本文選題：長鏈非編碼RNA + H19�。� 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：目的長鏈非編碼RNA(long non-coding RNA,lnc RNA)在腫瘤發(fā)生發(fā)展中扮演重要作用。lnc RNA H19在實體腫瘤中的研究越來越多,但是其在血液疾病中的表達態(tài)勢和臨床意義還有待研究。本文擬檢測急性髓系白血病(acute myeloid leukemia,AML)、慢性髓系白血病(chronic myeloid leukemia,CML)和骨髓增生異常綜合征(myelodysplastic syndrome,MDS)患者骨髓單個核細(xì)胞中H19表達水平并探討其臨床意義及潛在的致病機制。方法應(yīng)用實時定量PCR檢測36例正常對照,161例AML、78例CML和43例MDS患者的骨髓單個核細(xì)胞中H19 m RNA表達水平并分析臨床意義。實時定量甲基化特異性PCR及亞硫酸氫鹽測序方法檢測三種髓系腫瘤中H19甲基化態(tài)勢,并應(yīng)用去甲基化藥物處理白血病細(xì)胞株(THP-1和K562),評價H19甲基化是否為其異常表達的調(diào)控機理。采用si RNA沉默K562細(xì)胞中H19表達,通過細(xì)胞計數(shù)、流式細(xì)胞術(shù)檢測對細(xì)胞增殖、凋亡的影響,并結(jié)合文獻資料及生物信息學(xué)網(wǎng)站預(yù)測下游潛在靶基因。結(jié)果三種髓系腫瘤患者較對照組均存在不同程度的H19高表達,且H19通過調(diào)節(jié)下游靶基因ID2可影響白血病細(xì)胞的增殖和凋亡。(1)AML中H19基因表達AML患者中H19表達水平顯著高于對照組(P=0.003)。H19高表達組患者的發(fā)病年齡和白細(xì)胞計數(shù)(white blood cell,WBC)明顯大于H19低表達組(P=0.009和0.004)。H19高表達組良好核型的頻率相對較低(P=0.013),而中等核型頻率較高(P=0.002)。具體的核型分析顯示,H19高表達組t(15;17)染色體異位的發(fā)生率低(P=0.008),而正常核型的發(fā)生率相對較高(P=0.017)。此外,H19高表達組FLT3-ITD和DNMT3A的突變率也明顯高于H19低表達組(P=0.053和0.025)。AML中H19高表達組患者的完全緩解(complete remission,CR)率和總體生存(overall survival,OS)均顯著低于H19低表達組患者(P=0.039和0.020)。正常核型AML中,H19高表達組患者的無白血病生存時間略短于H19低表達組患者(P=0.072)。Cox多因素分析提示:H19高表達在非APL AML中趨近于是患者預(yù)后不良的獨立影響因素(P=0.063)。通過生物信息學(xué)分析TCGA數(shù)據(jù)和GEP數(shù)據(jù)發(fā)現(xiàn):H19高表達組患者的OS均顯著短于低表達組患者。此外,H19表達水平在CR期下調(diào),而復(fù)發(fā)后又顯著上調(diào)(P0.001)。盡管臨床樣本中H19表達和其甲基化水平并無相關(guān)性,但是THP-1細(xì)胞經(jīng)去甲基化藥物處理后,H19表達和未甲基化水平均升高,提示AML中H19表達可能部分受甲基化調(diào)控。此外,H19第一外顯子編碼的mi R-675表達態(tài)勢與H19并不一致。(2)CML中H19基因表達CML患者H19表達水平較對照組顯著升高(P0.001),且急變期(blast crisis,BC)H19表達水平高于慢性期(chronic phase,CP)、加速期(acute phase,AP)及CP/AP期(P=0.074、0.115和0.061)。H19高表達組WBC數(shù)和BCR-ABL1水平略高于H19低表達組(P=0.088和0.086)。檢測5例患者具有不同臨床分期的配對標(biāo)本發(fā)現(xiàn)AP/BC期H19表達量顯著高于CP期,提示H19表達可能與CML疾病進展相關(guān)。隨訪分析顯示CML患者完全分子緩解期H19表達也下調(diào)(P0.001)。臨床資料相關(guān)分析及K562加藥實驗揭示:CML中H19表達與H19未甲基化相關(guān)(R=0.259,P=0.042),且隨著加藥濃度的增加,H19表達和未甲基化水平均升高,提示CML中H19表達可能受甲基化調(diào)控。(3)MDS中H19基因表達MDS患者H19表達水平顯著高于對照組(P0.001),且H19表達與常見的臨床參數(shù)均沒有發(fā)現(xiàn)明顯相關(guān)性。通過Kaplan-Meier生存分析發(fā)現(xiàn):H19高表達組患者的OS較H19低表達組縮短,但并未達到顯著統(tǒng)計學(xué)差異(P=0.184)。Cox多因素分析也證明H19表達不是MDS患者預(yù)后的獨立影響因素(P0.05)。(4)H19體外生物學(xué)功能研究沉默K562細(xì)胞H19基因后,干擾組細(xì)胞的增殖速度較對照組減慢、且總體凋亡率顯著增高(P=0.002和0.014)。兩組細(xì)胞對去甲基化藥物的半數(shù)致死濃度并無明顯差異(P=0.203)。此外,干擾組中H19靶基因ID2表達量下調(diào),且AML臨床樣本中H19表達與ID2表達呈正相關(guān)(R=0.262,P=0.002)。結(jié)論H19過表達在髓系腫瘤中是常見的分子事件,且H19與mi R-675表達態(tài)勢并不一致。H19高表達與AML患者預(yù)后不良相關(guān),并可用于AML復(fù)發(fā)及CML疾病進展的監(jiān)測。沉默H19可下調(diào)ID2表達,減慢白血病細(xì)胞增殖,并且誘導(dǎo)細(xì)胞凋亡,提示H19/ID2異�？赡軈⑴c髓系腫瘤的發(fā)生發(fā)展。
[Abstract]:Objective the role of long chain non coding RNA (long non-coding RNA, LNC RNA) plays an important role in the development of cancer in the development of.Lnc RNA H19 in solid tumors, but its expression and clinical significance in blood diseases are still to be studied. This paper is to detect acute myeloid leukemia (acute myeloid leukemia,), chronic myeloid system The level of H19 expression in bone marrow mononuclear cells of patients with chronic myeloid leukemia (CML) and myelodysplastic syndrome (MDS) and its clinical significance and potential pathogeny mechanism. Methods real-time quantitative PCR was used to detect 36 normal controls, 161 AML, 78 CML, and 43 MDS patients' bone marrow mononuclear. The expression level of H19 m RNA in cells and analysis of clinical significance. Real-time quantitative methylation specific PCR and hydrogen sulfite sequencing method for detection of H19 methylation in three myeloid tumors, and using demethylation drugs to treat leukemic cell lines (THP-1 and K562), and evaluate whether H19 methylation is a regulatory mechanism for its abnormal expression. Si RNA is used. The expression of H19 in the silent K562 cells, the cell count, the effect of flow cytometry on cell proliferation and apoptosis, and the prediction of the potential target genes in the downstream with the literature and bioinformatics website. Results the three myeloid tumor patients have different levels of H19 high expression compared with the control group, and H19 can be influenced by the regulation of the downstream target gene ID2. The proliferation and apoptosis of leukemic cells (1) the expression level of H19 in AML patients with H19 gene expression in AML was significantly higher than that in the control group (P=0.003).H19 high expression group (white blood cell, WBC) was significantly greater than that of H19 low expression group (P=0.009 and 0.004), and the frequency of good karyotype in the high expression group was relatively low. The moderate karyotype frequency was higher (P=0.002). The specific karyotype analysis showed that the incidence of T (15; 17) in the high expression group of H19 was low (P=0.008), while the incidence of normal karyotype was relatively high (P=0.017). Moreover, the mutation rate of FLT3-ITD and DNMT3A in the H19 high expression group was also significantly higher than that in the H19 low expression group (P=0.053 and 0.025).AML in H19 high expression group. The total remission (complete remission, CR) rate and overall survival (overall survival, OS) were significantly lower than those of the H19 low expression group (P=0.039 and 0.020). In the normal karyotype AML, the non leukemia survival time in the H19 high expression group was shorter than that in the H19 low expression group (P= 0.072). The independent influence factor (P=0.063) was close to the patient's poor prognosis. Through bioinformatics analysis of TCGA data and GEP data, it was found that the OS in the H19 high expression group was significantly shorter than the low expression group. In addition, the H19 expression level was downregulated in the CR phase, and the recurrence was significantly up-regulated (P0.001). Although the expression of H19 and its methylation level in the clinical samples There was no correlation, but the expression of H19 and the level of unmethylation in THP-1 cells increased, suggesting that the expression of H19 in AML may be partly controlled by methylation. Furthermore, the expression of MI R-675 encoded by H19 first exon is not consistent with H19. (2) the H19 expression level of H19 gene expression in CML is significantly higher than that of the control group. The expression level of high (P0.001) and blast crisis (BC) H19 was higher than that of chronic phase (chronic phase, CP), acceleration period (acute phase, AP) and CP/AP phase (0.061) were slightly higher than that of low expression group (0.086). The expression of H19 was significantly higher than that of CP, suggesting that H19 expression may be related to the progression of CML disease. Follow-up analysis showed that the expression of H19 in complete molecular remission of CML was also downregulated (P0.001). Clinical data related analysis and K562 addition experiment revealed that H19 expression in CML was associated with H19 methylation (R= 0.259), and as the concentration increased, the expression was expressed. H19 expression in CML may be regulated by methylation. (3) the expression level of H19 in MDS patients with H19 gene expression in MDS is significantly higher than that of the control group (P0.001), and there is no significant correlation between the expression of H19 and the common clinical parameters. The group shortened, but did not achieve significant statistical difference (P=0.184).Cox multifactor analysis also proved that H19 expression was not an independent factor in the prognosis of MDS patients (P0.05). (4) after the study of H19 in vitro biological function of K562 cell H19 gene, the proliferation rate of the interference group was slower than the control group, and the overall apoptosis rate was significantly higher (P=0.002 and 0.). 014). There was no significant difference in the median lethal concentration of demethylation drugs in the two groups (P=0.203). In addition, the expression of H19 target gene ID2 expression in the interference group was down, and the expression of H19 in the AML clinical samples was positively correlated with the expression of ID2 (R=0.262, P=0.002). Conclusion H19 overexpression is a common molecular event in myeloma and H19 and MI R-675. The high expression of.H19 is not associated with poor prognosis in AML patients and can be used to monitor the recurrence of AML and the progression of CML disease. Silent H19 can downregulate the expression of ID2, slow down the proliferation of leukemic cells, and induce apoptosis, suggesting that H19/ID2 may be involved in the development of myeloid tumors.

【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R733

【參考文獻】

相關(guān)期刊論文 前1條

1 顧婷婷;張忠明;鄭鵬生;;DNA甲基化研究方法的回顧與評價[J];中國婦幼健康研究;2006年06期

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本文編號：1773696