A microRNA panel that regulates proinflammatory cytokines as diagnostic and prognosis biomarkers in colon cancer

Colon cancer (CC) is the third most common neoplasm and the fourth cause of cancer-related death worldwide in both sexes. It has been established that inflammation plays a critical role in tumorigenesis and progression of CC. Immune, stromal and tumor cells supply the tumor microenvironment with pro-inflammatory cytokines such as interleukin 1β, TNFα, IL-6 and IL-11, to hyperactivate signaling pathways linked to cancerous processes. Recent findings suggest a putative role of microRNAs (miRNAs) in the progression and management of the inflammatory response in intestinal diseases. Moreover, miRNAs are able to regulate expression of molecular mediators that are linking inflammation and cancer. In this work a miRNA panel differentially expressed between healthy, inflammatory bowel disease (IBD) and CC tissue was established. Identified miRNAs regulate signaling pathways related to inflammation and cancer progression. An inflammation associated-miRNA panel composed of 11-miRNAs was found to be overexpressed in CC but not in inflamed or normal tissues (miR-21-5p, miR-304-5p, miR-577, miR-335-5p, miR-21-3p, miR-27b-5p, miR-335-3p, miR-215-5p, miR-30b-5p, miR-192-5p, miR-3065-5p). The association of top hit miRNAs, miR-3065-5p and miR-30b-5p expression with overall survival of CC patients was demonstrated using Kaplan-Meier tests. Finally, differential miRNA expression was validated using an inflammation-associated CC model induced by Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) to compare miRNA expression in normal and inflamed tissue versus CC tissues. Based on these findings we propose the identified inflammatory miRNA panel as a potent diagnostic tool for CC determination.     

Circulating microRNAs associated with liver fibrosis in chronic hepatitis C patients

A major challenge in hepatitis C research is the detection of early potential for progressive liver disease. MicroRNAs (miRNAs) are small RNAs that regulate gene expression and can be biomarkers of pathological processes. In this study, we compared circulating miRNAs identified in hepatitis C virus (HCV)-infected patients presenting two extremes of liver disease: mild/moderate fibrosis and cirrhosis. The patients in the cirrhosis group subsequently developed hepatocellular carcinoma (HCC). We identified 163 mature miRNAs in the mild/moderate fibrosis group and 171 in the cirrhosis group, with 144 in common to both groups. Differential expression analysis revealed 5 upregulated miRNAs and 2 downregulated miRNAs in the cirrhosis group relative to the mild/moderate fibrosis group. Functional analyses of regulatory networks (target gene and miRNA) identified gene categories involved in cell cycle biological processes and metabolic pathways related to cell cycle, cancer, and apoptosis. These results suggest that the differentially expressed circulating miRNAs observed in this work (miR-215-5p, miR-483-5p, miR-193b-3p, miR-34a-5p, miR-885-5p, miR-26b-5p and miR -197-3p) may be candidates for biomarkers in the prognosis of liver disease.     

miR-145-5p affects the differentiation of gastric cancer by targeting KLF5 directly

Krüppel-like factor 5 (KLF5) takes part in the pathologic processes of many types of cancer; however, its expression and roles in the biological behavior of gastric cancer remain unknown. TargetScan suggested that miR-145-5p is the predicted effective and conserved microRNA (miRNA) that binds to KLF5 through its 3′-untranslated region (UTR). We investigated the expression of KLF5 and miR-145-5p messenger RNA (mRNA) in gastric cancer and then analyzed its role in the biological behavior of gastric cancer cells. Our results indicated that KLF5 expression was detected by immunohistochemistry in 39.7% of the gastric cancer cases and was increased compared with that of the corresponding noncancerous normal mucosa (0.01 < p < 0.05). The poorly differentiated subtype showed positive KLF5 expression, whereas the differentiated subtype showed negative KLF5 expression (p < 0.05). Dual-luciferase reporter assay suggested KLF5 3′-UTR was the direct target of miR-145-5p. Compared with the differentiated gastric cancer, miR-145-5p was downregulated in undifferentiated gastric cancer (p < 0.05). The downregulation of KLF5 expression and differentiation of MGC-803 and BGC-823 caused by siKLF5 or miR-145-5p mimic transfection. Our results indicated that miR-145-5p/KLF5 3′-UTR affected the differentiation of gastric cancer. miR-145-5p was able to promote gastric cancer differentiation by targeting KLF5 3′-UTR directly. Our data suggest a novel mechanism for cancer differentiation and a new facet to the role of miR-145-5p/KLF5 in gastric cancer.     

Coordinated aberrant expression of miRNAs in colon cancer

Applying the method of multiple parallel sequencing on the MiSeq platform (Illumina, United States), a comparative analysis of miRNA expression in tumor and normal colon tissue cells was performed. Forty miRNAs aberrantly expressed in cancer were detected. Among them, 15 and 25 miRNAs showed increased and decreased expression, respectively, for all or most of the cases. Sixteen miRNA clusters were identified, which showed a coordinated or incompletely coordinated aberrant expression in colorectal cancer cells. In two (miR-183/182 and miR-106b/25) and four (miR-143/145, miR-497/195, miR-30e/30c-1, and miR-30a/30c-2) miRNA clusters, respectively, a statistically significant coordinated increase or decrease in expression was registered for all miRNAs within the corresponding cluster. Three aberrantly expressed well-known miRNAs (miR-100-5p, miR-30d-5p, and miR-204-5p) were identified, which, however, had never before been associated with colorectal cancer. The obtained results demonstrate the potential and promising application of 6 miRNA clusters with coordinated aberrant expression as markers for colorectal cancer.     

MiR-192-5p inhibits proliferation,migration, and invasion in papillary thyroid carcinoma cells by regulation of SH3RF3

Background: The decreased level of miR-192-5p has been reported in several kinds of cancers, including bladder, colon, ovarian, and non-small cell lung cancer. However, the expression and function of miR-192-5p in papillary thyroid carcinoma/cancer (PTC) remains unknown.Objective: The present study aimed to explore the function and underlying mechanism of miR-192-5p in PTC development.Methods: PTC tissues and relative normal controls from PTC patients were collected. qRT-PCR analysis was performed to measure miR-192-5p and SH3RF3 mRNA level in PTC tissues and cell lines. CCK-8 method and FCM assay were used to test cell proliferation and apoptosis in TPC-1 cells, respectively. The abilities of cell migration and invasion were detected by wound healing and transwell assays, respectively. The protein expression was evaluated by Western blot. The interaction between miR-192-5p and Src homology 3 (SH3) domain containing ring finger 3 (SH3RF3) were confirmed by dual-luciferase reporter assay.Results: MiR-192-5p level was obviously decreased in PTC tissues and cell lines. Overexpression of miR-192-5p suppressed proliferation, migration, invasion, and EMT process, while induced apoptosis in TPC-1 cells. In addition, miR-192-5p negatively modulated SH3RF3 expression by binding to its 3′-untranslated region (3′UTR). Silencing SH3RF3 inhibited the migration, invasion, and EMT of TPC-1 cells. In the meantime, matrine, an alkaloid extracted from herb, exerted its anti-cancer effects in PTC cells dependent on increase in miR-192-5p expression and decrease in SH3RF3 expression.Conclusion: We firstly declared that miR-192-5p played a tumor suppressive role in PTC via targeting SH3RF3. Moreover, matrine exerted its anti-cancer effects in PTC via regulating miR-192-5p/SH3RF3 pathway.     

miR-204-5p regulates cell proliferation,invasion, and apoptosis by targeting IL-11 in esophageal squamous cell carcinoma

Esophageal cancer (EC) is the world's eighth most common malignant neoplasm and is ranked as the sixth leading cause of death related to cancer. Aberrant microRNA (miRNA) expression has been reported to be associated with esophageal squamous cell carcinoma. However, the molecular mechanism of miR-204-5p in esophageal squamous cell carcinoma (ESCC) is not clear. Therefore, the aim of this study was to investigate the potential role of miR-204-5p in ESCC. In the present study, we found that miR-204-5p could affect ESCC proliferation, invasion, apoptosis, and cell cycle in cell and mouse models. A dual-luciferase reporter assay showed that miR-204-5p expression was negatively correlated with interleukin-11 (IL-11) expression. IL-11 overexpression reversed the suppressive effects of miR-204-5p in the cell lines. These results indicated that miR-204-5p functions as a tumor suppressor by directly targeting IL-11 in ESCC.     

Alterations in microRNA expression profile in HCV-infected hepatoma cells: Involvement of miR-491 in regulation of HCV replication via the PI3 kinase/Akt pathway

The aim of this study was to investigate the role of microRNA (miRNA) on hepatitis C virus (HCV) replication in hepatoma cells. Using miRNA array analysis, miR-192/miR-215, miR-194, miR-320, and miR-491 were identified as miRNAs whose expression levels were altered by HCV infection. Among them, miR-192/miR-215 and miR-491 were capable of enhancing replication of the HCV replicon as well as HCV itself. HCV IRES activity or cell proliferation was not increased by forced expression of miR-192/miR-215 or miR-491. Investigation of signaling pathways revealed that miR-491 specifically suppressed the phosphoinositol-3 (PI3) kinase/Akt pathway. Under inhibition of PI3 kinase by LY294002, the suppressive effect of miR-491 on HCV replication was abolished, indicating that suppression of HCV replication by miR-491 was dependent on the PI3 kinase/Akt pathway. miRNAs altered by HCV infection would then affect HCV replication, which implies a complicated mechanism for regulating HCV replication. HCV-induced miRNA may be involved in changes in cellular properties including hepatocarcinogenesis.     

MicroRNA-524-5p suppresses the progression of papillary thyroid carcinoma cells via targeting on FOXE1 and ITGA3 in cell autophagy and cycling pathways

MicroRNAs are beneficial for cancer therapy as they can simultaneously downregulate multiple targets involved in diverse biological pathways related to tumor development. In papillary thyroid cancer, many microRNAs were identified as differentially expressed factors in tumor tissues. In another way, recent studies revealed cell proliferation, cell cycling, apoptosis, and autophagy are critical pathways controlling papillary thyroid cancer development and progression. As miR-524-5p was approved as a cancer suppressor targeting multiple genes in several types of cancer cells, this study aims to characterize the role of miR-524-5p in the thyroid cancer cell. The expression of miR-524-5p was decreased in the papillary thyroid cancer tissues and cell lines, while forkhead box E1 (FOXE1) and ITGA3 were increased. In the clinical case, expression of miR-524-5p, FOXE1, and ITGA3 were significantly correlated with papillary thyroid cancer development and progression. FOXE1 and ITGA3 were approved as direct targets of miR-524-5p. miR-524-5p could inhibit papillary thyroid cancer cell viability, migration, invasion, and apoptosis through targeting FOXE1 and ITGA3. Cell cycling and autophagy pathways were disturbed by downregulation of FOXE1 and ITGA3, respectively. Collectively, miR-524-5p targeting on FOXE1 and ITGA3 prevents thyroid cancer progression through different pathways including cell cycling and autophagy.     

Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis

Of the over 200 identified mammalian microRNAs (miRNAs), only a few have known biological activity. To gain a better understanding of the role that miRNAs play in specific cellular pathways, we utilized antisense molecules to inhibit miRNA activity. We used miRNA inhibitors targeting miR-23, 21, 15a, 16 and 19a to test efficacy of antisense molecules in reducing miRNA activity on reporter genes bearing miRNA-binding sites. The miRNA inhibitors de-repressed reporter gene activity when a miRNA-binding site was cloned into its 3′-untranslated region. We employed a library of miRNA inhibitors to screen for miRNA involved in cell growth and apoptosis. In HeLa cells, we found that inhibition of miR-95, 124, 125, 133, 134, 144, 150, 152, 187, 190, 191, 192, 193, 204, 211, 218, 220, 296 and 299 caused a decrease in cell growth and that inhibition of miR-21 and miR-24 had a profound increase in cell growth. On the other hand, inhibition of miR-7, 19a, 23, 24, 134, 140, 150, 192 and 193 down-regulated cell growth, and miR-107, 132, 155, 181, 191, 194, 203, 215 and 301 increased cell growth in lung carcinoma cells, A549. We also identified miRNA that when inhibited increased the level of apoptosis (miR-1d, 7, 148, 204, 210, 216 and 296) and one miRNA that decreased apoptosis (miR-214) in HeLa cells. From these screens, we conclude that miRNA-mediated regulation has a complexity of cellular outcomes and that miRNAs can be mediators of regulation of cell growth and apoptosis pathways.     

U6 is not a suitable endogenous control for the quantification of circulating microRNAs

Recently, microRNAs have been detected in serum and plasma, and circulating microRNA (miRNA) profiles have now been associated with many diseases such as cancers and heart disease, as well as altered physiological states. Because of their stability and disease resistance, circulation miRNAs appear to be an ideal material for biomarkers of diseases and physiological states in blood. However, the lack of a suitable internal reference gene (internal reference miRNA) has hampered research and application of circulating miRNAs. Currently, U6 and miR-16 are the most common endogenous controls in the research of miRNAs in tissues and cells. We performed microarray-based serum miRNA profiling on the serum of 20 nasopharyngeal carcinoma patients and 20 controls to detect the expressions of U6 and miRNAs. Profiling was followed by real-time quantitative Polymerase Chain Reaction (qPCR) in 80 patients (20 each with gastric cancer, nasopharyngeal carcinoma, colorectal cancer, and breast cancer) and 30 non-cancerous controls. qPCR was also performed to detect miRNAs in serum with repeated freezing and thawing. The results of microarray showed that with the exception of U6, Ct values of miR-16, miR-24, miR-142-3p, miR-19b and miR-192 in serum samples of nasopharyngeal carcinoma were greater than control samples. The results of 110 cases showed large fluctuations in U6 expression. The difference between the greatest and the least levels of expression was 3.29 for delta Ct values, and 1.23 for miR-16. The expressions of U6, miR-16 and miR-24 in serum subjected to different freeze–thaw cycles showed that U6 expression gradually decreased after 1, 2, and 4 cycles of freezing and thawing, while the expression of miR-16 and miR-24 remained relatively stable. Collectively, our results suggested that U6 is unsuitable as an internal reference gene in the research of circulating miRNAs.     

The loss of MiR-139-5p promotes colitis-associated tumorigenesis by mediating PI3K/AKT/Wnt signaling

MiR-139-5p down-regulation has frequently been implicated in colorectal carcinoma. However, there is little known about its biological function between inflammation and cancer in vivo. Here, a transgenic murine model of colorectal carcinoma was used to investigate pathogenetic role of miR-139-5p in colitis and colitis-associated tumorigenesis. We showed that miR-139-5p knockout mice were higher sensitive to DSS-induced colitis and enhanced formation of intestinal neoplasia was observed when mice were exposed to AOM/DSS treatment. MiR-139-5p knockout mice exhibited an increased expression of genes involved in Wnt pathway. Such genes are closely associated with cell proliferation and differentiation, promoting the β-catenin nuclear accumulation. Furthermore, biochemical studies in HCT-116 cells revealed that the over-expression of miR-139-5p inhibited the crosstalk between PI3K/AKT and Wnt pathway mediated by IGF-1R. Collectively, these findings indicate that miR-139-5p plays a crucial role in the development and progression of colitis-associated tumorigenesis and suggest that miR-139-5p may serve as a potential therapeutic target for the treatment of colitis-associated cancer in the future.     

胃癌与癌旁组织miRNA差异表达谱的研究

目的研究胃癌中表达失调的miRNA及其生物学功能,从而进一步阐明miRNA在胃癌发生中的分子机制。方法将总RNA加ploy（A）尾后进行反转录PCR扩增特异miRNA,使用QuantityOne软件进行定量分析,计算每对样本胃癌与癌旁组织比值（T／NRatio）,使用SAM软件进行统计分析。MTT法检测miR-21和miR-17-5p对胃癌细胞系生长的影响。结果在8对胃癌及癌旁组织样本中对237个miRNA进行了表达谱分析。对于检测到表达的161个miRNA,使用SAM软件进行统计分析,确认22个在胃癌中表达上调,2个在胃癌中表达下调（FDR=0．0963）。进一步通过生长抑制试验证实在胃癌组织中表达异常增高的miR-21和miR-17-5p对胃癌细胞的生长有明显的促进作用。结论这些在胃癌中异常表达的miRNAs具有成为新一代胃癌标记物的潜力,能够为胃癌的精确诊断分型提供依据,同时针对这些靶点可以开发新的核酸治疗技术,通过抑制或增强其功能来达到治疗胃癌的目的。     

MiR-153-5p promotes sensibility of colorectal cancer cells to oxaliplatin via targeting Bcl-2-mediated autophagy pathway

ABSTRACT

Oxaliplatin (L-OHP) is one of the effective chemotherapeutic drugs for colorectal cancer (CRC). Further investigation into the molecular mechanism of chemoresistance could improve outcomes for patients with colorectal cancer. Recently, microRNAs have been reported as a key in drug resistance of tumors. In this study, we aimed to investigate the effects of miR-153-5p in L-OHP-resistant CRC cells, and its underlying mechanism. Downregulation of miR-153-5p was observed in CRC cells, while upregulation of miR-153-5p enhances the chemosensitivity of CRC/L-OHP cells. The autophagy of CRC/L-OHP cells was markedly increased after exposure to L-OHP but abolished by the upregulation of miR-153-5p. Dual-luciferase reporter assays validated that Bcl-2 was a direct target of miR-153-5p. In conclusion, our data suggested that miR-153-5p was a mediator of cisplatin resistance in colorectal cancer by affecting Bcl-2-mediated autophagy, indicating a new therapeutic target for CRC treatment.     

Identification of candidate microRNA biomarkers in renal fibrosis: a meta-analysis of profiling studies

Abstract

The prognostic, diagnostic and therapeutic value of microRNA (miRNA) expression aberrations in renal fibrosis has been studied in recent years. However, the miRNA expression profiling efforts have led to inconsistent results between the studies. The aim of this study was to perform a meta-analysis on the renal fibrosis miRNA expression profiling studies to identify candidate diagnostic biomarkers. We performed comprehensive literature searches in several databases to identify miRNA expression studies of renal fibrosis in animal models and humans. The miRNAs expression data were extracted from 20 included studies, and both miRNA vote-counting strategy and Robust Rank Aggregation method were utilized to identify significant miRNA meta-signatures. The predicted and validated targets of miRNA meta-signature were obtained by using MultiMiR package in 11 databases. Then a gene set enrichment analysis (KEGG, PANTHER pathways and GO processes) were carried out with GeneCodis web tool to recognize pathways that are most strongly influenced by modified expressions of these miRNAs. We recognized in both meta-analysis approaches a significant miRNA meta-signature of five up-regulated (miR-142-3p, miR-223-3p, miR-21-5p, miR-142-5p and miR-214-3p) and two down-regulated (miR-29c-3p and miR-200a-3p) miRNAs. Enrichment analysis confirmed that miRNA meta-signature cooperatively target functionally related genes in signalling and developmental pathways in renal fibrosis. This meta-analysis identified seven highly significant and consistently dysregulated miRNAs from 20 datasets, as the focus of future investigations to discover their potential influence to renal fibrosis and their clinical utility as biomarkers and/or as therapeutic mediators against chronic kidney disease..     

miR-155-3p: processing by-product or rising star in immunity and cancer?

MicroRNAs (miRNAs) are key players in gene regulation that target specific mRNAs for degradation or translational repression. Each miRNA is synthesized as a miRNA duplex comprising two strands (5p and 3p). However, only one of the two strands becomes active and is selectively incorporated into the RNA-induced silencing complex in a process known as miRNA strand selection. Recently, significant progress has been made in understanding the factors and processes involved in strand selection. Here, we explore the selection and functionality of the miRNA star strand (either 5p or 3p), which is generally present in the cell at low levels compared to its partner strand and, historically, has been thought to possess no biological activity. We also highlight the concepts of miRNA arm switching and miRNA isomerism. Finally, we offer insights into the impact of aberrant strand selection on immunity and cancer. Leading us through this journey is miR-155, a well-established regulator of immunity and cancer, and the increasing evidence that its 3p strand plays a role in these arenas. Interestingly, the miR-155-5p/-3p ratio appears to vary dependent on the timing of the immune response, and the 3p strand seems to play a regulatory role upon its partner 5p strand.