發(fā)布時(shí)間：2020-11-03 02:23

　　 慢性髓系白血病(chronic myelogenous leukemia,CML)是由多能造血干細(xì)胞異常增殖所致的骨髓增殖性腫瘤,t(9;22)(q34;q11)易位產(chǎn)生的BCR-ABL1融合基因所編碼的P210蛋白具有高度酪氨酸激酶活性,致使細(xì)胞增殖及凋亡異常。甲磺酸伊馬替尼(Imatinib Mesylate,IM)作為第一個應(yīng)用于臨床的靶向治療藥物,為CML治療開創(chuàng)了新的時(shí)代。但仍有一部分患者存在著原發(fā)或者繼發(fā)性耐藥,影響患者的預(yù)后及生活質(zhì)量。MicroRNA(miRNA)是一類長約18-25個核苷酸的非編碼小RNA,通過靶向作用于靶基因的3’非編碼區(qū)(3’-untranslated region,3’-UTR),在轉(zhuǎn)錄后水平抑制靶基因的表達(dá),包括阻止靶基因的翻譯及加速靶基因的降解。miRNA不僅參與了CML發(fā)生和發(fā)展,而且在IM耐藥方面也發(fā)揮了重要作用。很多研究已經(jīng)證實(shí)在IM治療前后多種miRNA表達(dá)存在變化,同樣在耐藥的CML患者中也存在著miRNA的差異性表達(dá),本研究通過高通量測序檢測了mi RNA在CML的IM耐藥及敏感的細(xì)胞株中的差異性表達(dá),進(jìn)一步選取了差異性較大的miR-202-5p作為研究對象,探討其在CML細(xì)胞IM耐藥中發(fā)揮的作用及機(jī)制。去泛素化酶(Deubiquitinating enzymes,DUBs)可將泛素化的靶蛋白上的泛素解離下來,阻止蛋白質(zhì)被降解,參與多種重要生命活動,包括細(xì)胞周期調(diào)控、基因轉(zhuǎn)錄、激酶活化、蛋白質(zhì)降解、DNA修復(fù)等。泛素羧基末端水解酶15(Ubiquitin-specific proteases 15,USP15),是USP家族的成員,在不同的腫瘤中發(fā)揮不同的生物學(xué)作用。已有研究證實(shí)USP15參與了腫瘤對紫杉醇的耐藥過程,但USP15在CML中的作用及調(diào)控IM耐藥的機(jī)制尚不清楚,我們的實(shí)驗(yàn)證實(shí)miR-202-5p靶向抑制USP15的表達(dá),通過miR-202-5p/USP15軸調(diào)控了CML對IM的耐藥,為進(jìn)一步研究CML的IM耐藥及臨床克服CML耐藥提供實(shí)驗(yàn)基礎(chǔ)。第一部分miR-202-5p在不同CML細(xì)胞株及CML患者中的表達(dá)差異目的:篩選CML耐藥細(xì)胞株中差異性表達(dá)miRNA及驗(yàn)證mi R-202-5p在CML患者及不同細(xì)胞株中表達(dá)差異。方法:1.CCK-8方法檢測K562/K562G細(xì)胞對IM的IC_(50);RT-q PCR檢測K562/K562G細(xì)胞耐藥基因的表達(dá);Western blot檢測K562/K562G的P-糖蛋白(P-gp)表達(dá)。2.高通量測序檢測K562細(xì)胞及K562G細(xì)胞中miRNA表達(dá)差異。3.實(shí)時(shí)定量PCR(RT-qPCR)及原位雜交(FISH)方法檢測mi R-202-5p在CML細(xì)胞中的表達(dá)。4.RT-qPCR及FISH方法檢測miR-202-5p在CML患者中的表達(dá)。結(jié)果:1.K562G細(xì)胞株為IM耐藥細(xì)胞株應(yīng)用CCK-8方法檢測了K562/K562G細(xì)胞對IM的IC_(50),在K562G細(xì)胞中IM的IC_(50)約為K562細(xì)胞的30倍;RT-qPCR方法證明K562G細(xì)胞中ABCB1基因表達(dá)水平高于K562細(xì)胞;Western blot證實(shí)在K562G細(xì)胞中,P-gp蛋白表達(dá)水平明顯上調(diào)。表明K562G細(xì)胞為IM耐藥細(xì)胞株。2.高通量測序檢測miRNA在K562細(xì)胞與K562G細(xì)胞中的表達(dá)差異高通量測序結(jié)果顯示,與K562細(xì)胞相比,在K562G細(xì)胞中有102種已知的miRNA表達(dá)上調(diào),92種miRNA表達(dá)下調(diào);差異性表達(dá)的miRNA的靶基因在多條信號通路上存在富集。3.mi R-202-5p在IM耐藥的CML細(xì)胞株中表達(dá)增高RT-qPCR方法及FISH結(jié)果顯示,miR-202-5p在耐藥細(xì)胞株K562G中的表達(dá)量最高,在正常外周血白細(xì)胞中表達(dá)最低。4.mi R-202-5p在ABCB1(+)CML患者中的表達(dá)上調(diào)RT-qPCR及FISH結(jié)果顯示ABCB1(+)CML患者的miR-202-5p的表達(dá)高于ABCB1(-)CML患者,并且CML患者中miR-202-5p的表達(dá)高于健康供者的miR-202-5p的表達(dá)。小結(jié):miR-202-5p在CML耐藥細(xì)胞株及ABCB1(+)CML患者中表達(dá)增加。第二部分miR-202-5p調(diào)控CML細(xì)胞對甲磺酸伊馬替尼的敏感性目的:驗(yàn)證miR-202-5p是否直接參與了CML細(xì)胞對IM的耐藥。方法:1.RT-qPCR方法檢測不同濃度IM刺激后miRNA-202-5p的表達(dá)。2.K562/K562G細(xì)胞過表達(dá)或敲低miR-202-5p,CCK-8檢測細(xì)胞對IM對IC_(50),AnnexinV/PI流式凋亡檢測IM刺激后的細(xì)胞凋亡,Western blot檢測Cyclin D1和Caspase-3的表達(dá)。結(jié)果:1.IM可下調(diào)CML細(xì)胞株miR-202-5p表達(dá)在K562/K562G細(xì)胞中,IM可降低miR-202-5p表達(dá)。2.過表達(dá)miR-202-5p可降低K562細(xì)胞對IM的敏感性與對照組相比,K562細(xì)胞過表達(dá)miR-202-5p后,IM的IC_(50)上升;給與IM刺激后,過表達(dá)miR-202-5p的K562細(xì)胞凋亡率下降。Western blot顯示,過表達(dá)mi R-202-5p能夠逆轉(zhuǎn)由IM應(yīng)用所帶來對Cyclin D1的下降及Caspase-3的增加。3.敲低miR-202-5p可增加K562G細(xì)胞對IM敏感性與對照組相比,K562G細(xì)胞敲低miR-202-5p后,IM的IC_(50)下降;給與IM刺激后,敲低miR-202-5p的K562G細(xì)胞凋亡率增加。Western blot顯示,敲低miR-202-5p能夠增加由IM應(yīng)用所帶來的Cyclin D1的下降及Caspase-3的增加。小結(jié):miR-202-5p調(diào)控CML細(xì)胞對IM耐藥。第三部分mi R-202-5p靶向抑制去泛素化酶USP15表達(dá)調(diào)節(jié)CML細(xì)胞對甲磺酸伊馬替尼的敏感性目的:證實(shí)mi R-202-5p靶向抑制USP15調(diào)控CML細(xì)胞株對IM耐藥性的作用機(jī)制。方法:1.生物信息學(xué)預(yù)測miR-202-5p靶基因,RT-qPCR、雙熒光素酶報(bào)基因,Western blot檢測USP15為miR-202-5p的靶基因。2.RT-qPCR,Western blot及免疫熒光染色方法檢測了CML患者中USP15的表達(dá)水平以及IM對USP15表達(dá)的影響。3.CML細(xì)胞過表達(dá)或敲低USP15,或同時(shí)過表達(dá)或敲低上述兩者,然后CCK-8檢測細(xì)胞IM的IC_(50);AnnexinV/PI流式凋亡檢測IM刺激后的細(xì)胞凋亡;Western blot檢測Cyclin D1、Caspase-3及USP15的表達(dá)。結(jié)果:1.在CML細(xì)胞中USP15是mi R-202-5p的靶基因與轉(zhuǎn)染mimic-NEG組相比,K562細(xì)胞中過表達(dá)miR-202-5p下調(diào)USP15的mRNA表達(dá)水平。雙熒光素酶報(bào)告基因測試顯示mi R-202-5p mimic能靶向USP15的3'UTR序列。Western blot結(jié)果顯示,轉(zhuǎn)染miR-202-5p模擬物下調(diào)USP15蛋白的表達(dá)水平,而miR-202-5p抑制物提高USP15蛋白的表達(dá)水平。說明USP15是miR-202-5p的靶基因。2.USP15在K562G細(xì)胞中表達(dá)下降與K562細(xì)胞相比,USP15的mRNA及蛋白質(zhì)表達(dá)水平在K562G細(xì)胞中顯著下降,免疫熒光染色得到相同的結(jié)果,而且USP15主要定位在細(xì)胞質(zhì)中。3.USP15在CML患者中低表達(dá),且ABCB1(+)CML患者中的表達(dá)更低RT-qPCR及Western blot結(jié)果顯示,與正常供者PBMCs相比,USP15的mRNA及蛋白質(zhì)表達(dá)水平在CML患者PBMCs表達(dá)是明顯下降的;免疫熒光染色得到相同的結(jié)果;而且在ABCB1(+)CML患者PBMCs表達(dá)更低。4.敲低USP15降低,增加K 562細(xì)胞對IM的敏感性CCK-8結(jié)果顯示,與對照組相比,敲低USP15后K562細(xì)胞對IM的IC_(50)上升;而IM處理上述細(xì)胞后,Western blot結(jié)果顯示,敲低USP15能夠逆轉(zhuǎn)由IM應(yīng)用所帶來的Cyclin D1的下降及Caspase-3的增加。5.過表達(dá)USP15可增加K562G細(xì)胞對IM的敏感性CCK-8結(jié)果顯示,與對照組相比,過表達(dá)USP15后K562G細(xì)胞IM的IC_(50)上升;而IM處理上述細(xì)胞后,Western blot結(jié)果顯示,過表達(dá)USP15能夠增加由IM應(yīng)用所帶來對Cyclin D1的下降及Caspase-3的增加。6.mi R-202-5p/USP15軸調(diào)控CML細(xì)胞對IM的耐藥K562細(xì)胞中共轉(zhuǎn)染miR-202-5p mimic和pcDNA3.1-USP15,Western blot結(jié)果顯示USP15蛋白表達(dá)增加,過表達(dá)USP15消除miR-202-5p對K562細(xì)胞的耐藥性。在K562G共轉(zhuǎn)染miR-202-5p inhibitor與si-USP15,Western blot結(jié)果顯示USP15蛋白表達(dá)下降。用CCK-8方法檢測共轉(zhuǎn)染細(xì)胞與單轉(zhuǎn)染miR-202-5p inhibitor細(xì)胞對IM的IC_(50),共轉(zhuǎn)染組IC_(50)下降。這些結(jié)果表明USP15介導(dǎo)miR-202-5p對CML細(xì)胞IM耐藥。小結(jié):mi R-202-5p靶向抑制USP15表達(dá)調(diào)節(jié)CML細(xì)胞株對IM耐藥。第四部分桑黃素通過調(diào)控miR-188-5p/PTEN軸誘導(dǎo)CML細(xì)胞凋亡及增加CML細(xì)胞對甲磺酸伊馬替尼的敏感性目的:證實(shí)桑黃素是否過通過miR-188-5p/PTEN軸誘導(dǎo)CML細(xì)胞凋亡及增加CML細(xì)胞對IM的敏感性。方法:1 CCK-8方法檢測桑黃素或與IM聯(lián)合用藥處理后細(xì)胞的增殖,AnnexinV/PI流式凋亡檢測細(xì)胞凋亡率。2桑黃素處理細(xì)胞,RT-qPCR檢測PTEN基因表達(dá),Western blot檢測PTEN蛋白及下游通路蛋白表達(dá)。3生物信息學(xué)預(yù)測可與PTEN基因靶向作用的miRNA,RT-qPCR檢測桑黃素處理后miR-188-5p表達(dá);雙熒光素酶報(bào)告基因、Western blot檢測PTEN是否是mi R-188-5p的靶基因。結(jié)果:1.桑黃素能夠抑制CML細(xì)胞增殖,誘導(dǎo)CML細(xì)胞凋亡桑黃素抑制CML細(xì)胞株增殖,并誘導(dǎo)CML細(xì)胞凋亡,隨著桑黃素濃度增大,細(xì)胞凋亡明顯增多。2.桑黃素可以增加CML細(xì)胞對IM敏感性CCK-8檢測結(jié)果顯示,與對照組相比,桑黃素與IM聯(lián)合用藥可降低IM的IC_(50),且聯(lián)合用藥可明顯增加細(xì)胞的凋亡率。3.桑黃素通過增加PTEN蛋白表達(dá),抑制PI3K/AKT通路激活,促進(jìn)細(xì)胞凋亡RT-qPCR結(jié)果顯示,桑黃素處理CML細(xì)胞,PTEN的mRNA水平稍有增加;Western blot方法檢測PTEN蛋白質(zhì)表達(dá)增高,p-AKT蛋白質(zhì)表達(dá)下降,Caspase-3蛋白質(zhì)表達(dá)增加,BAX在蛋白水平和mRNA水平增加,BCL-2的蛋白水平和mRNA水平均明顯下降。4.桑黃素可降低CML細(xì)胞中mi R-188-5p表達(dá),從而上調(diào)PTEN的表達(dá)生物信息學(xué)預(yù)測miR-188-5p可靶向作用PTEN基因;RT-qPCR結(jié)果顯示,桑黃素處理后miR-188-5p表達(dá)明顯下降;熒光報(bào)告基因顯示,轉(zhuǎn)染miR-188-5p模擬物后,包含有PTEN野生型3'UTR組的熒光活性減少了33%;Western blot顯示,在轉(zhuǎn)染miR-188-5p模擬物,PTEN蛋白質(zhì)的表達(dá)量下降,而miR-188-5p抑制物處理組,PTEN蛋白質(zhì)的表達(dá)上升。桑黃素處理K562細(xì)胞后又同時(shí)轉(zhuǎn)染miR-188-5p模擬物,發(fā)現(xiàn)與單加桑黃素相比,轉(zhuǎn)染miR-188-5p模擬物后可明顯逆轉(zhuǎn)桑黃素增加的PTEN表達(dá)量。以上結(jié)果提示,在K562細(xì)胞中mi R-188-5p直接靶向PTEN 3'UTR抑制其蛋白質(zhì)的表達(dá)。5.桑黃素通過抑制miR-188-5p表達(dá)誘導(dǎo)CML細(xì)胞凋亡,增加CML細(xì)胞對IM的敏感性CCK-8檢測結(jié)果顯示,敲低miR-188-5p組,IM的IC_(50)下降;轉(zhuǎn)染miR-188-5p抑制物組與對照組相比,凋亡率明顯增高;CCK-8方法檢測細(xì)胞增殖率及IM的IC_(50),結(jié)果顯示,通過轉(zhuǎn)染miR-188-5p模擬物,可以逆轉(zhuǎn)桑黃素與IM協(xié)同作用。以上結(jié)果說明桑黃素增能增加CML細(xì)胞敏感性是通過抑制miR-188-5p表達(dá)實(shí)現(xiàn)的。小結(jié):桑黃素通過調(diào)控miR-188-5p/PTEN軸誘導(dǎo)CML細(xì)胞凋亡及增加CML細(xì)胞對IM的敏感性。結(jié)論:1.mi R-202-5p在CML耐藥細(xì)胞株及ABCB1(+)CML患者中表達(dá)上調(diào)。2.miR-202-5p調(diào)控CML細(xì)胞對IM耐藥3.miR-202-5p靶向抑制USP15表達(dá)調(diào)節(jié)CML細(xì)胞株對IM耐藥4.桑黃素通過調(diào)控miR-188-5p/PTEN軸誘導(dǎo)CML細(xì)胞凋亡及增加CML細(xì)胞對IM的敏感性
【學(xué)位單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位年份】：2018
【中圖分類】：R733.72
【部分圖文】：

圖3 miR-202-5p在甲磺酸伊馬替尼耐藥的CML細(xì)胞株中表達(dá)增高Fig. 3 miR-202-5p is upregulated in imatinib resistance CML cell line. (A)Comparison of miRNA expression in K562 cells and K562G cells i by usingthe high-thoughput sequencing. The heatmap represents the miRNA with : 1.Log2（Fold-Change）≥2 or≤-2；2. P value≤0.05；3. count≥100. (B)RT-qPCR detected the expression of miR-202-5p in K562 cells and K562Gcells. The relative levels were normalized to U6 . (P < 0.001) (C) RT-qPCRdetected the expression of miR-202-5p in CML cell line and normal personPBMCs. Expressions of miR-202-5p in normal person PBMCs were lowercompared to CML cell line. (P<0.001) (D) Fluorescence in situ hybridization(FISH) staining was performed on sections from K562 cells , K562G cells andnormal person PBMCs. Red and blue staining indicates miR-202-5p, andDAPI. Bar = 40 μm.

圖 2 過表達(dá) miR-202-5p 可降低 K562 細(xì)胞對 IM 的敏感性Fig. 2 Upregulation of miR-202-5p decreases the sensitive of IM in K562 cells.(A). qRT-PCR was performed to measure miR-202-5p levels in K562 cellstransfected with the mimic-NEG or miR-202-5p mimic. Increased miR-202-5plevels were observed in the miR-202-5p transfected cells compared to themimic-NEG (P<0.001). (B) K562 cells were treated with IM (0.1μM) for 48 h.Cell viability was determined using a CCK8 assay. More cells survived inmiR-202-5p mimic transfected group. Upregulation of miR-202-5p increasedIC50 values of K562 cells to IM. (C) K562 cells were treated with IM（0.1μM）followed by analysis of apoptosis. There were fewer cells undergoingapoptosis in the miR-202-5p mimic transfected group. (D) Western blotting

圖 3 敲低 miR-202-5p 可增加 K562G 細(xì)胞對甲磺酸伊馬替尼的敏感Fig. 3 Knockdown of miR-202-5p promotes sensitive of IM in K562G (A) RT-qPCR was performed to measure miR-202-5p levels in K562Gtransfected with the inhibitor-NEG or miR-202-5p inhibitor. DecremiR-202-5p levels were observed in the miR-202-5p inhibitor transfectedcompared to the inhibitor-NEG.( B) K562G cells were treated with IM (3for 48 h. Cell viability was determined using a CCK8 assay. Less survived in miR-202-5p mimic transfected group. Knockdown of miR-20decreased IC50 values of K562G cells to IM. (C) K562G cells were trwith IM（3μM）followed by analysis of apoptosis. There were more undergoing apoptosis in the miR-202-5p inhibitor transfected group (P<0(D) Western blotting for Caspas-3 and CyclinD1 in K562G cells transfwith miR-202-5p inhibitor or inhibitor-NEG and treated with IM (3μM
【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Amit K.Tiwari;Hsiang-Chun Wu;;Overexpression of P-glycoprotein induces acquired resistance to imatinib in chronic myelogenous leukemia cells[J];癌癥;2012年02期

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