髓系肿瘤中长链非编码RNA H19表达的临床意义及生物学功能研究

发布时间：2018-04-19 15:38

本文选题：长链非编码RNA + H19　；参考：《江苏大学》2017年硕士论文

【摘要】：目的长链非编码RNA(long non-coding RNA,lnc RNA)在肿瘤发生发展中扮演重要作用。lnc RNA H19在实体肿瘤中的研究越来越多,但是其在血液疾病中的表达态势和临床意义还有待研究。本文拟检测急性髓系白血病(acute myeloid leukemia,AML)、慢性髓系白血病(chronic myeloid leukemia,CML)和骨髓增生异常综合征(myelodysplastic syndrome,MDS)患者骨髓单个核细胞中H19表达水平并探讨其临床意义及潜在的致病机制。方法应用实时定量PCR检测36例正常对照,161例AML、78例CML和43例MDS患者的骨髓单个核细胞中H19 m RNA表达水平并分析临床意义。实时定量甲基化特异性PCR及亚硫酸氢盐测序方法检测三种髓系肿瘤中H19甲基化态势,并应用去甲基化药物处理白血病细胞株(THP-1和K562),评价H19甲基化是否为其异常表达的调控机理。采用si RNA沉默K562细胞中H19表达,通过细胞计数、流式细胞术检测对细胞增殖、凋亡的影响,并结合文献资料及生物信息学网站预测下游潜在靶基因。结果三种髓系肿瘤患者较对照组均存在不同程度的H19高表达,且H19通过调节下游靶基因ID2可影响白血病细胞的增殖和凋亡。(1)AML中H19基因表达AML患者中H19表达水平显著高于对照组(P=0.003)。H19高表达组患者的发病年龄和白细胞计数(white blood cell,WBC)明显大于H19低表达组(P=0.009和0.004)。H19高表达组良好核型的频率相对较低(P=0.013),而中等核型频率较高(P=0.002)。具体的核型分析显示,H19高表达组t(15;17)染色体异位的发生率低(P=0.008),而正常核型的发生率相对较高(P=0.017)。此外,H19高表达组FLT3-ITD和DNMT3A的突变率也明显高于H19低表达组(P=0.053和0.025)。AML中H19高表达组患者的完全缓解(complete remission,CR)率和总体生存(overall survival,OS)均显著低于H19低表达组患者(P=0.039和0.020)。正常核型AML中,H19高表达组患者的无白血病生存时间略短于H19低表达组患者(P=0.072)。Cox多因素分析提示:H19高表达在非APL AML中趋近于是患者预后不良的独立影响因素(P=0.063)。通过生物信息学分析TCGA数据和GEP数据发现:H19高表达组患者的OS均显著短于低表达组患者。此外,H19表达水平在CR期下调,而复发后又显著上调(P0.001)。尽管临床样本中H19表达和其甲基化水平并无相关性,但是THP-1细胞经去甲基化药物处理后,H19表达和未甲基化水平均升高,提示AML中H19表达可能部分受甲基化调控。此外,H19第一外显子编码的mi R-675表达态势与H19并不一致。(2)CML中H19基因表达CML患者H19表达水平较对照组显著升高(P0.001),且急变期(blast crisis,BC)H19表达水平高于慢性期(chronic phase,CP)、加速期(acute phase,AP)及CP/AP期(P=0.074、0.115和0.061)。H19高表达组WBC数和BCR-ABL1水平略高于H19低表达组(P=0.088和0.086)。检测5例患者具有不同临床分期的配对标本发现AP/BC期H19表达量显著高于CP期,提示H19表达可能与CML疾病进展相关。随访分析显示CML患者完全分子缓解期H19表达也下调(P0.001)。临床资料相关分析及K562加药实验揭示:CML中H19表达与H19未甲基化相关(R=0.259,P=0.042),且随着加药浓度的增加,H19表达和未甲基化水平均升高,提示CML中H19表达可能受甲基化调控。(3)MDS中H19基因表达MDS患者H19表达水平显著高于对照组(P0.001),且H19表达与常见的临床参数均没有发现明显相关性。通过Kaplan-Meier生存分析发现:H19高表达组患者的OS较H19低表达组缩短,但并未达到显著统计学差异(P=0.184)。Cox多因素分析也证明H19表达不是MDS患者预后的独立影响因素(P0.05)。(4)H19体外生物学功能研究沉默K562细胞H19基因后,干扰组细胞的增殖速度较对照组减慢、且总体凋亡率显著增高(P=0.002和0.014)。两组细胞对去甲基化药物的半数致死浓度并无明显差异(P=0.203)。此外,干扰组中H19靶基因ID2表达量下调,且AML临床样本中H19表达与ID2表达呈正相关(R=0.262,P=0.002)。结论H19过表达在髓系肿瘤中是常见的分子事件,且H19与mi R-675表达态势并不一致。H19高表达与AML患者预后不良相关,并可用于AML复发及CML疾病进展的监测。沉默H19可下调ID2表达,减慢白血病细胞增殖,并且诱导细胞凋亡,提示H19/ID2异常可能参与髓系肿瘤的发生发展。
[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.

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733

【参考文献】