動(dòng)物microRNA-181的功能與應(yīng)用前景

常楊,穆偉濤,滿朝來

哈爾濱師范大學(xué)生命科學(xué)與技術(shù)學(xué)院,黑龍江省分子細(xì)胞遺傳與遺傳育種重點(diǎn)實(shí)驗(yàn)室,哈爾濱 150025

動(dòng)物microRNA(miRNA)是一類長度約17～25個(gè)核苷酸、在進(jìn)化上高度保守的非編碼RNA。miRNA在多種細(xì)胞活動(dòng)中通過阻止靶mRNA的翻譯或互補(bǔ)結(jié)合誘導(dǎo)其降解,在轉(zhuǎn)錄后水平調(diào)節(jié)基因的表達(dá)。miRNA 通常結(jié)合在靶基因的 3′-非翻譯區(qū)(3′-UTR)發(fā)揮對靶基因的抑制作用,但也發(fā)現(xiàn)miRNA能夠靶向基因的編碼區(qū)(Coding sequence,CDS),如Huang等[1]研究表明miR-181a就是通過直接靶向鋅指蛋白mRNA的編碼序列來抑制其表達(dá),進(jìn)而實(shí)現(xiàn)轉(zhuǎn)錄后調(diào)控。miR-181以其在多種組織臟器中選擇性表達(dá)(如小鼠miR-181在胸腺、腦和肺等組織臟器中選擇性的高表達(dá)[2]),并具有功能多樣、生物學(xué)效應(yīng)顯著等特點(diǎn),近年來逐漸成為人們關(guān)注的焦點(diǎn)之一。

人和小鼠miR-181家族都包括4類高度保守的成員,即 miR-181a、miR-181b、miR-181c和 miR-181d,至今已鑒定出它們的種子區(qū)域能夠靶向上百種mRNA。miR-181對于細(xì)胞的生長分化和機(jī)體的免疫功能調(diào)節(jié)至關(guān)重要,并且在多種疾病中發(fā)現(xiàn)了miR-181表達(dá)異常。本文從細(xì)胞增殖、凋亡、分化、免疫和腫瘤等幾個(gè)方面綜述了動(dòng)物miR-181的研究進(jìn)展,以期為miR-181的深入研究提供參考。

1 miR-181與細(xì)胞增殖和凋亡

細(xì)胞的增殖與凋亡是動(dòng)物機(jī)體正常生長代謝的必然過程,該過程被眾多細(xì)胞信號分子以精確機(jī)制網(wǎng)絡(luò)調(diào)控。其中,miRNA作為轉(zhuǎn)錄后調(diào)控的主要表觀調(diào)控機(jī)制之一,miR-181家族成員參與多種細(xì)胞的增殖和凋亡過程,在細(xì)胞的生長代謝中發(fā)揮關(guān)鍵的調(diào)控作用(表1)。

首先,miR-181能夠抑制多種細(xì)胞的增殖過程。例如,Zhang等[3]研究發(fā)現(xiàn)ActivinA能以劑量和時(shí)間依賴性方式抑制miR-181a在小鼠顆粒細(xì)胞(Granulosa cell)中的表達(dá); 如果 miR-181a過表達(dá),則會(huì)抑制其靶標(biāo) ActivinⅡA 型受體(ActRⅡA)的翻譯,進(jìn)而導(dǎo)致細(xì)胞周期蛋白 D2(CyclinD2)和增殖細(xì)胞核抗原(Proliferating cell nuclear antigen,PCNA)表達(dá)的下調(diào),從而阻止該細(xì)胞的增殖,所以miR-181a在功能上能夠負(fù)向調(diào)控顆粒細(xì)胞的增殖。Liu等[4]研究發(fā)現(xiàn)miR-181a也可以抑制間充質(zhì)干細(xì)胞(Mesenchymal stem cell,MSC)的增殖,卻不影響該細(xì)胞的凋亡; 在MSC細(xì)胞中過表達(dá)miR-181a,能夠阻止TGF-β信號路徑的激活,并且下調(diào)該路徑中的靶基因Tgfbr1(Transforming growth factor beta receptor 1)和Tgfbrap1(Transforming growth factor beta receptor associated protein 1)表達(dá)活性,進(jìn)而實(shí)現(xiàn) miR-181a抑制MSC細(xì)胞增殖的調(diào)控作用。此外,Dahlhaus等[5]研究發(fā)現(xiàn),在急性髓系細(xì)胞白血病(Acute myelocytic leukemia,AML)細(xì)胞中過表達(dá)miR-181a會(huì)顯著降低該細(xì)胞的增殖和代謝活性,而抑制miR-181a表達(dá)對AML細(xì)胞增殖和代謝活性卻沒有產(chǎn)生影響。所以推測miR-181a調(diào)控細(xì)胞增殖的途徑可能很多,有些細(xì)胞效應(yīng)可能無法用單一信號機(jī)制來說明。例如,miRNA的表達(dá)變化可能會(huì)改變細(xì)胞內(nèi)環(huán)境,進(jìn)而誘導(dǎo)細(xì)胞多種功能的改變,綜合效果是干擾細(xì)胞的增殖過程。

miR-181a除了能夠抑制多種細(xì)胞增殖外,也可以促進(jìn)多種細(xì)胞凋亡。Zhu等[6]研究發(fā)現(xiàn)順氯氨鉑(Cisplatin)處理過的人腎近曲小管上皮細(xì)胞(HK-2)凋亡率明顯上升,而且miR-181a的表達(dá)活性也顯著升高,進(jìn)一步研究表明：miR-181a能夠下調(diào)抗凋亡蛋白 BCL-2表達(dá)活性,上調(diào)促凋亡基因Bax,從而促進(jìn) HK-2細(xì)胞凋亡。Chen等[7]在研究人惡性膠質(zhì)瘤細(xì)胞(U87MG)對放射物質(zhì)的敏感性時(shí),亦發(fā)現(xiàn)在輻射處理后的 U87MG細(xì)胞中,BCL-2上調(diào),miR-181a下調(diào); 而過表達(dá)miR-181a則會(huì)下調(diào)BCL-2蛋白。Ouyang等[8]利用熒光素酶檢測系統(tǒng)也進(jìn)一步證明了Bcl-2和Mcl-1是miR-181a的靶標(biāo),他們發(fā)現(xiàn)在星狀膠質(zhì)細(xì)胞中下調(diào) miR-181a能夠減少缺糖損傷誘導(dǎo)的細(xì)胞凋亡、線粒體功能紊亂和線粒體膜電位消失,所以調(diào)控miR-181a的表達(dá)對臨床提高惡性膠質(zhì)瘤的治療效率可能具有重要參考價(jià)值。

表1 miR-181在細(xì)胞增生、凋亡與分化中的作用

此外,人們發(fā)現(xiàn)miR-181b也與細(xì)胞增殖和凋亡密切相關(guān),miR-181a和miR-181b雖為同一家族成員,但在相同組織細(xì)胞中的生物學(xué)作用卻大相徑庭。例如,Chen等[9]發(fā)現(xiàn)在AML細(xì)胞中miR-181b是高表達(dá)的,miR-181b通過靶向Mlk2基因來促進(jìn)AML細(xì)胞的增殖,所以miR-181b 在AML中起著促進(jìn)細(xì)胞增殖的調(diào)控作用,這與miR-181a抑制AML細(xì)胞增殖的功能相反。有趣的是,既是同一 miR-181在不同組織細(xì)胞中的功能作用也會(huì)存在差異。例如 Shi等[10]研究發(fā)現(xiàn)人miR-181a和miR-181b在神經(jīng)膠質(zhì)瘤細(xì)胞中的功能均表現(xiàn)為抑制作用,即抑制腫瘤細(xì)胞生長、誘導(dǎo)凋亡和抑制轉(zhuǎn)移,而且miR-181b的抑制效應(yīng)強(qiáng)于miR-181a,這與miR-181b在AML細(xì)胞中的促進(jìn)作用相反。

2 miR-181與細(xì)胞分化

細(xì)胞分化在動(dòng)物機(jī)體生長發(fā)育和損傷修復(fù)中發(fā)揮著重要作用,研究發(fā)現(xiàn) miR-181對多種細(xì)胞分化具有促進(jìn)作用(表1)。

首先,miR-181對人胚胎干細(xì)胞(Human embryonic stem cells,hESCs)的分化具有促進(jìn)作用。例如,Xu等[11]研究發(fā)現(xiàn)共激活因子相關(guān)的精氨酸甲基轉(zhuǎn)移酶1(Coactivator-associated arginine methyltransferase1,CARM1)是miR-181c的靶基因,而CARM1可以催化組蛋白甲基化[12,13],在抑制 hESCs分化中起著積極的作用,所以 miR-181c過表達(dá)能夠促進(jìn)hESCs細(xì)胞的分化。Kane等[14]發(fā)現(xiàn)在hESCs向血管內(nèi)皮細(xì)胞(Endothelial cells,ECs)分化的過程中,miR-181a和 miR-181b以時(shí)間依賴性和分化依賴性的方式逐漸增加,在向 ECs細(xì)胞分化的hESCs(hESC-ECs)中過表達(dá)miR-181a和b能夠增加血管內(nèi)皮細(xì)胞特異的標(biāo)志分子血小板內(nèi)皮細(xì)胞黏附分子-1(Platelet endothelial cell adhesion molecule-1,PECAM-1)和血管內(nèi)皮鈣粘蛋白(Vascular endothelial cadherin,VE-cadherin)的表達(dá)活性,增加NO的產(chǎn)量,促進(jìn)hESCs向ECs分化。O'Loghlen等[15]發(fā)現(xiàn)CBX7(Chromobox protein homolog 7)在ESC細(xì)胞分化中發(fā)揮著關(guān)鍵作用,并且 miR-181可以通過調(diào)控 CBX7進(jìn)而間接調(diào)控ESC細(xì)胞的分化。這些研究雖然發(fā)現(xiàn)了miR-181以不同的分子機(jī)制調(diào)控ESC細(xì)胞的分化,但研究結(jié)果都一致表明miR-181在ESC細(xì)胞分化調(diào)控中發(fā)揮著關(guān)鍵作用。

其次,miR-181對造血干細(xì)胞的分化具有促進(jìn)作用。Mintz等[16]發(fā)現(xiàn)在 CD34+造血干細(xì)胞中,miR-181a*能夠靶向Nanog基因的3′-UTR抑制該基因表達(dá),而NANOG、SOX2和OCT4等是維持細(xì)胞自我更新未分化狀態(tài)的重要因子[17～19],所以miR-181a*能夠促進(jìn) CD34+細(xì)胞的分化。Li等[20]以人 CD34+造血干細(xì)胞作為研究模型,發(fā)現(xiàn) Lin28是miR-181a的一個(gè)直接靶基因,而且miR-181起著“分子開關(guān)”的作用,提高miR-181a表達(dá)水平會(huì)首先下調(diào)Lin28,然后上調(diào)let-7,進(jìn)而促進(jìn)巨核細(xì)胞的分化。

最后,miR-181對成肌細(xì)胞和成骨細(xì)胞的分化具有促進(jìn)作用。Naguibneva等[21]發(fā)現(xiàn),在肌纖維分化期間的兩個(gè)主要階段里,miR-181是終末分化時(shí)上調(diào)水平最顯著的 miRNA之一,在肌肉肌酸激酶(Muscle creatine kinase,MCK)等分化特異蛋白表達(dá)前或表達(dá)同時(shí)被上調(diào)。在小鼠成肌細(xì)胞(C2C12)體外分化期間,MyoD誘導(dǎo)Myogenin生成并促進(jìn)整個(gè)分化過程,在缺少miR-181的細(xì)胞中 MyoD表達(dá)受到抑制。進(jìn)一步研究發(fā)現(xiàn)miR-181能夠下調(diào)同源盒蛋白HOX-A11(分化過程的一個(gè)阻遏物),而HOX-A11會(huì)抑制MyoD表達(dá),因此,miR-181表達(dá)上調(diào)會(huì)促進(jìn)MyoD表達(dá),進(jìn)而促進(jìn)C2C12細(xì)胞分化。Li等[22]在大鼠成肌細(xì)胞(L6)中,利用病毒載體介導(dǎo) miR-181a表達(dá)下調(diào)后,發(fā)現(xiàn)能夠顯著促進(jìn)L6細(xì)胞的增生而不發(fā)生分化,這也進(jìn)一步印證了 miR-181對成肌細(xì)胞的分化促進(jìn)作用。在miR-181促進(jìn)成骨細(xì)胞分化方面,Bhushan等[23]研究發(fā)現(xiàn) miR-181a 在 BMP(Bone morphogenetic protein)誘導(dǎo)的 C2C12和 MC3T3細(xì)胞成骨分化的過程中顯著上調(diào),miR-181a可以通過靶向成骨細(xì)胞分化的負(fù)向調(diào)控子(RGS4 和 GATA6)來抑制TGF-β信號分子,進(jìn)而促進(jìn)成骨細(xì)胞的分化。

3 miR-181與免疫

miR-181在淋巴細(xì)胞的增生分化與發(fā)育、自身免疫、炎癥、抗病毒及免疫調(diào)節(jié)等多個(gè)方面都具有重要的調(diào)控作用(表2)。

首先,miRNA在造血干細(xì)胞向各種血細(xì)胞分化的過程中扮演著重要的角色。Chen等[2]研究發(fā)現(xiàn)miR-181在小鼠骨髓B淋巴細(xì)胞中被優(yōu)先表達(dá),并且miR-181在造血干細(xì)胞中的表達(dá)也會(huì)促進(jìn)向B細(xì)胞系分化,miR-181在促進(jìn)B淋巴細(xì)胞分化方面發(fā)揮著重要的調(diào)控作用。

表2 miR-181在免疫中的作用

其次,miR-181在T細(xì)胞的發(fā)育過程中發(fā)揮著關(guān)鍵作用。Chen等[24]研究表明 mir-181a-1可以促進(jìn)CD4-和CD8-T細(xì)胞向CD4+和CD8+T細(xì)胞的分化,而miR-181c并無此功能。Li等[25]將miR-181a在T細(xì)胞中過表達(dá),利用抗原刺激后檢測到細(xì)胞內(nèi)鈣離子增加,IL-2表達(dá)量較對照組也提高了兩倍,并且提高了T細(xì)胞受體(T cell receptor,TCR)介導(dǎo)的CD4+T細(xì)胞的活性,因?yàn)樵?T細(xì)胞發(fā)育的整個(gè)時(shí)期,不同發(fā)育階段的T細(xì)胞對抗原的敏感性不同,miR-181a能夠下調(diào)TCR信號路徑的多個(gè)負(fù)向調(diào)節(jié)物[26],進(jìn)而提高TCR對抗原的敏感性,所以miR-181a在T細(xì)胞發(fā)育期間起到“信號變阻器”的作用。

有趣的是,miR-181c對T細(xì)胞的調(diào)控卻表現(xiàn)出與 miR-181a相反的作用。Xue等[27]研究發(fā)現(xiàn)miR-181c的表達(dá)在T細(xì)胞活化過程中被下調(diào),轉(zhuǎn)染miR-181c模擬物會(huì)部分抑制Jurkat 細(xì)胞和人外周血單核細(xì)胞(Periphera blood mononuclear cell,PBMC)CD4+T細(xì)胞的活化,并且降低已激活CD4+T細(xì)胞的增殖能力。他們進(jìn)一步研究發(fā)現(xiàn)IL-2是miR-181c的直接靶標(biāo),miR-181c能夠抑制 IL-2的表達(dá),而IL-2能夠促進(jìn)T細(xì)胞的增生,所以miR-181c過表達(dá)會(huì)抑制外周血CD4+T細(xì)胞的活化與增殖。

再次,miR-181能夠促進(jìn)NK細(xì)胞(Natural killer cell)的發(fā)育,提高NK細(xì)胞的功能活性。Frank等[28]研究發(fā)現(xiàn) miR-181的表達(dá)水平影響 CD34+造血干細(xì)胞向NK細(xì)胞的分化和IFN-γ在初級CD56+NK細(xì)胞中的表達(dá)量。當(dāng)敲除miR-181a或miR-181b,會(huì)導(dǎo)致成熟的CD56+NK細(xì)胞比例降低,而過表達(dá)miR-181a或miR-181b會(huì)導(dǎo)致CD56+NK細(xì)胞明顯增多,同時(shí)顯著提高 NK細(xì)胞的 IFN-γ產(chǎn)量。進(jìn)一步研究發(fā)現(xiàn)負(fù)向調(diào)節(jié) Norch信號的 NLK(Nemo-like kinase)是miR-181的直接靶標(biāo),而活化的Norch信號通路能夠促進(jìn) NK細(xì)胞的發(fā)育,所以 miR-181是通過下調(diào)NLK這一機(jī)制來促進(jìn)NK細(xì)胞的發(fā)育。此外,Zi?tara等[29]和 Henao-Mejia等[30]的研究都一致表明miR-181是早期NKT細(xì)胞發(fā)育生物合成代謝所必需的,miR-181可以調(diào)控磷酸酶 PTEN的表達(dá)來控制PI3K信號通路,而該信號通路是免疫細(xì)胞合成代謝的主要激活途徑。

另外,miR-181與自身免疫反應(yīng)也具有密切關(guān)系。Lashine等[31]研究發(fā)現(xiàn)miR-181a在系統(tǒng)性紅斑狼瘡(Systemic lupus erythematosus,SLE)患者中表現(xiàn)為顯著下調(diào),而 PCAF(p300-CBP-associated factor)的表達(dá)量卻顯著上調(diào),這是因?yàn)镻CAF是miR-181a的靶標(biāo),又是p53調(diào)控相關(guān)的關(guān)鍵蛋白,PCAF可以通過乙酰化 p53使其穩(wěn)定,并泛素化 p53的負(fù)向調(diào)控子HDM2,使更多的p53釋放出來促進(jìn)細(xì)胞凋亡。細(xì)胞凋亡紊亂與自身免疫病的發(fā)生密切相關(guān),雖然目前通過改變 miRNA的表達(dá)活性實(shí)現(xiàn)對相應(yīng)疾病的治療仍有待于深入研究,但miR-181在SLE中的調(diào)控機(jī)制給自身免疫病的治療提供了一個(gè)新的探索思路。

最后,miR-181在炎癥反應(yīng)中也扮演著重要角色,既能表現(xiàn)出抑制炎癥的功能,也能發(fā)揮對炎癥反應(yīng)的促進(jìn)作用。Wu等[32]報(bào)道了miR-181a負(fù)向調(diào)節(jié)氧化型低密度脂蛋白(Oxidized low density lipoprotein,Ox-LDL)誘導(dǎo)樹突狀細(xì)胞(Dendritic cells,DCs)的炎癥反應(yīng)。他們在DCs細(xì)胞中過表達(dá)miR-181a,會(huì)誘導(dǎo)下調(diào)CD83和CD40,減少Ox-LDL刺激下的CD83和 CD40的上調(diào),還可以抑制炎癥因子 TNF-α和IL-6的表達(dá),并提高抗炎因子IL-10的表達(dá); 而抑制內(nèi)源 miR-181a的表達(dá),則會(huì)增加 Ox-LDL誘導(dǎo)的CD83和 CD40的表達(dá)。進(jìn)一步研究發(fā)現(xiàn) miR-181a是通過靶向下調(diào)關(guān)鍵的炎癥相關(guān)轉(zhuǎn)錄因子 c-Fos的表達(dá),進(jìn)而實(shí)現(xiàn)炎癥抑制效應(yīng)。Hutchison等[33]也進(jìn)一步驗(yàn)證了miR-181的抗炎作用,發(fā)現(xiàn) miR-181家族成員在星狀膠質(zhì)細(xì)胞中是受發(fā)育調(diào)控和高表達(dá)的,封閉 miR-181的表達(dá)會(huì)增強(qiáng) LPS(Lipopolysac-charides)誘導(dǎo)的星狀膠質(zhì)細(xì)胞促炎因子(TNF-a、IL-6、IL-1b、IL-8)和 HMGB1(High mobility group box-1 protein)的生成,而過表達(dá) miR-181則會(huì)導(dǎo)致抗炎因子 IL-10表達(dá)的顯著上調(diào)。進(jìn)一步研究發(fā)現(xiàn)甲基CpG結(jié)合蛋白2(MeCP2)和X連鎖凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)mRNA是miR-181的作用靶標(biāo),所以miR-181在星狀膠質(zhì)細(xì)胞炎癥的分子反應(yīng)中發(fā)揮重要的抗炎作用。而Feng等[34]發(fā)現(xiàn)miR-181在哮喘小鼠模型中卻發(fā)揮著促進(jìn)炎癥反應(yīng)的作用,研究結(jié)果表明,在哮喘病發(fā)作的前期,哮喘組小鼠脾 CD4+T 細(xì)胞miR-181a的表達(dá)水平比對照組顯著提高,5 d后降至與對照組相同的水平,并且miR-181a表達(dá)量與炎性細(xì)胞的數(shù)量呈正向線性相關(guān)。

值得一提的是,miR-181能夠靶向病毒復(fù)制所需的胞內(nèi)因子或者直接靶向病毒基因組 RNA來抑制病毒的復(fù)制,進(jìn)而參與宿主的抗病毒免疫應(yīng)答。Guo等[35]研究發(fā)現(xiàn)miR-181家族成員能特異結(jié)合在豬繁殖和呼吸綜合癥病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)基因組的ORF4基因下游的一個(gè)高度保守區(qū)上,以特異性和劑量依賴性方式直接抑制 PRRSV的復(fù)制,其中以 miR-181c表現(xiàn)出最強(qiáng)的抑制效應(yīng)。Gao等[36]研究發(fā)現(xiàn)miR-181能夠靶向細(xì)胞CD163 mRNA的3'-UTR,下調(diào)豬血單核細(xì)胞和肺巨噬細(xì)胞(Pulmonary alveolar macrophages,PAMs)中PRRSV受體CD163的表達(dá),CD163下調(diào)后會(huì)抑制PRRSV進(jìn)入PAMs的感染效率,所以miR-181c可以被用于抗 PRRSV感染的治療靶標(biāo),在抗PRRSV感染方面具有潛在的應(yīng)用價(jià)值。

4 miR-181與腫瘤

蛋白編碼基因(如癌基因和抑癌基因)在揭示腫瘤發(fā)生和發(fā)展中起著關(guān)鍵作用,而近來越來越多的證據(jù)表明：miRNA的功能失調(diào)也與腫瘤發(fā)生密切相關(guān)。Ji等[37,38]研究發(fā)現(xiàn) miR-181能夠直接靶向肝細(xì)胞分化轉(zhuǎn)錄調(diào)控子(如尾型同源盒轉(zhuǎn)錄因子 2(Caudal type homeobox transcription factor 2,CDX2)和GATA結(jié)合蛋白6(GATA6))和Wnt/β-catenin 信號通路抑制子(NLK))。進(jìn)一步研究發(fā)現(xiàn),miR-181家族成員的表達(dá)與 β-catenin表達(dá)成正相關(guān),而且 miR-181a-2和 miR-181b-2基因啟動(dòng)子區(qū)域存在 7個(gè)β-catenin/Tcf4結(jié)合位點(diǎn),表明 miR-181可以被原發(fā)性肝癌(Hepatocellular carcinoma,HCC)中 Wnt/βcatenin信號路徑激活,這說明 miR-181、NLK 和β-catenin存在相互調(diào)控關(guān)系,為將來治療HCC提供了一個(gè)有效的切入點(diǎn)。此外,Meng等[39]研究發(fā)現(xiàn)在肝癌干細(xì)胞(Hepatocellular cancer stem cells,HSCs)中l(wèi)et-7 和miR-181家族成員的表達(dá)上調(diào)。抑制let-7能夠上調(diào)HSCs對 sorafenib 和 doxorubicin的化療敏感性,沉默miR-181的表達(dá)會(huì)導(dǎo)致HSCs遷移和侵襲能力降低; 如果敲除 HSCs細(xì)胞的 IL-6和 Twist基因會(huì)顯著降低let-7 和miR-181的表達(dá),并會(huì)抑制化療抗性和細(xì)胞侵襲。他們還發(fā)現(xiàn) let-7直接靶向SOCS-1和caspase-3,而miR-181直接靶向RASSF1A、TIMP3 和 nemo-like kinase(NLK),這進(jìn)一步表明let-7和miR-181可能會(huì)成為治療肝癌的分子靶標(biāo)。

在肝癌的發(fā)生方面,Song等[40]研究發(fā)現(xiàn)當(dāng)肝癌細(xì)胞(HepG2)暴露于多環(huán)芳烴(PAHs)的苯并(a)蒽(Benzo[a]anthracene,BA)和苯并(k)熒蒽(Benzo[k]fluoranthene,BF)下,miR-181a、-181b 和-181d 的表達(dá)會(huì)顯著上調(diào),miR-181的有效靶標(biāo)MAPK磷酸酶-5(MAPK phosphatase-5,MKP-5)被顯著抑制,而此時(shí) p38 MAPK的磷酸化水平卻迅速提高。封閉miR-181表達(dá)后能夠增強(qiáng)MKP-5的表達(dá)和抑制p38 MAPK的磷酸化,并且能夠抑制癌細(xì)胞的遷移,表明 miR-181是通過靶向 MKP-5進(jìn)而來調(diào)控 p38 MAPK活性,并在PAH誘導(dǎo)的肝癌發(fā)生中起著關(guān)鍵作用。Wang等[41]利用飼喂缺乏膽堿和氨基酸食物(Choline-deficient and amino acid defined,CDAA)的小鼠培育65周齡的癌前病變和84周齡的肝癌模型,結(jié)果表明飼喂 CDAA食物的小鼠在早期階段miR-181b上調(diào),miR-181b通過靶向下調(diào)金屬蛋白酶3(Tissue inhibitor of metalloprotease-3,TIMP3)進(jìn)而促進(jìn)肝癌發(fā)生。這些研究數(shù)據(jù)表明：miR-181不但是肝癌發(fā)生的誘因,也可能成為肝癌治療的有力武器。

不同miR-181家族成員生物學(xué)功能的差異在腫瘤疾病中也得到了有力體現(xiàn)。例如,miR-181a能夠促進(jìn)胃癌的發(fā)生發(fā)展,而miR-181b卻能夠抑制胃癌細(xì)胞的增生。Zhang等[42]研究發(fā)現(xiàn)miR-181a在人胃癌組織中過表達(dá),并且miR-181a過表達(dá)會(huì)促進(jìn)癌細(xì)胞的增殖、轉(zhuǎn)移和入侵,抑制胃癌細(xì)胞的凋亡。進(jìn)一步研究發(fā)現(xiàn) miR-181a是阻止了抑癌基因Klf6(Kruppel-like factor 6)的表達(dá),進(jìn)而對胃癌實(shí)現(xiàn)正向調(diào)控。而Chen等[43]發(fā)現(xiàn)miR-181b在人胃腺癌組織中顯著下調(diào),這是因?yàn)閙iR-181b能夠下調(diào)cAMP反應(yīng)元件結(jié)合蛋白(CREB1)的表達(dá),所以miR-181b過表達(dá)會(huì)阻抑胃癌細(xì)胞的增殖。

近年來,循環(huán)miRNA在疾病診斷中的地位越來越受到人們的關(guān)注,其中 miR-181在乳腺癌的發(fā)生和早期篩檢中也表現(xiàn)出了潛在的應(yīng)用價(jià)值。例如,Neel等[44]研究發(fā)現(xiàn)在乳腺癌細(xì)胞中,miR-181被activin 和 TGF-β共同調(diào)控,并在這兩個(gè)生長因子的下游發(fā)揮著腫瘤促進(jìn)作用。Guo等[45]采用實(shí)時(shí)定量 PCR技術(shù)檢測,發(fā)現(xiàn)乳腺癌患者血清中的 miR-181a水平顯著低于正常人,他們利用miR-181a作為標(biāo)記物用于乳腺癌的診斷,診斷結(jié)果的敏感性和特異性分別為70.7%和59.9%,這顯著高于傳統(tǒng)的腫瘤標(biāo)記物 CA153和 CEA(敏感性分別為 10.53%和9.21%),所以血清 miR-181作為一個(gè)腫瘤標(biāo)記物,在早期乳腺癌診斷和治療靶標(biāo)的選擇中可能具有較好的應(yīng)用前景。

miR-181不但在腫瘤診斷中具有潛在的應(yīng)用價(jià)值,而且在腫瘤的預(yù)后中也具有積極的開發(fā)前景。Nishimura等[46]利用實(shí)時(shí)定量 PCR技術(shù)分析了 162例結(jié)腸癌(Colorectal cancer,CRC)病人,發(fā)現(xiàn)正常結(jié)腸組織與結(jié)腸癌組織中 miR-181a的表達(dá)并無顯著差異; 但是,根據(jù) miR-181a的表達(dá)水平,把癌組織樣本分為低水平表達(dá)和高水平表達(dá)兩組,通過對這兩組的臨床病理因子和預(yù)后的比較,發(fā)現(xiàn) miR-181a高水平表達(dá)組比低水平表達(dá)組有著顯著的預(yù)后不良(P=0.011)。多變量分析表明miR-181a高表達(dá)對CRC而言是一個(gè)獨(dú)立的重要預(yù)后因子,但是miR-181a表達(dá)與臨床病理參數(shù)間沒有觀察到相關(guān)性。Pichler 等[47]的研究結(jié)果也表明miR-181a的表達(dá)水平與CRC病人的低存活率相關(guān),表明 miR-181a也可能會(huì)成為CRC病人預(yù)后診斷中一個(gè)新的預(yù)后因子。在其他疾病方面,Lin等[48]在血液惡性腫瘤中發(fā)現(xiàn)miR-181a/b的表達(dá)水平與總存活率(Overall survival,OS)顯著相關(guān),miR-181a/b高表達(dá)組較比低表達(dá)水平組能夠延長血液腫瘤病人的OS,這表明miR-181也可能成為血液腫瘤疾病的一個(gè)重要預(yù)后因子。

總之,越來越多的數(shù)據(jù)表明 miR-181家族成員在參與腫瘤發(fā)生、轉(zhuǎn)移、診斷和預(yù)后等方面都發(fā)揮著重要作用(表3),其中以miR-181a和b生物學(xué)功能表現(xiàn)最為活躍,相信隨著研究的深入,miR-181在腫瘤中的更多功能和作用機(jī)制會(huì)被逐漸發(fā)現(xiàn),并且miR-181在腫瘤疾病的診斷和治療中應(yīng)用價(jià)值能夠逐漸得以實(shí)現(xiàn)。

表3 miR-181在腫瘤中的作用

5 展 望

miR-181家族成員在細(xì)胞增生、分化與凋亡、免疫和炎癥反應(yīng)、腫瘤發(fā)生和治療等諸多領(lǐng)域都具有重要的調(diào)控作用,雖然目前關(guān)于 miR-181的研究還大多停留在實(shí)驗(yàn)室階段,但是 miR-181在醫(yī)學(xué)和畜牧業(yè)上已經(jīng)初步展露出其潛在的應(yīng)用價(jià)值。例如,測定血清 miR-181a水平能夠提高乳腺癌早期診斷的準(zhǔn)確性[45],miR-181c具有抗豬PRRSV感染和繁殖方面的能力[35,36],miR-181a的調(diào)控作用對于治療SLE具有意義[31,32],以及miR-181a對結(jié)腸癌患者具有預(yù)后價(jià)值[46]等等,這些研究都表明：miR-181無論作為腫瘤的分子標(biāo)記物用于疾病的診斷和預(yù)后,還是作為藥物作用的靶標(biāo)用于畜牧業(yè)上的抗病毒治療,都具有巨大的潛在開發(fā)前景。

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