RNA-Seq analysis miR-455-3p overexpression effect on breast cancer cell line
ABSTRACT: MiR-455-3p has been reported to suppress the expression of ROCK2, HOXB5, hTERT, and RUNX2, which are involved in multiple biological processes including renal fibrosis, chondrogenic development and differentiation, Alzheimer’s disease, preeclampsia, and tumorigenesis. Here, we showed that miR-455-3p broadly inhibited TGF-beta-induced EMT by repressing the expression of Smad2, ZEB1, and HDAC2 in breast cancer cells, and that miR-455-3p loss-of-function promotes the acquisition of cell invasive properties and the ability to metastasize. These results indicate that miR-455-3p represents an important node that inhibits metastasis of breast cancer. Overall design: MiR-455-3p mimics or controls were transfected into MCF-7 cells (n=3 for each group) for 48 hours. Total RNA was isolated using Trizol reagent (Invitrogen, USA). Agilent 2100 Bioanalyzer (Agilent Technologies, USA) was used to evaluate the RNA quality.
Project description:GATA3 is a basic and essential transcription factor that regulates many pathophysiological processes and is required for the development of mammary luminal epithelial cells. Loss-of-function GATA3 alterations in breast cancer are associated with poor prognosis. Here, we sought to understand the tumor-suppressive functions GATA3 normally performs. We discovered a role for GATA3 in suppressing epithelial-to-mesenchymal transition (EMT) in breast cancer by activating miR-455-3p expression. Enforced expression of miR-455-3p alone partially prevented EMT induced by transforming growth factor ? (TGF-?) both in cells and tumor xenografts by directly inhibiting key components of TGF-? signaling. Pathway and biochemical analyses showed that one miRNA-455-3p target, the TGF-?-induced protein ZEB1, recruits the Mi-2/nucleosome remodeling and deacetylase (NuRD) complex to the promotor region of miR-455 to strictly repress the GATA3-induced transcription of this microRNA. Considering that ZEB1 enhances TGF-? signaling, we delineated a double-feedback interaction between ZEB1 and miR-455-3p, in addition to the repressive effect of miR-455-3p on TGF-? signaling. Our study revealed that a feedback loop between these two axes, specifically GATA3-induced miR-455-3p expression, could repress ZEB1 and its recruitment of NuRD (MTA1) to suppress miR-455, which ultimately regulates TGF-? signaling. In conclusion, we identified that miR-455-3p plays a pivotal role in inhibiting the EMT and TGF-? signaling pathway and maintaining cell differentiation. This forms the basis of that miR-455-3p might be a promising therapeutic intervention for breast cancer.
Project description:Breast cancer is one of the most malignant diseases in women worldwide. Serum microRNAs (miRNAs), with the characteristics of high sensitivity and specificity, have recently attracted more attentions to serve as potential biomarkers for tumor diseases. In this study, 194 breast cancer patients' serum samples were collected before surgery and enrolled into different groups based on their diagnostic information. To search for breast cancer diagnostic biomarkers, serum miRNAs were screened by microarray in pooled samples of healthy volunteers and breast cancer patients in different clinical stages. The miRNAs were further verified in each individual patient's serum samples in diagnostic and predictive sets. The serum level of miR-1915-3p was upregulated and miR-455-3p was downregulated significantly in breast cancer patients compared with healthy volunteers. Furthermore, the patients with infiltrating carcinoma or lymph node metastasis had a higher serum level of miR-1915-3p and lower serum level of miR-455-3p than patients with the carcinoma in situ or patients without lymph node metastasis. ROC analysis suggested that miR-1915-3p and miR-455-3p had the potential as a promising serum diagnostic and predictive biomarkers of breast cancer. miR-1915-3p was over-expressed in certain human breast cancer cells. Functional experiments in vitro showed that miR-1915-3p enhanced cell proliferative and migrational abilities. Overexpression of miR-1915-3p repressed target gene DUSP3 and activated ERK1/2. Collectively, this study provided a new insight that miR-1915-3p might play a role in the development of breast cancer and that serum miR-1915-3p and miR-455-3p could serve as diagnostic and predictive biomarkers for breast cancer.
Project description:Lacking of treatment methods for the patients with triple negative breast cancer (TNBC) underscores the pivotal needs to further understand its biology as well as to find better biomarkers and develop novel therapeutic strategies. Increasing evidences support that aberrantly expressed microRNAs (miRNAs) are involved in tumorigenesis and may serve as biomarkers for diagnostic and prognostic purposes of various cancers. In current study, we found that miR-455-3p and miR-196a-5p were intensively overexpressed in TNBC compared with the hormone receptor (HR) positive breast cancer whereas miR-425-5p was down-regulated by miRNA microarray analysis. qRT-PCR analysis confirmed that the expression of miR-455-3p in TNBC cell lines MDA-MB-231 and MDA-MB-468 was higher than that in HR positive breast cancer cell line MCF-7(p<0.01). Functional experiments in vitro showed that miR-455-3p enhanced cell proliferative, invasive and migrational abilities in TNBC cell lines. miRNA targets prediction showed SMAD2, LTBR and etoposide induced 2.4 (EI24) were potential target genes of miR-455-3p, and then it was confirmed by qRT-PCR assay. Dual luciferase reporter assay showed the specific binding of miR-455-3p to 3' UTR of EI24 in TNBC. Then we found miR-455-3p inhibited the EI24 expression at the levels of mRNA and protein. Through small interfering RNA (siRNA) targeting EI24 gene, there were strengthened capabilities of invasion and migration of TNBC cells, and increased expression of EI24 had the inverse effects. In conclusion, the data suggest that miRNA455-3p promotes invasion and migration by targeting tumor suppressor EI24 and might be a potential prognostic biomarker and therapeutic target in TNBC.
Project description:The aim of this work was to determine whether miR-455-3p regulates DNA methylation during chondrogenic differentiation of hMSCs. The expression of miR-455-3p and de novo methyltransferase DNMT3A was assessed in micromass culture of hBMSCs, which induced chondrogenic differentiation in vitro, and in E16.5 mice in vivo. A luciferase reporter assay was used to confirm whether miR-455-3p directly targets DNMT3A by interaction with the 3'-UTR. Using an Illumina Infinium Methylation EPIC microarray, genome-wide DNA methylation of hBMSCs with or without overexpressed miR-455-3p was examined for 28 days during induced chondrogenic differentiation. Here, we showed that miR-455-3p was more expressed during the middle stage of hBMSC chondrogenic differentiation, and less expressed in the late stage. DNMT3A was less expressed in the middle stage and more expressed in the late stage, and was also more expressed in the palms of miR-455-3p deletion mice compared to those of wild-type mice. The luciferase reporter assay demonstrated that miR-455-3p directly targets DNMT3A 3'-UTR. miR-455-3p overexpression inhibits the degenerate process during chondrogenic differentiation, while deletion of miR-455-3p in mice accelerated cartilage degeneration. Genome-wide DNA methylation analysis showed miR-455-3p overexpression regulates DNA methylation of cartilage-specific genes. GO analysis revealed PI3K-Akt signaling pathway was most hypomethylated. Our data show that miR-455-3p can regulate hMSC chondrogenic differentiation by affecting DNA methylation. Overexpression of miR-455-3p and DNA methylation inhibitors can thus potentially be utilized to optimize chondrogenic differentiation.
Project description:Long non-coding RNAs (lncRNAs) play pivotal roles in diseases such as osteoarthritis (OA). However, knowledge of the biological roles of lncRNAs is limited in OA. We aimed to explore the biological function and molecular mechanism of HOTTIP in chondrogenesis and cartilage degradation. We used the human mesenchymal stem cell (MSC) model of chondrogenesis, in parallel with, tissue biopsies from normal and OA cartilage to detect HOTTIP, CCL3, and miR-455-3p expression in vitro. Biological interactions between HOTTIP and miR-455-3p were determined by RNA silencing and overexpression in vitro. We evaluated the effect of HOTTIP on chondrogenesis and degeneration, and its regulation of miR-455-3p via competing endogenous RNA (ceRNA). Our in vitro ceRNA findings were further confirmed within animal models in vivo. Mechanisms of ceRNAs were determined by bioinformatic analysis, a luciferase reporter system, RNA pull-down, and RNA immunoprecipitation (RIP) assays. We found reduced miR-455-3p expression and significantly upregulated lncRNA HOTTIP and CCL3 expression in OA cartilage tissues and chondrocytes. The expression of HOTTIP and CCL3 was increased in chondrocytes treated with interleukin-1β (IL-1β) in vitro. Knockdown of HOTTIP promoted cartilage-specific gene expression and suppressed CCL3. Conversely, HOTTIP overexpression reduced cartilage-specific genes and increased CCL3. Notably, HOTTIP negatively regulated miR-455-3p and increased CCL3 levels in human primary chondrocytes. Mechanistic investigations indicated that HOTTIP functioned as ceRNA for miR-455-3p enhanced CCL3 expression. Taken together, the ceRNA regulatory network of HOTTIP/miR-455-3p/CCL3 plays a critical role in OA pathogenesis and suggests HOTTIP is a potential target in OA therapy.
Project description:The aims of the present study are to determine whether hydrogen sulfide (H2S) is involved in the expression of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production, and to identify the role of microRNA-455-3p (miR-455-3p) during those processes. In cultured human umbilical vein endothelial cells (HUVECs), the expression of miR-455-3p, eNOS protein and the NO production was detected after administration with 50??M NaHS. The results indicated that H2S could augment the expression of miR-455-3p and eNOS protein, leading to the increase of NO level. We also found that overexpression of miR-455-3p in HUVECs increased the protein levels of eNOS whereas inhibition of miR-455-3p decreased it. Moreover, H2S and miR-455-3p could no longer increase the protein level of eNOS in the presence of proteasome inhibitor, MG-132. In vivo, miR-455-3p and eNOS expression were considerably increased in C57BL/6 mouse aorta, muscle and heart after administration with 50??mol/kg/day NaHS for 7 days. We also identified that H2S levels and miR-455-3p expression increased in human atherosclerosis plaque while H2S levels decreased in plasma of atherosclerosis patients. Our data suggest that the stability of eNOS protein and the NO production could be regulated by H2S through miR-455-3p.
Project description:A non-invasive and early-detectable peripheral biomarker is urgently needed for Alzheimer's disease (AD). The present study is a step forward to verify the biomarker properties of human microRNA-455-3p (Hsa-miR-455-3p) in AD patients. Our previous findings on mild cognitive impaired subjects, AD patients and AD cells and mouse models unveiled the miR-455-3p as a potential peripheral biomarker for AD. In the current study, we verified the differential expression of miR-455-3p in postmortem AD brains obtained from NIH NeuroBioBank, and fibroblasts and B-lymphocytes from both familial and sporadic AD patients from Coriell Cell Repository of National Institutes on Aging. Total RNA was extracted from the fibroblasts, B-lymphocytes and AD postmortem brains, and expression of miR-455-3p was measured by real-time reverse-transcriptase RT-PCR. Our real-time RT-PCR analysis showed a significant (P = 0.0002) upregulation of miR-455-3p expression in AD postmortem brains compared to healthy control samples. Expression of miR-455-3p was also upregulated in the fibroblasts from AD patients, however a significant difference in miR-455-3p level was observed in the cells from sporadic AD patients (P = 0.014) compared to healthy controls. Similarly, in B-lymphocytes, miR-455-3p level was also higher (P = 0.044) especially in sporadic AD cases compared to controls. Receiver operating characteristic (ROC) curve analysis indicated the significant area under ROC curve (AUROC) value of miR-455-3p in AD postmortem brain (AUROC = 0.792; P = 0.001) and AD fibroblasts cells (AUROC = 0.861; P = 0.03), whereas in B-lymphocytes AUROC value of miR-455-3p was not significant. Further, in-silico analysis for miRNA targets predictions showed the binding capacity of miR-455-3p with several AD associated key genes such as APP, NGF, USP25, PDRG1, SMAD4, UBQLN1, SMAD2, TP73, VAMP2, HSPBAP1, and NRXN1. Hence, these observations further revealed that miR-455-3p is a potential biomarker for AD and its possible therapeutic target for AD.
Project description:Lacking of both prognostic biomarkers and therapeutic targets, triple-negative breast cancer (TNBC) underscores pivotal needs to uncover novel biomarkers and viable therapies. MicroRNAs have broad biological functions in cancers and may serve as ideal biomarkers. In this study, by data mining of the Cancer Genome Atlas database, we screened out 4 differentially-expressed microRNAs (DEmiRNAs) between TNBC and normal samples: miR-135b-5p, miR-9-3p, miR-135b-3p and miR-455-5p. They were specially correlated with the prognosis of TNBC but not non-TNBC. The weighted correlation network analysis (WGCNA) for potential target genes of 3 good prognosis-related DEmiRNAs (miR-135b-5p, miR-9-3p, miR-135b-3p) identified 4 hub genes with highly positive correlation with TNBC subtype: FOXC1, BCL11A, FAM171A1 and RGMA. The targeting relationships between miR-9-3p and FOXC1/FAM171A1, miR-135b-3p and RGMA were validated by dual-luciferase reporter assays. Importantly, the regulatory functions of 4 DEmiRNAs and 3 verified target genes on cell proliferation and migration were explored in TNBC cell lines. In conclusion, we shed lights on these 4 DEmiRNAs (miR-135b-5p, miR-9-3p, miR-135b-3p, miR-455-5p) and 3 hub genes (FOXC1, FAM171A1, RGMA) as specific prognostic biomarkers and promising therapeutic targets for TNBC.
Project description:The purpose of our study is to understand the protective role of miR-455-3p against abnormal amyloid precursor protein (APP) processing, amyloid beta (A?) formation, defective mitochondrial biogenesis/dynamics and synaptic damage in AD progression. In-silico analysis of miR-455-3p has identified the APP gene as a putative target. Using mutant APP cells, miR-455-3p construct, biochemical and molecular assays, immunofluorescence and transmission electron microscopy (TEM) analyses, we studied the protective effects of miR-455-3p on - 1) APP regulation, amyloid beta (A?)(1-40) & (1-42) levels, mitochondrial biogenesis & dynamics; 3) synaptic activities and 4) cell viability & apoptosis. Our luciferase reporter assay confirmed the binding of miR-455-3p at the 3'UTR of APP gene. Immunoblot, sandwich ELISA and immunostaining analyses revealed that the reduced levels of the mutant APP, A?(1-40) & A?(1-42), and C99 by miR-455-3p. We also found the reduced levels of mRNA and proteins of mitochondrial biogenesis (PGC1?, NRF1, NRF2, and TFAM) and synaptic genes (synaptophysin and PSD95) in mutant APP cells; on the other hand, mutant APP cells that express miR-455-3p showed increased mRNA and protein levels of biogenesis and synaptic genes. Additionally, expression of mitochondrial fission proteins (DRP1 and FIS1) were decreased while the fusion proteins (OPA1, Mfn1 and Mfn2) were increased by miR-455-3p. Our TEM analysis showed a decrease in mitochondria number and an increase in the size of mitochondrial length in mutant APP cells transfected with miR-455-3p. Based on these observations, we cautiously conclude that miR-455-3p regulate APP processing and protective against mutant APP-induced mitochondrial and synaptic abnormalities in AD.
Project description:Background:Numerous circular RNAs (circRNAs) are functionally investigated in various human cancers, including colorectal cancer (CRC). In this study, we explored the function of circCSNK1G1 and mechanism of action in CRC, aiming to provide evidence for circCSNK1G1 involving in CRC pathogenesis. Methods:The expression of circCSNK1G1, miR-455-3p and Myosin VI (MYO6) were examined using quantitative real-time polymerase chain reaction (qRT-PCR). The functions of circCSNK1G1 on cell proliferation, apoptosis, cycle and migration/invasion were investigated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, flow cytometry assay and transwell assay, respectively. The targeted relationship between miR-455-3p and circCSNK1G1 or MYO6 predicted by bioinformatics analysis was validated using dual-luciferase reporter assay and RNA pull-down assay. The role of circCSNK1G1 was also explored in nude mice in vivo. Results:The expression of circCSNK1G1 and MYO6 was elevated, while the expression of miR-455-3p was declined in CRC tissues and cells. Silencing circCSNK1G1 inhibited CRC cell proliferation, migration and invasion and induced cell apoptosis and cell cycle arrest. MiR-455-3p was a target of circCSNK1G1, and miR-455-3p could bind to MYO6. CircCSNK1G1 positively regulated MYO6 expression by targeting miR-455-3p. Inhibition of miR-455-3p reversed the effects of circCSNK1G1 silencing in CRC cells. Besides, miR-455-3p restoration blocked CRC cell growth and metastasis, which were abolished by MYO6 overexpression. Moreover, circCSNK1G1 regulated the miR-455-3p/MYO6 axis to block tumor growth in vivo. Conclusion:CircCSNK1G1 participated in the progression of CRC partly by modulating the miR-455-3p/MYO6 network, which provided a theoretical basis for circCSNK1G1 involving in CRC pathogenesis, hinting that circCSNK1G1 might be a useful biomarker for CRC treatment.