Downregulation of miR?193a?3p via targeting cyclin D1 in thyroid cancer.
ABSTRACT: Thyroid cancer (TC) is a frequently occurring malignant tumor with a rising steadily incidence. microRNA (miRNA/miR)?193a?3p is an miRNA that is associated with tumors, playing a crucial role in the genesis and progression of various cancers. However, the expression levels of miR?193a?3p and its molecular mechanisms in TC remain to be elucidated. The present study aimed to probe the expression of miR?193a?3p and its clinical significance in TC, including its underlying molecular mechanisms. Microarray and RNA sequencing data gathered from three major databases, specifically Gene Expression Omnibus (GEO), ArrayExpress and The Cancer Genome Atlas (TCGA) databases, and the relevant data from the literature were used to examine miR?193a?3p expression. Meta?analysis was also conducted to evaluate the association between clinicopathological parameters and miR?193a?3p in 510 TC and 59 normal samples from the TCGA database. miRWalk 3.0, and the TCGA and GEO databases were used to predict the candidate target genes of miR?193a?3p. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes and protein?protein interaction network enrichment analyses were conducted by using the predicted candidate target genes to investigate the underlying carcinogenic mechanisms. A dual luciferase assay was performed to validate the targeting regulatory association between the most important hub gene cyclin D1 (CCND1) and miR?193a?3p. miR?193a?3p expression was considerably downregulated in TC compared with in the non?cancer controls (P<0.001). The area under the curve of the summary receiver operating characteristic was 0.80. Downregulation of miR?193a?3p was also significantly associated with age, sex and metastasis (P=0.020, 0.044 and 0.048, respectively). Bioinformatics analysis indicated that a low miR?193a?3p expression may augment CCND1 expression to affect the biological processes of TC. In addition, CCND1, as a straightforward target, was validated through a dual luciferase assay. miR?193a?3p and CCND1 may serve as prognostic biomarkers of TC. Finally, miR?193a?3p may possess a crucial role in the genesis and progression of TC by altering the CCND1 expression.
Project description:Background:Hepatocellular carcinoma (HCC) is the second-highest cause of malignancy-related death worldwide, and many physiological and pathological processes, including cancer, are regulated by microRNAs (miRNAs). miR-193a-3p is an anti-oncogene that plays an important part in health and disease biology by interacting with specific targets and signals. Methods:In vitro assays were performed to explore the influences of miR-193a-3p on the propagation and apoptosis of HCC cells. The sequencing data for HCC were obtained from The Cancer Genome Atlas (TCGA), and the expression levels of miR-193a-3p in HCC and non-HCC tissues were calculated. The differential expression of miR-193a-3p in HCC was presented as standardized mean difference (SMD) with 95% confidence intervals (CIs) in Stata SE. The impact of miR-193a-3p on the prognoses of HCC patients was determined by survival analysis. The potential targets of miR-193a-3p were then predicted using miRWalk 2.0 and subjected to enrichment analyses, including Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Protein-Protein Interaction (PPI) network analysis. The interaction between miR-193a-3p and one predicted target, Cyclin D1 (CCND1), was verified by dual luciferase reporter assays and Pearson correlation analysis. Results:MiR-193a-3p inhibited the propagation and facilitated the apoptosis of HCC cells in vitro. The pooled SMD indicated that miR-193a-3p had a low level of expression in HCC (SMD: -0.88, 95% CI [-2.36 -0.59]). Also, HCC patients with a higher level of miR-193a-3p expression tended to have a favorable overall survival (OS: HR = 0.7, 95% CI [0.43-1.13], P = 0.14). For the KEGG pathway analysis, the most related pathway was "proteoglycans in cancer", while the most enriched GO term was "protein binding". The dual luciferase reporter assays demonstrated the direct interaction between miR-193a-3p and CCND1, and the Pearson correlation analysis suggested that miR-193a-3p was negatively correlated with CCND1 in HCC tissues (R = - 0.154, P = 0.002). Conclusion:miR-193a-3p could suppress proliferation and promote apoptosis by targeting CCND1 in HCC cells. Further, miR-193a-3p can be used as a promising biomarker for the diagnosis and treatment of HCC in the future.
Project description:miR-193a-3p is a tumor-related miRNA playing an essential role in tumorigenesis and progression of non-small cell lung cancer (NSCLC). The objective of the present study was to investigate the relationship between miR-193a-3p expression and clinical value and to further explore the potential signaling of miR-193a-3p in the carcinogenesis of NSCLC. RNA-sequencing and microarray data were collected from the databases GEO, ArrayExpress and The Cancer Genome Atlas (TCGA). Furthermore, in silico assessments were performed to analyze the prospective pathways and networks of the target genes of miR-193a-3p. In total, 453 cases of NSCLC patients and 476 normal controls were included in blood samples, while 920 cases of NSCLC patients and 406 normal controls were included in tissue samples. The pooled positive likelihood ratio, the pooled negative likelihood ratio and the pooled diagnostic odds ratio were calculated to reflect the diagnostic value of miR-193a-3p in blood and tissue samples. Moreover, the areas under the curve of the summary receiver operating characteristic curve of blood and tissue were 0.64 and 0.79, respectively. In addition, we found a lower level of miR-193a in NSCLC tissues than in non-cancerous controls based on TCGA. A gene ontology (GO) enrichment analysis demonstrated that miR-193a-3p could be related to key signaling pathways in NSCLC. Also, several vital pathways were illustrated by KEGG. Lower expression of miR-193a-3p in tissue samples of NSCLC may be associated with tumorigenesis and be a predictor of deterioration of NSCLC patients, and pathway analysis revealed crucial signaling pathways correlated with the incidence and progress of NSCLC.
Project description:MicroRNAs (miRNAs) are small non-coding RNAs that affect various biological processes by altering the expression of a target gene. An miRNA microarray analysis has previously revealed a significant decrease in miR-193a-3p levels in prostate cancer tissues compared with that in their benign prostate hyperplasia counterparts. However, the role of miR-193a-3p has yet to be elucidated. In the present study, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to evaluate the expression levels of miR-193a-3p in two human prostate cancer cell lines. Forced overexpression of miR-193a-3p was established by transfecting mimics into DU-145 and PC3 cell lines. Cell proliferation and the cell cycle were assessed using a cell viability assay, flow cytometry and a colony formation assay. In addition, the target gene of miR-193a-3p was determined by a luciferase assay, RT-qPCR and western blot analysis. The regulation of the cell cycle by miR-193a-3p was also evaluated by western blotting. The results demonstrated that miR-193a-3p expression levels were lower in prostate cancer cell lines as compared with the RWPE normal prostate epithelium cell line. Subsequent gain-of-function studies revealed that stable miR-193a-3p transfection inhibited cell viability, proliferation and colony formation, and induced G1 phase arrest in prostate cancer cells. A luciferase assay and western blot analysis identified cyclin D1 (CCND1) as a direct target gene of miR-193a-3p. In addition, the forced expression of CCND1 was able to counter the inhibitory effects of miR-193a-3p transfection in the prostate cancer cells. In summary, the results suggest that miR-193a-3p may inhibit the viability, proliferation and survival of prostate cancer cells by regulating the expression profile of CCND1, and that miR-193a-3p may be a novel therapeutic biomarker for prostate cancer.
Project description:Long intergenic non-coding RNA 00152 (LINC00152) is aberrantly expressed in various human malignancies and plays an important role in the pathogenesis. Here, we found that LINC00152 is upregulated in hepatocellular carcinoma (HCC) tissues as compared to adjacent non-neoplastic tissues; gain-and-loss-of-function analyses in vitro showed that LINC00152 facilitates HCC cell cycle progression through regulating the expression of CCND1. LINC00152 knockdown inhibits tumorigenesis in vivo. MS2-RIP analysis indicated that LINC00152 binds directly to miR-193a/b-3p, as confirmed by luciferase reporter assays. Furthermore, ectopic expression of LINC00152 partially halted the decrease in CCND1 expression and cell proliferation capacity induced by miR-193a/b-3p overexpression. Thus, LINC00152 acts as a competing endogenous RNA (ceRNA) by sponging miR-193a/b-3p to modulate its target gene, CCND1. Our findings establish a ceRNA mechanism regulating cell proliferation in HCC via the LINC00152/miR-193a/b-3p/CCND1 signalling axis, and identify LINC00152 as a potential therapeutic target for HCC.
Project description:MicroRNAs (miRNAs) are short noncoding RNAs derived from the 3' and 5' ends of the same precursor. However, the biological function and mechanism of miRNA arm expression preference remain unclear in breast cancer. We found significant decreases in the expression levels of miR-193a-5p but no significant differences in those of miR-193a-3p in breast cancer. MiR-193a-3p suppressed breast cancer cell growth and migration and invasion abilities, whereas miR-193a-5p suppressed cell growth but did not influence cell motility. Furthermore, NLN and CCND1, PLAU, and SEPN1 were directly targeted by miR-193a-5p and miR-193a-3p, respectively, in breast cancer cells. The endogenous levels of miR-193a-5p and miR-193a-3p were significantly increased by transfecting breast cancer cells with the 3'UTR of their direct targets. Comprehensive analysis of The Cancer Genome Atlas database revealed significant differences in the arm expression preferences of several miRNAs between breast cancer and adjacent normal tissues. Our results collectively indicate that the arm expression preference phenomenon may be attributable to the target gene amount during breast cancer progression. The miRNA arm expression preference may be a means of modulating miRNA function, further complicating the mRNA regulatory network. Our findings provide a new insight into miRNA regulation and an application for breast cancer therapy.
Project description:Increasing evidence has demonstrated that microRNA (miR)?133a?3p is an important regulator of hepatocellular carcinoma (HCC). In the present study, the diagnostic role of miR?133a?3p in HCC, and the potential functional pathways, were both explored based on publicly available data. Eligible microarray datasets were collected from NCBI Gene Expression Omnibus (GEO) database and ArrayExpress database. The data related to HCC and matched adjacent normal tissues were also downloaded from The Cancer Genome Atlas (TCGA). Published studies reporting the association between miR?133a?3p expression and HCC were reviewed from multiple databases. By combining the data derived from three sources (GEO, TCGA and published studies), the authors analyzed the comprehensive relationship between miR?133a?3p expression and clinicopathological features of HCC. Eventually, putative targets of miR?133a?3p in HCC were selected for further bioinformatics prediction. A total of eight published microarray datasets were gathered, and the pooled results demonstrated that the expression of miR?133a?3p in the tumor group was lower than that in normal groups [standardized mean difference (SMD)=?0.54; 95% confidence interval (CI), ?0.74 to ?0.35; P<0.001]. Consistently, the level of miR?133a?1 in HCC was reduced markedly compared to normal tissues (P<0.001) based on TCGA data, and the AUC value of low miR?133a?1 expression for HCC diagnosis was 0.670 (P<0.001). Furthermore, the combined SMD of all datasets (GEO, TCGA and literature) suggested that significant difference was observed between the HCC group and the normal control group, and lower miR?133a?3p expression in HCC group was noted (SMD=?0.69; 95% CI, ?1.10 to ?0.29; P=0.001). In addition, the authors discovered five key genes of the calcium signaling pathway (NOS1, ADRA1A, ADRA1B, ADRA1D and TBXA2R) that may probably be targeted by miR?133a?3p in HCC. The study reveals that miR?133a?3p may function as a tumor suppressor in HCC. The prospective novel pathways and key genes of miR?133a?3p could offer potential biomarkers for HCC; however, the predictions require further confirmation.
Project description:BACKGROUND:Remarkable deregulation of several microRNAs (miRNAs) is demonstrated in cutaneous melanoma. hsa-miR-193a-3p is reported to be under-expressed in tissues and in plasma of melanoma patients, but the role of both miR-193a arms in melanoma is not known yet. METHODS:After observing the reduced levels of miR-193a arms in plasma exosomes of melanoma patients, the effects of hsa-miR-193a-3p and -5p transfection in cutaneous melanoma cell lines are investigated. RESULTS:In melanoma cell lines A375, 501Mel, and MeWo, the ectopic over-expression of miR-193a arms significantly reduced cell viability as well as the expression of genes involved in proliferation (ERBB2, KRAS, PIK3R3, and MTOR) and apoptosis (MCL1 and NUSAP1). These functional features were accompanied by a significant downregulation of Akt and Erk pathways and a strong increase in the apoptotic process. Since in silico databases revealed TROY, an orphan member of the tumor necrosis receptor family, as a potential direct target of miR-193a-5p, this possibility was investigated using the luciferase assay and excluded by our results. CONCLUSIONS:Our results underline a relevant role of miR-193a, both -3p and -5p, as tumor suppressors clarifying the intracellular mechanisms involved and suggesting that their ectopic over-expression could represent a novel treatment for cutaneous melanoma patients.
Project description:Emerging evidence has demonstrated that microRNAs (miRNAs/miRs) have various biological functions in the development of human epidermal growth factor receptor 2 (HER2) positive breast cancer. The aim of the present study is to reveal the mechanism of miR‑193a‑3p inhibiting the progress of HER2 positive breast cancer. The expression of miR‑193a‑3p was evaluated by quantitative polymerase chain reaction (PCR). The methylation status of miR‑193a‑3p was evaluated by PCR and pyrosequencing analysis. Overexpression of miR‑193a‑3p and growth factor receptor bound protein 7 (GRB7) combined with in vitro tumorigenic assays were conducted to determine the carcinostatic capacities of miR‑193a‑3p in HER2 positive breast cancer cells. The association between miR‑193a‑3p and GRB7 was determined by luciferase reporter assay. Protein level was evaluated using western blot analysis. miR‑193a‑3p was downregulated in HER2 positive breast cancer cells and clinical tissues. Methylation‑mediated silencing led to decreased expression of miR‑193a‑3p in HER2 positive breast cancer. Overexpression of miR‑193a‑3p could inhibit proliferation, migration and invasion of breast cancer cells. Overexpression of GRB7 could abolish this effect. miR‑193a‑3p could directly target the 3' untranslated region of GRB7. miR‑193a‑3p could directly or indirectly target extracellular signal‑regulated kinase 1/2 (ERK1/2) and forkhead box M1 (FOXM1) signaling. In conclusion, it was identified that silencing of miR‑193a‑3p through hypermethylation can promote HER2 positive breast cancer progress by targeting GRB7, ERK1/2 and FOXM1 signaling. The function of miR‑193a‑3p in HER2 positive breast cancer implicates its potential application in therapy.
Project description:MicroRNAs (miRNAs) have been confirmed to participate in liver fibrosis progression and activation of hepatic stellate cells (HSCs). In this study, the role of miR-193a/b-3p in concanavalin A (ConA)-induced liver fibrosis in mice was evaluated. According to the results, the expression of miR-193a/b-3p was down-regulated in liver tissues after exposure to ConA. Lentivirus-mediated overexpression of miR-193a/b-3p reduced ConA-induced liver injury as demonstrated by decreasing ALT and AST levels. Moreover, ConA-induced liver fibrosis was restrained by the up-regulation of miR-193a/b-3 through inhibiting collagen deposition, decreasing desmin and proliferating cell nuclear antigen (PCNA) expression and lessening the content of hydroxyproline, transforming growth factor-β1 (TGF-β1) and activin A in liver tissues. Furthermore, miR-193a/b-3p mimics suppressed the proliferation of human HSCs LX-2 via inducing the apoptosis of LX-2 cells and lowering the levels of cell cycle-related proteins Cyclin D1, Cyclin E1, p-Rb and CAPRIN1. Finally, TGF-β1 and activin A-mediated activation of LX-2 cells was reversed by miR-193a/b-3p mimics via repressing COL1A1 and α-SMA expression, and restraining the activation of TGF-β/Smad2/3 signalling pathway. CAPRIN1 and TGF-β2 were demonstrated to be the direct target genes of miR-193a/b-3p. We conclude that miR-193a/b-3p overexpression attenuates liver fibrosis through suppressing the proliferation and activation of HSCs. Our data suggest that miR-193a-3p and miR-193b-3p may be new therapeutic targets for liver fibrosis.
Project description:Human growth factor receptor-bound protein-7 (GRB7) is a pivotal mediator involved in receptor tyrosine kinase signaling and governing diverse cellular processes. Aberrant upregulation of GRB7 is frequently associated with the progression of human cancers. However, the molecular mechanisms leading to the upregulation of GRB7 remain largely unknown. Here, we propose that the epigenetic modification of GRB7 at the post-transcriptional level may be a crucial factor leading to GRB7 upregulation in ovarian cancers. Methods: The upstream miRNA regulators were predicted by in silico analysis. Expression of GRB7 was examined by qPCR, immunoblotting and immunohistochemical analyses, while miR-193a-3p levels were evaluated by qPCR and in situ hybridization in ovarian cancer cell lines and clinical tissue arrays. MS-PCR and pyrosequencing analyses were used to assess the methylation status of miR-193a-3p. Stable overexpression or gene knockdown and Tet-on inducible approaches, in combination with in vitro and in vivo tumorigenic assays, were employed to investigate the functions of GRB7 and miR-193a-3p in ovarian cancer cells. Results: Both miR-193a-3p and its isoform, miR-193b-3p, directly targeted the 3' UTR of GRB7. However, only miR-193a-3p showed a significantly inverse correlation with GRB7-upregulated ovarian cancers. Epigenetic studies revealed that methylation-mediated silencing of miR-193a-3p led to a stepwise decrease in miR-193a-3p expression from low to high-grade ovarian cancers. Intriguingly, miR-193a-3p not only modulated GRB7 but also ERBB4, SOS2 and KRAS in the MAPK/ERK signaling pathway to enhance the oncogenic properties of ovarian cancer cells in vitro and in vivo. Conclusion: These findings suggest that epigenetic silencing of miR-193a-3p by DNA hypermethylation is a dynamic process in ovarian cancer progression, and miR-193a-3p may be explored as a promising miRNA replacement therapy in this disease.