邵耀中,齊 瑩,郭 鑫,蔣樹娟,黃郁晶,柳中洋,阮 強,解立怡

(西安交通大學(xué)第一附屬醫(yī)院腎臟內(nèi)科,陜西 西安 710061;*通訊作者,E-mail:275573101@qq.com)

人巨細(xì)胞病毒微小RNA miR-US4-5p靶向抑制PAK2的表達

邵耀中,齊 瑩,郭 鑫,蔣樹娟,黃郁晶,柳中洋,阮 強,解立怡*

(西安交通大學(xué)第一附屬醫(yī)院腎臟內(nèi)科,陜西 西安 710061;*通訊作者,E-mail:275573101@qq.com)

目的 尋找人巨細(xì)胞病毒微小RNA miR-US4-5p調(diào)控的靶mRNA,檢測miR-US4-5p對靶mRNA PAK2蛋白質(zhì)表達的調(diào)節(jié)作用。 方法 采用Hybrid-PCR方法及Targetscan軟件分析從人巨細(xì)胞病毒感染的人胚肺成纖維細(xì)胞總RNA中篩選候選靶mRNA;應(yīng)用熒光素酶實驗,通過構(gòu)建PMIR-PAK2報告基因,將其和miR-US4-5p、miRNA陰性對照共轉(zhuǎn)染到HEK293細(xì)胞中,驗證miR-US4-5p與候選靶mRNA的結(jié)合能力;應(yīng)用Western blot方法,通過將miR-US4-5p、miRNA陰性對照分別轉(zhuǎn)染到HEK293細(xì)胞、HELF細(xì)胞、THP-1細(xì)胞中,驗證轉(zhuǎn)染miR-US4-5p對靶mRNA蛋白質(zhì)表達的調(diào)節(jié)作用。 結(jié)果 通過Hybrid-PCR方法及Targetscan篩選出12個miR-US4-5p的待選靶mRNA;熒光素酶實驗表明:與轉(zhuǎn)染miRNA陰性對照相比,轉(zhuǎn)染miR-US4-5p可以顯著地下調(diào)PMIR-PAK2的熒光素酶活性,從而驗證了miR-US4-5p與PAK2的結(jié)合性,在HEK293細(xì)胞、HELF、THP-1細(xì)胞中PAK2可以被過表達的miR-US4-5p造成不同程度的下調(diào)。 結(jié)論 人巨細(xì)胞病毒表達的miR-US4-5p在三種不同細(xì)胞系中具備抑制PAK2蛋白質(zhì)表達的能力。

人巨細(xì)胞病毒; 微小RNAs; p21激活激酶

人巨細(xì)胞病毒(human cytomegalovirus,HCMV)是一種普遍存在的β-皰疹病毒。在免疫力低下的患者中,有較高的發(fā)病率和死亡率[1]。人巨細(xì)胞病毒具有230 kb的基因組,編碼來自16個前體的至少26個成熟miRNA[2]。miRNA對它們靶mRNA的轉(zhuǎn)錄后調(diào)控被認(rèn)為是通過miRNA 5′末端的2-7個核苷酸(被稱為種子區(qū))和位于mRNA 3′非編碼區(qū)的結(jié)合位點之間的結(jié)合來實現(xiàn)。這種結(jié)合導(dǎo)致所涉及的靶mRNA的翻譯抑制和降解[3]。越來越多的證據(jù)表明,病毒的miRNA可與免疫逃避、潛伏感染及細(xì)胞凋亡相關(guān)[4-6]。作為一個從人巨細(xì)胞病毒miR-US4前體來源的miRNA,人巨細(xì)胞病毒的miR-US4-5p是從HCMV感染的人胚肺成纖維細(xì)胞(HELF)小RNA的深度測序分析中首次鑒定的[7]。然而,它的功能目前還不清楚。

因此,基于miRNA在病毒和宿主生物學(xué)功能中所發(fā)揮的巨大作用,本研究通過Hybrid-PCR的方法篩選人巨細(xì)胞病毒miR-US4-5p的待選靶基因,通過熒光素酶試驗對待選靶基因進一步驗證,并應(yīng)用Western blot的方法在多種細(xì)胞系中驗證miR-US4-5p對待選靶基因蛋白表達的影響。這對進一步研究miR-US4-5p對宿主疾病發(fā)生的機制、探究其生物學(xué)功能均具有重要意義。

1 材料與方法

1.1 病毒株、細(xì)胞株

實驗所用的HCMV Han株是本實驗室保存的臨床分離株。人胚肺成纖維細(xì)胞(HELF)、人胚腎293細(xì)胞(HEK293)、人外周血單核細(xì)胞THP-1來自于中國科學(xué)院上海細(xì)胞所。

1.2 感受態(tài)細(xì)菌、質(zhì)粒、抗體

Top10感受態(tài)細(xì)菌購自北京天根生化科技有限公司;使用含15%胎牛血清的MEM培養(yǎng)HELF。使用含10%胎牛血清的DMEM培養(yǎng)HEK293細(xì)胞。使用含10%胎牛血清的1640培養(yǎng)THP-1細(xì)胞。PCR2.1載體購自Invitrogen公司(美國);熒光素酶報告質(zhì)粒購自Applied Biosystems公司(美國);熒光素酶活性檢測試劑購自Promega公司(美國);蛋白質(zhì)提取和檢測試劑購自Thermo公司(美國);抗體購自Abcam公司(美國);PCR試劑、限制性內(nèi)切酶、T4 DNA連接酶購自TakaRa公司(中國);、miR-US4-5p的成熟體、抑制劑及陰性對照成熟體購自RiboBio公司(中國)。

1.3 hybrid-PCR和Targetscan軟件預(yù)測篩選miR-US4-5p的候選靶mRNA

Hybrid PCR是快速有效篩選任意miRNA候選靶mRNA的方法[8]。Hybrid PCR基于3′-Full RACE的實驗方法,根據(jù)miRNA序列(5′-UGGACGUGCAGGGGGAUGUCUG-3′)設(shè)計miR-US4-5p的雜交引物(5′-CRGRCRTCCCCCTGCRCGTCCR-3′)。以HCMV感染細(xì)胞的晚期(L)總RNA為模板,使用miR-US4-5p的雜交引物與oligo dT的通用內(nèi)外引物,進行半巢式擴增,得到候選靶基因的信息池。具體操作步驟如下:培養(yǎng)HELF,密度為100%時接種HCMV Han株。在接毒后72 h,用Trizol法提取HCMV感染晚期總RNA。用TaKaRa公司的3’Full RACE試劑盒中的oligo dT引物及逆轉(zhuǎn)錄酶將mRNA反轉(zhuǎn)錄成cDNA。設(shè)計并使用miR-US4-5p的雜交引物作為上游引物,oligo dT引入的通用引物“ACCGTCGTTCCACTAGTGATTT”作為下游引物,以cDNA為模板進行PCR擴增,引物由上海invitrogen公司合成。PCR產(chǎn)物經(jīng)過純化后(A9280,Promega,美國),用TA克隆的辦法連接到PCR2.1載體(TakaRa,中國)。將連接產(chǎn)物轉(zhuǎn)化到感受態(tài)細(xì)菌中,隨機挑取陽性克隆進行DNA測序。測序結(jié)果經(jīng)過NCBI的blast軟件比對得到同源的mRNA序列,作為待選的靶mRNA(http://www.ncbi.nlm.nih.gov/blast)。同時也使用Targetscan(http://www.targetscan.org/)在線進行miR-US4-5p靶基因的預(yù)測。選取這兩個靶基因庫的交集。

1.4 熒光素酶實驗驗證miR-US4-5p與候選PAK2 mRNA的結(jié)合能力

用逆轉(zhuǎn)錄PCR方法擴增PAK2 mRNA與miR-US4-5p結(jié)合位點附近300-500 bp的序列。在擴增引物的5′末端分別加入了SpeⅠ和HindⅢ酶切位點序列。純化PCR產(chǎn)物,進行SpeⅠ和HindⅢ雙酶切。將酶切產(chǎn)物連接到pMIR report載體上熒光素酶基因的3′非翻譯區(qū)(3′UTR區(qū))的相應(yīng)位點,得到“pMIR-PAK2”表達載體。培養(yǎng)HEK293細(xì)胞,采用lip3000共轉(zhuǎn)染pMIR-PAK2、海腎熒光素酶質(zhì)粒(TK)以及miR-US4-5p的成熟體(銳博公司,廣州)到HEK293細(xì)胞。每個候選靶mRNA做3個復(fù)孔。用線蟲的一種非特異性靶向miRNA作為陰性對照。用pMIR空載體作為系統(tǒng)誤差對照。貼壁細(xì)胞通過裂解釋放熒光素酶(Dual-Luciferase? Reporter Assay E1960,promega,美國),進行熒光素酶活性檢測(LB9507,Berthhold Technologies,德國)。先檢測每一樣本的螢火蟲熒光素酶熒光強度,后檢測海腎熒光素酶熒光強度。用“螢火蟲熒光素酶熒光值/海腎熒光素酶熒光值”得到消除了組內(nèi)轉(zhuǎn)染誤差的終數(shù)據(jù)。以每個樣本的3個復(fù)孔所測終數(shù)據(jù)的平均值進行對比分析。

1.5 Western blot實驗分析miR-US4-5p對PAK2蛋白質(zhì)表達的調(diào)控作用

通過Western blot實驗,分析不同細(xì)胞系中過表達miR-US4-5p對PAK2蛋白質(zhì)表達是否有下調(diào)作用。將miR-US4-5p成熟體轉(zhuǎn)染至HEK293、HELF、THP-1細(xì)胞中,分為陰性對照組和轉(zhuǎn)染miR-US4-5p成熟體組。用線蟲的一種非特異性靶向miRNA成熟體作為微小RNA陰性對照,使用甘油醛-3-磷酸脫氫酶(GAPDH)作為組內(nèi)對照消除轉(zhuǎn)染誤差。轉(zhuǎn)染24 h后收集蛋白質(zhì)(M-PER?Mammalian Protein Extraction Reagent, PIERCE,美國)進行Western blot檢測。依次使用兔源多克隆抗體(Abcam公司,英國)和山羊抗兔的二抗(中杉金橋公司,北京)進行反應(yīng)。使用ChemiDocTMMP System(Biorad,美國)進行成像,使用儀器配套軟件image lab進行實驗結(jié)果灰度分析處理。

2 結(jié)果

2.1 Hybrid-PCR及Targetscan篩選結(jié)果

從19個hybrid-PCR克隆中獲得了4個cDNA序列(見表1)。同時通過Targetscan (http://www.targetscan.org/)預(yù)測了9個待選靶基因(見表2)。所有靶基因均來自人基因組。PAK2是唯一同時存在于兩種預(yù)測方式中的靶基因。通過生物信息學(xué)軟件RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/submission.html)繪制了miR-US4-5p與PAK2 3'-UTR方向互補示意圖,并得出最小解離自由能為-33.7 kcal/mol(見圖1)。這些靶基因涉及到細(xì)胞凋亡、HCMV感染與復(fù)制、細(xì)胞能量代謝、生物大分子合成等方面。

表1 Hybrid-PCR篩選獲得的HCMV miR-US4-5p候選靶mRNAs

Table 1 Putative targets of hcmv-miR-US4-5p identified by hybrid-PCR

mRNAnamesAccessionNo.Homosapiensmortalityfactor4like2(MORF4L2)NM-001142426HomosapiensRNAbindingprotein,fox-1homolog(C.elegans)2(RBFOX2)NM-001082577HomosapiensnuclearfactorI/X(CCAAT-bindingtranscriptionfactor)(NFIX)NM-002501Homosapiensp21protein(Cdc42/Rac)-activatedkinase2(PAK2)NM-002577

表2 Targetscan軟件預(yù)測獲得的HCMV miR-US4-5p候選靶mRNAs

Table 2 Putative targets of hcmv-miR-US4-5p predicted by Targetscan(http://www.targetscan.org/)

mRNAnamesAccessionNo.HomosapiensKCNE1-like(KCNE1L)NM-012282Homosapiensp21protein(Cdc42/Rac)-activatedkinase2(PAK2)NM-002577HomosapiensXK,Kellbloodgroupcomplexsubunit-relatedfamily,member5(XKR5)NM-001289973Homosapiensmembraneassociatedguanylatekinase,WWandPDZdomaincontaining2(MA-GI2)NM-001301128HomosapiensSp6transcriptionfactor(SP6)NM-199262Homosapienscalmodulin1(phosphorylasekinase,delta)(CALM1)NM-006888Homosapiensneuro-oncologicalventralantigen1(NOVA1)NM-002515Homosapiensproteinphosphatase3(formerly2B),regulatorysubunitB,alphaisoform(PPP3R1)NM-000945Homosapienszincfinger,BED-typecontaining4(ZBED4)NM-014838

將終止密碼子后的第一個堿基位置定義為“1”,預(yù)測的miR-US4-5p種子區(qū)與PAK2的結(jié)合位點在PAK2 3′非編碼區(qū)2240-2246的位置,其最小解離自由能為-33.7 kCal/mol圖1 PAK2 3′-UTR與miR-US4-5p結(jié)合靶位點示意圖Figure 1 A schematic diagram of predicted binding site of miR-US4-5p in the PAK2 3′UTR

2.2 熒光素酶實驗檢測結(jié)果

將PAK2 3′-UTR構(gòu)建到pMIR-REPORTERTMLuciferase載體,轉(zhuǎn)染HEK293后,通過雙熒光素酶檢測系統(tǒng)檢測miR-US4-5p對PAK2 3′-UTR的調(diào)控作用。熒光素酶活性分析結(jié)果顯示,與miRNA的陰性對照相比較,miR-US4-5p組PAK2 3′-UTR的熒光值顯著降低(P<0.05,見圖2), miR-US4-5p可以下調(diào)構(gòu)建有PAK2 3′非編碼區(qū)的PMIR質(zhì)粒(PMIR-PAK2)約50%的熒光素酶活性。

與miRNA陰性對照比較，*P<0.05圖2 驗證miR-US4-5p靶向PAK2 mRNA的熒光素酶實驗檢測結(jié)果Figure 2 Result of Luciferase assay to validate miR-US4-5p targets at PAK2

2.3 從蛋白水平驗證miR-US4-5p靶向下調(diào)PAK2表達的Western blot結(jié)果

Western blot檢測了miR-US4-5p對PAK2蛋白表達水平的影響，結(jié)果發(fā)現(xiàn),PAK2 siRNA有效地抑制了3種不同細(xì)胞系內(nèi)源表達的PAK2蛋白表達水平。與之相似,3種不同細(xì)胞系內(nèi)源表達的PAK2蛋白表達水平同時也被miR-US4-5p不同程度下調(diào)(見圖3)。

3 討論

隨著HCMV編碼的miRNA的發(fā)現(xiàn)[7,9-11],關(guān)于這些miRNAs的生物學(xué)功能逐漸被研究,包括miR-UL112-1[5,12-14]、miR-US25-1[14,15]、miR-US25-2-3p[16]、miR-UL36[17]、miR-UL70-3p以及miR-UL148D[2,18]。諸多證據(jù)表明,HCMV利用自身編碼的miRNAs來調(diào)節(jié)自身及宿主細(xì)胞的基因差異表達進一步完成免疫逃避、細(xì)胞進程調(diào)節(jié)、病毒DNA復(fù)制以及對細(xì)胞凋亡的調(diào)節(jié)。

PAK2即p21活化激酶,是一個廣泛存在于所有組織和細(xì)胞系中的62 kDa大小的蛋白。一些研究表明,不像PAK1,PAK2在細(xì)胞程序性凋亡,在不同的背景下發(fā)揮著雙重的作用。碳末端催化的36 kDa大小的PAK2片段(PAK2p34)是從PAK2裂解衍生/活化而來,與Fas和神經(jīng)酰胺誘導(dǎo)的Jurkat細(xì)胞凋亡,TNF-α誘導(dǎo)的MCF-7細(xì)胞凋亡和紫外線誘導(dǎo)的A431細(xì)胞的凋亡相關(guān)[19,20],這表明裂解衍生/活化的PAK2具有促凋亡功能。除了裂解衍生/活化的PAK2p34具有的促凋亡功能,全長的PAK2具有抗凋亡功能。體外表達持續(xù)活性PAK2-T402E,其模仿活化全長PAK2,似乎消除了PAK2p34對腫瘤壞死因子α,生長因子的撤出以及紫外線對于BALB3T3成纖維細(xì)胞的促凋亡作用。其機制似乎是對促細(xì)胞凋亡Bcl-2家族蛋白Bad的磷酸化以及調(diào)節(jié)應(yīng)激誘導(dǎo)的ERK,JNK和p38途徑[21]。此外,這種觀點被最近的觀察所支持,在用紫杉醇刺激的SKOV3-TR30細(xì)胞中過表達miR-134通過下調(diào)PAK2表達表現(xiàn)出了促凋亡的作用。這可能與把Bad磷酸化為Bad-112和Bad-136有關(guān)[22]。

1.negative control；2.hcmv-miR-US4-5P；與陰性對照比較，*P<0.05圖3 miR-US4-5p對三種細(xì)胞中PAK2蛋白表達的影響的免疫印跡結(jié)果Figure 3 PAK2 expression in three kinds of cells after treated with miR-US4-5p by Western blot

HCMV包含了許多抗凋亡的基因,如US28,US4,UL37,UL38和UL144[23-28]。另外,越來越多的研究表明,HCMV編碼的miRNA可以調(diào)節(jié)細(xì)胞凋亡。hcmv-miR-UL148D和hcmv-miR-UL36-5p被報道在感染細(xì)胞中有抑制凋亡的能力[18,29]。hcmv-miR-US25-1可以促進由氧化低密度脂蛋白所介導(dǎo)的內(nèi)皮細(xì)胞的凋亡[30]。在本研究中,過表達miR-US4-5p可以下調(diào)多種細(xì)胞系中PAK2的表達,因此推測miR-US4-5p在自然感染狀態(tài)下的后期階段可以通過抑制PAK2表達發(fā)揮促凋亡作用,從而達到相應(yīng)的生物學(xué)效應(yīng),例如促進病毒釋放。盡管如此,細(xì)胞凋亡受到很多因子調(diào)節(jié),例如BAD、TRAIL、NF-κb、Bcl-2和Bax。盡管過表達miR-US4-5p表現(xiàn)出抑制PAK2表達,但是這個對凋亡的確切作用還需要進一步的研究。可以假定,在和其他的促凋亡的HCMV的基因和miRNA一起的作用下,miR-US4-5p可能在調(diào)節(jié)自然感染狀態(tài)下細(xì)胞凋亡并在感染晚期宿主細(xì)胞和病毒之間建立平衡發(fā)揮著作用。

[1] Kesson AM, Kakakios A.Immunocompromised children: conditions and infectious agents[J]. Paediatr Respir Rev, 2007, 8:231-239.

[2] Babu SG, Pandeya A, Verma N,etal.Role of HCMV miR-UL70-3p and miR-UL148D in overcoming the cellular apoptosis[J]. Mol Cell Biochem, 2014, 393: 89-98.

[3] Fabian MR, Sonenberg N, Filipowicz W. Regulation of mRNA translation and stability by microRNAs[J]. Ann Rev Biochem, 2010, 79: 351-379.

[4] Cullen BR. Viral and cellular messenger RNA targets of viral microRNAs[J]. Nature, 2009, 457: 421-425.

[5] Huang T, Cui Y, Zhang X.Involvement of viral microRNA in the regulation of antiviral apoptosis in shrimp[J]. J Virol, 2014, 88: 2544-2554.

[6] Skalsky RL, Cullen BR. Viruses, microRNAs, and host interactions[J]. Ann Revi Microbiol, 2010, 64: 123-141.

[7] Stark TJ, Arnold JD, Spector DH,etal. High-resolution profiling and analysis of viral and host small RNAs during human cytomegalovirus infection[J]. J Virol, 2012, 86:226-235.

[8] Huang Y, Qi Y, Ruan Q,etal. A rapid method to screen putative mRNA targets of any known microRNA[J]. Virol J, 2011, 8: 8.

[9] Dunn W, Trang P, Zhong Q,etal. Human cytomegalovirus expresses novel microRNAs during productive viral infection[J]. Cell Microbiol, 2005, 7:1684-1695.

[10] Grey F, Antoniewicz A, Allen E,etal. Identification and characterization of human cytomegalovirus-encoded microRNAs[J]. J Virol, 2005, 79:12095-12099.

[11] Pfeffer S, Sewer A, Lagos-Quintana M,etal. Identification of microRNAs of the herpesvirus family[J]. Nature Methods, 2005, 2:269-276.

[12] Huang Y, Qi Y, Ma Y,etal. The expression of interleukin-32 is activated by human cytomegalovirus infection and down regulated by hcmv-miR-UL112-1[J]. Virol J, 2013, 10:51.

[13] Stern-Ginossar N, Elefant N, Zimmermann A,etal. Host immune system gene targeting by a viral miRNA[J]. Science, 2007, 317:376-381.

[14] Stern-Ginossar N, Saleh N, Goldberg MD,etal. Analysis of human cytomegalovirus-encoded microRNA activity during infection[J]. J Virol, 2009, 83:10684-10693.

[15] Grey F, Tirabassi R, Meyers H,etal. A viral microRNA down-regulates multiple cell cycle genes through mRNA 5′UTRs[J]. PLoS Pathog, 2010, 6:e1000967.

[16] Qi M, Qi Y, Ma Y,etal. Over-expression of human cytomegalovirus miR-US25-2-3p downregulates eIF4A1 and inhibits HCMV replication[J]. FEBS Lett, 2013, 587:2266-2271.

[17] Huang Y, Qi Y, Ma Y,etal. Down-regulation of human cytomegalovirus UL138, a novel latency-associated determinant, by hcmv-miR-UL36[J]. J Biosci, 2013, 38:479-485.

[18] Wang YP, Qi Y, Huang YJ,etal. Identification of immediate early gene X-1 as a cellular target gene of hcmv-mir-UL148D[J]. Int JMol Med, 2013,31:959-966.

[19] Rudel T, Bokoch GM. Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2[J]. Science, 1997, 276:1571-1574.

[20] Tang TK, Chang WC, Chan WH,etal. Proteolytic cleavage and activation of PAK2 during UV irradiation-induced apoptosis in A431 cells[J]. J Cell Biochem, 1998, 70:442-454.

[21] Jakobi R, Moertl E, Koeppel MA. p21-activated Protein Kinase-PAK Suppresses Programmed Cell Death of BALB3T3 Fibroblasts[J]. J Biol Chem, 2001, 276:16624-16634.

[22] Shuang T, Wang M, Shi C,etal. Down-regulated expression of miR-134 contributes to paclitaxel resistance in human ovarian cancer cells[J]. FEBS Lett, 2015, 589:3154-3164.

[23] Arnoult D, Bartle LM, Skaletskaya A,etal. Cytomegalovirus cell death suppressor vMIA blocks Bax-but not Bak-mediated apoptosis by binding and sequestering Bax at mitochondria[J]. ProcNatl Acad Sci US A, 2004, 101: 7988-7993.

[24] Goldmacher VS, Bartle LM, Skaletskaya A,etal. A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2[J]. Proc Natl Acad Sci USA, 1999, 96: 12536-12541.

[25] Pleskoff O, Casarosa P, Verneuil L,etal. The human cytomegalovirus-encoded chemokine receptor US28 induces caspase-dependent apoptosis[J]. FEBS J, 2005, 272: 4163-4177.

[26] Skaletskaya A, Bartle LM, Chittenden T,etal. A cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase-8 activation[J]. Proc Natl Acad Sci USA, 2001. 98(14): 7829-7834.

[27] Smith W, Tomasec P, Aicheler R,etal. Human cytomegalovirus glycoprotein UL141 targets the TRAIL death receptors to thwart host innate antiviral defenses[J]. Cell Host Microbe, 2013, 13(3): 324-335.

[28] Terhune S, Torigoi E, Moorman N,etal. Human cytomegalovirus UL38 protein blocks apoptosis[J]. J Virol, 2007, 81(7): 3109-3123.

[29] Guo X, Huang Y, Qi Y,etal. Human cytomegalovirus miR-UL36-5p inhibits apoptosis via downregulation of adenine nucleotide translocator 3 in cultured cells[J]. Arch Virol, 2015, 160(10): 2483-2490.

[30] Fan J, Zhang W, Liu Q. Human cytomegalovirus-encoded miR-US25-1 aggravates the oxidised low density lipoprotein-induced apoptosis of endothelial cells[J]. Biomed Res Int, 2014, 2014: 531979.

Human cytomegalovirus miRNA miR-US4-5p inhibits expression of PAK2

SHAO Yaozhong,QI Ying,GUO Xin,JIANG Shujuan,HUANG Yujing,LIU Zhongyang, RUAN Qiang,XIE Liyi*

(DepartmentofNephrology,FirstAffiliatedHospital,Xi’anJiaotongUniversity,Xi’an710061,China;*Correspondingauthor,E-mail:275573101@qq.com)

ObjectiveTo find the target message RNAs(mRNA) of human cytomegalovirus(HCMV) microRNA(miRNA) miR-US4-5p, and to explore the effects of miR-US4-5p on protein expression of the target mRNA PAK2.MethodsHybrid-PCR and Targetscan were used to screen the candidate target mRNAs in the pool of human embryo lung fibroblast(HELF) total RNAs. Luciferase report assay was used to validate the specific banding of miR-US4-5p to PAK2 mRNA after PMIR-PAK2 was constructed and contransfected with miR-US4-5p and miRNA negative control to HEK293 cells. Western blot was used to validate the regulation effects of miR-US4-5p on the protein expression of target mRNA after miR-US4-5p, miRNA negative control transfected to HEK293 cells, HELF cells, THP-1 cells.ResultsTwelve mRNAs, including PAK2(p21-activated Kinase 2), were identified as the targets of miR-US4-5p. Dual-luciferase assay showed the luciferase activity of PMIR-PAK2 was significantly down-regulated after transfection of miR-US4-5p compared to the miRNA negative control, which validated the binding of miR-US4-5p and PAK2. PAK2 expression was down-regulated by over-expressed miR-US4-5p in HEK293, HELF and THP-1 cells.ConclusionHCMV miR-US4-5p may have the ability to inhibit the protein expression of its target mRNA, PAK2, during infection.

human cytomegalovirus; microRNAs; PAK2

國家自然科學(xué)基金資助項目(81171580)；教育部高等學(xué)校博士學(xué)科點專項科研基金資助項目(20112104110012)

邵耀中,男,1988-05生,博士,助理研究員,E-mail:asdsaosao@163.com

2017-04-10

Q78

A

1007-6611(2017)08-0808-05

10.13753/j.issn.1007-6611.2017.08.011