本文關(guān)鍵詞： 微RNA- 遺傳載體 基因 c-myc 乙型肝炎相關(guān)性肝腫瘤 HepG..細(xì)胞　出處：《重慶醫(yī)學(xué)》2016年12期 　論文類型：期刊論文

【摘要】：目的構(gòu)建人微小RNA-21(microRNA-21,miRNA-21)真核過表達(dá)載體pmR-21,探討其在HepG2.2.15細(xì)胞中對(duì)c-myc基因表達(dá)的調(diào)控作用。方法 PCR擴(kuò)增miRNA-21的前體基因片段(pre-miRNA-21),雙酶切后連接到pmR-mCherry載體上,通過雙酶切和測序驗(yàn)證重組載體的準(zhǔn)確性;將重組載體轉(zhuǎn)染到HepG2.2.15細(xì)胞中為實(shí)驗(yàn)組,另設(shè)空載體組(轉(zhuǎn)染pmRmCherry空質(zhì)粒組),空白組(未轉(zhuǎn)染組),陽性對(duì)照組(HepG2細(xì)胞),24h后觀察載體熒光蛋白表達(dá)情況,流式細(xì)胞術(shù)檢測轉(zhuǎn)染效率,實(shí)時(shí)熒光定量PCR評(píng)估m(xù)iRNA-21的表達(dá)情況;轉(zhuǎn)染72h后,RT-PCR和Western blot檢測c-myc mRNA及蛋白表達(dá)水平;CCK-8法檢測各組細(xì)胞增殖情況。結(jié)果經(jīng)雙酶切和測序驗(yàn)證,目的基因片段插入載體中;實(shí)驗(yàn)組及空載體組轉(zhuǎn)染24h后細(xì)胞內(nèi)可見強(qiáng)熒光,轉(zhuǎn)染效率大于50%;實(shí)驗(yàn)組細(xì)胞miRNA-21表達(dá)較空載體組、空白組水平升高;轉(zhuǎn)染72h后實(shí)驗(yàn)組細(xì)胞c-myc mRNA表達(dá)較空載體組、空白組升高;實(shí)驗(yàn)組細(xì)胞增殖快于空載體組及空白組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論成功構(gòu)建miRNA-21真核過表達(dá)載體pmR-21,該重組載體可穩(wěn)定表達(dá)miRNA-21;miRNA-21可上調(diào)c-myc基因的表達(dá),c-myc基因是miRNA-21發(fā)揮促癌作用的靶點(diǎn)之一。
[Abstract]:Objective to construct the eukaryotic expression vector pmR-21 of human minimal RNA-21 microRNA-21 miRNA-21, and to investigate its role in regulating c-myc gene expression in HepG2.2.15 cells. Methods Pre-miRNA-21, a precursor gene fragment of miRNA-21 amplified by PCR, was ligated to pmR-mCherry vector after double enzyme digestion. The accuracy of the recombinant vector was verified by double enzyme digestion and sequencing. The recombinant vector was transfected into HepG2.2.15 cells as experimental group. In addition, the expression of fluorescent protein was observed 24 hours after transfection of pmRmCherry empty plasmid group, blank group (without transfection group, positive control group). Flow cytometry was used to detect transfection efficiency and real-time fluorescence quantitative PCR was used to evaluate the expression of miRNA-21. After 72 hours of transfection, c-myc mRNA and protein expression level were detected by RT-PCR and Western blot. The results showed that the target gene fragment was inserted into the vector by double enzyme digestion and sequencing. The expression of c-myc mRNA in the experimental group was higher than that in the blank vector group 72 hours after transfection, and the cell proliferation in the experimental group was faster than that in the empty vector group and blank group, and the expression of c-myc mRNA in the experimental group was higher than that in the blank vector group. Conclusion miRNA-21 eukaryotic expression vector pmR-21 can stably express miRNA-21 miRNA-21 can up-regulate the expression of c-myc gene and c-myc gene is one of the targets of miRNA-21 for carcinogenesis.
【作者單位】： 南方醫(yī)科大學(xué)附屬廣州軍區(qū)廣州總醫(yī)院兒科;南方醫(yī)科大學(xué)附屬廣州軍區(qū)廣州總醫(yī)院檢驗(yàn)科;
【基金】：廣州市科技計(jì)劃基金資助項(xiàng)目(2013J4100116)
【分類號(hào)】：R3416

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