Circulating miR-129-3p in combination with clinical factors predicts vascular calcification in hemodialysis patients.

Background: Vascular calcification (VC) commonly occurs and seriously increases the risk of cardiovascular events and mortality in patients with hemodialysis. For optimizing individual management, we will develop a diagnostic multivariable prediction model for evaluating the probability of VC. Methods: The study was conducted in four steps. First, identification of miRNAs regulating osteogenic differentiation of vascular smooth muscle cells (VSMCs) in calcified condition. Second, observing the role of miR-129-3p on VC in vitro and the association between circulating miR-129-3p and VC in hemodialysis patients. Third, collecting all indicators related to VC as candidate variables, screening predictors from the candidate variables by Lasso regression, developing the prediction model by logistic regression and showing it as a nomogram in training cohort. Last, verifying predictive performance of the model in validation cohort. Results: In cell experiments, miR-129-3p was found to attenuate vascular calcification, and in human, serum miR-129-3p exhibited a negative correlation with vascular calcification, suggesting that miR-129-3p could be one of the candidate predictor variables. Regression analysis demonstrated that miR-129-3p, age, dialysis duration and smoking were valid factors to establish the prediction model and nomogram for VC. The area under receiver operating characteristic curve of the model was 0.8698. The calibration curve showed that predicted probability of the model was in good agreement with actual probability and decision curve analysis indicated better net benefit of the model. Furthermore, internal validation through bootstrap process and external validation by another independent cohort confirmed the stability of the model. Conclusion: We build a diagnostic prediction model and present it as an intuitive tool based on miR-129-3p and clinical indicators to evaluate the probability of VC in hemodialysis patients, facilitating risk stratification and effective decision, which may be of great importance for reducing the risk of serious cardiovascular events.

MiR-129-3p regulates ferroptosis in the liver of Selenium-deficient broilers by targeting SLC7A11.

Selenium (Se) has been proven to be an essential trace element for organism. Se deficiency in poultry can cause widespread damage, such as exudative diathesis. The liver is not only the main organ of metabolism, but also one of the organs with high Se content in organism. Recent studies have shown that solute carrier family 7 member 11 (SLC7A11) plays a key role in the negative regulation of ferroptosis. In order to explore the mechanism of Se deficiency induces liver ferroptosis in broilers, and the role of microRNAs (miRNAs) in this process, we divided broilers into 2 groups: control group (0.2 mg/kg Se) and Se deficiency group (0.03 mg/kg Se). Hematoxylin-Eosin staining detected liver tissue damage in broilers. Predicted and verified the targeting relationship between miR-129-3p and SLC7A11 through miRDB and dual luciferase report experiments. The genes related to ferroptosis were detected by qRT-PCR and Western Blot. The results showed that the expression level of miR-129-3p mRNA in Se-deficient liver was significantly increased. To understand whether the miR-129-3p/SLC7A11 axis could involve in the process of ferroptosis, our further research showed that overexpression of miR-129-3p could reduce the expression of SLC7A11 and its downstream GCL, GSS, and GPX4, thereby inducing ferroptosis. These data indicates that miR-129-3p affected ferroptosis under Se deficiency conditions through the SLC7A11 pathway. Our research provides a new perspective for the mechanism of Se deficiency on the liver damage.

CircSETDB1 knockdown inhibits the malignant progression of serous ovarian cancer through miR-129-3p-dependent regulation of MAP3K3.

BACKGROUND: Circular RNA (circRNA) is recently found to participate in the regulation of tumor progression, including ovarian cancer. However, the application of circRNA SET domain bifurcated histone lysine methyltransferase 1 (circSETDB1) as a therapeutic target in serous ovarian cancer (SOC) remains to be elucidated. Herein, circSETDB1 role in SOC malignant progression and underlying mechanism are revealed. METHODS: The expression of circSETDB1, microRNA-129-3p (miR-129-3p) and mitogen-activated protein kinase kinase kinase 3 (MAP3K3) messenger RNA (mRNA) was detected by quantitative real-time polymerase chain reaction. Protein abundance was determined by western blot analysis. Cell proliferation, apoptosis, invasion and migration were demonstrated by cell counting kit-8 and 5-Ethynyl-29-deoxyuridine assays, flow cytometry analysis, transwell invasion assay and wound-healing assay, respectively. The interaction between miR-129-3p and circSETDB1 or MAP3K3 was predicted by online database, and identified by mechanism assays. The effect of circSETDB1 knockdown on tumor formation in vivo was unveiled by mouse model experiment. RESULTS: CircSETDB1 and MAP3K3 expression were apparently upregulated, whereas miR-129-3p expression was downregulated in SOC tissues and cells in comparison with normal fallopian tube tissues or normal ovarian epithelial cells. CircSETDB1 knockdown inhibited cell proliferation, invasion and migration, but induced cell apoptosis in SOC cells. Additionally, miR-129-3p inhibitor impaired circSETDB1 silencing-mediated SOC malignant progression. MiR-129-3p repressed SOC cell processes via binding to MAP3K3. Furthermore, circSETDB1 knockdown suppressed tumor growth in vivo. CONCLUSION: CircSETDB1 silencing repressed SOC malignant progression through miR-129-3p/MAP3K3 pathway. This study supports circSETDB1 as a new therapeutic target for SOC.

1. CircSETDB1 expression was increased in SOC tissues and cells.2. CircSETDB1 silencing repressed the malignancy of SOC cells.3. CircSETDB1 mediated SOC malignant progression by interacting with miR-129-3p.4. MAP3K3 served as a target gene of miR-129-3p.5. CircSETDB1 knockdown inhibited tumor formation in vivo.

LncRNA SNHG1 regulates immune escape of renal cell carcinoma by targeting miR-129-3p to activate STAT3 and PD-L1.

Immune escape of renal cell carcinoma (RCC) impacts patient survival. However, the molecular mechanism of long noncoding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) in RCC immune escape remains unclear. Quantitative real-time PCR and western blotting results revealed that the expression of lncRNA SNHG1 and STAT3 were upregulated in RCC tissues and cells and that the expression of miR-129-3p was downregulated. Enzyme-linked immunosorbent assay results revealed the increased levels of immune-related factors (interferon-γ, tumour necrosis factor α, and interleukin-2) in RCC tissues. SNHG1 knockdown or miR-129-3p overexpression inhibited the proliferation and invasion of A498 and 786-O cells, while the proliferation and cytotoxicity of CD8+ T cells increased, which promoted the secretion of immune-related factors. STAT3 overexpression decreased the protective effect of miR-129-3p overexpression on RCC cell immune escape. In addition, miR-129-3p knockdown and STAT3 overexpression decreased the protective effect of lncRNA SNHG1 knockdown on RCC cell immune escape. In addition, PD-L1 expression was downregulated after lncRNA SNHG1 knockdown but upregulated after miR-129-3p knockdown and STAT3 overexpression. Dual-luciferase assays showed that lncRNA SNHG1 targets miR-129-3p, and miR-129-3p targets STAT3. RNA pull-down and RNA immunoprecipitation assays verified the regulatory relationship between SNHG1 and STAT3. In vivo, shSNHG1 prolonged the overall survival of RCC tumour model mice and inhibited RCC tumour growth and immune escape but increased CD8+ T cell infiltration in mice. Our findings provide an experimental basis for elucidating the molecular mechanisms of immune escape by RCC and reveal a novel target to treat this disease.

miR-129-3p Targeting of MCU Protects Against Glucose Fluctuation-Mediated Neuronal Damage via a Mitochondrial-Dependent Intrinsic Apoptotic Pathway.

INTRODUCTION: Glucose fluctuations have an adverse effect on several diabetes-related complications, especially for the nervous system, but the underlying mechanisms are not clear. MicroRNAs are critical regulators of posttranscription in many physiological processes, such as apoptosis. Our study clarified the neuroprotective effects of miR-129-3p targeting mitochondrial calcium uniporter (MCU) in glucose fluctuation-mediated neuronal damage and the specific mechanisms involved. METHODS: The expression of MCU and miR-129-3p was examined by real-time PCR and Western blot in the glucose fluctuation cell model. Dual-luciferase reporter assay was performed to confirm the transcriptional regulation of miR-129-3p by MCU. Fluorescent probe and assay kit assay was used to determine oxidative stress condition. Mitochondrial-dependent intrinsic apoptotic factors were examined by flow cytometry assay, enzyme-linked immunosorbent assay (ELISA), and gene and protein expression assays. RESULTS: We found an upregulation of MCU and downregulation of miR-129-3p in glucose fluctuation-treated primary hippocampal neuronal cells, and miR-129-3p directly targeted MCU. miR-129-3p overexpression produced a dramatic reduction in calcium overload, reactive oxygen species (ROS) generation, GSH-to-GSSG ratio, MMP-2 expression in the mitochondrial-dependent intrinsic apoptosis pathway and an increase in MnSOD activity. Increasing MCU expression rescued the effects of miR-129-3p overexpression. miR-129-3p downregulation produced a significant increase in calcium overload, reactive oxygen species (ROS) generation, MMP-2 expression, cytochrome c release and cell apoptosis, and antioxidant N-acetyl cysteine (NAC) rescued the effects of miR-129-3p downregulation. CONCLUSION: Therefore, miR-129-3p suppressed glucose fluctuation-mediated neuronal damage by targeting MCU via a mitochondrial-dependent intrinsic apoptotic pathway. The miR-129-3p/MCU axis may be a promising therapeutic target for glucose fluctuation-mediated neuronal damage.

miR-129-3p alleviates chondrocyte apoptosis in knee joint fracture-induced osteoarthritis through CPEB1.

BACKGROUND: Osteoarthritis (OA), a refractory disease, is one of the leading contributors for disability worldwide. Since chondrocyte is the only resident cell in cartilage, this study aims to explore the roles of miR-129-3p and CPEB1 in chondrocyte apoptosis in knee joint fracture-induced OA. METHODS: Cartilage was collected from 20 OA patients who underwent total knee replacement (OA group) and 20 patients with knee contusion (normal group). Then, miR-129-3p and CPEB1 levels in the cartilage were quantified by qRT-PCR. Primary rat chondrocytes in the knee were isolated and identified by toluidine blue staining and immunofluorescent staining of type II collagen. OA cellular models were induced by TNF-α treatment, in which miR-129-3p and CPEB1 expressions were assessed. Subsequently, cell viability, apoptosis, and the expression levels of apoptotic protein and caspase-3 were measured. Dual luciferase reporter assay identified the interaction between miR-129-3p and CPEB1. RESULTS: Patients in the OA group had decreased miR-129-3p expression and increased CPEB1 expression than those in the normal group. TNF-α treatment successfully induced the OA cellular model. Downregulated miR-129-3p and upregulated CPEB1 expressions were found in OA-treated chondrocytes. miR-129-3p overexpression or CPEB1 knockdown improved chondrocyte viability and attenuated apoptosis, and vice versa. miR-129-3p negatively regulated CPEB1, thus ameliorating apoptosis and enhancing cell viability. CONCLUSION: miR-129-3p negatively targeted CPEB1 to facilitate chondrocyte viability and hamper apoptosis.

Effects of miR-129-3p on biological functions of prostate cancer cells through targeted regulation of Smad3.

Effects of miR-129-3p on the biological functions of prostate cancer cells through the targeted regulation of Smad3 were investigated. RT-PCR was used to detect the expression levels of miR-129-3p in prostate cancer tissues and cells and its target gene Smad3 mRNA determined by bioinformatics prediction. Correlation between miR-129-3p and Smad3 was analyzed. MTT assay, cell invasion detection, and apoptosis detection were conducted to detect the effects of miR-129-3p and Smad3 on the proliferation, invasion, and apoptosis of prostate cancer cells. The results of RT-qPCR showed that the expression level of miR-129-3p decreased but that of Smad3 increased in the prostate cancer tissue, and the expression levels of the two were significantly and negatively correlated. Additionally, the expression levels were closely related to the degree of tumor differentiation, TNM staging, and lymph node metastasis (P<0.05). Bioinformatics prediction and subsequent experiments proved that Smad3 was the direct target gene of miR-129-3p. Cell detection confirmed that the overexpression of miR-129-3p or the inhibition of Smad3 expression inhibited the proliferation and invasion of prostate cancer cells, promoting apoptosis, and increased the expression level of pro-apoptotic protein Bax, as well as decreased the expression level of anti-apoptotic protein Bcl-2. Inhibition of miR-129-3p expression had the opposite effect to overexpression. miR-129-3p, which may be a new and potential target for the treatment of prostate cancer, can inhibit the proliferation and invasion of prostate cancer cells and promote their apoptosis by directly targeting Smad3.

Nrf2-miR-129-3p-mTOR Axis Controls an miRNA Regulatory Network Involved in HDACi-Induced Autophagy.

Histone deacetylase inhibitors (HDACis) are the recommended treatment for many solid tumors; however, resistance is a major clinical obstacle for their efficacy. High levels of the transcription factor nuclear factor erythroid 2 like-2 (Nrf2) in cancer cells suggest a vital role in chemoresistance, and regulation of autophagy is one mechanism by which Nrf2 mediates chemoresistance. Although the molecular mechanisms underlying this activity are unclear, understanding them may ultimately improve therapeutic outcomes following HDACi treatment. In this study, we found that HDACi treatment increased Nrf2 mRNA and protein levels and enhanced Nrf2 transcriptional activity. Conversely, Nrf2 knockdown or inhibition blocked HDACi-induced autophagy. In addition, a microRNA (miRNA) array identified upregulation of miR-129-3p in response to Nrf2 overexpression. Chromatin immunoprecipitation assays confirmed miR-129-3p to be a direct Nrf2 target. RepTar and RNAhybrid databases indicated mammalian target of rapamycin (mTOR) as a potential miR-129-3p target, which we experimentally confirmed. Finally, Nrf2 inhibition or miR-129-3p in combination with HDACis increased cell death in vitro and in vivo. Collectively, these results demonstrated that Nrf2 regulates mTOR during HDACi-induced autophagy through miRNA-129-3p and inhibition of this pathway could enhance HDACi-mediated cell death.

Avenanthramide A Induces Cellular Senescence via miR-129-3p/Pirh2/p53 Signaling Pathway To Suppress Colon Cancer Growth.

Cellular senescence is the state of irreversible cell cycle arrest that provides a blockade during oncogenic transformation and tumor development. Avenanthramide A (AVN A) is an active ingredient exclusively extracted from oats, which possesses antioxidant, anti-inflammatory, and anticancer activities. However, the underlying mechanism(s) of AVN A in the prevention of cancer progression remains unclear. In the current study, we revealed that AVN A notably attenuated tumor formation in an azoxymethane/dextran sulfate sodium (AOM/DSS) mouse model. AVN A treatment triggered cellular senescence in human colon cancer cells, evidenced by enlarging cellular size, upregulating ß-galactosidase activity, γ-H2AX positive staining, and G1 phase arrest. Moreover, AVN A treatment significantly increased the expression of miR-129-3p, which markedly repressed the E3 ubiquitin ligase Pirh2 and two other targets, IGF2BP3 and CDK6. The Pirh2 silencing by miR-129-3p led to a significant increase in protein levels of p53 and its downstream target p21, which subsequently induced cell senescence. Taken together, our data indicate that miR-129-3p/Pirh2/p53 is a critical signaling pathway in AVN A induced cellular senescence and AVN A could be a potential chemopreventive strategy for cancer treatment.

Tetrahydroxy stilbene glucoside alleviates palmitic acid-induced inflammation and apoptosis in cardiomyocytes by regulating miR-129-3p/Smad3 signaling.

OBJECTIVE: Tetrahydroxy stilbene glucoside (TSG) has been reported to exert a cytoprotective effect against various toxicants. However, the function and mechanism of TSG in palmitic acid (PA)-induced inflammation and apoptosis in cardiomyocytes are still unknown. The present study was designed to investigate the post-transcriptional mechanism in TSG-treated cardiomyocytes' inflammation and apoptosis induced by PA. METHODS: The mRNA and protein levels were assayed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting, respectively. The targeted genes were predicted by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay. Cell proliferation was analyzed by CCK-8 assay. Annexin V-fluorescein isothiocyanate/polyimide (annexin V-FITC/PI) staining was used to evaluate apoptosis using flow cytometry. RESULTS: TSG restricted the detrimental effects, including the activated inflammatory response and apoptosis, of PA in cardiomyocytes, as well as the up-regulation of miR-129-3p and down-regulation of p-Smad3 expression. In addition, bioinformatics and experimental analysis suggested that Smad3 was a direct target of miR-129-3p, which could inhibit or enhance the expression of p-Smad by transfection with miR-129-3p mimics or inhibitors, respectively. Furthermore, our results demonstrated that overexpression of Smad3 reversed the inhibition of inflammation and apoptosis by overexpression of miR-129-3p in PA-stimulated cardiomyocytes. CONCLUSION: TSG targeted to miR-129-3p/Smad3 signaling inhibited PA-induced inflammation and apoptosis in cardiomyocytes.

MiR-129-3p favors intracellular BCG survival in RAW264.7 cells by inhibiting autophagy via Atg4b.

Autophagy plays an important role in the fight against Mycobacterium tuberculosis infection. Massive researches proved that miRNAs could be the regulators of autophagy, which implied miRNAs could favor MTB invasion or latent infection. In our study, multiple bioinformatics databases and software were used to seek and lock the miRNAs associating with regulation of autophagy. Notably, a novel miR-129-3p was found and its target gene Atg4b showed grand potential in mediation of autophagy. Moreover, BCG infection triggered miR-129-3p overexpression in RAW264.7 cells. Up-regulation of miR-129-3p decreased mRNA or protein level of Atg4b and resulted in the inhibition of autophagy. The antagomir of miR-129-3p had the opposite impact. The LC3 puncta formation in RAW264.7 cells were also affected after transfection of miR-129-3p mimic or antagomir. The mRFP-GFP-LC3 analysis indicated that mimic of miR-129-3p impaired autophagic flux while antagomir improved autophagy. The CFU assay results showed that miR-129-3p promoted the intracellular survival of BCG in macrophages. Consequently, these data suggested that miR-129-3p could favor MTB survival by inhibiting autophagy via Atg4b.

Chidamide-induced ROS accumulation and miR-129-3p-dependent cell cycle arrest in non-small lung cancer cells.

BACKGROUND: Epigenetic therapy is a promising popular treatment modality for various cancers. Histone modification and miRNA should not be underestimated in lung cancer. This study aimed to investigate whether chidamide, a histone deacetylase inhibitor (HDACi), which inhibits telomerase activity and induces cell cycle arrest, influences ROS and miRNA production in non-small cell lung cancer (NSCLC) cells. METHODS: H1355 and A549 were treated with chidamide. The analysis of DNA content was measured by FACSCalibur equipped with a 488 nm laser. H1355 cells were transfected with miR-129-3p mimic by Lipofectamine2000. Telomerase activity was performed on the telomeric repeat amplification protocol (TRAP) assay. Detection of thymidylate synthase (TS), p21, p53, pRB, and ß-actin, were performed by western blot analysis. RESULTS: Our data showed that expression of TS, p21, and pRB were altered in the presence of chidamide by PCR and western blot. Using BrdU-incorporation analysis, we found that chidamide induced G1 arrest through the regulation of the TS gene by miR-129-3p. Chidamide was shown to suppress telomerase activity in the TRAP assay and reduced the expression of human telomerase reverse transcriptase (hTERT) by PCR and q-PCR in H1355 and A549 cells. Chidamide increased the generation of reactive oxygen species (ROS) by flow cytometry. N-acetyl cysteine (NAC), a ROS scavenger, attenuated chidamide-induced telomerase activity inhibition. CONCLUSION: Chidamide repressed telomerase activity through ROS accumulation and cell cycle arrest by miR-129-3p upregulation in both H1355 and A549 cells. This is the first study to demonstrate that chidamide induces miR-129-3p upregulation and ROS accumulation, leading to cell cycle arrest.

miR-129-5p and -3p co-target WWP1 to suppress gastric cancer proliferation and migration.

Gastric cancer (GC) is a worldwide health problem. Uncovering the underlining molecular mechanisms of GC is of vital significance. Here, we identified a novel oncogene WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) in GC. WWP1 could promote GC cell proliferation and migration in vitro and expedite GC growth in vivo. We also found out two microRNAs (miRNAs): miR-129-5p and -3p could both target WWP1. Interestingly, miR-129-5p bound to the CDS region of WWP1 mRNA. The miR-129 pairs (miR-129-5p and -3p) play pivotal roles in GC to suppress its proliferation and migration in vitro and slow down GC growth in vivo by repressing WWP1. In summary, we identified two tumor suppressive miRNAs which share the same precursor that could regulate the same oncogene WWP1 in GC. Our finding would add new route for GC research and treatment.

MicroRNA-129-3p functions as a tumor suppressor in serous ovarian cancer by targeting BZW1.

The aberrant expression of microRNAs (miRNAs) underlies a series of human diseases, including ovarian cancers. In our previous study, we found that miR-129-1-3p and miR-129-2-3p levels were significantly decreased in serous ovarian cancer via a microarray and quantitative PCR. In this study, we investigated the pathological role of miR-129-3p in an ovarian cancer cell line, SKOV3 cells. The results demonstrated that miR-129-3p overexpression distinctively inhibited the proliferation, migration, and invasion of ovarian cancer cells. The regulator of cell cycling, BZW1 (basic leucine zipper and W2 domains 1), was validated as a novel direct target of miR-129-3p. Specifically, miR-129-3p bound directly to the 3' untranslated region of BZW1 and suppressed its expression. Our results indicate that miR-129-3p serves as a tumor suppressor by targeting BZW1 in ovarian cancer cells and highlight that the restoration of miR-129-3p might be a novel therapeutic strategy for ovarian cancer.

Methylation-associated silencing of microRNA-129-3p promotes epithelial-mesenchymal transition, invasion and metastasis of hepatocelluar cancer by targeting Aurora-A.

Metastasis and recurrence has become one major obstacle for further improving the survival of hepatocelluar cancer (HCC) patients. Therefore, it is critical to elucidate the mechanisms involved in HCC metastasis. This study aimed to investigate the roles of microRNA (miR)-129-3p in HCC metastasis and its possible molecular mechanisms. By using microarray analysis to compare levels of different miRNAs in HCC tissues with or without lymph node metastasis (LNM), we showed that HCC tissues with LNM had reduced levels of miR-129-3p, which was related to its promoter hypermethylation and correlated with tumor metastasis, recurrence and poor prognosis. Gain - and loss - of - function assays indicated that re-expression of miR-129-3p could reverse epithelial-mesenchymal transition (EMT), and reduce in vitro invasion and in vivo metastasis of HCC cells. Aurora-A, a serine/threonine protein kinase, was identified as a direct target of miR-129-3p. Knockdown of Aurora-A phenocopied the effect of miR-129-3p overexpression on HCC metastasis. In addition, Aurora-A upregulation could partially rescue the effect of miR-129-3p. We further demonstrated that activation of PI3K/Akt and p38-MAPK signalings were involved in miR-129-3p-mediated HCC metastasis. These findings suggest that methylation-mediated miR-129-3p downregulation promotes EMT, in vitro invasion and in vivo metastasis of HCC cells via activation of PI3K/Akt and p38-MAPK signalings partially by targeting Aurora-A. Therefore, miR-129-3p may be a novel prognostic biomarker and potential therapeutic target for HCC.

miR-129-3p controls centrosome number in metastatic prostate cancer cells by repressing CP110.

The centrosome plays a key role in cancer invasion and metastasis. However, it is unclear how abnormal centrosome numbers are regulated when prostate cancer (PCa) cells become metastatic. CP110 was previously described for its contribution of centrosome amplification (CA) and early development of aggressive cell behaviour. However its regulation in metastatic cells remains unclear. Here we identified miR-129-3p as a novel metastatic microRNA. CP110 was identified as its target protein. In PCa cells that have metastatic capacity, CP110 expression was repressed by miR-129-3p. High miR-129-3p expression levels increased cell invasion, while increasing CP110 levels decreased cell invasion. Overexpression of CP110 in metastatic PCa cells resulted in a decrease in the number of metastasis. In tissues of PCa patients, low CP110 and high miR-129-3p expression levels correlated with metastasis, but not with the expression of genes related to EMT. Furthermore, overexpression of CP110 in metastatic PCa cells resulted in excessive-CA (E-CA), and a change in F-actin distribution which is in agreement with their reduced metastatic capacity. Our data demonstrate that miR-129-3p functions as a CA gatekeeper in metastatic PCa cells by maintaining pro-metastatic centrosome amplification (CA) and preventing anti-metastatic E-CA.