Knockdown of ABHD11­AS1 prevents the procession of TNBC by upregulating miR­199a­5p.

Breast cancer (BC) has become a threat to women's health. In addition, patients with triple-negative BC (TNBC) have the worst prognosis among all patients with BC. Furthermore, long non-coding RNA ABHD11-AS1 is aberrantly highly expressed in TNBC, suggesting that RNA ABHD11-AS1 may serve as an important role in the progression of TNBC. However, the detailed function of ABHD11-AS1 in TNBC remains largely unknown. The levels of ABHD11-AS1 in MDA-MB-231 cells were assessed by reverse transcription-quantitative PCR. To investigate the effect of ABHD11-AS1 on the progression of TNBC, a xenograft animal model was established. Knockdown of ABHD11-AS1 inhibited the epithelial-mesenchymal transition and migration of TNBC cells. In addition, ABHD11-AS1 promoted the viability and migration of TNBC cells by upregulating microRNA (miR)-199a-5p. Furthermore, knockdown of ABHD11-AS1 suppressed TNBC tumor growth in vivo by upregulating miR-199a-5p. In conclusion, knockdown of ABHD11-AS1 suppressed the progression of TNBC via upregulation of miR-199a-5p. The data of the present study may provide novel directions and a theoretical basis for TNBC treatment.

Cancer-associated fibroblast-released extracellular vesicles carrying miR-199a-5p induces the progression of​ gastric cancer through regulation of FKBP5-mediated AKT1/mTORC1 signaling pathway.

Accumulating evidence has unfolded the significance of extracellular vesicles (EVs) in diseases and cancers. Here, we attempted to discuss the role of cancer-associated fibroblasts (CAFs)-derived EVs containing miR-199a-5p in gastric tumorigenesis. Upregulated miR-199a-5p was first identified in cancer cells. Then, we selected CAFs for isolation of EVs which were co-cultured with AGS cells. We observed successful delivery of miR-199a-5p via CAF-derived EVs. Besides, miR-199a-5p promoted malignant properties of AGS cells. Moreover, miR-199a-5p downregulated FKBP5, leading to upregulated phosphorylation level of AKT1, which promoted the malignant phenotypes of AGS cells by activating mammalian target of rapamycin complex 1(mTORC1). Exosomal miR-199a-5p from CAFs promoted gastric tumorigenesis in vivo. Our findings point toward the critical role of CAFs-derived EVs carrying miR-199a-5p in gastric cancer progression.

Inhibitory effect of siRNA-KCNQ1OT1 on human lens epithelial cell apoptosis by targeting miR-199a-5p / 中华实验眼科杂志

Objective:To explore the inhibitory effect of long non-coding RNA (lncRNA) KCNQ1 overlapping transcript 1 (KCNQ1OT1) by targeting microRNA-199a-5p (miR-199a-5p) on the apoptosis of human lens epithelial cells (LECs).Methods:The anterior lens capsule tissue of 23 age-related cataract patients who underwent cataract surgery in Xinxiang First People's Hospital from December 2018 to August 2019 was collected.At the same time, anterior lens capsules from 20 healthy donor were collected.The expressions of KCNQ1OT1 and miR-199a-5p in the tissues were detected by real-time fluorescence PCR.Human LECs SRA01/04 cultured in vitro were divided into blank control group, model control group, small interfering RNA-negative control (siR-NC) group, siR-KCNQ1OT1 group, miR-NC group, miR-199a-5p group, siR-KCNQ1OT1+ anti-miR-NC group and siR-KCNQ1OT1+ anti-miR-199a-5p group.No intervention was administered to blank control group.Cells in model control group were cultured with 100 μmol/L H 2O 2 for 24 hours to establish oxidative stress injured model, and cells in the other six groups were transfected with corresponding transfection reagents for 6 hours by liposome method according to grouping, and then treated with 100 μmol/L H 2O 2 for 24 hours.The expressions of KCNQ1OT1 and miR-199a-5p in lens anterior capsule tissue and LECs cells were determined by real-time fluorescent quantitative PCR.Cell viability was detected with thiazolyl blue (MTT). Cell apoptosis was analyzed by flow cytometry.The expressions of B-cell lymphoma/leukemia-2 (bcl-2) and bcl-2 related X protein (Bax) proteins were assayed by Western blot.The superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured by enzyme-linked immunosorbent assay (ELISA). The targeting relationship between KCNQ1OT1 and miR-199a-5p was verified by dual luciferase reporter experiment.The study protocol was approved by an Ethics Committee of Xinxiang First People's Hospital (No.2019-001). Written informed consent was obtained from relatives of patient. Results:The relative expression of KCNQ1OT1 in the anterior capsule of patients with age-related cataract was 2.41±0.42, which was significantly higher than 0.97±0.19 of normal people, and the relative expression of miR-199a-5p in the capsule of patients with age-related cataract was 0.36±0.12, which was lower than 1.04±0.15 of normal people, and the differences were statistically significant ( t=14.112, 16.507; both at P<0.001). Compared with blank control group, the relative expressions of KCNQ1OT1 and bax protein, cell apoptosis rate and MDA content were significantly increased, and the relative expressions of miR-199a-5p and bcl-2 protein, cell viability and SOD activity were significantly reduced in model control group, showing statistically significant differences (all at P<0.001). Compared with siR-NC group, the relative expressions of KCNQ1OT1 and bax protein, cell apoptosis rate and MDA content in cells of siR-KCNQ1OT1 group were decreased, while the relative expression of bcl-2 protein, cell survival rate and SOD activity were increased, and the differences were statistically significant (all at P<0.05). Compared with miR-NC group, the KCNQ1OT1-wild type (WT) luciferase activity in miR-199a-5p group was significantly decreased, with a statistically significant difference ( t=21.131, P<0.001). The relative expression levels of miR-199a-5p and bcl-2 proteins, cell survival rate and SOD activity were significantly increased, and the relative expression of bax protein, cell apoptosis rate and MDA content were significantly decreased in miR-199a-5p group than those in miR-NC group, and the differences were statistically significant (all at P<0.05). The relative expression levels of miR-199a-5p and bcl-2 proteins, cell survival rate and SOD activity were significantly lower, and the cell apoptosis rate, relative expression of bax protein and MDA content were significantly higher in siR-KCNQ1OT1+ anti-miR-199a-5p group than those in siR-KCNQ1OT1+ anti-miR-NC group, and the differences were statistically significant (all at P<0.05). Conclusions:The inhibition of KCNQ1OT1 can promote the cell viability of human LECs, inhibit H 2O 2-induced cell apoptosis and oxidative stress, and the mechanism may be related to the up-regulation of miR-199a-5p.

Diverging targets mediate the pathological roleof miR-199a-5p and miR-199a-3p by promoting cardiac hypertrophy and fibrosis.

MicroRNA-199a-5p (miR-199a-5p) and -3p are enriched in the myocardium, but it is unknown whether miR-199a-5p and -3p are co-expressed in cardiac remodeling and what roles they have in cardiac hypertrophy and fibrosis. We show that miR-199a-5p and -3p are co-upregulated in the mouse and human myocardium with cardiac remodeling and in Ang-II-treated neonatal mouse ventricular cardiomyocytes (NMVCs) and cardiac fibroblasts (CFs). miR-199a-5p and -3p could aggravate cardiac hypertrophy and fibrosis in vivo and in vitro. PPAR gamma coactivator 1 alpha (Ppargc1a) and sirtuin 1 (Sirt1) were identified as target genes to mediate miR-199a-5p in promoting both cardiac hypertrophy and fibrosis. However, miR-199a-3p aggravated cardiac hypertrophy and fibrosis through targeting RB transcriptional corepressor 1 (Rb1) and Smad1, respectively. Serum response factor and nuclear factor κB p65 participated in the upregulation of miR-199a-5p and -3p in Ang-II-treated NMVCs and mouse CFs, and could be conversely elevated by miR-199a-5p and -3p. Together, Ppargc1a and Sirt1, Rb1 and Smad1 mediated the pathological effect of miR-199a-5p and -3p by promoting cardiac hypertrophy and fibrosis, respectively. This study suggests a possible new strategy for cardiac remodeling therapy by inhibiting miR-199a-5p and -3p.

Autophagy inhibition and microRNA­199a­5p upregulation in paclitaxel­resistant A549/T lung cancer cells.

Multidrug resistance (MDR) is one of the major reasons for the clinical failure of cancer chemotherapy. Autophagy activation serves a crucial role in MDR. However, the specific molecular mechanism linking autophagy with MDR remains unknown. The results of the present study demonstrated that autophagy was inhibited and microRNA (miR)­199a­5p levels were upregulated in MDR model lung cancer cells (A549/T and H1299/T) compared with those in the parental cell lines. Paclitaxel (PTX) treatment increased the expression levels of miR­199a­5p in parental lung cancer cells compared with those in PTX­untreated cells, and these expression levels were negatively correlated with PTX sensitivity of the cells. miR­199a­5p knockdown in A549/T cells induced autophagy and resensitized cells to multiple chemotherapeutic drugs including PTX, taxotere, topotecan, SN38, oxaliplatin and vinorelbine. By contrast, miR­199a­5p overexpression in A549 cells suppressed autophagy and desensitized cells to these chemotherapeutic drugs. Mechanistically, the results of the present study demonstrated that miR­199a­5p blocked autophagy by activating the PI3K/Akt/mTOR signaling pathway and inhibiting the protein expression of autophagy­related 5. Furthermore, p62 protein was identified as a direct target of miR­199a­5p; miR­199a­5p bound to p62 mRNA to decrease its mRNA and protein expression levels. In conclusion, the results of the present study suggested that miR­199a­5p may contribute to MDR development in lung cancer cells by inhibiting autophagy and targeting p62. The regulatory effect of miR­199a­5p on autophagy may provide novel insights for future multidrug­resistant lung cancer chemotherapy.

MicroRNA-199a-5p accelerates nucleus pulposus cell apoptosis and IVDD by inhibiting SIRT1-mediated deacetylation of p21.

Intervertebral disc degeneration (IVDD) is a multifactorial pathological process associated with low back pain in which nucleus pulposus cell senescence is disrupted. Increasing evidence reveals that IVDD can be modulated by microRNAs (miRNAs or miRs). In the current study, we set out to elucidate the role of miR-199a-5p in nucleus pulposus cell apoptosis and IVDD progression. After sample collection, we found highly expressed miR-199a-5p in nucleus pulposus tissues of both patients diagnosed with IVDD and in IVDD rat models. Next, normal and degenerated nucleus pulposus cells were isolated and transfected with miR-199a-5p mimic, miR-199a-5p inhibitor, overexpressed sirtuin 1 (oe-SIRT1), and oe-p21, followed by detection of nucleus pulposus cell apoptosis and proliferation. In addition, the binding of miR-199a-5p and SIRT1, the interaction between p21 and SIRT1, and the regulation of p21 acetylation by SIRT1 were analyzed. We found that miR-199a-5p overexpression promoted nucleus pulposus cell apoptosis and IVDD. Overexpression of SIRT1 countered the effect of miR-199a-5p overexpression, while overexpression of p21 reversed the effect of miR-199a-5p silencing. Also, miR-199a-5p inhibited SIRT1, promoted p21 acetylation, and upregulated p21 expression, thus accelerating nucleus pulposus cell apoptosis and IVDD. Overall, miR-199a-5p promotes nucleus pulposus cell apoptosis and IVDD by suppressing SIRT1-dependent deacetylation of p21.

Epigenetic silencing of microRNA-199a-5p promotes the proliferation of non-small cell lung cancer cells by increasing AKAP1 expression.

MicroRNA (miR)-199a-5p expression is downregulated in a variety of malignancies, including non-small cell lung cancer (NSCLC), and its low expression is associated with a poor prognosis. However, to the best of our knowledge, the mechanism underlying miR-199a-5p downregulation in NSCLC and its target effectors remain to be elucidated. The present study revealed the downregulation of miR-199a-5p expression in NSCLC tissues and cell lines compared with in para-carcinoma tissues and a lung epithelial cell line. Further experiments indicated that the methylation levels of the miR-199a promoter were markedly higher in NSCLC tissues compared with in para-carcinoma tissues. The DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine markedly increased the expression levels of miR-199a-5p in NSCLC cells. Furthermore, it was identified that miR-199a-5p mimics transfection decreased the expression levels of A-kinase anchoring protein 1 (AKAP1) at both the mRNA and protein levels by targeting the 3' untranslated region of AKAP1 mRNA. The in vitro experiments demonstrated that miR-199a-5p overexpression inhibited the proliferation and tumorigenicity of NSCLC cells, whereas overexpression of AKAP1 partially recovered the malignant phenotypes, suggesting that AKAP1 may be a downstream effector targeted by miR-199a-5p. Collectively, the present findings indicated that miR-199a-5p may be a novel regulator of AKAP1, and that miR-199a-5p may be a potential tumor suppressor in NSCLC.

MicroRNA-199a-5p suppresses the cell growth of colorectal cancer by targeting oncogene Caprin1.

MicroRNAs-199a-5p (miR-199a-5p) plays critical regulatory roles in various types of human cancers. However, the biological function and regulatory mechanisms of miR-199a-5p in colorectal cancer (CRC) remain unclear. The aim of this study was to investigate the role of miR-199a-5p in CRC and possible mechanisms of its action. The expression of miR-199a-5p in CRC tumor tissues was validated using quantitative real-time PCR (qRT-PCR). The effects of miR-199a-5p on cell proliferation and apoptosis were evaluated in vitro. Then, the association of miR-199a-5p and its downstream target was investigated in both cell line and clinical specimens. Furthermore, gain- and loss-of-function studies of cytoplasmic activation/proliferation-associated protein-1 (Caprin1) were performed to assess whether the suppressive effect of on CRC cells were via targeting Caprin1. Using a microarray platform, we focused on miR-199a-5p for further research, which was one of the most markedly downregulated miRNAs in CRC tumor tissues. Functionally, the overexpression of miR-199a-5p inhibited proliferation and induced apoptosis in both HTC116 and SW480 cells. Furthermore, cytoplasmic activation/proliferation-associated protein-1 (Caprin1), a well-known oncogene, was directly targeted by miR-199a-5p. It was also observed that Caprin1 was upregulated, and inversely correlated with miR-199a-5p levels in CRC tissues. Further investigations revealed that knockdown of Caprin1 by siRNA has similar role with miR-199a-5p overexpression in CRC cells, suggesting the oncogenic role of Caprin1 in CRC. In the contrast, we found that overexpression of Caprin1 reversed the suppressive effects of miR-199a-5p on CRC cells. Collectively, our study suggests that miR-199a-5p/Caprin1 axis may serve as potential therapeutic targets for the treatment of CRC.

miR-199a-5p stimulates ovarian granulosa cell apoptosis in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder and one of the most common causes of infertility in women. PCOS patients have been found with dysregulated miRNA, which is indicative of their roles as noninvasive biomarkers and novel therapeutic targets in PCOS. Herein, this study sets out to explore the mechanism of action of miR-199a-5p in PCOS in relation to the janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway via Wilms' tumor 1 (WT1) regulation in a rat model of PCOS. The expression of miR-199a-5p was highly expressed in ovarian cortical tissues and serum of PCOS patients, as examined by RT-qPCR. Ovarian granulosa cells (GCs) were harvested from PCOS rat model, followed by subsequent purification. Gain- and loss-of-function experiments of miR-199a-5p were performed to determine its functions in PCOS. Cell viability, cell apoptosis and serum hormone levels were assessed, the results of which showed that downregulation of miR-199a-5p contributed to the promotion of GC viability and inhibition of apoptosis, while simultaneously inducing the elevation of serum E2 level and reduction of serum AMH, PG, LH and FSH levels in the PCOS rat model. WT1 was identified as a target gene of miR-199a-5p by dual-luciferase reporter gene assay, and inhibition of miR-199a-5p resulted in the activation of WT1-mediated JAK/STAT3 pathway. The activated JAK/STAT3 pathway suppressed the development of PCOS by miR-199a-5p, indicating a mechanism by which miR-199a-5p could potentially prevent PCOS through the WT1-mediated JAK/STAT3 pathway.

miRNA-199a-5p suppresses proliferation and invasion by directly targeting NF-κB1 in human ovarian cancer cells.

The aberrant expression of microRNA (miRNA)-199a-5p has been frequently reported in a number of cancer types, but to the best of our knowledge, this has not been reported in ovarian cancer (OC). The role and the molecular mechanism of miR-199a-5p in OC have not been reported. Therefore, the present study investigated the effects of miR-199a-5p overexpression on the proliferation and invasion of OC cells. The level of miR-199a-5p in OC cell lines was determined by reverse transcription-quantitative polymerase chain reaction. The miR-199a-5p mimic was transiently transfected into OC cells using Lipofectamine™ 2000 reagent. Subsequently, the BrdU-ELISA results indicated that the exogenous expression of miR-199a-5p inhibited cell proliferation. In addition, miR-199a-5p overexpression was able to inhibit the invasion of HO-8910 and ES-2 cells. RT-qPCR was performed to determine the expression of matrix metalloproteinase (MMP)-2 and -9 in OC cells. NF-κB1 expression was reduced by upregulation of miR-199a-5p. Bioinformatics analysis predicted that NF-κB1 was a potential target of miR-199a-5p. Luciferase reporter assay further confirmed that miR-199a-5p was able to directly target the 3'UTR of NF-κB1. In conclusion, miRNA-199a-5p may suppress the proliferation and invasion of human ovarian cancer cells by directly targeting NF-κB1.

MicroRNA-199a-5p aggravates primary hypertension by damaging vascular endothelial cells through inhibition of autophagy and promotion of apoptosis.

The present study investigated the expression of microRNA (miRNA or miR)-199a-5p in the peripheral blood of patients with primary hypertension, and examined its mechanism of action in vascular endothelial cell injury induced by hypertension. A total of 57 patients with primary hypertension, who were treated at the Affiliated Hospital of Qingdao University (Qingdao, China) between December 2014 and November 2015 were included in the present study. Peripheral blood was collected from all patients. The expression of miR-199a-5p was measured using reverse-transcription quantitative polymerase chain reaction analysis. Human umbilical vein endothelial cells (HUVECs) were divided into negative control, miR-199a-5p mimics and rescue (co-transfected with miR-199a-5p mimics and inhibitor) groups. After transfection, the proliferation and apoptosis of HUVECs were evaluated by a Cell Counting Kit-8 assay, a bromodeoxyuridine incorporation assay and flow cytometry. Western blot analysis was used to determine the expression of proteins involved in autophagy-associated and adenosine monophosphate kinase (AMPK)/unc-51 like autophagy activating kinase 1 (ULK1) signaling pathways. Laser scanning confocal microscopy and electron microscopy were used to observe the autophagy of HUVECs. The expression of miR-199a-5p was elevated in peripheral blood of patients with hypertension, and was correlated with the progression of hypertension. Overexpression of miR-199a-5p inhibited the proliferation and promoted the apoptosis of HUVECs. Upon expression of miR-199a-5p, the transition between microtubule-associated proteins 1A/1B light chain 3B (LC3B)I and LC3BII proteins was inhibited, the expression of p62 protein was upregulated. In addition, miR-199a-5p decreased the numbers of autophagosomes and autolysosomes in HUVECs. The present study demonstrated that expression of miR-199a-5p is positively correlated with the severity of hypertension. Expression of miR-199a-5p aggravated vascular endothelial injury by inhibiting autophagy and promoting the apoptosis of HUVECs via downregulation of the AMPK/ULK1 signaling pathway.

MicroRNA-199a-5p Induced Autophagy and Inhibits the Pathogenesis of Ankylosing Spondylitis by Modulating the mTOR Signaling via Directly Targeting Ras Homolog Enriched in Brain (Rheb).

BACKGROUND/AIMS: Ankylosing spondylitis (AS) is an inflammatory and immune disease leading to disability. Autophagy has been identified as a potential player in understanding the pathogenesis of AS. METHODS: MiRNA-199a-5p and autophagy-related gene expression were determined by qRT-PCR or Western blot. Cytokine production was determined using ELISA assays. Proliferation was determined by MTT assay. MiRNA-199a-5p and Ras homolog enriched in brain (Rheb) were upregulated or downregulated by overexpression of plasmid or siRNA transfection. RESULTS: Expression of miRNA-199a-5p, and autophagy-related genes LC3, beclin1, and ATG5 was significantly decreased in T cells of AS patients. Serum concentrations of TNF-α, IL-17, and IL-23 were promoted in AS patients, compared to healthy controls. MiRNA-199a-5p expression levels also showed significant negative correlations with the Ankylosing Spondylitis Disease Activity Score (ASDAS) and modified Stoke Ankylosing Spon dylitis Spinal Score (mSASSS) of AS patients. In Jurkat T cells and T cells isolated from AS patients, miRNA-199a-5p overexpression promoted autophagy-related genes expression and decreased TNF-α, IL-17, and IL-23 levels, whereas inhibition of miRNA-199a-5p attenuated these effects. As a direct target of miRNA-199a-5p, Rheb inhibition led to a striking decrease in the phosphorylation of the mechanistic target of rapamycin (mTOR) and induced autophagy. Moreover, pcDNA3.1-Rheb effectively reduced the inhibiting effects of mTOR signaling caused by miRNA-199a-5p overexpression. All effects were offset by pretreating with rapamycin (an mTOR antagonist). CONCLUSIONS: AS patients with advanced spinal damage had decreased autophagy levels and that miRNA-199a-5p may induce autophagy and inhibit the pathogenesis of AS by modulating the mTOR signaling via direct targeting Rheb.

miR-199a-5p enhances expression of myocardial fibrosis-related genes by targeting SIRT1 / 中国病理生理杂志

AIM:To investigate the role of microRNA (miR)-199a-5p in myocardial fibrosis and the potential target of miR-199a-5p.METHODS:C57BL/6 mouse cardiac fibroblasts were isolated and cultured for cellular experimen-tal study.Dual-luciferase reporter assay was performed to confirm the interaction between miR-199a-5p and the 3'-untrans-lated region (3'-UTR) of silent information regulator 1 (SIRT1).The expression of SIRT1 and fibrosis markers collagen (Col) 1a1, Col3a1 and α-smooth muscle actin (α-SMA) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively.RESULTS:The expression levels of miR-199a-5p, Col1a1, Col3a1 andα-SMA were marked-ly increased in cardiac fibroblasts after treatment with angiotensin Ⅱ(AngⅡ).The over-expression of miR-199a-5p signif-icantly increased the expression of Col1a1, Col3a1 andα-SMA in cardiac fibroblasts.Moreover, the results of dual-lucifer-ase reporter assay revealed that miR-199a-5p interacted with the 3'-UTR of SIRT1.miR-199a-5p inhibited SIRT1 expres-sion at post-transcriptional level.Meanwhile, miR-199a-5p mimic, in parallel to SIRT1 siRNA, inhibited SIRT1 expres-sion, increased the expression of Col1a1, Col3a1 and α-SMA in cardiac fibroblasts.Inactivation of NF-κB signaling con-tributed to the decrease in miR-199a-5p in Ang II-treated cardiac fibroblasts .CONCLUSION:SIRT1 is a target gene of miR-199a-5p, which mediates the pro-fibrotic effect of miR-199a-5p on cardiac fibroblasts .

miR-199a-5p enhances expression of myocardial fibrosis-related genes by targeting SIRT1 / 中国病理生理杂志

AIM:To investigate the role of microRNA (miR)-199a-5p in myocardial fibrosis and the potential target of miR-199a-5p.METHODS:C57BL/6 mouse cardiac fibroblasts were isolated and cultured for cellular experimen-tal study.Dual-luciferase reporter assay was performed to confirm the interaction between miR-199a-5p and the 3'-untrans-lated region (3'-UTR) of silent information regulator 1 (SIRT1).The expression of SIRT1 and fibrosis markers collagen (Col) 1a1, Col3a1 and α-smooth muscle actin (α-SMA) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively.RESULTS:The expression levels of miR-199a-5p, Col1a1, Col3a1 andα-SMA were marked-ly increased in cardiac fibroblasts after treatment with angiotensin Ⅱ(AngⅡ).The over-expression of miR-199a-5p signif-icantly increased the expression of Col1a1, Col3a1 andα-SMA in cardiac fibroblasts.Moreover, the results of dual-lucifer-ase reporter assay revealed that miR-199a-5p interacted with the 3'-UTR of SIRT1.miR-199a-5p inhibited SIRT1 expres-sion at post-transcriptional level.Meanwhile, miR-199a-5p mimic, in parallel to SIRT1 siRNA, inhibited SIRT1 expres-sion, increased the expression of Col1a1, Col3a1 and α-SMA in cardiac fibroblasts.Inactivation of NF-κB signaling con-tributed to the decrease in miR-199a-5p in Ang II-treated cardiac fibroblasts .CONCLUSION:SIRT1 is a target gene of miR-199a-5p, which mediates the pro-fibrotic effect of miR-199a-5p on cardiac fibroblasts .

MicroRNA-199a-5p inhibits cisplatin-induced drug resistance via inhibition of autophagy in osteosarcoma cells.

Osteosarcoma (OS) is the most common cancer of the bone. Chemotherapy is commonly used for the clinical treatment of OS. However, chemoresistance to cisplatin [also known as diamminedichloridoplatinum (II) (DDP)] is a major obstacle for OS therapy, the underlying mechanism of which is not fully understood. The present study aimed to investigate the role of microRNA (miR)-199a-5p in the regulation of chemoresistance to DDP in OS cells. Reverse transcription-quantitative polymerase chain reaction demonstrated that the expression level of miR-199a-5p was significantly reduced in human OS MG63 cells. In addition, DDP treatment also upregulated the protein levels of light chain 3 (LC3)-II and Beclin1 as well as the ratio of LC3-II vs. LC3-I in MG63 cells, indicating that autophagy was activated. Restoration of miR-199a-5p expression promoted DDP-induced inhibition of MG63 cell proliferation and inhibited DDP-induced autophagy, as indicated by the reduced protein levels of LC3-II and Beclin1 and the ratio of LC3-II vs. LC3-I. Finally, luciferase reporter assay data revealed that miR-199a-5p directly targeted Beclin1 and negatively mediated Beclin1 expression at a post-transcriptional level in MG63 cells. In conclusion, our study suggests that miR-199a-5p promotes the cytotoxicity of DDP in OS cells via inhibition of autophagy. Therefore, miR-199a-5p/autophagy signaling is involved in chemoresistance and may become a potential target for the treatment of DDP-resistant OS.

Identification of targeting relationship between mouse lncRNA-H19 and miR-199 a-5 p by dual-luciferase reporter assay / 中国病理生理杂志

AIM:To validate the association between long noncoding (lncRNA)-H19 and microRNA-199a-5p (miR-199a-5p) through the dual-luciferase reporter gene system by construction of a luciferase reporter vector containing the gene of lncRNA-H19.METHODS:The potential complementary binding sites of lncRNA-H19 and miR-199a-5p were predicted by RegRNA 2.0.The H19 gene or its mutant ( Mut) fragment was cloned into luciferase reporter vector psi-CHECK-2.Restriction enzyme analysis and sequence analysis were used to identify whether the recombinant plasmids of the H19 and H19-Mut were successfully constructed .miR-199a-5p mimics, miR-199a-5p inhibitor, miR-199a-5p mimics neg-ative control or miR-199a-5p inhibitor negative control was co-transfected into the 293T cells with the luciferase reporters containing H19 or H19-Mut.Dual-luciferase reporter assay was performed to detect the luciferase activity in different groups in order to verify the relationship between lncRNA-H19 and miR-199a-5p.RESULTS:The results of double enzyme diges-tion and DNA sequencing showed that the sequence of luciferase reporter vector was correct .The results of dual-luciferase reporter assay indicated that the H 19 reporter gene luciferase activity significantly decreased in miR-199a-5p mimics group by 49%(P<0.01), and the H19 reporter gene luciferase activity was obviously upregulated in miR-199a-5p inhibitor group compared with miR-199a-5p mimics group ( P<0.01).However, miR-199a-5p mimics, miR-199a-5p inhibitor, miR-199a-5p mimics negative control and miR-199a-5p inhibitor negative control showed no effect at H 19-Mut reporter gene.CONCLUSION:lncRNA-H19 binds to miR-199a-5p to exert an inhibitory effect at transcriptional level .

Glucocorticoid inhibits cell proliferation in differentiating osteoblasts by microRNA-199a targeting of WNT signaling.

The inhibition of osteoblast proliferation by glucocorticoids (GCs) is very important in the etiology of GC-induced osteoporosis. The mechanisms of this process are still not fully understood. The results of recent studies have indicated an important role for microRNAs in GC-mediated responses in various cellular processes, including cell proliferation and apoptosis. Therefore, we developed the hypothesis that these regulatory molecules might be involved in GC-decreased osteoblast proliferation. Western blotting, quantitative real-time PCR, cell proliferation assays, and luciferase assays were employed to investigate the role of miRNAs in GC-inhibited osteoblast proliferation. microRNA-199a-5p was significantly increased in osteoblasts treated with dexamethasone (Dex). To delineate the role of microRNA-199a-5p, we silenced and overexpressed microRNA-199a-5p in osteoblasts. We found that overexpressing microRNA-199a-5p remarkably increased the inhibition effect of Dex on osteoblast proliferation, and depleting microRNA-199a-5p significantly attenuated Dex-inhibited osteoblast proliferation. Results of mechanistic studies indicated that microRNA-199a-5p inhibited FZD4 and WNT2 expression through a microRNA-199a-5p binding site within the 3'-UTR of FZD4 and WNT2. The post-transcriptional repression of FZD4 and WNT2 were further confirmed by luciferase reporter assay. These results indicated that microRNA-199a-5p may play a significant role in GC-inhibited osteoblast proliferation by regulating the WNT signaling pathway.