發(fā)布時間：2018-07-17 07:48

【摘要】：背景化療藥物耐受嚴重影響腫瘤臨床的治療療效。MicroRNAs(miRNAs)在癌細胞化療耐藥性的多種生物學(xué)過程起到關(guān)鍵作用。目的分析mir-34a-5p和AGTR1在骨肉瘤細胞化療耐受中的作用機制。方法1.依據(jù)RNA-Seq和miR-omic組學(xué)分析,結(jié)合mi RBase、Targetscan和CHIPBase三個數(shù)據(jù)庫對mi R-34a-5p的下游靶基因進行預(yù)測,尋找可能受到miR-34a-5p調(diào)控的基因。2.采用qRT-PCR、western blot技術(shù)檢測骨肉瘤(OS)化療敏感細胞株G-292和化療耐受細胞株SJSA-1細胞中mir-34a-5p和血管緊張素Ⅱ1型受體(AGTR1)的表達水平。3.采用雙熒光素酶報告系統(tǒng)檢測mir-34a-5p是否與AGTR1的3’-UTR序列結(jié)合,驗證AGTR1是否為mir-34a-5p的直接靶基因。4.在G-292細胞中轉(zhuǎn)染mir-34a-5p的mimic和si-AGTR1,并在SJSA-1細胞中轉(zhuǎn)染mir-34a-5p的antagomir和GFP-AGTR1。應(yīng)用qRT-PCR分析,驗證AGTR1在細胞內(nèi)的表達是否受到mi R-34a-5p mimic和antagomir轉(zhuǎn)染的影響。5.通過骨肉瘤化療敏感性檢測(MTT)、細胞凋亡、細胞增殖和裸鼠移植瘤實驗驗證mir-34a-5p和AGTR1在骨肉瘤細胞化療耐受中的作用機制。結(jié)果1.RNA-Seq和mi R-omic組學(xué)數(shù)據(jù)均表明mir-34a-5p在耐受細胞株SJSA-1中的表達水平明顯高于敏感細胞株G-292細胞中的表達水平,而AGTR1的表達水平相反,提示AGTR1可能是mir-34a-5p下游靶基因之一。2.qRT-PCR、western blot結(jié)果表明,mir-34a-5p在SJSA-1中的表達水平明顯高于G-292細胞中的表達水平,而AGTR1的表達水平相反,在耐受細胞株SJSA-1中的表達水平明顯低于敏感細胞株G-292細胞中的表達水平。3.雙熒光素酶報告系統(tǒng)檢測結(jié)果發(fā)現(xiàn),mir-34a-5p可與AGTR1的3’-UTR序列結(jié)合,降低細胞內(nèi)熒光活性,證明AGTR1是mir-34a-5p的靶基因。4.在G-292細胞中轉(zhuǎn)染mir-34a-5p的mimic,si-AGTR1,AGTR1的m RNA和蛋白表達下調(diào);在SJSA-1細胞中轉(zhuǎn)染mir-34a-5p的antagomir,GFP-AGTR1 AGTR1的mRNA和蛋白表達上調(diào)。結(jié)果表明mir-34a-5p負調(diào)控靶基因AGTR1。5.骨肉瘤細胞化療敏感性檢測(MTT)結(jié)果表明,mir-34a-5p的mimic能夠促進骨肉瘤細胞的化療耐受,而mir-34a-5p的antagomir能夠降低骨肉瘤細胞的化療耐受,si-AGTR1干擾后化療敏感性的MTT檢測結(jié)果與mir-34a-5p的mimic結(jié)果一致,能提高骨肉瘤細胞的化療敏感性,而GFP-AGTR1與mir-34a-5p的antagomir的結(jié)果一致,能降低骨肉瘤細胞的化療敏感性。該結(jié)果與細胞凋亡實驗和裸鼠移植瘤實驗保持一致。結(jié)論mir-34a-5p是通過AGTR1基因參與調(diào)控骨肉瘤細胞化療耐受的,預(yù)示mir-34a-5p可以作為評估骨肉瘤患者預(yù)后的分子指標。
[Abstract]:Background chemotherapeutic drug tolerance seriously affects the therapeutic efficacy of tumor. MicroRNAs (miRNAs) play a key role in the biological process of chemotherapeutic resistance of cancer cells. Objective to investigate the role of mir-34a-5p and AGTR1 in chemotherapy tolerance of osteosarcoma cells. Method 1. Based on RNA-Seq and miR-omic analysis, the downstream target genes of mi R-34a-5p were predicted with the combination of three databases: mi RBasesi-Targetscan and CHIPBase, and the gene .2which might be regulated by miR-34a-5p was found. The expression levels of mir-34a-5p and angiotensin 鈪，

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