scholarly journals MALAT1 Modulates TGF-β1-Induced Endothelial-to-Mesenchymal Transition through Downregulation of miR-145

2017 ◽  
Vol 42 (1) ◽  
pp. 357-372 ◽  
Author(s):  
Yin Xiang ◽  
Yachen Zhang ◽  
Yong Tang ◽  
Qianhui Li
Keyword(s):  
Target Genes ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Neointimal Hyperplasia ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Mesenchymal Transition ◽  
Endothelial To Mesenchymal Transition ◽  
Tgf Β1

Background/Aims: Endothelial-to-mesenchymal transition (EndMT) plays significant roles under various pathological conditions including cardiovascular diseases, fibrosis, and cancer. EndMT of endothelial progenitor cells (EPCs) contributes to neointimal hyperplasia following cell therapy Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA (lncRNA) that promotes metastasis and cancer. MicroRNA-145 (miR-145) is a tumor suppressor that has been reported to inhibit SMAD3-mediated epithelial-to-mesenchymal transition (EMT) of cancer cells. In the present study, we investigated the role of MALAT1 and miR-145 in EndMT of human circulating EPCs induced by transforming growth factor beta1 (TGF-β1). Methods: Human circulating EPCs were isolated and characterized by fluorescence-activated cell sorting (FACS). Expression levels of EndMT markers were assessed by qRT-PCR and western blotting. Alpha-smooth muscle actin (α-SMA) expression was measured by cell immunofluorescence staining. The regulatory relationship between MALAT1 and miR-145 and its target genes, TGFBR2 (TGFβ receptortype II) and SMAD3 (mothers against decapentaplegic homolog 3) was analyzed using the luciferase reporter assay. Results: We found that EndMT of EPCs induced by TGF-β1 is accompanied by increased MALAT1 expression and decreased miR-145 expression, and MALAT1 and miR-145 directly bind and reciprocally repress each other in these cells. Dual-Luciferase Reporter assay indicated that miR-145 inhibits TGF-β1-induced EndMT by directly targeting TGFBR2 and SMAD3. Conclusions: MALAT1 modulates TGF-β1-induced EndMT of EPCs through regulation of TGFBR2 and SMAD3 via miR-145. Thus, the MALAT1-miR-145-TGFBR2/SMAD3 signaling pathway plays a key role in TGF-β1-induced EndMT.

scholarly journals Regulation of CTGF Expression by miR-133b for the Treatment of Renal Interstitial Fibrosis in Old UUO Rats

2020 ◽  
Author(s):  
Dan Cao ◽  
Yuan Wang ◽  
Yingjie Zhang ◽  
Yinping Zhang ◽  
Qi Huang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Target Genes ◽  
Interstitial Fibrosis ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Renal Interstitial Fibrosis ◽  
Hk2 Cells ◽  
Tgf Β1 ◽  
Dual Luciferase Reporter Assay

Abstract Introduction: Renal interstitial fibrosis, an important pathological feature of kidney aging and chronic renal failure, is regulated by mesenchymal stem cells (MSCs). We have previously demonstrated the high expression of miR-133b in MSC-derived extracellular vesicles (MSC-EVs) from old rats, which mediated the inhibition of epithelial-mesenchymal transition (EMT) of renal tubules induced by transforming growth factor-β1 (TGF-β1). We investigated the effect of miR-133b for the treatment of geriatric renal interstitial fibrosis and evaluated its target genes.Methods: miR-133b expression induced during the EMT of HK2 cells by TGF-β1 at different concentrations (0, 6, 8, and 10 ng/mL) and time points (0, 24, 48, and 72 h) was detected using real-time polymerase chain reaction. The target genes of miR-133b were validated using a dual-luciferase reporter assay. In vitro experiments were performed to observe mRNA and protein expression of miR-133b targets, E-cadherin, α-smooth muscle actin (SMA), fibronectin, and collagen 3A1 (Col3A1), in HK2 cells transfected with miR-133b under TGF-β1 stimulation. A 24-week-old unilateral ureteral obstruction (UUO) mouse model was established and injected with transfection reagent and miR-133b into the caudal vein. miR-133b、 target gene and other indexes mentioned above mRNA and protein levels and renal interstitial fibrosis were detected at 7 and 14 days.Results: miR-133b expression gradually decreased with an increase in TGF-β1 concentration and treatment time, and miR-133b mimic downregulated connective tissue growth factor (CTGF) expression. Dual-luciferase reporter assay confirmed CTGF as a direct target of miR-133b. miR-133b mimic transfection inhibited the TGF-β1-induced EMT of HK2 cells; this effect was reversed by CTGF overexpression. miRNA-133b expression significantly increased (approximately 70-100 times) in mouse kidneys after injection of the miRNA-133b overexpression complex, significantly alleviating renal interstitial fibrosis in UUO mice.Conclusion: miR-133b exerted targeted inhibitory effects on CTGF expression, consequently reducing the TGF-β1-induced EMT of HK2 cells and renal interstitial fibrosis in old UUO mice.

scholarly journals Hsa_circ_0123190 acts as a competitive endogenous RNA to regulate APLNR expression by sponging hsa-miR-483-3p in lupus nephritis

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Chunyi Zhang ◽  
Congcong Gao ◽  
Xueqi Di ◽  
Siwan Cui ◽  
Wenfang Liang ◽  
...  
Keyword(s):  
Lupus Nephritis ◽  
Lupus Erythematosus ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β1 ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Reporter Assay ◽  
Clinical Value ◽  
Chronicity Index

Abstract Background Lupus nephritis (LN) is one of the most severe complications of systemic lupus erythematosus (SLE). Circular RNAs (circRNAs) can act as competitive endogenous RNAs (ceRNAs) to regulate gene transcription, which is involved in mechanism of many diseases. However, the role of circRNA in lupus nephritis has been rarely reported. In this study, we aim to investigate the clinical value of circRNAs and explore the mechanism of circRNA involvement in the pathogenesis of LN. Methods Renal tissues from three untreated LN patients and three normal controls (NCs) were used to identify differently expressed circRNAs by next-generation sequencing (NGS). Validated assays were used by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The interactions between circRNA and miRNA, or miRNA and mRNA were further determined by luciferase reporter assay. The extent of renal fibrosis between the two groups was assessed by Masson-trichome staining and immunohistochemistry (IHC) staining. Results 159 circRNAs were significantly dysregulated in LN patients compared with NCs. The expression of hsa_circ_0123190 was significantly decreased in the renal tissues of patients with LN (P = 0.014). Bio-informatics analysis and luciferase reporter assay illustrated that hsa_circ_0123190 can act as a sponge for hsa-miR-483-3p, which was also validated to interact with APLNR. APLNR mRNA expression was related with chronicity index (CI) of LN (P = 0.033, R2 = 0.452). Moreover, the fibrotic-related protein, transforming growth factor-β1 (TGF-β1), which was regulated by APLNR, was more pronounced in the LN group (P = 0.018). Conclusion Hsa_circ_0123190 may function as a ceRNA to regulate APLNR expression by sponging hsa-miR-483-3p in LN.

scholarly journals miR-33a Mediates the Anti-Tumor Effect of Lovastatin in Osteosarcoma by Targeting CYR61

2018 ◽  
Vol 51 (2) ◽  
pp. 938-948 ◽  
Author(s):  
Yazeng Huang ◽  
Jun Zhang ◽  
Haiyu Shao ◽  
Jianwen Liu ◽  
Mengran Jin ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Invasion ◽  
Epithelial To Mesenchymal Transition ◽  
Regulatory Element ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Osteosarcoma Cell ◽  
Reporter Assay ◽  
Related Protein ◽  
Mesenchymal Transition

Background/Aims: Preventing cell metastasis is an effective therapeutic strategy to treat osteosarcoma and improve prognosis. Statins have been found to have anticancer effects in addition to their cholesterol-lowering action. As a new target of statins, cysteine-rich 61 (CYR61) was recently identified to promote cell migration and metastasis in osteosarcoma. However, the underlying mechanisms mediating the regulation of CYR61 expression by statins remain unknown. Methods: Human osteosarcoma cell lines MG63 and SaOS2 were used to clarify the effect of lovastatin on CYR61 expression. Real-time PCR was performed to detect mRNA or microRNA (miRNA) levels and western blot was performed to detect protein levels. Cell invasive ability was determined using Transwell assays. Lentivirus encoding CYR61 cDNA or sterol regulatory element-binding protein 2 (SREBP-2) shRNA was used to upregulate CYR61 expression or downregulate SREBP-2 expression. Binding of the CYR61 3’ untranslated region (UTR) and miR-33a was analyzed by luciferase reporter assay. Results: We found that lovastatin treatment decreased CYR61 expression, inhibited cell invasion and altered epithelial-to-mesenchymal-transition (EMT)-related protein expression, while CYR61 overexpression abolished the effect of lovastatin. Moreover, lovastatin increased the expression of SREBP-2 and miR-33a, which were then downregulated by SREBP-2 silencing. Bioinformatics analysis indicated that the CYR61 3′UTR harbored a potential miR-33a binding site and luciferase reporter assay demonstrated that CYR61 was a target of miR-33a in osteosarcoma cells. Furthermore, miR-33a could inhibit cell invasion and alter EMT-related protein expression. SREBP-2 silencing or miR-33a inhibitor upregulated CYR61 expression and reversed the effects of lovastatin on cell invasion and EMT-related proteins. Conclusion: Our findings suggest lovastatin suppresses osteosarcoma cell invasion through the SREBP-2/miR-33a/CYR61 pathway.

scholarly journals TGF-β and the Smad Signaling Pathway Support Transcriptomic Reprogramming during Epithelial-Mesenchymal Cell Transition

2005 ◽  
Vol 16 (4) ◽  
pp. 1987-2002 ◽  
Author(s):  
Ulrich Valcourt ◽  
Marcin Kowanetz ◽  
Hideki Niimi ◽  
Carl-Henrik Heldin ◽  
Aristidis Moustakas
Keyword(s):  
Target Genes ◽  
Transforming Growth Factor ◽  
Epithelial Mesenchymal Transition ◽  
Smooth Muscle Actin ◽  
Expression Patterns ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Type I ◽  
Mesenchymal Transition ◽  
Smad Signaling

Epithelial-mesenchymal transition (EMT) contributes to normal tissue patterning and carcinoma invasiveness. We show that transforming growth factor (TGF)-β/activin members, but not bone morphogenetic protein (BMP) members, can induce EMT in normal human and mouse epithelial cells. EMT correlates with the ability of these ligands to induce growth arrest. Ectopic expression of all type I receptors of the TGF-β superfamily establishes that TGF-β but not BMP pathways can elicit EMT. Ectopic Smad2 or Smad3 together with Smad4 enhanced, whereas dominant-negative forms of Smad2, Smad3, or Smad4, and wild-type inhibitory Smad7, blocked TGF-β–induced EMT. Transcriptomic analysis of EMT kinetics identified novel TGF-β target genes with ligand-specific responses. Using a TGF-β type I receptor that cannot activate Smads nor induce EMT, we found that Smad signaling is critical for regulation of all tested gene targets during EMT. One such gene, Id2, whose expression is repressed by TGF-β1 but induced by BMP-7 is critical for regulation of at least one important myoepithelial marker, α-smooth muscle actin, during EMT. Thus, based on ligand-specific responsiveness and evolutionary conservation of the gene expression patterns, we begin deciphering a genetic network downstream of TGF-β and predict functional links to the control of cell proliferation and EMT.

Heptachlor-induced epithelial to mesenchymal transition in HK-2 cells mediated via TGF-β1/Smad signalling

2019 ◽  
Vol 38 (5) ◽  
pp. 567-577 ◽  
Author(s):  
N Singh ◽  
M Siddarth ◽  
R Ghosh ◽  
AK Tripathi ◽  
BD Banerjee
Keyword(s):  
Epithelial To Mesenchymal Transition ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Dependent Manner ◽  
Mesenchymal Transition ◽  
Epithelial Marker ◽  
Proximal Tubular ◽  
Renal Proximal Tubular ◽  
Smad 3 ◽  
Tgf Β1

This study investigated the effect of heptachlor-induced oxidative stress (OS) on transforming growth factor (TGF)-β1-mediated epithelial to mesenchymal transition (EMT) in human renal proximal tubular epithelial (HK-2) cells. Following treatment of HK-2 cells with an increasing concentration of heptachlor (0.01–10 µM) for 24 h, the intracellular reactive oxygen species and malondialdehyde level increased, whereas the glutathione-s-hydroxylase (GSH) level declined significantly in a dose-dependent manner. Pretreatment with N-acetyl cysteine attenuates the heptachlor-induced OS. In this study, we have shown that heptachlor-induced OS regulates the mRNA expression of TGF-β1-mediated Smad signalling genes accompanied by increased nuclear localization of phosphorylated Smad-2 and phosphorylated Smad-3. Furthermore, the m-RNA and protein level of epithelial marker, that is, E-cadherin decreased while the mesenchymal marker, that is, α-smooth muscle actin increased in heptachlor exposed HK-2 cells. In conclusion, heptachlor-induced OS might be responsible for the activation of TGF-β1/Smad signalling which ultimately leads to renal damage by means of EMT.

MKL1 mediates TGF-β1-induced α-smooth muscle actin expression in human renal epithelial cells

2008 ◽  
Vol 294 (5) ◽  
pp. F1116-F1128 ◽  
Author(s):  
Gerard Elberg ◽  
Lijuan Chen ◽  
Dorit Elberg ◽  
Michael D. Chan ◽  
Charlotte J. Logan ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Epithelial Cells ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Primary Cultures ◽  
Muscle Actin ◽  
Mesenchymal Transition ◽  
Endogenous Expression ◽  
Tgf Β1 ◽  
Stimulation Of

Transforming growth factor-β1 (TGF-β1) is known to induce epithelial-mesenchymal transition in the kidney, a process involved in tubulointerstitial fibrosis. We hypothesized that a coactivator of the serum response factor (SRF), megakaryoblastic leukemia factor-1 (MKL1), stimulates α-smooth muscle actin (α-SMA) transcription in primary cultures of renal tubular epithelial cells (RTC), which convert into myofibroblasts on treatment with TGF-β1. Herein, we study the effect of MKL1 expression on α-SMA in these cells. We demonstrate that TGF-β1 stimulation of α-SMA transcription is mediated through CC(A/T)6-rich GG elements known to bind to SRF. These elements also mediate the MKL1 effect that dramatically activates α-SMA transcription in serum-free media. MKL1 fused to green fluorescent protein localizes to the nucleus and induces α-SMA expression regardless of treatment with TGF-β1. Using proteasome inhibitors, we also demonstrate that the proteolytic ubiquitin pathway regulates MKL1 expression. These data indicate that MKL1 overexpression is sufficient to induce α-SMA expression. Inhibition of endogenous expression of MKL1 by small interfering RNA abolishes TGF-β1 stimulation of α-SMA expression. Therefore, MKL1 is also absolutely required for TGF-β1 stimulation of α-SMA expression. Western blot and immunofluorescence analysis show that overexpressed and endogenous MKL1 are located in the nucleus in non-stimulated RTC. Chromatin immunoprecipitation assay demonstrates that TGF-β1 induces binding of endogenous SRF and MKL1 to the α-SMA promoter in chromatin. Since MKL1 constitutes a potent factor regulating α-SMA expression, modulation of endogenous MKL1 expression or activity may have a profound effect on myofibroblast formation and function in the kidney.

scholarly journals Hsa_circ_0123190 acts as a competitive endogenous RNA to regulate APLNR expression by sponging hsa-miR-483-3p in lupus nephritis

2020 ◽  
Author(s):  
Chunyi Zhang ◽  
Congcong Gao ◽  
Xueqi Di ◽  
Siwan Cui ◽  
Wenfang Liang ◽  
...  
Keyword(s):  
Lupus Nephritis ◽  
Lupus Erythematosus ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β1 ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Reporter Assay ◽  
Clinical Value ◽  
Chronicity Index

Abstract Background Lupus nephritis (LN) is one of the most severe complications of systemic lupus erythematosus (SLE). Circular RNAs(circRNAs) can act as competitive endogenous RNAs (ceRNAs) to regulate gene transcription, which is involved in mechanism of many diseases. However, the role of circRNA in lupus nephritis has been rarely reported. In this study, we aim to investigate the clinical value of circRNAs and explore the mechanism of circRNA involvement in the pathogenesis of LN. Methods Renal tissues from three untreated LN patients and three normal controls (NCs) were used to identify differently expressed circRNAs by next-generation sequencing (NGS). Validated assays were used by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The interactions between circRNA and miRNA, or miRNA and mRNA were further determined by luciferase reporter assay. The extent of renal fibrosis between the two groups was assessed by Masson-trichome staining and immunohistochemistry (IHC) staining. Results 159 circRNAs were significantly dysregulated in LN patients compared with NCs. The expression of hsa_circ_0123190 was significantly decreased in renal tissues of patients with LN (P = 0.014). Bio-informatic analysis and luciferase reporter assay illustrated that hsa_circ_0123190 can act as a sponge for hsa-miR-483-3p, which was also validated to interact with APLNR. APLNR mRNA expression was related with chronicity index (CI) of LN (P = 0.033, R2 = 0.452). Moreover, the fibrotic-related protein, transforming growth factor-β1 (TGF-β1), which was regulated by APLNR, was more pronounced in the LN group (P = 0.018). Conclusion Hsa_circ_0123190 may function as a ceRNA to regulate APLNR expression by sponging hsa-miR-483-3p in LN.

scholarly journals LncRNA ZFPM2-AS1 Promotes Metastasis and Epithelial-mesenchymal Transition of Hepatocellular Carcinoma via Targeting miR-3612/ADAM15 Axis

2020 ◽  
Author(s):  
Nan Yang ◽  
Tianxiang Chen ◽  
Bowen Yao ◽  
Liang Wang ◽  
Runkun Liu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Biological Effects ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Mesenchymal Transition ◽  
Hcc Cells

Abstract Background: Long non-coding RNAs (lncRNAs) have obtained growing attention due to their potential effects as novel regulators in various tumors. This study aimed to investigate the expression and roles of lncRNA ZFPM2-AS1 in the progression of hepatocellular carcinoma (HCC). Methods: Transwell was used to determine migration and invasion of HCC cells in vitro. The lung metastasis mouse model was established to detect tumor metastasis of HCC in vivo. The direct binding of miR-3612 to 3'UTR of DAM15 was confirmed by luciferase reporter assay. The expression of ZFPM2-AS1 and miR-3612 in HCC specimens and cell lines were detected by real-time PCR. The correlation among ZFPM2-AS1 and miR-3612 were disclosed by a dual-luciferase reporter assay, RIP assay and biotin pull-down assay.Results: In present study, we found that ZFPM2-AS1 was up-regulated in HCC tissues and cells and its upregulation was associated with TNM stage, vascular invasion, and poor prognosis of HCC patients. Functionally, gain- and loss-of-function experiments indicated that ZFPM2-AS1 promoted cell migration, invasion and EMT progress in vitro and in vivo. ZFPM2-AS1 could function as a competing endogenous RNA (ceRNA) by sponging miR-3612 in HCC cells. Mechanically, miR-3612 inhibited HCC metastasis and alternation of miR-3612 reversed the promotive effects of ZFPM2-AS1 on HCC cells. In addition, we confirmed that ADAM15 was a direct target of miR-3612 in HCC and mediated the biological effects of miR-3612 and ZFPM2-AS1 in HCC. Curcumin, an active derivative from turmeric, exerts its anticancer effects through ZFPM2-AS1/miR-3612/ADAM15 pathway. Our data identified ZFPM2-AS1 as a novel oncogenic lncRNA and correlated malignant clinical outcomes in HCC patients. Conclusions: ZFPM2-AS1 performed as oncogenic role via targeting miR-3612 and subsequently promoted ADAM15 expression in HCC. Our results revealed that ZFPM2-AS1 could be a potential prognostic biomarker and therapeutic target for HCC.

scholarly journals LncRNA NKILA Promotes Epithelial-Mesenchymal Transition of Liver Cancer Cells by Targeting miR-485-5p

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Yuxu Wang ◽  
Chao Li ◽  
Yuyi Shi ◽  
Jing Kuai
Keyword(s):  
Liver Cancer ◽  
Normal Liver ◽  
Liver Cells ◽  
Luciferase Reporter Assay ◽  
Biological Behavior ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Mesenchymal Transition ◽  
The Relationship ◽  
Dual Luciferase Reporter Assay

Objective. Liver cancer (LC), one of the familiar malignancies, has a very high morbidity all over the world. The onset of the disease is hidden, and the patients usually do not express any special symptoms. Most of them will have been developed to the middle and later stage when they are diagnosed. This is one of the main reasons why the prognosis of LC is extremely pessimistic all the year round. Recently, researchers have focused mainly on molecular studies, among which LncRNA is a hot spot. This research aims to explore the biological behaviors of LncRNA NKILA and miR-485-5p in LC cells and verify the relationship between them, thereby providing a new theoretical basis for future prevention and treatment. Methods. Ninety-four early LC patients admitted to our hospital from January 2015 to January 2017 were regarded as the research objects. In addition, human LC cells SMMC-7721, HepG2, and normal liver cells HL-7702 were purchased. The LncRNA NKILA and miR-485-5p level in cancer and adjacent tissues, LC, and normal liver cells of patients was tested by PCR. Patients were followed up for 3 years. Then, LncRNA NKILA and miR-485-5p’s effects on prognosis and cell biological behavior were analyzed. At last, the relationship between LncRNA NKILA and miR-485-5p was assessed by a dual-luciferase reporter assay. Results. The LncRNA NKILA expression was high in LC tissues and cells ( P < 0.050 ), while miR-485-5p was low compared with the normal adjacent tissues ( P < 0.050 ). Prognostic follow-up manifested that high LncRNA NKILA or low miR-485-5p could predict the poor prognosis and high mortality risk of the patients ( P < 0.050 ). LC cells with downregulated LncRNA NKILA documented inhibited proliferation, invasion, and EMT, while the apoptosis level of the cells increased ( P < 0.050 ). The proliferation, invasion, and EMT were inhibited by miR-485-5p increase, while the apoptosis of the cells decreased after upregulating miR-485-5p ( P < 0.050 ). Online websites predicted that LncRNA NKILA had a binding site with miR-485-5p, and dual-luciferase reporter assay confirmed that LncRNA NKILA could directly target with miR-485-5p ( P < 0.050 ). The miR-485-5p in LC cells increased after LncRNA NKILA was silenced ( P < 0.050 ). The rescue experiment documented that LncRNA NKILA inhibition on LC cells was reversed by inhibiting miR-485-5p ( P < 0.050 ). Conclusion. The LncRNA NKILA with high expression advances LC cell proliferation, invasion, and EMT by targeting miR-485-5p.

miR-30a-5p Inhibits Epithelial-to-Mesenchymal Transition by Targeting CDK6 in Nasal Polyps

2020 ◽  
pp. 194589242093981 ◽  
Author(s):  
Ting Zhang ◽  
Yong Zhou ◽  
Bo You ◽  
Yiwen You ◽  
Yongbing Yan ◽  
...  
Keyword(s):  
Nasal Polyps ◽  
Epithelial To Mesenchymal Transition ◽  
Transforming Growth Factor ◽  
A549 Cells ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Aberrant Expression ◽  
Mesenchymal Transition ◽  
Emt Markers ◽  
Tgf Β1

Background Epithelial-to-Mesenchymal Transition (EMT) is considered as a crucial event in disease development and dysregulation of microRNAs (miRNAs) is involved in the regulation of EMT in various human diseases. Emerging evidences congregated over the years have demonstrated that miR-30a-5p was decreased in diseases and its overexpression inhibited the process of diseases via attenuating EMT. Although aberrant expression of miRNAs and occurrence of EMT were previously reported in Nasal Polyps (NPs), the role of miR-30a-5p in EMT of NPs is still remains unclear. Objective The purpose of our present study was to explore the expression and potential function of miR-30a-5p in EMT of NPs. Methods The expression of miR-30a-5p and mRNA expression level were detected by quantitative real-time PCR (qRT-PCR) in transforming growth factor β1 (TGF-β1) - induced EMT model and NPs patients. Western Blot (WB) and immunohistochemistry (IHC) were performed to evaluate the protein expression level of EMT markers. The cells mobility was assessed by Wound-Healing assay. Luciferase reporter assay was utilized to verify the relationship between Cyclin-dependent kinase 6 (CDK6) and miR-30a-5p. Results Firstly, we observed that miR-30a-5p was down-regulated notably, accompanying with the alteration of EMT markers expression in NPs tissues and EMT model induced by TGF-β1 in primary Human Nasal Epithelial Cells (pHNECs) and A549 cells in vitro. Moreover, the functional assays demonstrated that overexpression of miR-30a-5p significantly inhibited EMT and cells mobility. Subsequently, CDK6 was validated as a direct target of miR-30a-5p. Finally, we performed the rescue experiments indicating that overexpression of CDK6 eliminated the suppressive effects of miR-30a-5p in TGF-β1-induced EMT in pHNECs and A549 cells. Conclusion Taken together, our results suggested that EMT was involved in NPs, and overexpression of miR-30a-5p could attenuate EMT via repressing the expression of the CDK6 in pHNECs and A549 cells.

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