【摘要】：目的構(gòu)建has-miR-146a真核過表達(dá)載體pmR-146a,探究其在HepG2.2.15肝癌細(xì)胞中對(duì)c-Myc基因的表達(dá)調(diào)控作用。方法 PCR擴(kuò)增has-miR-146a的前體基因片段(pre-has-miR-146a),雙酶切后連接到pmR-mCherry載體上,通過菌落PCR、雙酶切和測(cè)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肝癌細(xì)胞中作為實(shí)驗(yàn)組,同時(shí)設(shè)空載體組(轉(zhuǎn)染pmR-mCherry空質(zhì)粒組),空白組(轉(zhuǎn)染試劑Lipofectamino 2000+PBS),24、48h后觀察載體熒光蛋白表達(dá)量,qPCR檢測(cè)各組細(xì)胞has-miR-146a表達(dá)情況;轉(zhuǎn)染24、48h后qPCR檢測(cè)c-Myc基因mRNA表達(dá)量,48h后Western blot檢測(cè)c-Myc蛋白表達(dá)水平。結(jié)果經(jīng)菌落PCR、雙酶切和測(cè)序證實(shí),pre-has-miR-146a基因片段插入pmR-mCherry載體中;實(shí)驗(yàn)組和空載體組轉(zhuǎn)染24、48h熒光顯微鏡觀察可見強(qiáng)熒光,與非熒光條件下作對(duì)比,轉(zhuǎn)染效率在50%~60%;實(shí)驗(yàn)組has-miR-146a表達(dá)量明顯高于空載體組和空白組(P0.01);轉(zhuǎn)染24、48h后實(shí)驗(yàn)組細(xì)胞c-Myc基因mRNA表達(dá)量較空載體組和空白組低(P0.05);轉(zhuǎn)染48h后,蛋白表達(dá)量較空載體組和空白組低(P0.01)。結(jié)論成功構(gòu)建has-miR-146a真核過表達(dá)載體pmR-has-146a,該重組載體可穩(wěn)定表達(dá)has-miR-146a;has-miR-146a可以下調(diào)c-Myc癌基因的表達(dá),可以作為治療原發(fā)性肝癌的潛在靶點(diǎn)之一。
[Abstract]:Objective to construct has-miR-146a eukaryotic expression vector pmR-146a, to investigate its role in regulating the expression of c-Myc gene in HepG2.2.15 hepatoma cells. Methods the has-miR-146a precursor gene fragment (pre-has-miR-146a) was amplified by PCR and ligated to the pmR-mCherry vector after double enzyme digestion. The accuracy of the recombinant vector was verified by colony PCR, digestion and sequencing, and the recombinant vector was transfected into HepG2.2.15 hepatoma cells as experimental group. At the same time, empty vector group (transfected with empty pmR-mCherry plasmid group) and blank group (Lipofectamino 2000 PBS), 24 h after transfection) were used to observe the expression of fluorescent protein and the expression of has-miR-146a was detected by qPCR. The expression of c-Myc gene mRNA was detected by qPCR 48 h after transfection, and c-Myc protein was detected by Western blot 48 h later. Results the pre-has-miR-146a gene fragment was inserted into the pmR-mCherry vector by double enzyme digestion and sequencing of colony PCR, and the strong fluorescence was observed by fluorescence microscope in the experimental group and the empty vector group for 48 h after transfection, and the results were compared with those in the non-fluorescent condition. The expression of has-miR-146a in the experimental group was significantly higher than that in the blank vector group and blank group (P0.01); the expression of c-Myc gene mRNA in the experimental group was lower than that in the empty vector group and blank group after 48 h transfection (P0.05); after 48 h transfection, the protein expression level was lower than that in the empty vector group and blank group (P0.01). Conclusion the successful construction of has-miR-146a eukaryotic expression vector pmR-has-146a, the recombinant vector can stably express has-miR-146a;has-miR-146a can down-regulate the expression of c-Myc oncogene, and can be used as a potential target for the treatment of primary liver cancer.
【作者單位】： 廣州軍區(qū)廣州總醫(yī)院兒科;廣州中醫(yī)藥大學(xué);廣州軍區(qū)廣州總醫(yī)院檢驗(yàn)科;
【基金】：廣東省科技項(xiàng)目(2013B03180009) 廣東省廣州市科技計(jì)劃項(xiàng)目(201607010123)
【分類號(hào)】：R3416

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