【摘要】：背景化療藥物耐受?chē)?yán)重影響腫瘤臨床的治療療效。MicroRNAs(miRNAs)在癌細(xì)胞化療耐藥性的多種生物學(xué)過(guò)程起到關(guān)鍵作用。目的分析mir-34a-5p和AGTR1在骨肉瘤細(xì)胞化療耐受中的作用機(jī)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(gè)數(shù)據(jù)庫(kù)對(duì)mi R-34a-5p的下游靶基因進(jìn)行預(yù)測(cè),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(cè)骨肉瘤(OS)化療敏感細(xì)胞株G-292和化療耐受細(xì)胞株SJSA-1細(xì)胞中mir-34a-5p和血管緊張素Ⅱ1型受體(AGTR1)的表達(dá)水平。3.采用雙熒光素酶報(bào)告系統(tǒng)檢測(cè)mir-34a-5p是否與AGTR1的3’-UTR序列結(jié)合,驗(yàn)證AGTR1是否為mir-34a-5p的直接靶基因。4.在G-292細(xì)胞中轉(zhuǎn)染mir-34a-5p的mimic和si-AGTR1,并在SJSA-1細(xì)胞中轉(zhuǎn)染mir-34a-5p的antagomir和GFP-AGTR1。應(yīng)用qRT-PCR分析,驗(yàn)證AGTR1在細(xì)胞內(nèi)的表達(dá)是否受到mi R-34a-5p mimic和antagomir轉(zhuǎn)染的影響。5.通過(guò)骨肉瘤化療敏感性檢測(cè)(MTT)、細(xì)胞凋亡、細(xì)胞增殖和裸鼠移植瘤實(shí)驗(yàn)驗(yàn)證mir-34a-5p和AGTR1在骨肉瘤細(xì)胞化療耐受中的作用機(jī)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(xì)胞株SJSA-1中的表達(dá)水平明顯高于敏感細(xì)胞株G-292細(xì)胞中的表達(dá)水平,而AGTR1的表達(dá)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(dá)水平明顯高于G-292細(xì)胞中的表達(dá)水平,而AGTR1的表達(dá)水平相反,在耐受細(xì)胞株SJSA-1中的表達(dá)水平明顯低于敏感細(xì)胞株G-292細(xì)胞中的表達(dá)水平。3.雙熒光素酶報(bào)告系統(tǒng)檢測(cè)結(jié)果發(fā)現(xiàn),mir-34a-5p可與AGTR1的3’-UTR序列結(jié)合,降低細(xì)胞內(nèi)熒光活性,證明AGTR1是mir-34a-5p的靶基因。4.在G-292細(xì)胞中轉(zhuǎn)染mir-34a-5p的mimic,si-AGTR1,AGTR1的m RNA和蛋白表達(dá)下調(diào);在SJSA-1細(xì)胞中轉(zhuǎn)染mir-34a-5p的antagomir,GFP-AGTR1 AGTR1的mRNA和蛋白表達(dá)上調(diào)。結(jié)果表明mir-34a-5p負(fù)調(diào)控靶基因AGTR1。5.骨肉瘤細(xì)胞化療敏感性檢測(cè)(MTT)結(jié)果表明,mir-34a-5p的mimic能夠促進(jìn)骨肉瘤細(xì)胞的化療耐受,而mir-34a-5p的antagomir能夠降低骨肉瘤細(xì)胞的化療耐受,si-AGTR1干擾后化療敏感性的MTT檢測(cè)結(jié)果與mir-34a-5p的mimic結(jié)果一致,能提高骨肉瘤細(xì)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(xì)胞的化療敏感性。該結(jié)果與細(xì)胞凋亡實(shí)驗(yàn)和裸鼠移植瘤實(shí)驗(yàn)保持一致。結(jié)論mir-34a-5p是通過(guò)AGTR1基因參與調(diào)控骨肉瘤細(xì)胞化療耐受的,預(yù)示mir-34a-5p可以作為評(píng)估骨肉瘤患者預(yù)后的分子指標(biāo)。
[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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