Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells

Specific types of human papillomaviruses (HPVs) cause cervical cancer. Cervical cancers exhibit aberrant cellular microRNA (miRNA) expression patterns. By genome-wide analyses, we investigate whether the intracellular and exosomal miRNA compositions of HPV-positive cancer cells are dependent on endogenous E6/E7 oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that E6/E7 silencing significantly affects ten of the 52 most abundant intracellular miRNAs in HPV18-positive HeLa cells, downregulating miR-17-5p, miR-186-5p, miR-378a-3p, miR-378f, miR-629-5p and miR-7-5p, and upregulating miR-143-3p, miR-23a-3p, miR-23b-3p and miR-27b-3p. The effects of E6/E7 silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The E6/E7-regulated miRNAs are enriched for species involved in the control of cell proliferation, senescence and apoptosis, suggesting that they contribute to the growth of HPV-positive cancer cells. Consistently, we show that sustained E6/E7 expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative p21 gene in HPV-positive cancer cells. In exosomes secreted by HeLa cells, a distinct seven-miRNA-signature was identified among the most abundant miRNAs, with significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p, upon E6/E7 silencing. Several of the E6/E7-dependent exosomal miRNAs have also been linked to the control of cell proliferation and apoptosis. This study represents the first global analysis of intracellular and exosomal miRNAs and shows that viral oncogene expression affects the abundance of multiple miRNAs likely contributing to the E6/E7-dependent growth of HPV-positive cancer cells.     

肝细胞癌患者血清外泌体microRNA表达鉴定

目的:探讨肝癌细胞外泌体中差异表达的microRNAs(miRNAs)在肝细胞癌(HCC)诊断中的应用价值。方法:通过高通量测序筛选肝癌细胞外泌体中差异表达的miRNAs。实时定量PCR验证差异表达分子;检测差异表达的miRNAs在健康人(Health)、慢性乙型肝炎患者(CHB)、肝硬化患者(LC)及乙型肝炎病毒阳性的肝细胞癌患者(HCC)血清外泌体中的表达。结果:高通量测序筛选到肝癌细胞外泌体中差异表达的miRNA共88种,其中58种表达上调,30种表达下调。选择其中8种差异表达的miRNAs进行q RT-PCR验证,结果显示,此8种miRNAs在细胞上清外泌体、细胞内、癌与癌旁组织中的表达趋势与测序结果一致。miR-221-3p和miR-224-5p在HCC组外泌体中的表达水平显著高于Health组、CHB组和LC组(P0.01),miR-124-3p和let-7a-5p在HCC组外泌体中的表达水平显著低于其他各组(P0.05)。四个组中,miR-21-5p、miR-191-5p、miR-34a-5p和miR-122-5p的表达水平不存在显著性差异(P0.05)。结论:血清外泌体中的miR-221-3p、miR-224-5p、miR-124-3p和let-7a-5p可能成为肝细胞癌的候选标志物。     

Upregulated microRNAs in membranous glomerulonephropathy are associated with significant downregulation of IL6 and MYC mRNAs

Membranous glomerulonephropathy (MGN) is a glomerulopathy characterized by subepithelial deposits of immune complexes on the extracapillary side of the glomerular basement membrane. Insertion of C5b-9 (complement membrane-attack complex) into the membrane leads to functional impairment of the glomerular capillary wall. Knowledge of the molecular pathogenesis of MGN is actually scanty. MicroRNA (miRNA) profiling in MGN and unaffected tissues was performed by TaqMan Low-Density Arrays. Expression of miRNAs and miRNA targets was evaluated in Real-Time polymerase chain reaction (PCR). In vitro transient silencing of miRNAs was achieved through transfection with miRNA inhibitors. Ten miRNAs (let-7a-5p, let-7b-5p, let-7c-5p, let-7d-5p, miR-107, miR-129-3p, miR-423-5p, miR-516-3p, miR-532-3p, and miR-1275) were differentially expressed (DE) in MGN biopsies compared to unaffected controls. Interleukin 6 (IL6) and MYC messenger RNAs (mRNAs; targets of DE miRNAs) were significantly downregulated in biopsies from MGN patients, and upregulated in A498 cells following let-7a-5p or let-7c-5p transient silencing. Gene ontology analysis showed that DE miRNAs regulate pathways associated with MGN pathogenesis, including cell cycle, proliferation, and apoptosis. A significant correlation between DE miRNAs and mRNAs and clinical parameters (i.e., antiphospholipid antibodies, serum creatinine, estimated glomerular filtration, proteinuria, and serum cholesterol) has been detected. Based on our data, we propose that DE miRNAs and their downstream network may be involved in MGN pathogenesis and could be considered as potential diagnostic biomarkers of MGN.     

Identification of Differential MicroRNAs in Cerebrospinal Fluid and Serum of Patients with Major Depressive Disorder

Major depression is a debilitating disease. To date, the development of biomarkers of major depressive disorder (MDD) remains a challenge. Recently, alterations in the expression of microRNAs (miRNAs) from post-mortem brain tissue and peripheral blood have been linked to MDD. The goals of this study were to detect the differential miRNAs in cerebrospinal fluid (CSF) and serum of MDD patients. First, the relative expression levels of 179 miRNAs (relative high levels in serum) were analyzed by miRNA PCR Panel in the CSF of MDD patients. Then, the differentially altered miRNAs from CSF were further assessed by qRT-PCR in the serum of the same patients. Finally, the serum differentially altered miRNAs were further validated by qRT-PCR in the serum of another MDD patients. The CSF-results indicated that 11 miRNAs in MDD patients were significantly higher than these in control subjects, and 5 miRNAs were significantly lower than these in control subjects. The serum-results from the same patients showed that 3 miRNAs (miR-221-3p, miR-34a-5p, and let-7d-3p) of the 11 miRNAs were significantly higher than these in control subjects, and 1 miRNA (miR-451a) of 5 miRNAs was significantly lower than these in control subjects. The up-regulation of miR-221-3p, miR-34a-5p, let-7d-3p and down-regulation of miR-451a was further validated in another 32 MDD patients. ROC analysis showed that the area under curve of let-7d-3p, miR-34a-5p, miR-221-3p and miR-451a was 0.94, 0.98, 0.97 and 0.94, with specificity of 90.48%, 95.24%, 90.48% and 90.48%, and sensitivity of 93.75%, 96.88%, 90.63% and 84.85%, respectively. In addition, target gene prediction found that the altered miRNAs are involved in affecting some important genes and pathway related to MDD. Our results suggested that differentially altered miRNAs in CSF might be involved in MDD, and serum miR-221-3p, miR-34a-5p, let-7d-3p, and miR-451a might be able to serve as biomarkers for MDD.     

Changes in microRNA abundance may regulate diapause in the flesh fly,Sarcophaga bullata

Diapause, an alternative developmental pathway characterized by changes in developmental timing and metabolism, is coordinated by molecular mechanisms that are not completely understood. MicroRNA (miRNA) mediated gene silencing is emerging as a key component of animal development and may have a significant role in initiating, maintaining, and terminating insect diapause. In the present study, we test this possibility by using high-throughput sequencing and qRT-PCR to discover diapause-related shifts in miRNA abundance in the flesh fly, Sarcophaga bullata. We identified ten evolutionarily conserved miRNAs that were differentially expressed in diapausing pupae compared to their nondiapausing counterparts. miR-289-5p and miR-1-3p were overexpressed in diapausing pupae and may be responsible for silencing expression of candidate genes during diapause. miR-9c-5p, miR-13b-3p, miR-31a-5p, miR-92b-3p, miR-275-3p, miR-276a-3p, miR-277-3p, and miR-305-5p were underexpressed in diapausing pupae and may contribute to increased expression of heat shock proteins and other factors necessary for the enhanced environmental stress-response that is a feature of diapause. In S. bullata, a maternal effect blocks the programming of diapause in progeny of females that have experienced pupal diapause, and in this study we report that several miRNAs, including miR-263a-5p, miR-100-5p, miR-125-5p, and let-7-5p were significantly overexpressed in such nondiapausing flies and may prevent entry into diapause. Together these miRNAs appear to be integral to the molecular processes that mediate entry into diapause.     

circPVT1通过miR-24-3p/let-7a-5p/FSCN1轴促进鼻咽癌细胞侵袭迁移

目的 鼻咽癌是一种来源于鼻咽上皮的恶性肿瘤,其临床特征之一是易发生淋巴转移,但是目前鼻咽癌转移的分子机制尚未阐明。circPVT1是由PVT1基因2号外显子反向拼接形成的环状RNA (circRNA),在多种肿瘤中表达上调,本文探讨了circPVT1在鼻咽癌侵袭迁移中的作用和分子机制。方法 通过RT-qPCR检测circPVT1及其下游miRNA和FSCN1在鼻咽癌细胞的表达情况,Transwell和划痕愈合实验检测circPVT1对鼻咽癌细胞侵袭迁移的影响,RNA pull-down实验检测circPVT1结合的miRNA,双荧光素酶报告实验检测miR-24-3p和let-7a-5p靶向抑制FSCN1 mRNA表达。结果 在鼻咽癌细胞中过表达circPVT1可以促进鼻咽癌细胞侵袭迁移,而敲低circPVT1则可以抑制鼻咽癌细胞的侵袭迁移。进一步研究发现,circPVT1可以通过竞争性吸附miR-24-3p和let-7a-5p,上调FSCN1的表达,从而促进鼻咽癌细胞的侵袭迁移。结论 circPVT1通过miR-24-3p/let-7a-5p/FSCN1轴促进鼻咽癌细胞侵袭迁移,证实c...     

Profiles of serum microRNAs; miR-125b-5p and miR223-3p serve as novel biomarkers for HBV-positive hepatocellular carcinoma

Recently, circulating miRNAs have been reported as promising biomarkers for various pathologic conditions including cancer. Certain microRNAs (miRNAs) have been shown early diagnostic potential for many types of cancer. The objective of this study was to investigate the potential of certain serum/plasma miRNAs as novel non-invasive biomarkers for early diagnosis of hepatitis B virus (HBV) related hepatocellular carcinoma (HCC). For this reason, the expression levels of 24 miRNA (let-7c, miR-92a-3p, 423-5p, 150-5p, 223-3p, 125b-5p, 342-3p, miR-206, 122-5p, 375, 223-5p, 10a-5p, 23b-5p, 99a-5p, 23a-5p, 10a-3p, 122-3p, 125b-1-3p, 23b-3p, 125b-2-3p, 23a-3p, 92a-1-5p, 92a-2-5p, 99a-3p) were analyzed in plasma of patients with chronic hepatitis B, HBV-positive cirrhosis and HBV-positive HCC and compared with control group samples. Totally 94 plasma samples; 28 control and 66 patient plasma (24 CHB, 22 HBV-positive cirrhosis, 20 HBV-positive HCC) and were included in this study. The expression levels of 24 miRNAs were detected for all control and patient group plasma samples by qRT-PCR using BioMark? 96.96 Dynamic Array (Fluidigm Corporation) system. The expression levels of miR-125b-5p were detected 2.85 fold, 2.46 fold and 1.89 fold (p = 0.01513, p = 0.0009440, p = 0.0001446) up regulated in CHB, HBV-positive cirrhosis and HBV-positive HCC, respectively when compared versus control group individually by Mann–Whitney U test. The expression levels of miR-223-3p were detected 5.55 fold, 13.88 fold and 12.65 fold (p = 0.01513, p = 0.0009440, p = 0.0001446) down regulated in same comparisons. When all groups were compared versus control group by one-way ANOVA test, the expression levels of miR-223-3p were also found statistically significant (p < 0.05). Although not statistically significant, miR-125b-5p tended to be upregulated. (p = 0.07192). These results significantly imply that miR-125b-5p and miR223-3p could be used as novel non-invasive biomarkers of HBV-positive HCC in very early, even at CHB stage of liver disease.     

MicroRNA-27a-3p inhibits lung and skin fibrosis of systemic sclerosis by negatively regulating SPP1

ObjectiveTo investigate the role and mechanism of microRNAs (miRNAs) in fibrotic processes involved in the pathology of systemic sclerosis (SSc).MethodsR language and bioinformatics methods were used to identify differential miRNAs and mRNAs and analyze their biological functions. Transfection experiments were performed to evaluate the function and regulatory mechanism of miR-27a-3p in vitro. Levels of fibrosis-related genes, SPP1 and cell proliferation were assessed.ResultsMiR-27a-3p is reduced both in SSc lung and skin tissues. Overexpression of miR-27a-3p significantly inhibited fibrosis-related genes expression and protein abundance and cell proliferation, whereas inhibition of miR-27a-3p significantly enhanced these phenomena. Moreover, miR-27a-3p exerts its anti-fibrosis effect by negatively regulating SPP1 and ERK signal, more prominent in fibroblasts.ConclusionsOur findings show that miR-27a-3p regulates a common mechanism in the process of SSc skin and lung fibrosis. MiR-27a-3p/SPP1/ERK1/2 axis may be an important target for delaying the progression of SSc fibrosis.     

Effect of valproic acid on miRNAs affecting histone deacetylase in a model of anaplastic thyroid cancer

Background

Thyroid cancer is the most common malignant tumor of the endocrine system seen in the thyroid gland. More than 90% of thyroid cancers comprise papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC). Although anaplastic thyroid carcinoma (ATC) accounts for less than 2% of thyroid cancer. But patients’ lifespan after diagnosis is about 6 months. Surgical interventions, radioactive iodine use, and chemotherapy are not sufficient in the treatment of ATC, so alternative therapies are needed.

Methods and results

The WST-1 assay test was performed to evaluate the anti-proliferative effects of Valproic acid (VPA). Also, the effect of VPA on miRNAs affecting histone deacetylase was determined by Quantitative RT-PCR. In the SW1736 cell line, IC50 dose for VPA was found 1.6 mg/ml. In our study, the level of oncogenic genes expression in cells treated with VPA, including miR-184, miR-222-5p, miR-124-3p, and miR-328-3p, decreased. Also, the expression of tumor inhibitory genes including miR-323-5p, miR-182-5p, miR-138-5p, miR-217, miR-15a-5p, miR-29b-3p, miR-324-5p and miR-101-5p increased significantly.

Conclusions

VPA can ad-just countless gene expression patterns, including microRNAs (miRNAs), by targeting histone deacetylase (HDAC). However, further studies are required for more accurate results.

     

Individual and combined effects of DNA methylation and copy number alterations on miRNA expression in breast tumors

Background

The global effect of copy number and epigenetic alterations on miRNA expression in cancer is poorly understood. In the present study, we integrate genome-wide DNA methylation, copy number and miRNA expression and identify genetic mechanisms underlying miRNA dysregulation in breast cancer.

Results

We identify 70 miRNAs whose expression was associated with alterations in copy number or methylation, or both. Among these, five miRNA families are represented. Interestingly, the members of these families are encoded on different chromosomes and are complementarily altered by gain or hypomethylation across the patients. In an independent breast cancer cohort of 123 patients, 41 of the 70 miRNAs were confirmed with respect to aberration pattern and association to expression. In vitro functional experiments were performed in breast cancer cell lines with miRNA mimics to evaluate the phenotype of the replicated miRNAs. let-7e-3p, which in tumors is found associated with hypermethylation, is shown to induce apoptosis and reduce cell viability, and low let-7e-3p expression is associated with poorer prognosis. The overexpression of three other miRNAs associated with copy number gain, miR-21-3p, miR-148b-3p and miR-151a-5p, increases proliferation of breast cancer cell lines. In addition, miR-151a-5p enhances the levels of phosphorylated AKT protein.

Conclusions

Our data provide novel evidence of the mechanisms behind miRNA dysregulation in breast cancer. The study contributes to the understanding of how methylation and copy number alterations influence miRNA expression, emphasizing miRNA functionality through redundant encoding, and suggests novel miRNAs important in breast cancer.     

MiR-517a-3p accelerates lung cancer cell proliferation and invasion through inhibiting FOXJ3 expression

Aims

Aberrant expression of microRNAs (miRNAs) results in alterations of various biological processes (e.g., cell cycle, cell differentiation, and apoptosis) and cell transformation. Altered miRNAs expression was associated with lung carcinogenesis and tumor progression. This study aimed to investigate the function and underlying molecular events of miR-517a-3p on regulation of lung cancer cell proliferation and invasion.

Main methods

Transfected miR-517a-3p mimics or inhibitors into 95D and 95C cells respectively, the effects of miR-517a-3p on lung cancer cell proliferation, migration, and invasion were detected. Bioinformatics software forecasted potential target genes of miR-517a-3p and dual luciferase reporter gene system and western blot verified whether miR-517a-3p regulates FOXJ3 expression directly.

Key findings

MiR-517a-3p was differentially expressed in lung cancer 95D and 95C cell lines that have different metastatic potential. Manipulation of miR-517a-3p expression changed lung cancer cell proliferation, migration and invasion capacity. MiR-517a-3p directly regulated FOXJ3 expression by binding to FOXJ3 promoter.

Significance

This study demonstrated that miR-517a-3p promoted lung cancer cell proliferation and invasion by targeting of FOXJ3 expression.     

miR-199a-3p负调控CBX7影响肺癌细胞NCI-H460的生物学行为研究

目的:探讨mi R-199a-3p负调控CBX7影响肺癌细胞NCI-H460的生物学行为。方法:qRT-PCR法检测并比较肺癌组织、癌旁正常组织、肺癌细胞、正常肺上皮细胞中的mi R-199a-3p m RNA相对表达量。比较远处转移肺癌组织、未转移肺癌组织中mi R-199a-3p m RNA相对表达量。qRT-PCR法、Western Blot法检测并比较肺癌组织、癌旁正常组织中的CBX7 m RNA及蛋白的表达水平。荧光素酶活性法检测mi R-199a-3p与靶基因CBX7的结合。比较mi R-199a-3p模拟物转染组与阴性对照组的肺癌细胞中的CBX7 m RNA相对表达量及CBX7蛋白表达水平。CCK8实验检测mi R-199a-3p对肺癌细胞增殖的促进作用。Tranwell实验检测mi R-199a-3p对肺癌细胞侵袭与迁移能力的影响。结果:肺癌组织中mi R-199a-3p明显高于癌旁正常组织,发生远处转移的肺癌组织中mi R-199a-3p m RNA的表达量明显高于未发生转移的肺癌组织,差异有统计学意义(P<0.001)。肺癌组织中CBX7m RNA、CBX7蛋白表达水平均明显低于癌旁正常组织,差异有统计学意义(P<0.001)。荧光素酶活性法证实mi R-199a-3p可与靶基因CBX7结合抑制CBX7的表达。肺癌细胞中mi R-199a-3p m RNA的相对表达量明显高于正常肺上皮细胞,CBX7 m RNA相对表达量明显低于正常肺上皮细胞(P<0.05)。对于肺癌细胞,mi R-199a-3p模拟物转染组的CBX7 m RNA相对表达量及CBX7蛋白表达水平均明显低于阴性对照组(P<0.001)。CCK8实验证实mi R-199a-3p能够促进肺癌细胞的增殖,Tranwell实验证实mi R-199a-3p对肺癌细胞侵袭与迁移具有积极的促进作用。结论:mi R-199a-3p在肺癌的发生发展过程中发挥重要作用,能够通过抑制CBX7基因的表达,促进肺癌细胞的增殖、侵袭和转移。     

Estimation of time-dependent microRNA expression patterns in brain tissue,leukocytes, and blood plasma of rats under photochemically induced focal cerebral ischemia

miRNA expression over different time periods (24 and 48 h) using the quantitative RT-PCR and deep sequencing has been evaluated in a model of photochemically induced thrombosis. A combination of two approaches allowed us to determine the miRNA expression patterns caused by ischemia. Nine miRNAs, including let-7f-5p, miR-221-3p, miR-21-5p, miR-30c-5p, miR-30a-3p, miR-223-3p, miR-23a-3p, miR-22-5p, and miR-99a-5p, were differentially expressed in brain tissue and leukocytes of rats 48 h after onset of ischemia. In addition, six miRNAs were differentially expressed in the brain tissue and blood plasma of rats 24 h after exposure, among which miR-145-3p and miR-375-3p were downregulated and miR-19a-3p, miR-92a-3p, miR-188-5p, and miR-532-5p were upregulated. In our opinion, miR-188-5p and miR-532-5p may be considered to be new potential markers of ischemic injury. The level of miRNA expression tended to increase 48 h after the onset of ischemia in brain tissue and leukocytes, which reflects not only the local response in brain tissue due to inflammation, vascular endothelial dysfunction, and disorders of the permeability of the blood–brain barrier, but also the systemic response of the organism to multifactor molecular processes induced by ischemic injury.     

Genome-Wide Analyses of Radioresistance-Associated miRNA Expression Profile in Nasopharyngeal Carcinoma Using Next Generation Deep Sequencing

Background

Rapidly growing evidence suggests that microRNAs (miRNAs) are involved in a wide range of cancer malignant behaviours including radioresistance. Therefore, the present study was designed to investigate miRNA expression patterns associated with radioresistance in NPC.

Methods

The differential expression profiles of miRNAs and mRNAs associated with NPC radioresistance were constructed. The predicted target mRNAs of miRNAs and their enriched signaling pathways were analyzed via biological informatical algorithms. Finally, partial miRNAs and pathways-correlated target mRNAs were validated in two NPC radioreisitant cell models.

Results

50 known and 9 novel miRNAs with significant difference were identified, and their target mRNAs were narrowed down to 53 nasopharyngeal-/NPC-specific mRNAs. Subsequent KEGG analyses demonstrated that the 53 mRNAs were enriched in 37 signaling pathways. Further qRT-PCR assays confirmed 3 down-regulated miRNAs (miR-324-3p, miR-93-3p and miR-4501), 3 up-regulated miRNAs (miR-371a-5p, miR-34c-5p and miR-1323) and 2 novel miRNAs. Additionally, corresponding alterations of pathways-correlated target mRNAs were observed including 5 up-regulated mRNAs (ICAM1, WNT2B, MYC, HLA-F and TGF-β1) and 3 down-regulated mRNAs (CDH1, PTENP1 and HSP90AA1).

Conclusions

Our study provides an overview of miRNA expression profile and the interactions between miRNA and their target mRNAs, which will deepen our understanding of the important roles of miRNAs in NPC radioresistance.     

Circulating adiposity-related microRNAs as predictors of the response to a low-fat diet in subjects with obesity

Recent studies have revealed the critical role of several microRNAs (miRNAs) in energy homeostasis and metabolic processes and suggest that circulating miRNAs can be used as early predictors of weight loss in the design of precision nutrition. Thus, the aim of this study was to investigate circulating adiposity-related miRNAs as biomarkers of the response to two specific weight loss dietary treatments. The expression of 86 miRNAs was investigated in plasma of 78 subjects with obesity randomized to two different diets [moderately high-protein diet (n = 38) and low-fat diet (n = 40)] and in 25 eutrophic controls (BMI ≤ 25 kg/m²). Bioinformatic analyses were performed to explore the target genes and biological pathways regulated by the dysregulated miRNAs. As results, 26 miRNAs were found differently expressed in eutrophic and volunteers with obesity. Moreover, 7 miRNAs (miR-130a-3p, miR-142-5p, miR-144-5p, miR-15a-5p, miR-22-3p, miR-221-3p and miR-29c-3p) were differentially expressed between responders and non-responders to a low-fat diet. Furthermore, after adjustment for basal glucose levels, 1-SD increase in miR-22-3p expression was associated with reduction in the risk of non-response to low-fat diet [OR = 0.181, 95% CI (0.084-0.947), P = .043]. Bioinformatic analyses evidenced that these 7 miRNAs regulate the expression of genes participating in important metabolic pathways. Conclusively, 7 circulating miRNAs related to adiposity could be used for predicting the response to a low-fat diet intervention prescribed to lose weight.