miRNA—133b—3p對PC12細胞P2rx4的表達調(diào)控及細胞鈣活動的實驗觀察

何則平+高楊+王銳

【摘要】 目的：從離體細胞水平觀察miRNA-133b-3p調(diào)控P2rx4表達對神經(jīng)元鈣活動的影響。方法：用轉(zhuǎn)染試劑將miRNA-133b-3p mimic和miRNA-133b-3p inhibitor轉(zhuǎn)入PC12細胞中。轉(zhuǎn)染48 h后，用熒光定量PCR的方法檢測PC12細胞中miRNA-133b-3p的含量，P2rx4 mRNA的表達變化；用免疫印跡的方法檢測P2rx4受體蛋白的表達變化；用胞內(nèi)鈣成像技術(shù)檢測ATP孵育后細胞中鈣離子濃度的變化。結(jié)果：轉(zhuǎn)染48 h后，與陰性對照組相比，加入miRNA-133b-3p mimic后PC12細胞中miRNA-133b-3p的含量明顯增加，P2rx4 mRNA和P2rx4蛋白的表達減少，ATP孵育后細胞中鈣離子濃度的升高明顯減弱。轉(zhuǎn)染inhibitor后，細胞內(nèi)miRNA-133b-3p的含量明顯減少，P2rx4 mRNA和P2rx4蛋白的表達增加，ATP孵育后胞內(nèi)鈣離子濃度的升高明顯增強。結(jié)論：miRNA-133b-3p通過負向調(diào)控P2rx4的表達來影響細胞鈣活動。

【關(guān)鍵詞】 PC12細胞； miRNA-133b-3p； P2rx4； 鈣活動； 表達調(diào)控

【Abstract】 Objective：To observe the effect of miRNA-133b-3p on the calcium mobilization of neurons by regulating the expression of P2rx4.Method：Transfection reagents and miRNA-133b-3p mimic or inhibitor were added into the culture medium of PC12 cell.After 48 hours of transfection，the expression of miRNA-133b-3p

and P2rx4 mRNA were measured by real-time PCR.Western blot method was used for quantitative analysis on P2rx4 protein.The mobilization of intracellular calcium was measured with calcium imaging technique.Result：After 48 hours of transfection，compared with the negative control group，after adding miRNA-133b-3p mimic，the expression of miRNA-133b-3p in PC12 cells was significantly increased， the expressions of P2rx4 mRNA and P2rx4 protein were significantly decreased，and calcium ion concentration increase induced by ATP was decreased obviously.After adding miRNA-133b-3p inhibitor，the expression of miRNA-133b-3p was decreased significantly，and the expressions of P2rx4 mRNA and P2rx4 protein were significantly increased，the calcium ion concentration increase induced by ATP was enhanced obviously.Conclusion：miRNA-133b-3p downregulation of P2rx4 affectes intracellular calcium activity and is involved in chronic pain.

【Key words】 PC12 cell； miRNA-133b-3p； P2rx4； Calcium mobilization； Regulation of expression

First-authors address：Shanxi Medical University，Taiyuan 030001，China

doi：10.3969/j.issn.1674-4985.2017.29.006

疼痛是大多數(shù)疾病共有的癥狀，慢性疼痛及嚴重疼痛已經(jīng)構(gòu)成一個單獨的疾病[1]。不同類型疼痛的共同過程主要包括痛刺激對傷害性感受器的激活，傷害性信息的傳遞，痛信息在不同中樞的處理，并最終產(chǎn)生痛覺[2-3]。在這個過程發(fā)揮最基本、最重要作用的因素是各種痛相關(guān)的神經(jīng)活性物質(zhì)的產(chǎn)生。因此，探討不同條件下痛相關(guān)物質(zhì)的產(chǎn)生以及表達調(diào)控是疼痛尤其是慢性疼痛機制研究中最有可能獲得具有應(yīng)用價值成果的領(lǐng)域。

microRNA（miRNA）是一類單鏈非編碼RNA，可以對基因組的大部分進行轉(zhuǎn)錄后調(diào)節(jié)[4]。哺乳動物體內(nèi)超過60%的基因是miRNAs的靶基因，miRNAs在轉(zhuǎn)錄后水平調(diào)節(jié)蛋白質(zhì)的表達，在疼痛信號產(chǎn)生與傳遞過程發(fā)揮了重要作用[5]。課題組在前期工作中，利用miRNA芯片，初步明確了CFA致大鼠慢性炎性疼痛模型DRG中miRNAs的表達譜?！?br>