Circular RNA ZNF800 (hsa_circ_0082096) regulates cancer stem cell properties and tumor growth in colorectal cancer.

BACKGROUND: Cancer stem cells form a rare cell population in tumors that contributes to metastasis, recurrence and chemoresistance in cancer patients. Circular RNAs (circRNAs) are post-transcriptional regulators of gene expression that sponge targeted microRNA (miRNAs) to affect a multitude of downstream cellular processes. We previously showed in an expression profiling study that circZNF800 (hsa_circ_0082096) was up-regulated in cancer stem cell-enriched spheroids derived from colorectal cancer (CRC) cell lines. METHODS: Spheroids were generated in suspension spheroidal culture. The ZNF800 mRNA, pluripotency stem cell markers and circZNF800 levels were determined by quantitative RT-PCR. CircZNF800-miRNA interactions were shown in RNA pulldown assays and the miRNA levels determined by stem-loop qRT-PCR. The effects of circZNF800 on cell proliferation were tested by EdU staining followed by flowcytometry. Expression of stem cell markers CD44/CD133, Lgr5 and SOX9 was demonstrated in immunofluorescence microscopy. To manipulate the cellular levels of circZNF800, circZNF800 over-expression was achieved via transfection of in vitro synthesized and circularized circZNF800, and knockdown attained using a CRISPR-Cas13d-circZNF800 vector system. Xenografted nude mice were used to demonstrate effects of circZNF800 over-expression and knockdown on tumor growth in vivo. RESULTS: CircZNF800 was shown to be over-expressed in late-stage tumor tissues of CRC patients. Data showed that circZNF800 impeded expression of miR-140-3p, miR-382-5p and miR-579-3p while promoted the mRNA levels of ALK/ACVR1C, FZD3 and WNT5A targeted by the miRNAs, as supported by alignments of seed sequences between the circZNF800-miRNA, and miRNA-mRNA paired interactions. Analysis in CRC cells and biopsied tissues showed that circZNF800 positively regulated the expression of intestinal stem cell, pluripotency and cancer stem cell markers, and promoted CRC cell proliferation, spheroid and colony formation in vitro, all of which are cancer stem cell properties. In xenografted mice, circZNF800 over-expression promoted tumor growth, while circZNF800 knockdown via administration of CRISPR Cas13d-circZNF800 viral particles at the CRC tumor sites impeded tumor growth. CONCLUSIONS: CircZNF800 is an oncogenic factor that regulate cancer stem cell properties to lead colorectal tumorigenesis, and may be used as a predictive marker for tumor progression and the CRISPR Cas13d-circZNF800 knockdown strategy for therapeutic intervention of colorectal cancer.

Establishment of a schizophrenia classifier based on peripheral blood signatures and investigation of pathogenic miRNA-mRNA regulation.

To date, the diagnosis of schizophrenia (SCZ) mainly relies on patients' or guardians' self-reports and clinical observation, and the pathogenesis of SCZ remains elusive. In this study, we sought to develop a reliable classifier for diagnosing SCZ patients and provide clues to the etiology and pathogenesis of SCZ. Based on the high throughput sequencing analysis of peripheral blood miRNA expression profile and weighted gene co-expression network analysis (WGCNA) in our previous study, we selected eleven hub miRNAs for validation by qRT-PCR in 51 SCZ patients and 51 controls. miR-939-5p, miR-4732-3p let-7d-3p, and miR-142-3p were confirmed to be significantly up-regulated, and miR-30e-3p and miR-23a-3p were down-regulated in SCZ patients. miR-30e-3p with the most considerable fold change and statistically significance was selected for targeting validation. We first performed bioinformatics prediction followed by qRT-PCR and verified the up-regulation of potential target mRNAs (ABI1, NMT1, HMGB1) expression. Next, we found that the expression level of ABI1 was significantly up-regulated in SH-SY5Y cells transfected with miR-30e-3p mimics. Lastly, we conducted a luciferase assay in 293T cells confirming that miR-30e-3p could directly bind with the 3'untranslated region (3'-UTR) of ABI1, revealing that miR-30e-3p might play a role in the polymerization of neuronal actin and the reconstruction of the cytoskeleton via the downstream regulation of ABI1. In addition, we constructed a classifier by a series of bioinformatics algorithms and evaluated its diagnostic performance. It appears that the classifier consists of miRNAs and mRNAs possess a better discrimination performance than individual miRNA or mRNA in SCZ.

Value of negatively correlated miR-205-5p/HMGB3 and miR-96-5p/FOXO1 on the diagnosis of breast cancer and benign breast diseases.

Background: MicroRNA (miRNA) and mRNA levels in matching specimens were used to identify miRNA-mRNA interactions. We aimed to integrate transcriptome, immunophenotype, methylation, mutation, and survival data analyses to examine the profiles of miRNAs and target mRNAs and their associations with breast cancer (BC) diagnosis. Methods: Based on the Gene Expression Omnibus (GEO) database and The Cancer Genome Atlas (TCGA), differentially expressed miRNAs and targeted mRNAs were screened from experimentally verified miRNA-target interaction databases using Pearson's correlation analysis. We used real-time quantitative reverse transcription polymerase chain reaction to verify BC and benign disease samples, and logistic regression analysis was used to establish a diagnostic model based on miRNAs and target mRNAs. Receiver operating characteristic curve analysis was performed to test the ability to recognize the miRNA-mRNA pairs. Next, we investigated the complex interactions between miRNA-mRNA regulatory pairs and phenotypic hallmarks. Results: We identified 27 and 359 dysregulated miRNAs and mRNAs, respectively, based on the GEO and TCGA databases. Using Pearson's correlation analysis, 10 negative miRNA-mRNA regulatory pairs were identified after screening both databases, and the related miRNA and target mRNA levels were assessed in 40 BC tissues and 40 benign breast disease tissues. Two key regulatory pairs (miR-205-5p/High mobility group box 3 (HMGB3) and miR-96-5p/Forkhead Box O1 (FOXO1)) were selected to establish the diagnostic model. They also had utility in survival and clinical analyses. Conclusions: A diagnostic model including two miRNAs and their respective target mRNAs was established to distinguish between BC and benign breast diseases. These markers play essential roles in BC pathogenesis.

Identify miRNA-mRNA regulation pairs to explore potential pathogenesis of lung adenocarcinoma.

PURPOSE: MicroRNA (miRNA) function via base-pairing with complementary sequences within mRNA molecules. This study aims to identify critical miRNA-mRNA regulation pairs contributing to lung adenocarcinoma (LUAD) pathogenesis. PATIENTS AND METHODS: MiRNA and mRNA microarray and RNA-sequencing datasets were downloaded from gene expression omnibus (GEO) and the cancer genome atlas (TCGA) databases. Differential miRNAs (DE-miRNAs) and mRNAs (DE-mRNAs) were screened by the GEO2R tool and R packages. DAVID, DIANA, and Hiplot tools were used to perform gene enrichment analysis. The pairs of miRNA-mRNA were screened from the experimentally validated miRNA-target interactions databases (miRTarBase and TarBase). External validation was carried out in 30 pairs of LUAD tissues by quantitative reverse transcription and polymerase chain reaction (qRT-PCR). The diagnostic value of the miRNA-mRNA regulation pairs was evaluated by receiver operating characteristic curve (ROC) and decision curve analysis (DCA). Biological function assay was were also performed to confirm the function of miRNA-mRNA axis in LUAD progression. The study also performed the clinical, survival and tumor-associated phenotypic analysis of miRNA-mRNA pairs. RESULTS: A total of 7 miRNA and 13 mRNA expression datasets from GEO were analyzed, and 11 DE-miRNAs (5 down-regulated and 6 up-regulated in LUAD tissues) and 128 DE-mRNAs (30 up-regulated and 98 down-regulated in LUAD tissues) were identified. The pairs of miR-1-3p(down) and CENPF(up) and miR-126-5p(down) and UGT8(up) were verified in the external validation cohort (30 LUAD vs. 30 NC) using qRT-PCR. Areas under the ROC curve of the two miRNA-mRNA regulation pairs panel were 0.973 in TCGA-LUAD and 0.771 in the external validation. The DCA also showed that the miRNA-mRNA regulation pairs had an excellent diagnostic performance distinguishing LUAD from normal controls. The expression of the regulation pairs is different in different ages, TNM stages, and gender. The overexpression of miR-1-3p and miR-126-5p significantly inhibited the proliferation and migration of LUAD cells. Correlation analysis showed that CENPF correlated with prognosis and tumor immunity. CONCLUSIONS: The research identified potential miRNA-mRNA regulation pairs, providing a new idea for exploring the genesis and development of LUAD.

Global analysis of miRNA-mRNA regulation pair in bladder cancer.

PURPOSE: MicroRNA (miRNA) is a class of short non-coding RNA molecules that functions in RNA silencing and post-transcriptional regulation of gene expression. This study aims to identify critical miRNA-mRNA regulation pairs contributing to bladder cancer (BLCA) pathogenesis. PATIENTS AND METHODS: MiRNA and mRNA microarray and RNA-sequencing datasets were downloaded from gene expression omnibus (GEO) and the cancer genome atlas (TCGA) databases. The tool of GEO2R and R packages were used to screen differential miRNAs (DE-miRNAs) and mRNAs (DE-mRNAs) and DAVID, DIANA, and Hiplot tools were used to perform gene enrichment analysis. The miRNA-mRNA regulation pair were screened from the experimentally validated miRNA-target interactions databases (miRTarbase and TarBase). Twenty-eight pairs of BLCA tissues were used to further verify the screened DE-miRNAs and DE-mRNAs by quantitative reverse transcription and polymerase chain reaction (qRT-PCR). The diagnostic value of the miRNA-mRNA regulation pairs was evaluated by receiver operating characteristic curve (ROC) and decision curve analysis (DCA). The correlation analysis between the selected miRNA-mRNAs regulation pair and clinical, survival and tumor-related phenotypes was performed in this study. RESULTS: After miRTarBase, the analysis of 2 miRNA datasets, 6 mRNA datasets, and TCGA-BLCA dataset, a total of 13 miRNAs (5 downregulated and 8 upregulated in BLCA tissues) and 181 mRNAs (72 upregulated and 109 downregulated in BLCA tissues) were screened out. The pairs of miR-17-5p (upregulated in BLCA tissues) and TGFBR2 (downregulated in BLCA tissues) were verified in the external validation cohort (28 BLCA vs. 28 NC) using qRT-PCR. Areas under the ROC curve of the miRNA-mRNA regulation pair panel were 0.929 (95% CI: 0.885-0.972, p < 0.0001) in TCGA-BLCA and 0.767 (95% CI: 0.643-0.891, p = 0.001) in the external validation. The DCA also showed that the miRNA-mRNA regulation pairs had an excellent diagnostic performance distinguishing BLCA from normal controls. Correlation analysis showed that miR-17-5p and TGFBR2 correlated with tumor immunity. CONCLUSIONS: The research identified potential miRNA-mRNA regulation pairs, providing a new idea for exploring the genesis and development of BLCA.

Comparative analysis of the miRNA-mRNA regulation networks in turbot (Scophthalmus maximus L.) following Vibrio anguillarum infection.

MicroRNAs could not only regulate posttranscriptional silencing of target genes in eukaryotic organisms, but also have positive effect on their target genes as well. These microRNAs have been reported to be involved in mucosal immune responses to pathogen infection in teleost. Therefore, we constructed the immune-related miRNA-mRNA networks in turbot intestine following Vibrio anguillarum infection. In our results, 1550 differentially expressed (DE) genes and 167 DE miRNAs were identified. 113 DE miRNAs targeting 89 DE mRNAs related to immune response were used to construct miRNA-mRNA interaction networks. Functional analysis showed that target genes were associated with synthesis and degradation of ketone bodies, mucin type O-Glycan biosynthesis, homologous recombination, biotin metabolism, and intestinal immune network for IgA production that were equivalent to the function of IgT and IgM in fish intestine. Finally, 10 DE miRNAs and 7 DE mRNAs were selected for validating the accuracy of high-throughput sequencing results by qRT-PCR. The results of this study will provide valuable information for the elucidation of the regulation mechanisms of miRNA-mRNA interactions involved in disease resistance in teleost mucosal immune system.

Immune Infiltration of CD8+ T Cells in Patients With Diabetic Pancreatic Cancer Reduces the Malignancy of Cancer Tissues: An In Silico Study.

Background: Although the functional damage of the diabetic pancreas can affect the postoperative recovery of pancreatic cancer patients, there is no significant difference in the prognosis of pancreatic cancer patients with a history of diabetes and ordinary pancreatic cancer patients. There is still no practical theory to explain this phenomenon. Materials and Method: The mRNA expression profile data of 141 cases and 51 cases with clinical data of diabetes status were obtained from the TCGA database and the GEO database, respectively. The CRA001160 data set was obtained in the TISCH database. The Seurat was used to process single-cell expression profile sequencing data. The Cibersortx was used to construct a feature matrix of single-cell sequencing data and to deconvolve Bulk-RNAseq data to obtain each pancreatic cancer patients' tumour invasion score. TIDE was used to assess the immune escape potential of the tumour. MiRNet was used to construct the miRNA-mRNA regulatory network. Result: Compared with regular pancreatic cancer patients, the immune-related signal transduction pathways in diabetic pancreatic cancer patients are in an activated state. In patients with diabetic pancreatic cancer, the infiltration score of CD8+ T cells is high, and the infiltration score of corresponding malignant tumour cells is low. The Bayesian classifier can distinguish diabetic pancreatic cancer patients from non-diabetic pancreatic cancer patients based on 10 signature genes. The miRNA-mRNA regulatory network suggests that regulation by miRNA can influence mRNA expression and thus prognostic survival of pancreatic cancer patients. Conclusion: The activation of inflammatory-related signalling pathways in diabetic pancreatic cancer patients increases the immune infiltration of CD8+ T cells in cancer patients and reduces the development of malignant tumour tissues. The expression of 10 signature genes allowed the diagnosis of diabetic and non-diabetic pancreatic cancer patients. The miRNA-mRNA regulatory network may be the main cause of the differences in the tumour inflammatory microenvironment between the two groups of patients. These findings help us further understand the immune microenvironment of patients with diabetic pancreatic cancer.

Identifying miRNA-mRNA regulation network of major depressive disorder in ovarian cancer patients.

Major depression disorder (MDD) has become increasingly common in patients with ovarian cancer, which complicates the treatment course. The microRNA (miRNA)-mRNA regulation network may help elucidate the potential mechanism of MDD in ovarian cancer. The differentially expressed microRNAs (DEmiRs) and mRNAs (DEmRNAs) were therefore identified from the GSE61741, GSE58105 and GSE9116 ovarian cancer datasets using GEO2R. The target genes of the DEmiRs were then obtained using the TargetScan, microRNAorg, microT-CDS, miRDB and miRTarBase prediction tools. The DAVID program was used to identify the KEGG pathways of target genes, and the core genes of major depressive disorder (MDD) were identified using the Kaplan-Meier Plotter for ovarian cancer. A total of 5 DEmiRs (miR-23b-3p, miR-33b-3p, miR-1265, miR-933 and miR-629-5p) were obtained from GSE61741 and GSE58105. The target genes of these DEmiRs were enriched in pathways that were considered high risk for developing MDD in ovarian cancer. A total of 11 risk genes were selected from these pathways as the core genes in the miRNA-mRNA network of MDD in ovarian cancer, and eventually identified the following 12 miRNA-mRNAs pairs: miR-629-5p-FGF1, miR-629-5p-AKT3, miR-629-5p-MAGI2, miR-933-BDNF, miR-933-MEF2A, miR-23b-3p-TJP1, miR-23b-3p-JMJD1, miR-23b-3p-APAF1, miR-23b-3p-CAB39, miR-1265-CDKN1B, miR-33b-3p-CDKN1B, and miR-33b-3p-F2R. These results may provide novel insights into the mechanisms of developing MDD in ovarian cancer patients.

Dynamic miRNA-mRNA regulations are essential for maintaining Drosophila immune homeostasis during Micrococcus luteus infection.

Pathogen bacteria infections can lead to dynamic changes of microRNA (miRNA) and mRNA expression profiles, which may control synergistically the outcome of immune responses. To reveal the role of dynamic miRNA-mRNA regulation in Drosophila innate immune responses, we have detailedly analyzed the paired miRNA and mRNA expression profiles at three time points during Drosophila adult males with Micrococcus luteus (M. luteus) infection using RNA- and small RNA-seq data. Our results demonstrate that differentially expressed miRNAs and mRNAs represent extensively dynamic changes over three time points during Drosophila with M. luteus infection. The pathway enrichment analysis indicates that differentially expressed genes are involved in diverse signaling pathways, including Toll and Imd as well as orther signaling pathways at three time points during Drosophila with M. luteus infection. Remarkably, the dynamic change of miRNA expression is delayed by compared to mRNA expression change over three time points, implying that the "time" parameter should be considered when the function of miRNA/mRNA is further studied. In particular, the dynamic miRNA-mRNA regulatory networks have shown that miRNAs may synergistically regulate gene expressions of different signaling pathways to promote or inhibit innate immune responses and maintain homeostasis in Drosophila, and some new regulators involved in Drosophila innate immune response have been identified. Our findings strongly suggest that miRNA regulation is a key mechanism involved in fine-tuning cooperatively gene expressions of diverse signaling pathways to maintain innate immune response and homeostasis in Drosophila. Taken together, the present study reveals a novel role of dynamic miRNA-mRNA regulation in immune response to bacteria infection, and provides a new insight into the underlying molecular regulatory mechanism of Drosophila innate immune responses.

Computational analysis reveals microRNA-mRNA regulatory network in esophageal squamous cell carcinoma.

MicroRNAs (miRNAs) are known to regulate post-transcriptional gene expression. They are involved in carcinogenesis and tumor progression. The aim of this study was to explore the microRNA-mRNA regulatory network in esophageal squamous cell carcinoma (ESCC) using comprehensive computational approaches. In this study we have selected a total of 11 miRNAs from one previously reported study in ESCC. The mRNA targets of these miRNAs were predicted using various algorithms. The expression profiles of these mRNA targets were identified on DNA microarray experiment dataset across ESCC tissue samples. Based on the miRNA-mRNA regulatory relationships, the network was inferred. A total of 23 miRNA-mRNA regulatory interactions, with 11 miRNAs and 13 mRNA targets, were inferred in ESCC. The miRNA-mRNA regulatory network with increased confidence provides insights into the progression of ESCC and may serve as a biomarker for prognosis or the aggressiveness of ESCC. However, the results should be examined with further experimental validation.

Computational analysis reveals microRNA-mRNA regulatory network in esophageal squamous cell carcinoma / 华中科技大学学报（医学）（英德文版）

MicroRNAs (miRNAs) are known to regulate post-transcriptional gene expression. They are involved in carcinogenesis and tumor progression. The aim of this study was to explore the microRNA-mRNA regulatory network in esophageal squamous cell carcinoma (ESCC) using comprehensive computational approaches. In this study we have selected a total of 11 miRNAs from one previously reported study in ESCC. The mRNA targets of these miRNAs were predicted using various algorithms. The expression profiles of these mRNA targets were identified on DNA microarray experiment dataset across ESCC tissue samples. Based on the miRNA-mRNA regulatory relationships, the network was inferred. A total of 23 miRNA-mRNA regulatory interactions, with 11 miRNAs and 13 mRNA targets, were inferred in ESCC. The miRNA-mRNA regulatory network with increased confidence provides insights into the progression of ESCC and may serve as a biomarker for prognosis or the aggressiveness of ESCC. However, the results should be examined with further experimental validation.

Comparative RNA-sequencing analysis of myocardial and circulating small RNAs in human heart failure and their utility as biomarkers.

Heart failure (HF) is associated with high morbidity and mortality and its incidence is increasing worldwide. MicroRNAs (miRNAs) are potential markers and targets for diagnostic and therapeutic applications, respectively. We determined myocardial and circulating miRNA abundance and its changes in patients with stable and end-stage HF before and at different time points after mechanical unloading by a left ventricular assist device (LVAD) by small RNA sequencing. miRNA changes in failing heart tissues partially resembled that of fetal myocardium. Consistent with prototypical miRNA-target-mRNA interactions, target mRNA levels were negatively correlated with changes in abundance for highly expressed miRNAs in HF and fetal hearts. The circulating small RNA profile was dominated by miRNAs, and fragments of tRNAs and small cytoplasmic RNAs. Heart- and muscle-specific circulating miRNAs (myomirs) increased up to 140-fold in advanced HF, which coincided with a similar increase in cardiac troponin I (cTnI) protein, the established marker for heart injury. These extracellular changes nearly completely reversed 3 mo following initiation of LVAD support. In stable HF, circulating miRNAs showed less than fivefold differences compared with normal, and myomir and cTnI levels were only captured near the detection limit. These findings provide the underpinning for miRNA-based therapies and emphasize the usefulness of circulating miRNAs as biomarkers for heart injury performing similar to established diagnostic protein biomarkers.