A novel vector-based method for exclusive overexpression of star-form microRNAs

The roles of microRNAs (miRNAs) as important regulators of gene expression have been studied intensively. Although most of these investigations have involved the highly expressed form of the two mature miRNA species, increasing evidence points to essential roles for star-form microRNAs (miRNA*), which are usually expressed at much lower levels. Owing to the nature of miRNA biogenesis, it is challenging to use plasmids containing miRNA coding sequences for gain-of-function experiments concerning the roles of microRNA* species. Synthetic microRNA mimics could introduce specific miRNA* species into cells, but this transient overexpression system has many shortcomings. Here, we report that specific miRNA* species can be overexpressed by introducing artificially designed stem-loop sequences into short hairpin RNA (shRNA) overexpression vectors. By our prototypic plasmid, designed to overexpress hsa-miR-146b-3p, we successfully expressed high levels of hsa-miR-146b-3p without detectable change of hsa-miR-146b-5p. Functional analysis involving luciferase reporter assays showed that, like natural miRNAs, the overexpressed hsa-miR-146b-3p inhibited target gene expression by 3'UTR seed pairing. Our demonstration that this method could overexpress two other miRNAs suggests that the approach should be broadly applicable. Our novel strategy opens the way for exclusively stable overexpression of miRNA* species and analyzing their unique functions both in vitro and in vivo.     

Host microRNAs exhibit differential propensity to interact with SARS-CoV-2 and variants of concern

A significant number of SARS-CoV-2-infected individuals naturally overcome viral infection, suggesting the existence of a potent endogenous antiviral mechanism. As an innate defense mechanism, microRNA (miRNA) pathways in mammals have evolved to restrict viruses, besides regulating endogenous mRNAs. In this study, we systematically examined the complete repertoire of human miRNAs for potential binding sites on SARS-CoV-2 Wuhan-Hu-1, Beta, Delta, and Omicron. Human miRNA and viral genome interaction were analyzed using RNAhybrid 2.2 with stringent parameters to identify highly bonafide miRNA targets. Using publicly available data, we filtered for miRNAs expressed in lung epithelial cells/tissue and oral keratinocytes, concentrating on the miRNAs that target SARS-CoV-2 S protein mRNAs. Our results show a significant loss of human miRNA and SARS-CoV-2 interactions in Omicron (130 miRNAs) compared to Wuhan-Hu-1 (271 miRNAs), Beta (279 miRNAs), and Delta (275 miRNAs). In particular, hsa-miR-3150b-3p and hsa-miR-4784 show binding affinity for S protein of Wuhan strain but not Beta, Delta, and Omicron. Loss of miRNA binding sites on N protein was also observed for Omicron. Through Ingenuity Pathway Analysis (IPA), we examined the experimentally validated and highly predicted functional role of these miRNAs. We found that hsa-miR-3150b-3p and hsa-miR-4784 have several experimentally validated or highly predicted target genes in the Toll-like receptor, IL-17, Th1, Th2, interferon, and coronavirus pathogenesis pathways. Focusing on the coronavirus pathogenesis pathway, we found that hsa-miR-3150b-3p and hsa-miR-4784 are highly predicted to target MAPK13. Exploring miRNAs to manipulate viral genome/gene expression can provide a promising strategy with successful outcomes by targeting specific VOCs.     

Identification and bioinformatics analysis of microRNAs associated with stress and immune response in serum of heat-stressed and normal Holstein cows

MicroRNAs (miRNAs) are small single-stranded non-coding RNAs that have an important regulatory function in animal growth and developmental processes. However, the differential expression of miRNA and the role of these miRNAs in heat-stressed Holstein cows are still unknown. In this study, the profile of differentially expressed miRNAs and the target genes analysis in the serum of heat-stressed and normal Holstein cows were investigated by a Solexa deep-sequencing approach and bioinformatics. The data identified 52 differentially expressed miRNAs in 486 known miRNAs which were changed significantly between heat-stressed and normal Holstein cows (fold change >2, P < 0.001). Target genes analysis showed that at least 7 miRNAs (miR-19a, miR-19b, miR-146a, miR-30a-5p, miR-345-3p, miR-199a-3p, and miR-1246) were involved in the response to stress, oxidative stress, development of the immune system, and immune response among the identified 52 differentially expressed miRNAs. Five miRNAs (miR-27b, miR-181a, miR-181b, miR-26a, and miR-146b) were involved in stress and immune responses and the expression of five miRNAs was striking (P < 0.001). In addition, RT-qPCR and deep-sequencing methods showed that 8 miRNAs among the 12 selected miRNAs (miR-19a, miR-19b, miR-27b, miR-30a-5p, miR-181a, miR-181b, miR-345-3p, and miR-1246) were highly expressed in the serum of heat-stressed Holstein cows. GO and KEGG pathway analysis showed that these differentially expressed miRNAs were involved in a pathway that may differentially regulate the expression of stress response and immune response genes. Our study provides an overview of miRNAs expression profile and the interaction between miRNAs and their target genes, which will lead to further understanding of the important roles of miRNAs in heat-stressed Holstein cows.     

Identification of a MicroRNA Signature for the Diagnosis of Fibromyalgia

Background

Diagnosis of fibromyalgia (FM), a chronic musculoskeletal pain syndrome characterized by generalized body pain, hyperalgesia and other functional and emotional comorbidities, is a challenging process hindered by symptom heterogeneity and clinical overlap with other disorders. No objective diagnostic method exists at present. The aim of this study was to identify changes in miRNA expression profiles (miRNome) of these patients for the development of a quantitative diagnostic method of FM. In addition, knowledge of FM patient miRNomes should lead to a deeper understanding of the etiology and/or symptom severity of this complex disease.

Methods

Genome-wide expression profiling of miRNAs was assessed in Peripheral Blood Mononuclear Cells (PBMCs) of FM patients (N=11) and population-age-matched controls (N=10) using human v16-miRbase 3D-Gene microarrays (Toray Industries, Japan). Selected miRNAs from the screen were further validated by RT-qPCR. Participating patients were long term sufferers (over 10 years) diagnosed by more than one specialist under 1990 American College of Rheumatology criteria.

Results

Microarray analysis of FM patient PBMCs evidenced a marked downregulation of hsa-miR223-3p, hsa-miR451a, hsa-miR338-3p, hsa-miR143-3p, hsa-miR145-5p and hsa-miR-21-5p (4-fold or more). All but the mildest inhibited miRNA, hsa-miR-21-5p, were validated by RT-qPCR. Globally, 20% of the miRNAs analyzed (233/1212) showed downregulation of at least 2-fold in patients. This might indicate a general de-regulation of the miRNA synthetic pathway in FM. No significant correlations between miRNA inhibition and FM cardinal symptoms could be identified. However, the patient with the lowest score for mental fatigue coincided with the mildest inhibition in four of the five miRNAs associated with the FM-group.

Conclusions

We propose a signature of five strikingly downregulated miRNAs (hsa-miR223-3p, hsa-miR451a, hsa-miR338-3p, hsa-miR143-3p and hsa-miR145-5p) to be used as biomarkers of FM. Validation in larger study groups is required before the results can be transferred to the clinic.     

An integrated analysis of miRNA and mRNA expressions in non-small cell lung cancers

Using DNA microarrays, we generated both mRNA and miRNA expression data from 6 non-small cell lung cancer (NSCLC) tissues and their matching normal control from adjacent tissues to identify potential miRNA markers for diagnostics. We demonstrated that hsa-miR-96 is significantly and consistently up-regulated in all 6 NSCLCs. We validated this result in an independent set of 35 paired tumors and their adjacent normal tissues, as well as their sera that are collected before surgical resection or chemotherapy, and the results suggested that hsa-miR-96 may play an important role in NSCLC development and has great potential to be used as a noninvasive marker for diagnosing NSCLC. We predicted potential miRNA target mRNAs based on different methods (TargetScan and miRanda). Further classification of miRNA regulated genes based on their relationship with miRNAs revealed that hsa-miR-96 and certain other miRNAs tend to down-regulate their target mRNAs in NSCLC development, which have expression levels permissive to direct interaction between miRNAs and their target mRNAs. In addition, we identified a significant correlation of miRNA regulation with genes coincide with high density of CpG islands, which suggests that miRNA may represent a primary regulatory mechanism governing basic cellular functions and cell differentiations, and such mechanism may be complementary to DNA methylation in repressing or activating gene expression.     

Comparative microarray analysis of microRNA expression profiles in primary cutaneous malignant melanoma, cutaneous malignant melanoma metastases, and benign melanocytic nevi

Perturbations in microRNA (miRNA) expression profiles have been reported for cutaneous malignant melanoma (CMM) predominantly when examined in cell lines. Despite the rapidly growing number of newly discovered human miRNA sequences, the availability of up-to-date miRNA expression profiles for clinical samples of primary cutaneous malignant melanoma (PCMM), cutaneous malignant melanoma metastases (CMMM), and benign melanocytic nevi (BMN) is limited. Specimens excised from the center of tumors (lesional) from patients with PCMM (n=9), CMMM (n=4), or BMN (n=8) were obtained during surgery. An exploratory microarray analysis was performed by miRNA expression profiling based on Agilent platform screening for 1205 human miRNAs. The results from the microarray analysis were validated by TaqMan quantitative real-time polymerase chain reaction. In addition to several miRNAs previously known to be associated with CMM, 19 unidentified miRNA candidates were found to be dysregulated in CMM patient samples. Among the 19 novel miRNA candidates, the genes hsa-miR-22, hsa-miR-130b, hsa-miR-146b-5p, hsa-miR-223, hsa-miR-301a, hsa-miR-484, hsa-miR-663, hsa-miR-720, hsa-miR-1260, hsa-miR-1274a, hsa-miR-1274b, hsa-miR-3663-3p, hsa-miR-4281, and hsa-miR-4286 were upregulated, and the genes hsa-miR-24-1*, hsa-miR-26a, hsa-miR-4291, hsa-miR-4317, and hsa-miR-4324 were downregulated. The results of this study partially confirm previous CMM miRNA profiling studies identifying miRNAs that are dysregulated in CMM. However, we report several novel miRNA candidates in CMM tumors; these miRNA sequences require further validation and functional analysis to evaluate whether they play a role in the pathogenesis of CMM.     

MicroRNA Profiling in Human Colon Cancer Cells during 5-Fluorouracil-Induced Autophagy

Autophagy modulation is now recognized as a potential therapeutic approach for cancer (including colorectal cancer), yet the molecular mechanisms regulating autophagy in response to cellular stress are still not well understood. MicroRNAs (miRNAs) have been found to play important roles in controlling many cellular functions, including growth, metabolism and stress response. The physiological importance of the miRNA-autophagy interconnection is only beginning to be elucidated. MiRNA microarray technology facilitates analysis of global miRNA expression in certain situations. In this study, we explored the expression profile of miRNAs during the response of human colon cancer cells (HT29s) to 5-FU treatment and nutrient starvation using miRNA microarray analysis. The alteration of miRNA expression showed the same pattern under both conditions was further testified by qRT-PCR in three human colon cancer cell lines. In addition, bioinformatic prediction of target genes, pathway analysis and gene network analysis were performed to better understand the roles of these miRNAs in the regulation of autophagy. We identified and selected four downregulated miRNAs including hsa-miR-302a-3p and 27 upregulated miRNAs under these two conditions as having the potential to target genes involved in the regulation of autophagy in human colon cancer cells. They have the potential to modulate autophagy in 5-FU-based chemotherapy in colorectal cancer.     

通过miRNA基因表达谱的基因共表达网络构建对星形细胞瘤的基因靶标进行预测

星形细胞瘤为浸润性生长肿瘤,生长缓慢,多为隐形症状,难以早期发现。多数肿瘤切除后有复发可能,且复发后肿瘤可演变成间变性星形细胞瘤或多形性胶质母细胞瘤。因此寻找其生物标志物早期诊断,并研究新的治疗方法就十分重要。方法:通过选用GEO数据库的星形细胞瘤miRNA表达谱数据,进行差异分析以及靶基因预测,通过2者共表达网络的构建对筛选出的miRNA的研究价值进行探讨。结果:得出hsa-miR-29b-2-5p;hsa-miR-339-5p与hsa-miR-362-3p3个较为关键的miRNA及与3者关系密切的8个mRNA。结论:通过对8个mRNA在癌症中的作用的讨论,肯定了这3个关键miRNA作为潜在靶点的研究价值。     

Integrated miRNA and mRNA Expression Profiling in Inflamed Colon of Patients with Ulcerative Colitis

Background

Ulcerative colitis (UC) is associated with differential colonic expression of genes involved in immune response (e.g. IL8) and barrier integrity (e.g. cadherins). MicroRNAs (miRNAs) are regulators of gene expression and are involved in various immune-related diseases. In this study, we investigated (1) if miRNA expression in UC mucosa is altered and (2) if any of these changes correlate with mucosal mRNA expression. Integration of mRNA and miRNA expression profiling may allow the identification of functional links between dysregulated miRNAs and their target mRNA.

Methodology

Colonic mucosal biopsies were obtained from 17 UC (10 active and 7 inactive) patients and 10 normal controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively. Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. Microarray data were validated with qRT-PCR. Regulation of IL8 and CDH11 expression by hsa-miR-200c-3p was determined by luciferase reporter assays.

Results

When comparing active UC patients vs. controls, 51 miRNAs and 1543 gene probe sets gave significantly different signals. In contrast, in inactive UC vs. controls, no significant miRNA expression differences were found while 155 gene probe sets had significantly different signals. We then identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs. controls and found a highly significant inverse correlation between hsa-miR-200c-3p and IL8, an inflammatory marker, and between hsa-miR-200c-3p and CDH11, a gene related to intestinal epithelial barrier function. We could demonstrate that hsa-miR-200c-3p directly regulates IL8 and CDH11 expression.

Conclusion

Differential expression of immune- and barrier-related genes in inflamed UC mucosa may be influenced by altered expression of miRNAs. Integrated analysis of miRNA and mRNA expression profiles revealed hsa-miR-200c-3p for use of miRNA mimics as therapeutics.     

Dynamic expression of viral and cellular microRNAs in infectious mononucleosis caused by primary Epstein-Barr virus infection in children

Background

Epstein-Barr virus (EBV) was the first virus identified to encode microRNAs (miRNAs). Both of viral and human cellular miRNAs are important in EBV infection. However, the dynamic expression profile of miRNAs during primary EBV infection was unknown. This study aimed to investigate the dynamic expression profile of viral and cellular miRNAs in infectious mononucleosis (IM) caused by primary EBV infection.

Methods

The levels of viral and cellular miRNAs were measured in fifteen pediatric IM patients at three different time-points. Fifteen healthy children who were seropositive for EBV were enrolled in the control group. Relative expression levels of miRNAs were detected by quantitative real-time PCR (qPCR) assay.

Results

EBV-miR-BHRF1-1, 1-2-3P, miR-BART13-1, 19-3p, 11-3P, 12–1, and 16–1 in IM patients of early phase were significantly higher than in healthy children. Most cellular miRNAs of B cells, such as hsa-miR-155-5p, ?34a-5p, ?18b-5p, ?181a-5p, and ?142-5p were up-regulated; while most of cellular miRNAs of CD8?+?T cells, such as hsa-miR-223, ?29c-3p, ?181a, ?200a-3p, miR-155-5p, ?146a, and ?142-5p were down-regulated in IM patients. With disease progression, nearly all of EBV-miRNAs decreased, especially miR-BHRF1, but at a slower rate than EBV DNA loads. Most of the cellular miRNAs of B cells, including hsa-miR-134-5p, ?18b-5p, ?34a-5p, and -196a-5p increased with time. However, most of the cellular miRNAs of CD8?+?T cells, including hsa-let-7a-5p, ?142-3p, ?142-5p, and ?155-5p decreased with time. Additionally, hsa-miR-155-5p of B cells and hsa-miR-18b-5p of CD8+ T cells exhibited a positive correlation with miR-BHRF1-2-5P and miR-BART2-5P (0.96?≤?r?≤?0.99, P?<?0.05). Finally, hsa-miR-181a-5p of B cells had positive correlation with miR-BART4-3p, 4-5P, 16–1, and 22 (0.97?≤?r?≤?0.99, P?<?0.05).

Conclusions

Our study is the first to describe the expression profile of viral and cellular miRNAs in IM caused by primary EBV infection. These results might be the basis of investigating the pathogenic mechanism of EBV-related diseases and bring new insights into their diagnosis and treatment.

     

Identification of novel biomarkers and key pathways of condyloma acuminata

Condyloma acuminata (CA) is a prevalent sexually transmitted disease, associated with human papilloma viruses (HPV) infections and host immune status. In this present study, we aimed to explore immune landscape and biomarkers for CA prevention and treatment. We obtained differentially expressed genes (DEGs) of CA vs normal tissues in GSE140662 and screened out hub genes from the protein–protein interaction (PPI) network. Hub genes were then subjected to microRNA (miRNA) analysis. Besides, CCK-8, transwell, flow cytometry assays were employed to assess the cell proliferation, migration and apoptosis in Hela cells. ImmuCellAI was firstly applied to identify immune cell infiltration levels of CA. We obtained 275 DEGs, 23 hub genes and key miRNAs. Subsequently, we verified four up-regulated hub genes IFIT1, IFI27, OASL, SAMD9L and down-regulated mir-146a-5p in CA tissues by RT-qPCR. Moreover, over-expression of miR-146a-5p reduced Hela cells proliferation, migration, blocked cell cycle and induced apoptosis. Up-regulated miR-146a-5p attenuated PI3K/AKT and activated p38/ MAPK signaling pathway. Proportions of Monocyte, NK cells, Gamma delta cells, Th17 cells were relatively low, while Th1 and CD8+ T cells were relatively high in CA skin. Our study revealed that mir-146a-5p contribute to CA progression through PI3K/AKT and p38/MAPK signaling pathway.     

Unraveling the role of microRNA/isomiR network in multiple primary melanoma pathogenesis

Malignant cutaneous melanoma (CM) is a potentially lethal form of skin cancer whose worldwide incidence has been constantly increasing over the past decades. During their lifetime, about 8% of CM patients will develop multiple primary melanomas (MPMs), usually at a young age and within 3 years from the first tumor/diagnosis. With the aim of improving our knowledge on MPM biology and pathogenesis, we explored the miRNome of 24 single and multiple primary melanomas, including multiple tumors from the same patient, using a small RNA-sequencing approach. From a supervised analysis, 22 miRNAs were differentially expressed in MPM compared to single CM, including key miRNAs involved in epithelial–mesenchymal transition. The first and second melanoma from the same patient presented a different miRNA profile. Ten miRNAs, including miR-25-3p, 149-5p, 92b-3p, 211-5p, 125a-5p, 125b-5p, 205-5p, 200b-3p, 21-5p, and 146a-5p, were further validated in 47 single and multiple melanoma samples. Pathway enrichment analysis of miRNA target genes revealed a more differentiated and less invasive status of MPMs compared to CMs. Bioinformatic analyses at the miRNA isoform (isomiR) level detected a panel of highly expressed isomiRs belonging to miRNA families implicated in human tumorigenesis, including miR-200, miR-30, and miR-10 family. Moreover, we identified hsa-miR-125a-5p|0|−2 isoform as tenfold over-represented in melanoma than the canonical form and differentially expressed in MPMs arising in the same patient. Target prediction analysis revealed that the miRNA shortening could change the pattern of target gene regulation, specifically in genes implicated in cell adhesion and neuronal differentiation. Overall, we provided a putative and comprehensive characterization of the miRNA/isomiR regulatory network of MPMs, highlighting mechanisms of tumor development and molecular features differentiating this subtype from single melanomas.Subject terms: Small RNAs, Melanoma