In vitro knock-out of miR-155 suppresses leukemic and HCV virus loads in pediatric HCV-4–associated acute lymphoid leukemia: A promising target therapy

Hepatitis C virus (HCV) infection is a major public health problem, having a high prevalence in Egypt. Leukemia and lymphoma have been associated with HCV infection. MicroRNA-155 (miR-155) has been reported to play a regulatory role in cancer, inflammation, and immune response to infection. The expression level of miR-155 in HCV viremic patients is controversial; although high miR-155 levels were demonstrated in HCV genotypes 1,2, and 3, low levels of miR-155 were detected in Egyptian patients with HCV genotype 4. Several studies have investigated the correlation between the levels of miRNA-155 and the replication of HCV, others have evaluated miRNA-155 as a prognostic biomarker in different types of cancer. No studies have investigated the impact of miRNA-155 knockdown on HCV pediatric patients associated with childhood acute lymphoblastic leukemia (ALL). We knocked-out the miR_155a in cultured polymorphonuclear cells (PBMCs) obtained from 60 children with ALL; 30 were associated with HCV-4 infection and 30 were HCV negative. The miR_155a, HCV viral load, and cell proliferation werre assessed in treated and untreated cells using TaqMan assay quantitative polymerase chain reaction. We found that miRNA-155 was significantly upregulated by seven folds in the HCV-4 associated ALL group; while being linked to high HCV viral load and leukemic burden, miR_155a knock-out can improve the disease outcome. We conclude that miR-155 is a critical miRNA that is considered a therapeutic target in pediatric HCV leukemic patients.     

Role of microRNA-21 and microRNA-155 as biomarkers for bronchial asthma

MicroRNA (miRNA)-21 and miRNA-155 are important regulators of gene expression of different immunological molecules. This study aimed to investigate the role of miRNA-21 and miRNA-155 as biomarkers in asthma by comparing their serum expression levels in asthmatic patients to those in healthy controls and correlating their levels with serum IL-4. The expression levels of miRNA-21 and miRNA-155 were evaluated by quantitative RT-PCR. Serum levels of IL-4 were determined using ELISA. Asthmatic patients showed significantly higher serum miRNA-21 and miRNA-155 expression levels compared to controls. A statistically significant positive correlation between the expression levels of miRNA-21 and IL-4 serum levels in asthmatic patients was detected. Nonetheless, no correlation was detected between miRNA-155 expression and each of IL-4 and miRNA-21. A receiver operating characteristic curve analysis showed that at a cut-off value of 1.37, the sensitivity of miRNA-21 as an asthma biomarker was 100% and the specificity was 95%. At a cut-off value of 1.96, the sensitivity of miRNA-155 as an asthma biomarker was 100% and the specificity was 100%. It can be concluded that miRNA-21 and miRNA-155 are potential non-invasive biomarkers in the diagnosis of eosinophilic asthma and its response to therapy.     

miRNA-181a在胃癌细胞增殖和迁移中的作用

miRNA与恶性肿瘤患者的诊断和预后密切相关,为了考察miRNA-181a在胃癌细胞增殖和迁移中的作用,本研究检测了miRNA-181a在胃癌组织中的表达,并通过对人胃癌细胞系MGC-803转染miR-181a模拟物或抑制剂来考察miR-181a对细胞迁移和增殖的影响。RT-PCR显示,miRNA-181a在胃癌组织中的表达水平显著高于癌旁组织(p<0.05)。伤口愈合实验和Transwell实验显示,转染miR-181a抑制剂或TGF-β受体2(TGFβR2)过表达的pcDNA3.1质粒均可抑制MGC-803细胞的迁移。EdU实验和CCK-8实验显示,转染miR-181a抑制剂或TGFβR2过表达的pcDNA3.1质粒均可抑制MGC-803细胞的增殖。此外,miR-181a抑制剂处理可使TGFβR2蛋白表达明显升高。然而,miR-181a模拟物或抑制剂处理后TGFβR2mRNA水平没有显著变化。总之,本研究表明高表达的miR-181a通过在转录后抑制TGFβR2蛋白表达来促进胃癌细胞的迁移和增殖。miR-181a有望成为胃癌的潜在治疗靶点。     

急性心力衰竭患者血清miRNA-21、MYO、CK-MB与心功能和预后的关系

摘要 目的：探讨急性心力衰竭患者血清miRNA-21、肌红蛋白（MYO）、心肌肌酸激酶同工酶（CK-MB）与心功能和预后的关系。方法：选择2018年2月至2019年2月于我院接诊的95例急性心力衰竭患者为研究对象（病例组）,另选择同期在我院进行检查的80例健康人为对照组,分析两组患者血清miRNA-21、MYO、CK-MB、左心室射血分数（LVEF）、左心房内径（LAD）、左心室舒张末期内径（LVEd）水平变化情况及和预后相关性。结果：病例组患者血清miRNA-21、MYO、CK-MB水平显著高于对照组,差异显著（P＜0.05）;病例组患者LAD、LVEd水平显著高于对照组,LVEF水平显著低于对照组,差异显著（P＜0.05）;II级患者血清miRNA-21、MYO、CK-MB水平显著低于III级、IV级患者,III级患者血清miRNA-21、MYO、CK-MB水平显著低于IV级患者,差异显著（P＜0.05）;预后良好组患者血清miRNA-21、MYO、CK-MB水平显著低于预后不良组,差异显著（P＜0.05）;Spearman相关分析显示,血清miRNA-21、MYO及CK-MB和LVEF之间均呈负相关,血清miRNA-21、MYO及CK-MB和LAD、LVEd之间均呈正相关,（P＜0.05）;Logistic 回归分析结果显示,血清miRNA-21、MYO及CK-MB及LVEF、LAD、LVEd为急性心力衰竭患者预后的影响因素（P＜0.05）。结论：急性心力衰竭患者血清miRNA-21、MYO、CK-MB的表达升高,其与心功能和预后密切相关。     

microRNA-214在恶性肿瘤中的表达及相关性的研究进展

微小RNA(microRNA,miRNA)是广泛存在于动植物中的一类不编码蛋白质的短小的单链RNA分子,一般由22个核苷酸组成,它们可以特异性地结合mRNA并通过降解或抑制其翻译而在转录后水平调控基因表达。miRNA的表达及功能可影响许多表观遗传学特征,其功能涉及细胞的发生、生长、发育、分化和凋亡过程,在肿瘤的形成和进展过程中扮演重要角色。microRNA-214(miRNA-214,miR-214)参与肝癌、乳腺癌、宫颈癌、卵巢癌、恶性黑色素瘤、胃癌、胶质瘤、儿童骨肉瘤等恶性肿瘤的发生发展,以及与肿瘤细胞的侵袭及转移密切相关。miRNA-214在不同的肿瘤中表达水平并不相同,miRNA-214在不同肿瘤中的差异表达是通过调控某个或者某些癌基因及抑癌基因而实现其参与肿瘤的发生发展、侵袭及转移的作用。因此,本文主要通过阅读大量国内外文献,总结和概括了miRNA-214参与部分恶性肿瘤发生发展的机制。虽然目前对于miRNA的理论研究已经日渐完善和成熟,但是怎样将这些研究结果应用于临床,怎样能够更准确、更便捷的通过对miRNA的检测达到对疾病的诊断、治疗以及预后评估,想必一定会成为将来研究的热点,我们期待一种新型的恶性肿瘤的分子标志物会使越来越多的肿瘤患者获益。     

Anti-PD1 therapy induces lymphocyte-derived exosomal miRNA-4315 release inhibiting Bim-mediated apoptosis of tumor cells

Anti-PD1 immunotherapy, as a single agent or in combination with standard chemotherapies, has significantly improved the outcome of many patients with cancers. However, resistance to anti-PD1 antibodies often decreases the long-term therapeutic benefits. Despite this observation in clinical practice, the molecular mechanisms associated with resistance to anti-PD1 antibody therapy have not yet been elucidated. To identify the mechanisms of resistance associated with anti-PD1 antibody therapy, we developed cellular models including purified T cells and different cancer cell lines from glioblastoma, lung adenocarcinoma, breast cancer and ovarian carcinoma. A murine model of lung cancer was also used. Longitudinal blood samples of patients treated with anti-PD1 therapy were also used to perform a proof-of-concept study of our findings. We found that anti-PD1 exposure of T-cell promotes an enrichment of exosomal miRNA-4315. We also noted that exosomal miRNA-4315 induced a phenomenon of apopto-resistance to conventional chemotherapies in cancer cells receiving exosomal miRNA-4315. At molecular level, we discern that the apopto-resistance phenomenon was associated with the miRNA-4315-mediated downregulation of Bim, a proapoptotic protein. In cellular and mice models, we observed that the BH3 mimetic agent ABT263 circumvented this resistance. A longitudinal study using patient blood showed that miRNA-4315 and cytochrome c can be used to define the time period during which the addition of ABT263 therapy may effectively increase cancer cell death and bypass anti-PD1 resistance.This work provides a blood biomarker (exosomal miRNA-4315) for patient stratification developing a phenomenon of resistance to anti-PD1 antibody therapy and also identifies a therapeutic alternative (the use of a BH3 mimetic drug) to limit this resistance phenomenon.Subject terms: Cancer, miRNAs     

Aberrant Expression of Some Circulating miRNAs in Childhood Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is a heterogeneous cancer commonly affecting children due to dysregulation of miRNA expression. In the current study, authors investigated the expression profile for miRNA-125b-1 and miRNA-203 among childhood ALL. Blood samples were collected from newly diagnosed childhood ALL and healthy control children. The expression profile for candidate miRNAs was detected using quantitative RT-PCR analysis. Statistical analysis were performed using receiver operating characteristic curve (ROC) to examine the diagnostic efficacy of the two miRNA and their levels among ALL clinicopathological factors and phenotypes. The median expression level for miRNA-125b-1 was significantly high in childhood ALL; while miRNA-203 level was significantly low in childhood ALL as compared to control ones. MiRNA-125-1 reported significant increase in T-ALL as compared to other ALL phenotypes. Median miRNA-203 level was high in T-ALL followed by pre-B-ALL although no significant difference was reported. Clinicopathological factors did not emphasize significance with either detected miRNAs. Using ROC curve the diagnostic efficacy was significant with an area under the curve 0.858 for miRNA-125b-1 (83.72, 100%) and 0.878 for miRNA-203 (97.67, 86.96%). The combination of the two key miRNAs revealed absolute sensitivity (100%). MiRNA-125b-1 and miRNA-203 can be useful molecular markers for diagnosis of ALL. Further studies with large cohort are warranted to validate these results.     

Clinical significance of blood-based miRNAs as diagnostic and prognostic nucleic acid markers in breast cancer: Comparative to conventional tumor markers

microRNAs (miRNAs) are implicated in carcinogenesis and their expression in biological fluids offer great potential as nucleic acid markers for cancer detection and progression. Authors investigated the expression level of miRNAs (miRNA-21, miRNA-126, and miRNA-155) to evaluate their role as diagnostic and prognostic markers for breast cancer compared with other commonly used protein-based markers (CEA and CA15-3). Serum samples from patients with breast cancer (n = 96), patients with benign breast lesion (n = 47), and healthy individuals (n = 39) were enrolled for detection of miRNA expression levels and protein-based tumor markers using fluorescent real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Correlation among investigated markers with clinicopathological factors and clinical outcomes were determined. Expression of miRNA-21 and miRNA-155 revealed significant increases in patients with breast cancer compared with both benign and control groups, the same result was reported for tumor markers; on the other hand, miRNA-126 was significantly decreased in breast cancer group as compared with the other two groups. miRNA frequencies were significantly related to clinical staging and histological grading as compared with tumor markers. Patients with breast cancer with increased miRNA-21 and miRNA-155 and decreased miRNA-126 expressions had significantly worse disease-free survival, while only miRNA-21 and miRNA-126 showed poor OS (P< 0.005). In conclusion, investigated miRNAs were superior over tumor markers for the early stage of breast cancer especially those with high-risk factor and their assessment in blood facilitates their role as a potential prognostic molecular marker.     

LncRNA AWPPH and miRNA-21 regulates cancer cell proliferation and chemosensitivity in triple-negative breast cancer by interacting with each other

Long–non-coding RNAs (lncRNA) AWPPH promotes the progression of liver and bladder cancer, indicating its oncogenic role. The current study aimed to explore the involvement of AWPPH in triple-negative breast cancer (TNBC). In the current study, we found that plasma levels of lncRNA AWPPH and microRNA-21 (miRNA-21) were upregulated in patients with TNBC than in healthy controls, and the upregulation of plasma lncRNA AWPPH and miRNA-21 distinguished early-stage patients with TNBC from healthy controls. Plasma levels of lncRNA AWPPH and miRNA-21 were significantly and positively correlated in both patients with TNBC and healthy controls. LncRNA AWPPH and miRNA-21 overexpression led to promoted cancer cells proliferation and improved cancer cell viability under carboplatin treatment, while lncRNA AWPPH small interfering RNA (siRNA) silencing played an opposite role. In addition, miRNA-21 overexpression attenuated the effects of lncRNA AWPPH siRNA silencing on of cancer cell behaviors. LncRNA AWPPH overexpression led to upregulated miRNA-21 in TNBC cells, while miRNA-21 overexpression also led to significantly upregulated lncRNA AWPPH expression. Therefore, lncRNA AWPPH and miRNA-21 may regulate cancer cell proliferation and chemosensitivity in TNBC by interacting with each other.     

MicroRNA-98促进猪圆环病毒2型在3D4/21细胞中的复制

【目的】通过高通量测序的方法获得PCV2感染3D4/21细胞的miRNAs表达谱,并探讨miRNA-98在PCV2复制中的作用。【方法】本研究以猪肺泡巨噬细胞系3D4/21细胞为细胞模型,对PCV2感染过程中的3D4/21细胞进行miRNAs差异表达分析,筛选与病毒复制相关的特异性miRNAs,并探讨其在PCV2复制中的作用。【结果】经高通量测序,获得PCV2感染3D4/21细胞的miRNAs表达谱,结合实验室前期研究筛选获得miRNA-98。实验表明,miRNA-98的表达量随PCV2感染时间的延长而持续升高,其变化趋势与Cap蛋白表达变化基本一致,由此推测miRNA-98与PCV2复制正相关。过表达miRNA-98可显著上调Cap蛋白的表达量和PCV2的复制。进一步的研究表明,miRNA-98参与调节宿主免疫相关细胞因子的表达和PCV2的复制。【结论】miRNA-98可通过调节免疫相关细胞因子的表达调控宿主免疫功能,帮助PCV2逃逸宿主免疫,促进PCV2在3D4/21细胞中的复制。这些发现不仅为深入了解PCV2与宿主之间的关系提供了新视角,还有望为猪圆环病毒相关疾病的防控提供新的抗病毒策略。     

Identification of Drug Combinations Containing Imatinib for Treatment of BCR-ABL+ Leukemias

The BCR-ABL translocation is found in chronic myeloid leukemia (CML) and in Ph+ acute lymphoblastic leukemia (ALL) patients. Although imatinib and its analogues have been used as front-line therapy to target this mutation and control the disease for over a decade, resistance to the therapy is still observed and most patients are not cured but need to continue the therapy indefinitely. It is therefore of great importance to find new therapies, possibly as drug combinations, which can overcome drug resistance. In this study, we identified eleven candidate anti-leukemic drugs that might be combined with imatinib, using three approaches: a kinase inhibitor library screen, a gene expression correlation analysis, and literature analysis. We then used an experimental search algorithm to efficiently explore the large space of possible drug and dose combinations and identified drug combinations that selectively kill a BCR-ABL+ leukemic cell line (K562) over a normal fibroblast cell line (IMR-90). Only six iterations of the algorithm were needed to identify very selective drug combinations. The efficacy of the top forty-nine combinations was further confirmed using Ph+ and Ph- ALL patient cells, including imatinib-resistant cells. Collectively, the drug combinations and methods we describe might be a first step towards more effective interventions for leukemia patients, especially those with the BCR-ABL translocation.     

lncRNA RMRP knockdown suppress hepatocellular carcinoma biological activities via regulation miRNA-206/TACR1

Long noncoding RNA, RNA component of mitochondrial RNA processing endoribonuclease (RMRP) plays an important role in cancer development and is closely correlated with prognosis in cancer patients. However, whether RMRP affects prognosis in patients with hepatocellular carcinoma (HCC) remains unclear. The aim of the present study was to investigate the expression level of RMRP in HCC and its correlation with prognosis in patients with HCC and explain the effects and associated mechanisms by conducting an in vitro study. The high expression level of RMRP was correlated with poor prognosis in patients with HCC. Using in vitro analysis, RMRP knockdown suppressed HCC cell proliferation, invasion, and migration (P < .05). miRNA-206 overexpression had similar effects in HCC cell lines (Bel-7402 and Huh-7). Using Western blot analysis and cellular immunofluorescence detection, RMRP downregulation significantly suppressed TACR1/Erk1/2 pathway, while miRNA-206 was significantly upregulated (P < .05). RMRP downregulation inhibits HCC-related biological activities by the regulation of miRNA-206/TACR1.     

Quantitative evaluation of leukemic mitochondria with a computer-controlled image analyzer

Mitochondria from 25 patients with acute lymphoblastic leukemia (ALL) and 25 patients with acute myelogenous leukemia (AML) were compared in terms of their number, area, and shape index using a computer-controlled image analyzer. The number of mitochondria was greater in the AML than in the ALL patients. However, their size, as measured in electron micrographic profiles was similar in the two groups, in disagreement with conventional reports that mitochondria are small in granulocytes but large in lymphocytes. Two ALL patients had giant mitochondria. The mitochondria of the ALL cells were more irregular than those of the AML cells, and furthermore, within the ALL group, the degree of the irregularity was greater in those with a poor prognosis than in those in longstanding remission. The number of mitochondria was significantly greater in B-cell ALL than in null cell and T-cell ALL.     

Loss of TBL1XR1 Disrupts Glucocorticoid Receptor Recruitment to Chromatin and Results in Glucocorticoid Resistance in a B-Lymphoblastic Leukemia Model

Although great advances have been made in the treatment of pediatric acute lymphoblastic leukemia, up to one of five patients will relapse, and their prognosis thereafter is dismal. We have previously identified recurrent deletions in TBL1XR1, which encodes for an F-box like protein responsible for regulating the nuclear hormone repressor complex stability. Here we model TBL1XR1 deletions in B-precursor ALL cell lines and show that TBL1XR1 knockdown results in reduced glucocorticoid receptor recruitment to glucocorticoid responsive genes and ultimately decreased glucocorticoid signaling caused by increased levels of nuclear hormone repressor 1 and HDAC3. Reduction in glucocorticoid signaling in TBL1XR1-depleted lines resulted in resistance to glucocorticoid agonists, but not to other chemotherapeutic agents. Importantly, we show that treatment with the HDAC inhibitor SAHA restores sensitivity to prednisolone in TBL1XR1-depleted cells. Altogether, our data indicate that loss of TBL1XR1 is a novel driver of glucocorticoid resistance in ALL and that epigenetic therapy may have future application in restoring drug sensitivity at relapse.     

Rewired Metabolism in Drug-resistant Leukemia Cells: A METABOLIC SWITCH HALLMARKED BY REDUCED DEPENDENCE ON EXOGENOUS GLUTAMINE*

Cancer cells that escape induction therapy are a major cause of relapse. Understanding metabolic alterations associated with drug resistance opens up unexplored opportunities for the development of new therapeutic strategies. Here, we applied a broad spectrum of technologies including RNA sequencing, global untargeted metabolomics, and stable isotope labeling mass spectrometry to identify metabolic changes in P-glycoprotein overexpressing T-cell acute lymphoblastic leukemia (ALL) cells, which escaped a therapeutically relevant daunorubicin treatment. We show that compared with sensitive ALL cells, resistant leukemia cells possess a fundamentally rewired central metabolism characterized by reduced dependence on glutamine despite a lack of expression of glutamate-ammonia ligase (GLUL), a higher demand for glucose and an altered rate of fatty acid β-oxidation, accompanied by a decreased pantothenic acid uptake capacity. We experimentally validate our findings by selectively targeting components of this metabolic switch, using approved drugs and starvation approaches followed by cell viability analyses in both the ALL cells and in an acute myeloid leukemia (AML) sensitive/resistant cell line pair. We demonstrate how comparative metabolomics and RNA expression profiling of drug-sensitive and -resistant cells expose targetable metabolic changes and potential resistance markers. Our results show that drug resistance is associated with significant metabolic costs in cancer cells, which could be exploited using new therapeutic strategies.     

Identification of potential predictive markers of dexamethasone resistance in childhood acute lymphoblastic leukemia

Response to dexamethasone (DEXA), as a hallmark drug in the treatment of childhood acute lymphoblastic leukemia (ALL), is one of the pivotal prognostic factors in the prediction of outcome in ALL. Identification of predictive markers of chemoresistance is beneficial to selecting of the best therapeutic protocol with the lowest effect adverse. Hence, we aimed to find drug targets using the 2DE/MS proteomics study of a DEXA-resistant cell line (REH) as a model for poor DEXA responding patients before and after drug treatment. Using the proteomic methods, three differentially expressed proteins were detected, including voltage dependent anion channel 1 (VDAC1), sorting Nexin 3 (SNX3), and prefoldin subunit 6 (PFDN6). We observed low expression of three proteins after DEXA treatment in REH cells. We subsequently verified low expression of resulted proteins at the mRNA level using the quantitative PCR method. These proteins are promising proteins because of their important roles in drug resistance and regulation of apoptosis (VDAC1), protein trafficking (SNX3), and protein folding (PFDN6). Additionally, mRNA expression level of these proteins was assessed in 17 bone marrow samples from children with newly diagnosed ALL and 7 non-cancerous samples as controls. The results indicated that independent of the molecular subtypes of leukemia, mRNA expression of VDAC1, SNX3, and PFDN6 decreased in ALL samples compared with non-cancerous samples particularly in VDAC1 (p?<?0.001). Additionally, mRNA expression of three proteins was also declined in high-risk samples compared with standard risk cases. These results demonstrated diagnostic and prognostic value of these proteins in childhood ALL. Furthermore, investigation of protein-protein interaction using STRING database indicated that these proteins involved in the signaling pathway of NR3C1 as dexamethasone target. In conclusion, our proteomic study in DEXA resistant leukemic cells revealed VDAC1, SNX3, and PFDN6 are promising proteins that might serve as potential biomarkers of prognosis and chemotherapy in childhood ALL.