本文關(guān)鍵詞： 微小核糖核酸 肌營養(yǎng)素 核轉(zhuǎn)錄因子-κB　出處：《中國循環(huán)雜志》2016年12期 　論文類型：期刊論文

【摘要】：目的:探討心肌細胞中微小核糖核酸(mi R)let-7c(mi R-let-7c)對肌營養(yǎng)素基因表達是否具有調(diào)控作用以及可能的作用機制。方法:首先構(gòu)建攜帶肌營養(yǎng)素基因3'非編碼區(qū)(3'-UTR)片段的熒光素酶報告基因載體,與mi R-let-7c前體共轉(zhuǎn)染Hela細胞,雙熒光素酶報告基因系統(tǒng)檢測熒光素酶活性以驗證mi R-let-7c與肌營養(yǎng)素基因的靶向調(diào)控關(guān)系。進而培養(yǎng)大鼠心肌細胞H9c2,Taqman實時定量聚合酶鏈式反應(yīng)(PCR)法檢測mi R轉(zhuǎn)染效果,蛋白免疫印跡(Western blot)法檢測mi R-let-7c及mi R-let-7c抑制物轉(zhuǎn)染心肌細胞后肌營養(yǎng)素蛋白表達,以及心肌肥厚相關(guān)信號通路分子核轉(zhuǎn)錄因子-κB(NF-κB)的活性變化。結(jié)果:熒光素酶活性實驗結(jié)果表明,與重組熒光素酶報告基因表達載體(p MIR-MTPN)+mi R前體陰性對照組相比,p MIR-MTPN+mi R-let-7c前體組熒光素酶活性顯著降低[(59.30±9.90)%vs(98.10±15.10)%]。Western blot結(jié)果表明,mi R-let-7c前體組與mi R陰性對照組相比,肌營養(yǎng)素蛋白表達水平顯著降低([0.28±0.05)vs(0.90±0.09)],此外,NF-κB蛋白水平顯著降低([0.25±0.06)vs(0.75±0.07)];相反,mi R-let-7c抑制物組與抑制物陰性對照組相比,肌營養(yǎng)素蛋白表達水平顯著升高[(1.14±0.09)vs(0.44±0.09)],同時,NF-κB蛋白水平也顯著升高[(1.09±0.05)vs(0.71±0.06)],差異均有統(tǒng)計學意義(P0.05)。結(jié)論:miR-let-7c能夠通過作用于3'UTR區(qū)域,抑制肌營養(yǎng)素基因表達,并影響心肌肥厚關(guān)鍵信號通路分子NF-κB的活性。
[Abstract]:Objective: to study the microribonucleic acid (RRL) -7 ct mi R-let-7c (R let-7c) in cardiomyocytes. Methods: firstly, the luciferase reporter gene vector carrying the 3'non-coding region of mytrophin gene was constructed. Hela cells were co-transfected with mi R-let-7c precursor. Double luciferase reporter gene system was used to detect luciferase activity in order to verify the targeted regulatory relationship between mi R-let-7c and myotrophin gene, and then cultured rat cardiomyocytes H9c2. Taqman real-time quantitative polymerase chain reaction (PCR) was used to detect the transfection effect of miR. Western blotting was used to detect the expression of myotensin protein after mi R-let-7c and mi R-let-7c inhibitor were transfected into cardiomyocytes. Results: the activity of luciferase was determined by the changes of nuclear transcription factor-魏 B of NF- 魏 B in cardiac hypertrophy related signal pathway. Compared with the recombinant luciferase reporter gene expression vector (pMIR-MTPNNN) mi R precursor negative control group. Luciferase activity of p MIR-MTPN mi R-let-7c precursor group decreased significantly. [59.30 鹵9.90% vs 98.10 鹵15.10%. Western blot results showed that. Compared with the negative control group, the expression of myotrophin protein was significantly decreased in the precursor group of miR-let-7c (P < 0.05). [In addition, the level of NF- 魏 B protein decreased significantly (P < 0.05). [On the contrary, compared with the negative control group, the expression of mycotrophin protein in the R-let-7c inhibitor group was significantly higher than that in the negative control group (0.25 鹵0.06 vs 0.75 鹵0.07). [The protein level of NF- 魏 B also increased significantly. [1. 09 鹵0. 05 鹵0. 05 vs 0.71 鹵0. 06). Conclusion: 1 miR-let-7c can inhibit the expression of myotrophin gene by acting on 3 UTR region. It also affects the activity of NF- 魏 B, a key signal pathway in myocardial hypertrophy.
【作者單位】： 山西醫(yī)科大學基礎(chǔ)醫(yī)學院生物化學與分子生物學教研室;
【基金】：國家自然科學基金(81500364) 山西省基礎(chǔ)研究項目(2015021187) 山西省高等學�？萍紕�(chuàng)新項目(2015149)
【分類號】：R54
【正文快照】： 微小核糖核酸(miR)通過降解靶mR NA或抑制靶mR NA的翻譯來調(diào)控基因表達[1]。最近研究發(fā)現(xiàn)miR-let-7家族在心臟組織中高表達,參與許多重要的心臟功能調(diào)控[2],miR-let-7c可能在心肌肥厚中發(fā)揮重要作用,但其具體作用靶標和機制仍不清楚[3]。肌營養(yǎng)素(myotrophin,MTPN)是從原發(fā)性

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