合位點(diǎn)如表2所示。綜上所述,circLrp6具有與miRNA和蛋白結(jié)合的潛力。
Figure 4.The potential miRNAs and proteins interacting with circLrp6. A: the putative binding sites between circLrp6 and miR-330-5p or miR-326; B: Venn diagram revealing the overlap of the potential targeted proteins interacting with circLrp6 from CircInteractome, catRAPID and RBPsuite datasets.
表2 預(yù)測的與circLrp6結(jié)合的蛋白質(zhì)及其結(jié)合位點(diǎn)
臨床上,治療冠心病主要采用抗血栓藥物、經(jīng)皮冠狀動脈介入、搭橋手術(shù)等方法對閉塞動脈進(jìn)行血運(yùn)重建。然而,這些治療只能降低冠心病的嚴(yán)重程度,并不能恢復(fù)梗死心臟的收縮能力;同時(shí),缺血后的再灌注導(dǎo)致了嚴(yán)重的心功能障礙[2]。所以,尋找抑制心肌細(xì)胞死亡或刺激心臟再生的新型治療策略對臨床上治療冠心病具有深遠(yuǎn)的意義。
越來越多的研究發(fā)現(xiàn)circRNA參與心血管疾病的調(diào)控,具有作為血清診斷標(biāo)志物和治療藥物的潛力。例如,一項(xiàng)由642名急性心肌梗死患者參與的研究發(fā)現(xiàn)circRNA MICRA是左心室功能障礙的一個(gè)強(qiáng)有力的預(yù)測因子,MICRA表達(dá)量低的患者發(fā)生左心室功能障礙的風(fēng)險(xiǎn)較高[23];另一項(xiàng)研究發(fā)現(xiàn),hsa_circRNA_0124644是一種預(yù)測冠心病的潛在生物標(biāo)志物[24]。線粒體分裂和凋亡相關(guān)的circRNA MFACR通過結(jié)合miR-652-3p抑制基因的翻譯,進(jìn)而抑制線粒體分裂和細(xì)胞凋亡,最終抑制心肌梗死[25]。心肌纖維化是心肌肥大的重要病理特征,過表達(dá)circYAP可促進(jìn)TMP4和ACTG相互結(jié)合,抑制微絲聚集,進(jìn)而抑制纖維化的發(fā)生[26]。也有研究報(bào)道circPan3和circITCH參與調(diào)控阿霉素誘導(dǎo)的心肌毒性[27-28]。
本研究發(fā)現(xiàn)的circLrp6是一種來源于基因2號外顯子、長度為394 nt的circRNA,它在心肌細(xì)胞中高表達(dá)且定位于細(xì)胞核。通過構(gòu)建H2O2誘導(dǎo)的心肌細(xì)胞凋亡和小鼠心肌缺血再灌模型,發(fā)現(xiàn)circLrp6在凋亡發(fā)生后呈現(xiàn)時(shí)間依賴性下降。過表達(dá)和敲減H9c2細(xì)胞中的會對H2O2誘導(dǎo)的心肌細(xì)胞凋亡產(chǎn)生影響,表現(xiàn)為敲減可促進(jìn)H2O2誘導(dǎo)的心肌細(xì)胞凋亡,而過表達(dá)可抑制H2O2誘導(dǎo)的心肌細(xì)胞凋亡。
已有研究報(bào)道circLrp6在前列腺癌細(xì)胞和組織中高表達(dá),通過競爭性結(jié)合miR-330-5p而抑制NRBP1的降解,最終促進(jìn)前列腺癌細(xì)胞的增殖和侵襲,敲減可促進(jìn)癌細(xì)胞凋亡,抑制癌癥的發(fā)生[18]。另外,miR-330-5p在心血管疾病中也發(fā)揮重要的作用,沉默miR-330-5p可抑制阿霉素誘導(dǎo)的心肌毒性[28]。也有研究發(fā)現(xiàn)miR-326通過靶向MDK和抑制JAK/STAT和MAPK信號通路抑制心肌肥厚[29],還可以靶向Wnt1顯著增強(qiáng)內(nèi)皮祖細(xì)胞的血管生成能力[30]。除此之外,內(nèi)源性真核起始因子4A-III對維持體內(nèi)動脈粥樣硬化斑塊穩(wěn)定性[31]和缺氧誘導(dǎo)的H9c2細(xì)胞損傷都有影響[32]。LIN28A對糖尿病心肌病、缺血再灌注損傷和心肌肥大等多種心臟疾病具有調(diào)控作用[33-35]。這些預(yù)測的下游靶點(diǎn)都已經(jīng)被證明在心血管疾病中發(fā)揮調(diào)控作用,它們能否作為circLrp6的下游調(diào)控靶點(diǎn)在心肌缺血再灌注過程中影響心肌細(xì)胞凋亡將是我們下一步研究的方向。
我們首次研究了circLrp6在心血管疾病中的作用,證明circLrp6具有抑制心肌細(xì)胞凋亡的作用,通過生物信息手段預(yù)測到circLrp6可能通過與miRNA或蛋白質(zhì)結(jié)合而影響心肌細(xì)胞凋亡。circLrp6定位于細(xì)胞核中,這表明circLrp6可能通過調(diào)控DNA復(fù)制、基因轉(zhuǎn)錄等發(fā)生于細(xì)胞核的生物學(xué)過程影響細(xì)胞死亡,具體機(jī)制我們將在后續(xù)工作中進(jìn)行探索和驗(yàn)證,此外,circLrp6是否參與壞死、鐵死亡或焦亡等其他細(xì)胞死亡的調(diào)控,是否在心肌肥厚、心肌毒性、心律失常等其他心血管疾病中也發(fā)揮作用都值得進(jìn)一步研究。
綜上所述,本研究表明circLrp6在H2O2誘導(dǎo)心肌細(xì)胞凋亡和小鼠心肌缺血再灌模型中表達(dá)明顯下降,過表達(dá)circLrp6可抑制心肌細(xì)胞凋亡。
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Circular RNA Lrp6 regulates H2O2-induced apoptosis of H9c2 cardiomyocytes
DING Lin, LI Meng-yang, WANG Meng-yu, FA Hong-ge, FANG Xin-yu, WANG Jian-xun△
(,,266021,)
To investigate the effect of circular RNA Lrp6 (circLrp6) on hydrogen peroxide (H2O2)-induced cardiomyocyte apoptosis.The circular structure of circLrp6 was verified by Sanger sequencing and RNase R digestion. The subcellular localization of circLrp6 was detected by fluorescencehybridization (FISH). RT-qPCR was used to detect whether H2O2and myocardial ischemia-reperfusion influenced the expression of circLrp6 in cardiomyocytes. TUNEL staining was used to detect the apoptosis of cardiomyocytes. The downstream targets and binding sites of circLrp6 were predicted by bioinformatic methods.Circularly structural circLrp6 was localized in the nucleus. circLrp6 was down-regulated after treated with H2O2in H9c2 cells (<0.05). The expression of circLrp6 was also decreased in cardiac tissues of the mice with myocardial ischemia-reperfusion. TUNEL staining showed that overexpression of circLrp6 attenuated the apoptosis of H9c2 cells induced by H2O2. In addition, circLrp6 has the potential to combine with miRNA and protein.circLrp6 inhibits H2O2-induced cardiomyocyte apoptosis.
Circular RNA Lrp6; Cardiomyocytes; Hydrogen peroxide; Apoptosis
R363.2; R542.2
A
10.3969/j.issn.1000-4718.2022.03.004
1000-4718(2022)03-0412-08
2021-12-04
2022-01-24
[基金項(xiàng)目]國家自然科學(xué)基金資助項(xiàng)目(No. 81900259)
Tel: 0532-83780070; E-mail: wangjx@qdu.edu.cn
(責(zé)任編輯:盧萍,羅森)