High-glucose induced toxicity in HK-2 cells can be alleviated by inhibition of miRNA-320c.

Diabetic nephropathy (DN) is a major healthcare challenge worldwide. MiRNAs exert a regulatory effect on the progress of DN. Our study proposed to investigate the miR-320c expression and its function on the pathogenesis of DN in vitro. The level of miR-320c in HK-2 cells was quantified by RT-qPCR. Cell morphology, invasion, and migration were observed by optical microscope, Transwell invasion assay, and scratch wound assay. Then, the levels of PTEN, α-SMA, vimentin, E-cadherin, p-PI3K, PI3K, AKT, and p-AKT were analyzed through western blotting. A Dual-luciferase reporter assay was conducted to explore the target relationship between miR-320c and PTEN. It was discovered that miR-320c was over-expressed in high glucose (HG)-treated HK-2 cells. Furthermore, inhibition of miR-320c could alleviate the epithelial-mesenchymal transition (EMT) of HG-induced HK-2 cells and retain the normal morphology of HK-2 cells. Additionally, the miR-320c inhibitor decreased the invasiveness and migration of HG-treated HK-2 cells. Next, the target gene of miR-320c, PTEN, was identified, and the function of miR-320c was reversed by down-regulation of PTEN. Finally, we found inhibition of miR-320c restrained the PI3K/AKT pathway. Therefore, inhibition of miR-320c could alleviate toxicity of HK-2 cells induced by HG via targeting PTEN and restraining the PI3K/AKT pathway, illustrating that miR-320c may act as a new biomarker in the diagnosis of DN.

CircPDSS1 promotes the proliferation, invasion, migration, and EMT of breast cancer cell via regulating miR-320c/CKAP5 axis.

BACKGROUND: Breast cancer (BC) poses serious threats to women's health. A large number of reports have proved that circular RNAs (circRNAs) exert vital functions in human cancers, including BC. METHODS: The function of circPDSS1 in BC cells was tested by CCK-8, colony formation, TUNEL, transwell-invasion, wound healing, and IF assays. RNA pull down, luciferase reporter and RIP assays were employed to verify the relationship among circPDSS1, miR-320c and CKAP5. RESULTS: CircPDSS1 was upregulated in BC cells, and circPDSS1 knockdown repressed BC cell malignant behaviors. Further, circPDSS1 was found to bind to miR-320c in BC cells, and miR-320c overexpression suppressed malignant processes of BC cells. MiR-320c could also bind to CKAP5. Moreover, miR-320c inhibition increased the level of CKAP5, but circPDSS1 downregulation decreased the level of CKAP5. Finally, rescue experiments indicated that CKAP5 knockdown countervailed the promoting effect of miR-320c inhibition on the malignant behaviors of circPDSS1-depleted BC cells. CONCLUSIONS: CircPDSS1 promotes proliferation, invasion, migration as well as EMT of BC cells by modulating miR-320c/CKAP5 axis. Our finding may be useful for researchers to find new potential therapeutic or diagnostic targets for BC.

Expression of plasma miR-320c in patients with osteoarthritis and its effect on proliferation and apoptosis of chondrocyte / 中华老年医学杂志

Objective:To investigate the expression level of plasma miR-320c in patients with osteoarthritis(OA), and explore the clinical significance and the role in pathogenesis of OA.Methods:The clinical data and peripheral blood of 30 patients with OA, 30 patients with connective tissue diseases and 30 healthy control individuals were collected.The levels of plasma miR-320c were detected byfluorescentquantitative reverse transcription PCR(qRT-PCR). Correlation analysis was used to explore the correlation of plasma miR-320c level with knee X-ray data and VAS pain score in OA patients.Finally the miR-320c mimic, the miR-320c inhibitor, and the control material were transfected to the chondrocyte HC-a.The proliferative capacity of HC-a chondrocytes was examined at different time points as determined by the CCK-8 assay.Results:The expression level of plasma miR-320c was significantly higher in OA group(3.26±0.55)than that in the connective tissue diseases group(1.62±0.50)and in healthy control group(1.21±0.66)( F=107.66, P<0.001). Plasma miR-320c expression was positively correlated with radiographic grade( r=0.830, P<0.001), and had no correlation with VAS pain score in OA group( P>0.05). Through repeated measurement variance analysis, the time effect, the group effect and the interaction effect between group and time showed statistically significant differences in chondrocyte proliferation between NC mimic group and miR-320c mimic group( Ftime=5256.767, Fgroup=1947.436, Ftime×group=114.314, all P<0.001). The level of proliferation was significantly reduced.Apoptosis rate of chondrocytes was significantly increased in the group transfected with miR-320c( t=7.85, P<0.01). Conclusions:The expression level of plasma miR-320c is significantly higher in osteoarthritis patients and associated with knee radiographic severity grade.Furthermore, over-expression of miR-320c could suppress the proliferation of chondrocytes.Plasma miR-320c might be potential bio-marker for osteoarthritis knee severity assessment, and involves in regulating chondrocyte growth in the pathogenesis of osteoarthritis.

miR-320c Regulates SERPINA1 Expression and Is Induced in Patients With Pulmonary Disease / El miR-320c regula la expresión del gen SERPINA1 y se induce en pacientes con enfermedad pulmonar

Introduction: Alpha-1 antitrypsin deficiency (AATD) is a genetic condition resulting in lung and liver disease with a great clinical variability. MicroRNAs have been identified as disease modifiers; therefore miRNA deregulation could play an important role in disease heterogeneity. Members of miR-320 family are involved in regulating of multiple processes including inflammation, and have potential specific binding sites in the 3′UTR region of SERPINA1 gene. In this study we explore the involvement of miR-320c, a member of this family, in this disease. Methods: Firstly in vitro studies were carried out to demonstrate regulation of SERPINA1 gene by miR-320. Furthermore, the expression of miR-320c was analyzed in the blood of 98 individuals with different AAT serum levels by using quantitative PCR and expression was correlated to clinical parameters of the patients. Finally, HL60 cells were used to analyze induction of miR-320c in inflammatory conditions. Results: Overexpression of miR-320 members in human HepG2 cells led to inhibition of SERPINA1 expression. Analysis of miR-320c expression in patient's samples revealed significantly increased expression of miR-320c in individuals with pulmonary disease. Additionally, HL60 cells treated with the pro-inflammatory factor lipopolysaccharide (LPS) showed increase in miR-320c expression, suggesting that miR-320c responds to inflammation. Conclusion: Our findings demonstrate that miR-320c inhibits SERPINA1 expression in a hepatic cell line and its levels in blood are associated with lung disease in a cohort of patients with different AAT serum levels. These results suggest that miR-320c can play a role in AAT regulation and could be a biomarker of inflammatory processes in pulmonary diseases. (AU)

Introducción: La deficiencia de alfa-1 antitripsina (DAAT) es una condición genética que produce enfermedad pulmonar y hepática con una gran variabilidad clínica. Los microARN se han identificado como modificadores de la gravedad de algunas enfermedades y su desregulación podría desempeñar un papel en la heterogeneidad de esta enfermedad. Los miembros de la familia miR-320 regulan múltiples procesos, incluyendo la inflamación, y tienen lugares de unión en la región 3’UTR del gen SERPINA1. En este estudio exploramos la implicación del miR-320c, un miembro de esta familia, en la DAAT. Métodos: Primero se realizaron estudios in vitro para demostrar la regulación del gen SERPINA1 por parte del miR-320. Además, se analizó la expresión de miR-320c en la sangre de 98 individuos con diferentes niveles de AAT mediante PCR cuantitativa y se correlacionó con los parámetros clínicos. Por último, se utilizaron células HL60 para analizar la inducción de miR-320c en condiciones inflamatorias. Resultados: La sobreexpresión del miR-320 en células HepG2 inhibía la expresión del gen SERPINA1. El análisis de expresión de miR-320c en los pacientes reveló una expresión significativamente aumentada en los casos con enfermedad pulmonar. Por otro lado, las células HL60 tratadas con LPS como factor proinflamatorio mostraron un aumento de expresión de miR-320c, lo que sugiere que este miARN responde a procesos inflamatorios. Conclusión: Nuestros resultados demuestran que el miR-320c inhibe la expresión de SERPINA1 en células hepáticas y que sus niveles en sangre están asociados con la presencia de enfermedad pulmonar en pacientes con diferentes niveles de AAT. Esto sugiere que el miR-320c desempeña un papel en la regulación de los niveles de AAT y podría ser un biomarcador de inflamación en enfermedades pulmonares. (AU)

The Plasma Levels of hsa-miR-19b-3p, hsa-miR-125b-5p, and hsamiR- 320c in Patients with Asthma, COPD and Asthma-COPD Overlap Syndrome (ACOS).

BACKGROUND: Bronchial Asthma (BA) and Chronic Obstructive Pulmonary Disease (COPD) are chronic airway inflammation diseases. In recent years, patients with signs of both BA and COPD have been assigned to a separate group as Asthma-COPD Overlap Syndrome (ACOS). Free-circulating plasma microRNAs are considered as potential biomarkers of pulmonology diseases, including BA, COPD, and ACOS. OBJECTIVE: This study aimed to investigate the expression level of free-circulating plasma microRNAs, hsa-miR-19b-3p, hsa-miR-125b-5p, and hsa-miR-320c in patients with BA, COPD and ACOS for the detection and validation of new microRNAs as biomarkers for chronic lung diseases. METHODS: The relative expression levels of 720 microRNAs were evaluated by Real Time-Polymerase Chain Reaction (RT-PCR) in patients with COPD and BA. Three upregulated microRNAs (hsa-miR-19b-3p, hsa-miR-125b-5p and hsa-miR-320c) were selected for further study. The obtained data were analyzed using the microRNA PCR Array Data Analysis tool. The sensitivity and specificity were estimated using the area under the Receiver Operating Characteristics curve (ROC). RESULTS: The expression level of free-circulating hsa-miR-19b-3p was decreased in the blood plasma of patients with BA and ACOS, and increased in patients with COPD. hsa-miR-125b-5p was downregulated in the blood plasma of patients with COPD and upregulated in patients with BA and ACOS. hsa-miR-320c was downregulated in the blood plasma of patients with BA, and upregulated in patients with COPD and ACOS. The ROC curves of patients with BA for hsa-miR-19b-3p, patients with ACOS for hsa-miR-125b-5p, and patients with COPD for hsa-miR-320c revealed the probability of them as valuable biomarkers with AUCs of 0.824, 0.825, and 0.855, respectively. CONCLUSION: Our study revealed three promising biomarkers for the diagnosis of COPD, BA and ACOS.

Long noncoding RNA TUG1 regulates degradation of chondrocyte extracellular matrix via miR-320c/MMP-13 axis in osteoarthritis.

Osteoarthritis (OA) is a common chronic joint disease. This study aimed to explore the function of long noncoding RNA taurine-upregulated gene 1 (TUG1) in the progression and initiation of OA. Levels of TUG1, microRNA-320c (miR-320c) and fucosyltransferase 4 (FUT4) were examined via quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide and flow cytometry assays were used to detect cell viability and apoptosis, respectively. The expression of relative proteins was measured using Western blot. The interaction between miR-320c and TUG1 or FUT4 was confirmed utilizing dual-luciferase reporter and RNA immunoprecipitation assays. In this study, levels of TUG1 and FUT4 were distinctly upregulated, but miR-320c level significantly decreased in OA tissues and chondrocytes derived from OA tissues as well as in IL-1ß-stimulated C28/I2 cells. Mechanically, TUG1 sponged miR-320c and miR-320c targeted FUT4. In addition, TUG1 knockdown accelerated cell proliferation and repressed apoptosis and extracellular matrix (ECM) degradation in IL-1ß-induced C28/I2 cells, whereas these effects of TUG1 deletion were rescued by either miR-320c inhibitor or FUT4 upregulation. Meanwhile, TUG1 sponged miR-320c to regulate FUT4 expression in IL-1ß-induced C28/I2 cells. Collectively, TUG1 modulated cell proliferation, apoptosis and ECM degradation in IL-1ß-induced C28/I2 cells via the miR-320c/FUT4 axis, providing a new insight into the OA treatment.

MicroRNA-320c inhibits articular chondrocytes proliferation and induces apoptosis by targeting mitogen-activated protein kinase 1 (MAPK1).

AIM: To clarify the interaction of microRNA-320c (miR-320c) and mitogen-activated protein kinase 1 (MAPK1), and to investigate the effects of miR-320c on articular chondroctye proliferation and apoptosis. METHODS: Lentiviral expression vectors were constructed and dual luciferase assays containing MAPK1 3'-untranslated regions (3'-UTRs) were performed. Small hairpin RNA (shRNA) was utilized to modulate MAPK1 expression. The messenger RNA and protein expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting respectively. Cell Counting Kit-8 and flow cytometry were conducted to detect the proliferation and apoptosis of Human Chondrocyte-articular (HC-a) cells. Besides that, the influences of miR-320c and MAPK1 on MAPK pathway activation were also evaluated. RESULTS: Our data identified MAPK1 as a direct target gene of miR-320c, and miR-320c can negatively regulate MAPK1 expression by directly binding to MAPK1 3'-UTR in HC-a cells. Further functional study displayed that miR-320c overexpression and MAPK1 shRNA significantly suppressed the proliferation of HC-a cells and promoted cell apoptosis. Meanwhile, MAPK1 shRNA could attenuate miR-320c inhibitor promotive effects on HC-a cell proliferation and reverse its inhibitory effect on cell apoptosis. MAPK1 overexpression could rescue the inhibitory effect of miR-320c on HC-a cell proliferation, and weaken the accelerating effect of miR-320c on cell apoptosis. However, neither miR-320c or MAPK1 shRNA regulate the expression of c-JUN, JNK and c-Fos. CONCLUSION: miR-320c inhibits articular chondrocyte proliferation and induces apoptosis by targeting MAPK1, suggesting that miR-320c perhaps participates in the pathogenesis of osteoarthritis and acts as a potential target for the therapeutic treatment of osteoarthritis.

miR-320c Regulates SERPINA1 Expression and Is Induced in Patients With Pulmonary Disease.

INTRODUCTION: Alpha-1 antitrypsin deficiency (AATD) is a genetic condition resulting in lung and liver disease with a great clinical variability. MicroRNAs have been identified as disease modifiers; therefore miRNA deregulation could play an important role in disease heterogeneity. Members of miR-320 family are involved in regulating of multiple processes including inflammation, and have potential specific binding sites in the 3'UTR region of SERPINA1 gene. In this study we explore the involvement of miR-320c, a member of this family, in this disease. METHODS: Firstly in vitro studies were carried out to demonstrate regulation of SERPINA1 gene by miR-320. Furthermore, the expression of miR-320c was analyzed in the blood of 98 individuals with different AAT serum levels by using quantitative PCR and expression was correlated to clinical parameters of the patients. Finally, HL60 cells were used to analyze induction of miR-320c in inflammatory conditions. RESULTS: Overexpression of miR-320 members in human HepG2 cells led to inhibition of SERPINA1 expression. Analysis of miR-320c expression in patient's samples revealed significantly increased expression of miR-320c in individuals with pulmonary disease. Additionally, HL60 cells treated with the pro-inflammatory factor lipopolysaccharide (LPS) showed increase in miR-320c expression, suggesting that miR-320c responds to inflammation. CONCLUSION: Our findings demonstrate that miR-320c inhibits SERPINA1 expression in a hepatic cell line and its levels in blood are associated with lung disease in a cohort of patients with different AAT serum levels. These results suggest that miR-320c can play a role in AAT regulation and could be a biomarker of inflammatory processes in pulmonary diseases.

miR-320c Regulates SERPINA1 Expression and Is Induced in Patients With Pulmonary Disease.

INTRODUCTION: Alpha-1 antitrypsin deficiency (AATD) is a genetic condition resulting in lung and liver disease with a great clinical variability. MicroRNAs have been identified as disease modifiers; therefore miRNA deregulation could play an important role in disease heterogeneity. Members of miR-320 family are involved in regulating of multiple processes including inflammation, and have potential specific binding sites in the 3'UTR region of SERPINA1 gene. In this study we explore the involvement of miR-320c, a member of this family, in this disease. METHODS: Firstly in vitro studies were carried out to demonstrate regulation of SERPINA1 gene by miR-320. Furthermore, the expression of miR-320c was analyzed in the blood of 98 individuals with different AAT serum levels by using quantitative PCR and expression was correlated to clinical parameters of the patients. Finally, HL60 cells were used to analyze induction of miR-320c in inflammatory conditions. RESULTS: Overexpression of miR-320 members in human HepG2 cells led to inhibition of SERPINA1 expression. Analysis of miR-320c expression in patient's samples revealed significantly increased expression of miR-320c in individuals with pulmonary disease. Additionally, HL60 cells treated with the pro-inflammatory factor lipopolysaccharide (LPS) showed increase in miR-320c expression, suggesting that miR-320c responds to inflammation. CONCLUSION: Our findings demonstrate that miR-320c inhibits SERPINA1 expression in a hepatic cell line and its levels in blood are associated with lung disease in a cohort of patients with different AAT serum levels. These results suggest that miR-320c can play a role in AAT regulation and could be a biomarker of inflammatory processes in pulmonary diseases.

CDK6 and miR-320c Co-Regulate Chondrocyte Catabolism Through NF-κB Signaling Pathways.

BACKGROUND/AIMS: Cyclin-dependent kinase 6 (CDK6) regulates inflammatory response and cell differentiation. This study sought to determine whether CDK6 and miR-320c co-regulate chondrogenesis and inflammation. METHODS: Utilizing quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC), CDK6 and miR-320c expression were assessed in a micromass culture of human bone mesenchymal stem cells that underwent chondrogenesis in vitro as well as in chondrocytes from E16.5 mouse forelimbs. Normal chondrocytes were transfected with miR-320c mimic, miR-320c inhibitor, or CDK6-siRNA. Luciferase reporter assay results confirmed that miR-320c directly targets CDK6 by interacting with the 3'-untranslated region (3'-UTR) of its mRNA. qRT-PCR, Western blotting, and Cell Counting Kit-8 were subsequently used to evaluate the effects of miR-320c overexpression and CDK6 inhibition on inflammatory factor expression, as well as to investigate the effects of NF-kB and MAPK signaling pathway activation on IL-1ß-induced chondrocyte inflammation. RESULTS: Our results show that miR-320c expression increased during the middle stage and decreased during the late stage of hBMSC chondrogenic differentiation. In contrast, CDK6 expression decreased during the middle stage and increased during the late stage of hBMSC chondrogenic differentiation. Moreover, CDK6 expression increased in severe OA cartilage and in hypertrophic chondrocytes of mouse forelimbs at E16.5. Results of the luciferase reporter assay showed that miR-320c modulated CDK6 expression by binding to the 3'-UTR of its mRNA. miR-320c overexpression and CDK6 inhibition repressed IL-1ß-induced expression of inflammatory factors and regulated the NF-kB signaling pathway. CONCLUSION: CDK6 and miR-320c co-regulate hBMSC chondrogenesis and IL-1ß-induced chondrocyte inflammation through the NF-kB signaling pathway, suggesting that miR-320c and CDK6 inhibitors can be used to repress catabolism in human chondrocytes.