miR-133b Expression Research Articles

Dihydroartemisinin ameliorates palmitate-induced apoptosis in cardiomyocytes via regulation on miR-133b/Sirt1 axis

Excessive fat ectopically deposited in the non-adipose tissues is considered as one of the leading causes of myopathy. The aim of this study was to investigate the role of Dihydroartemisinin (DHA) in palmitate (PAL)-incubated H9c2 cells (lipotoxicity-induced cell injury model). Cell viability of PAL-treated cells was determined by MTT assay, and apoptotic regulators were examined by qRT-PCR and western blot analysis, in the absence or in the presence of DHA, respectively. Expression levels of miR-133b and Sirt1 were also evaluated by qRT-PCR and western blotting examination. PAL decreased the viability of H9c2 cells and enhanced the expression of apoptotic genes. DHA reversed the effect of PAL on cell viability and lowed the level of Caspase3 and Bax. It also lowered the expression of miR-133b, while enhanced the expression of Bcl-2. Sirt1 was revealed as target of miR-133b through transcriptional regulation and the process was affected by DHA. DHA partially protected against the PAL-induced lipotoxicity by influencing the expression of miR-133b that hindered the activity of Sirt1. DHA may be used as a potential treatment in clinical management for lipotoxicity induced heart complications.

Age-Related Exosomal and Endogenous Expression Patterns of miR-1, miR-133a, miR-133b, and miR-206 in Skeletal Muscles.

Skeletal muscle growth and maintenance depend on two tightly regulated processes, myogenesis and muscle regeneration. Both processes involve a series of crucial regulatory molecules including muscle-specific microRNAs, known as myomiRs. We recently showed that four myomiRs, miR-1, miR-133a, miR-133b, and miR-206, are encapsulated within muscle-derived exosomes and participate in local skeletal muscle communication. Although these four myomiRs have been extensively studied for their function in muscles, no information exists regarding their endogenous and exosomal levels across age. Here we aimed to identify any age-related changes in the endogenous and muscle-derived exosomal myomiR levels during acute skeletal muscle growth. The four endogenous and muscle-derived myomiRs were investigated in five skeletal muscles (extensor digitorum longus, soleus, tibialis anterior, gastrocnemius, and quadriceps) of 2-week–1-year-old wild-type male mice. The expression of miR-1, miR-133a, and miR-133b was found to increase rapidly until adolescence in all skeletal muscles, whereas during adulthood it remained relatively stable. By contrast, endogenous miR-206 levels were observed to decrease with age in all muscles, except for soleus. Differential expression of the four myomiRs is also inversely reflected on the production of two protein targets; serum response factor and connexin 43. Muscle-derived exosomal miR-1, miR-133a, and miR-133b levels were found to increase until the early adolescence, before reaching a plateau phase. Soleus was found to be the only skeletal muscle to release exosomes enriched in miR-206. In this study, we showed for the first time an in-depth longitudinal analysis of the endogenous and exosomal levels of the four myomiRs during skeletal muscle development. We showed that the endogenous expression and extracellular secretion of the four myomiRs are associated to the function and size of skeletal muscles as the mice age. Overall, our findings provide new insights for the myomiRs’ significant role in the first year of life in mice.

Effects of miR-133b on oxLDL-induced vascular endothelial cell injury by targeting SGTB

Objective: To investigate the effect of microRNA-133b (miR-133b) on oxidized low density lipoprotein (oxLDL) induced vascular endothelial cell injury by targeting small protein molecules rich in glutamine 34-tetrapeptide repeats (SGTB). Methods: Human umbilical vein endothelial cells (EVC-304) were induced by 100 μg/ml oxLDL for 24 h to construct a vascular endothelial cell injury model. EVC-304 cells were divided into control group, oxLDL group (oxLDL treatment), oxLDL+miR-NC group (transfectted with 20 nmol/L miR-NC+oxLDL treatment), oxLDL+miR-133b group (transfectted with 20 nmol/L miR-133b mimics +oxLDL treatment), oxLDL+si-NC group (transfectted with 20 nmol/L si-NC+oxLDL treatment), oxLDL+si-SGTB group (transfected with 20 nmol/L si-SGTB+oxLDL treatment), oxLDL+miR-133b+ pcDNA group (transfected with 20 nmol/L si-SGTB and pcDNA+oxLDL), oxLDL+miR-133b+pcDNA-SGTB group (transfected with 20 nmol/L si-SGTB and pcDNA-SGTB), 9 wells in each group. Real-time quantitative PCR (qRT-PCR) and Western blot were used to detect the expression levels of miR-133b and SGTB; flow cytometry was used to detect cell apoptosis; kits were used to detect malondialdehyde (MDA) content and the activities of superoxide disproportionation enzyme (SOD) and glutathione peroxidase (GSH-Px). The expression levels of Bcl-2 and Bax protein were detected by Western blot. The dual luciferase reporter gene assay and Western blot were used to verify the targeted and regulatory between miR-133b and SGTB. Results: Compared with the control group, the expressions of miR-133b and Bcl-2 in EVC-304 cells were decreased significantly after oxLDL induction, while the expression levels of SGTB and Bax were sincreased ignificantly (P＜0.05), the MDA content and apoptosis rate were increased significantly (P＜0.05), and the activities of SOD and GSH-Px were decreased significantly (P＜0.05). Over-expression of miR-133b or interfering with SGTB inhibited oxLDL-induced apoptosis and oxidative stress in EVC-304 cells (P＜ 0.05). miR-133b directly bound to SGTB, miR-133b overexpression significantly down-regulated SGTB expression (P＜0.05), miR-133b inhibition significantly up-regulated SGTB expression (P＜0.05) Over-expression of SGTB reversed the effect of over-expressing miR-133b on oxLDL-induced vascular endothelial cell injury (P＜0.05). Conclusion: miR-133b could attenuate oxidative stress damage and apoptosis induced by oxLDL in vascular endothelial cells by targeting and inhibiting SGTB expression.

MiR-133b regulates oxidative stress injury of trophoblasts in preeclampsia by mediating the JAK2/STAT3 signaling pathway.

Preeclampsia (PE) is a pregnancy-related syndrome. Aberrant placental microRNAs (miRNAs) expression might associate with PE, including miR-133b. However, its role in the pathogenesis of preeclampsia remains elusive. Therefore, this study explored the role of miR-133b in oxidative stress injury of trophoblasts in preeclampsia (PE) by mediating the JAK2/STAT3 signaling pathway. Placental tissues were collected from PE patients to detect the expression of miR-133b and JAK2/STAT3. Then, in vitro experiments were performed on human extravillous trophoblast-derived HTR-8/SVneo cells, which were divided into Normal, hypoxia/reoxygenation (H/R), H/R + miR-NC, H/R + miR-133b inhibitor, H/R + JAK2 siRNA and H/R + miR-133b inhibitor + JAK2 siRNA groups. Cell invasion and migration abilities were detected by Transwell and wound healing assays, while apoptosis was detected by flow cytometry. The intracellular oxidative stress levels were also measured. Furthermore, the expression of miR-133b and the JAK2/STAT3 pathway was determined by qRT-PCR and Western blotting. We found that miR-133b was up-regulated, with decreases in JAK2 and p-STAT3/STAT3 in placental tissues of PE patients. Additionally, HTR8/SVneo cells in the H/R group had decreased invasion and migration abilities with increased apoptotic rates and oxidative stress levels. Moreover, the expression of miR-133b was up-regulated with decreases in p-JAK2 and p-STAT3 in H/R-treated HTR8/SVneo cells. These indicators in the H/R + miR-133b inhibitor group were ameliorated in comparison with those in the H/R group but deteriorated in the H/R + JAK2 siRNA group. Moreover, JAK2 siRNA reversed the positive effect of the miR-133b inhibitor on the invasion and migration abilities of trophoblasts. In summary, inhibiting miR-133b may improve oxidative stress injury to promote the migration and invasion of trophoblasts and suppress apoptosis by activating the JAK2/STAT3 pathway.

LncRNA-ZFAS1 promotes the progression of endometrial carcinoma by targeting miR-34b to regulate VEGFA expression.

Zinc finger nuclear transcription factor, X-box binding 1-type containing 1 antisense RNA 1 (ZFAS1) functions as an oncogenic long noncoding RNA (lncRNA) to promote proliferation and metastasis of endometrial carcinoma cell; however, the underlying mechanism has not been fully understood. First, RT-qPCR analysis of endometrial carcinoma tissues and cells showed that ZFAS1 was enriched in endometrial carcinoma tissues and cells. miR-34b was reduced in endometrial carcinoma and suggested negative correlation with ZFAS1 in endometrial carcinoma. Second, functional assays demonstrated that siRNA-mediated silence of ZFAS1 suppressed endometrial carcinoma cell proliferation and metastasis. Third, ZFAS1 bind to miR-34b and negatively regulate expression of miR-34b in endometrial carcinoma cells. miR-34b also bind to and negatively regulate expression of vascular endothelial growth factor A (VEGFA) in endometrial carcinoma cells. Lastly, knockdown of miR-34b counteracted with the suppressive effects of ZFAS1 silence on endometrial carcinoma cell proliferation and metastasis. In conclusion, lncRNA ZFAS1 functioned as an oncogene to promote endometrial carcinoma cell proliferation and metastasis through miR-34b/VEGFA axis.

Dysregulation of myomiRs as common pathogenic feature associated with muscle atrophy in ALS, SMA and SBMA: Evidence from animal models and human patients

Motor neuron diseases (MNDs), including amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA) and spinal and bulbar muscular atrophy (SBMA), are a heterogeneous group of neurodegenerative disorders characterized by motor neuron loss. Despite variability in onset, progression and genetic causes, these disorders share a common involvement of skeletal muscle that is suspected to have a relevant role in MND pathogenesis. Due to the key role of muscle-specific microRNAs (myomiRs) in muscle development, here we investigated the expression of miR-206, miR-133a, miR-133b and miR-1 myomiRs, and their target genes, in G93A-SOD1 ALS, Δ7SMA and KI-SBMA mouse muscle during disease progression.

LncRNA TUG1 acts as a competing endogenous RNA to mediate CTGF expression by sponging miR-133b in myocardial fibrosis after myocardial infarction.

Myocardial fibrosis (MF) is one of the basic causes of many cardiovascular diseases. Noncoding RNAs (ncRNAs), including microRNA (miRNA) and long noncoding RNA (lncRNA), have been reported to play an indispensable role in MF. The current work is focused on investigating the biological role of lncRNA taurine upregulation gene 1 (TUG1) in activating cardiac myofibroblasts as well as the underlying mechanism. The outcome revealed that after myocardial infarction TUG1 expression increased and miR-133b expression decreased in the rat model of MF. The expression level of TUG1 increased following AngII treatment in cardiac myofibroblast. TUG1 knockdown inhibited the Ang-II induced cardiac myofibroblast activation and TUG1 overexpression increased proliferation and collagen generation of cardiac myofibroblasts. Bioinformatic prediction programs predicted that TUG1 had MRE directly combined with miR-133b seed sequence, luciferase activity, and RIP experiments indicated that TUG1, acted as a sponger and interacted with miR-133b in cardiac myofibroblasts. Furthermore, a target of miR-133b was CTGF and CTGF knockdown counteracted the promotion of MF by miR-133b knockdown. Collectively, our study suggested that TUG1 mediates CTGF expression by sponging miR-133b in the activation of cardiac myofibroblasts. The current work reveals a unique role of the TUG1/miR-133b/CTGF axis in MF, thus suggesting its immense therapeutic potential in the treatment of cardiac diseases.

Mir-221, mir-190b, mir-363-3p, mir-200c are involved in rat liver ischaemia-reperfusion injury through oxidative stress, apoptosis and endoplasmic reticulum stress.

In this study, it was aimed to investigate the relationship between expression levels of micro-RNAs, endoplasmic reticulum (ER) stress, apoptosis and oxidative stress markers in hepatic ischaemia-reperfusion (IR) injury. Sixteen rats were randomised into two groups: Sham and IR groups. In the IR group, portal vein and hepatic artery were totally clamped with an atraumatic microvascular clamp and 60minutes later unclamped and finally IR model was accomplished (60minutes ischaemia and 60minutes reperfusion). After sacrification, serum insulin-like growth factor-1 (IGF-1), tumour necrosis factor-α (TNF-α), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. Liver tissue samples were evaluated histopathologically. The expression levels of IR1-alpha, Perk, Catalase, Gpx-1, Caspase-3, Bcl-2 genes and miR-33a, miR-221, miR-190b, miR-363-3p, miR-200c, miR-223, miR-133b were measured by quantitative real-time polymerase chain reaction method. Biochemical parameters of the IR group showed significantly higher changes compared with the Sham group (P<.01). Histological tissue damage was significantly prominent in the IR group. ER stress, oxidative stress and apoptosis gene expression levels were significantly higher in the IR group (P<.01). Expression levels of miR-221, miR-190b, miR-363-3p and miR-200c were increased in the IR group compared with the Sham group. No significant difference was found between the two groups in terms of miR-33a, miR-133b and miR-223 expression levels (P>.05). There is a strong need to enlighten the physiopathological and molecular mechanisms of liver IR injury and to find more specific biomarkers for IR damage, and miR-221, miR-190b, miR-363-3p and miR-200c maybe used as potential biomarkers of hepatic IR injury.

Expression levels of serum circulating microRNAs in pediatric patients with ventricular and supraventricular arrhythmias

PurposeAberrant expression of various miRNA species has been implicated in numerous cardiac diseases, e.g., heart failure, hypertrophy, conduction disturbances, and arrhythmogenesis. The aim of this study was to determine whether miR-1, miR-133a, and miR-133b can serve as biomarkers in the diagnosis of ventricular (Va) and supraventricular (SVa) arrhythmias in pediatric patients. Materials and methodsMolecular analysis included 30 patients with SVa or Va (13–17.5 years; 14 boys/16 girls) and 20 non-arrhythmic controls. Arrhythmia was confirmed by 24-h Holter ECG recording. miRNA was extracted from serum using the miRNeasyR Serum/Plasma Kit. miScript SYBR Green PCR Kit (Qiagen) was used to quantify miRNA expression. ResultsThe levels of miR-1 and miR-133a expression were significantly higher in the SVa group than in the controls (p ​= ​0.0327 and p<0.0001, respectively). Additionally, both groups of patients with arrhythmia presented significantly lower expression levels of miR-133b than the controls (p<0.01 for both comparisons). The level of miR-133a expression in the SVa group was significantly higher than in the Va group (p ​= ​0.0124). ROC analysis demonstrated that the expressions of miR-1 and miR-133a could differentiate between the SVa patients and arrhythmia-free controls (AUC ​= ​0.7091, p ​= ​0.07 and AUC ​= ​0.8021, p ​= ​0.007, respectively). Furthermore, the expression of miR-133b was shown to distinguish patients with SVa and Va from the arrhythmia-free controls (AUC ​= ​0.7273, p ​= ​0.07 and AUC ​= ​0.8030, p ​= ​0.04, respectively). ConclusionsmiR-1, miR-133a, and miR-133b have the potential to become diagnostic biomarkers of arrhythmia in pediatric patients.

Identification of microRNAs and gene regulatory networks in cleft lip common in humans and mice.

The etiology of cleft lip with/without cleft palate (CL/P), one of the most frequent craniofacial birth defects worldwide, is complicated by contributions of both genetic and environmental factors. Understanding the etiology of these conditions is essential for developing preventive strategies. This study thus aims to identify regulatory networks of microRNAs (miRNAs), transcriptional factors (TFs) and non-TF genes associated with cleft lip (CL) that are conserved in humans and mice. Notably, we found that miR-27b, miR-133b, miR-205, miR-376b and miR-376c were involved in the regulation of CL-associated gene expression in both humans and mice. Among the candidate miRNAs, the overexpression of miR-27b, miR-133b and miR-205, but not miR-376b and miR-376c, significantly inhibited cell proliferation through suppression of CL-associated genes (miR-27b suppressed PAX9 and RARA; miR-133b suppressed FGFR1, PAX7, and SUMO1; and miR-205 suppressed PAX9 and RARA) in cultured human and mouse lip mesenchymal cells. Taken together, our results suggest that elevated expression of miR-27b, miR-133b and miR-205 may play a crucial role in CL through the suppression of genes associated with CL.

Acetoacetate promotes muscle cell proliferation via the miR-133b/SRF axis through the Mek-Erk-MEF2 pathway.

Acetoacetate (AA) is an important ketone body that is used as an oxidative fuel to supply energy for the cellular activities of various tissues, including the brain and skeletal muscle. We recently revealed a new signaling role for AA by showing that it promotes muscle cell proliferation in vitro, enhances muscle regeneration in vivo, and ameliorates the dystrophic muscle phenotype of Mdx mice. In this study, we provide new molecular insight into this function of AA. We show that AA promotes C2C12 cell proliferation by transcriptionally upregulating the expression of muscle-specific miR-133b, which in turn stimulates muscle cell proliferation by targeting serum response factor. Furthermore, we show that the AA-induced upregulation of miR-133b is transcriptionally mediated by MEF2 via the Mek-Erk1/2 signaling pathway. Mechanistically, our findings provide further convincing evidence that AA acts as signaling metabolite to actively regulate various cellular activities in mammalian cells.

Minor allele of rs55763075 located in MTHFR is associated with the risk of cognitive impairment after anesthesia via modulating miR-34b

This study aimed to investigate the association between cognitive impairment after general anesthesia and rs55763075 polymorphisms. We enrolled and grouped patients undergoing general anesthesia according to their genotypes of rs55763075 polymorphism. Mini–Mental State Examination (MMSE) scoring was performed to evaluate the cognitive status of patients. Quantitative real-time PCR was carried out to analyze the expression of methylenetetrahydrofolate reductase (MTHFR) mRNA and miR-34b while Western blot was performed to evaluate the expression of MTHFR protein. Furthermore, we studied the effect of rs55763075 polymorphism on the expression of MEHFR via luciferase assay. Accordingly, we found that the MMSE score in GG/GA groups was significantly higher than that in AA group. And a significant reduction of MTHFR mRNA expression was observed in the serum and peripheral blood mononuclear cells (PBMCs) of patients carrying AA genotype compared with the patients carrying GG/GA genotypes. Moreover, the MTHFR expression was much lower in the cultured AA-genotyped cells transfected with miR-34b. Luciferase assay results also showed that miR-34b transfection reduced luciferase activity in the cells carrying A allele but not in cells carrying G allele. In summary, the data of this study showed that minor allele (A) of rs55763075 polymorphisms in the 3'-untranslated region of MTHFR mRNA generated a potential binding site for miR-34b, which led to reduced level of folic acid in the patients carrying the AA genotype. Furthermore, we found that the MMSE score of AA-genotyped patients was lower than that of patients carrying GG/GA genotypes.

Expression levels of plasma exosomal miR-124, miR-125b, miR-133b, miR-130a and miR-125b-1-3p in severe asthma patients and normal individuals with emphasis on inflammatory factors

BackgroundIdentification of molecular markers, such as miRNAs is promising for the diagnosis of asthma and its clinical phenotypes. The aim of this study was to examine the changes in the expression of selected microRNAs in plasma exosomal fractions of severe asthma patients. The expression of miRNAs was determined in relation to the changes in inflammatory markers.MethodSevere asthma patients (n = 30) and healthy subjects (n = 30) were selected among the individuals referred to asthma and allergy clinic. Blood was collected from each participant to determine the serum high-sensitive C-reactive protein (hs-CRP) and total IgE. The exosomal fraction of plasma was isolated and processed for quantitation of miR-124, miR-125b, miR-133b, miR-130a and miR-125b-1-3p expression using quantitative real time-PCR (qRT-PCR).ResultsSerum hs-CRP and total IgE were significantly higher in asthma patients compared to controls. Expression of miR-124, miR-133b, and miR-130a was down-regulated in asthma patients as compared to controls (p < 0.0001). However, the expression of miR-125b was substantially higher in patients compared to controls (p < 0.0001). There was no significant difference in the expression of miR-125b-1-3p in the patients and controls. Data analysis revealed that among the miRNAs, changes in miR-125b in severe asthma patients were highly correlated with the serum levels of hs-CRP and IgE.ConclusionOverexpression of miR-125b in severe asthma which was associated with serum IgE and hs-CRP may suggest that this molecule is linked to inflammatory reactions. Up-regulation of miR-125b together with decreased expression of miR-124, miR-133b, and miR-130a may suggest that this miRNA profile is useful for diagnosis and discrimination of clinical phenotypes of asthma.

MiR-34c inhibits PDGF-BB-induced HAVSMCs phenotypic transformation and proliferation via PDGFR-\u03b2/SIRT1 pathway

The purpose of this study was to explore the effect of miR-34c on PDGF-BB-induced HAVSMCs phenotypic transformation and proliferation via PDGFR-β/SIRT1 pathway, so as to find a new method for early diagnosis and treatment of cardiovascular disease. HA-VSMCs were treated with platelet-derived growth factor-BB (PDGF-BB) at 0 h, 12 h, 24 h, 48 h or 36 h to explore the optimal time for phenotypic transformation of VSMCs. And then, PDGF-BB-induced HA-VSMCs were transfected with miR-34c mimics/mimics NC and pcDNA3.1-PDGFR-β/pcDNA3.1-NC to observe cell biological behaviour. CCK8 was used to detect cell proliferation activity. Transwell chamber assay was used to detect cell invasion. Early apoptosis was analyzed by flow cytometry. The expression of α-SMA and Smemb was detected by immunofluorescence staining. The expressions of PDGFR-β, IRF9, Acetyl-NF-κB/p65, Acetyl-p53 and CyclinD1 were analyzed by Western blot analysis. The expression of miR-34a, miR-34b and miR-34c was detected by RT-PCR, and the targeting relationship between miR-34c and PDGFR-β was detected by luciferase reporting assay. The results indicated the proliferation and migration of PDGF-BB-induced HA-VSMCs significantly increased, and apoptosis significantly decreased. Besides, α-SMA decreased significantly, while Smemb increased significantly. Furthermore, expressions of PDGFR-β, IRF9, Acetyl-NF-κB/p65, Acetyl-p53 and CyclinD1 increased significantly, and SIRT1 decreased significantly. Experimental results showed that, miR-34c mimics significantly inhibited cell proliferation and migration, and promoted cell apoptosis, and miR-34c inhibitor had the opposite effects. MiR-34c mimics significantly increased α-SMA expression and decreased Smemb expression, while the opposite effects were reflected after transfection with miR-34c inhibitor. Moreover, miR-34c mimics significantly decreased the expressions of PDGFR-β, IRF9, Acetyl-NF-κB/p65, Acetyl-p53 and CyclinD1, and significantly increased the expression of SIRT1, while miR-34c inhibitor had the opposite effects. Luciferase assay confirmed that PDGFR-β was a potential target of miR-34c. Subsequently, PDGF-BB-induced HA-VSMCs were co-transfected with miR-34c mimics and pcDNA3.1-PDGFR-β. The results indicated that PDGFR-β reversed the biological function of miR-34c mimic. The results revealed the potential application value of miR-34c as a marker molecule of phenotypic transformation, providing a potential target for improving phenotypic transformation.

MiR-133b inhibits cell proliferation, migration, and invasion of lung adenocarcinoma by targeting CDCA8

ObjectiveLung adenocarcinoma (LUAD) is the most common type of lung cancer. This study aims to explore the mechanism by which CDCA8 regulates cell proliferation, invasion, and migration of LUAD, and to generate novel insights into targeted therapy of LUAD. MethodsExpression profiles of mature microRNAs (miRNAs) and mRNAs, along with clinical data of LUAD were downloaded from TCGA database for differential analysis and survival analysis to mine differentially expressed mRNAs. qRT-PCR was used to detect the expression of CDCA8 and miR-133b in LUAD cell lines, and western blot was used to detect protein expression. The effects of CDCA8 on the proliferation, migration, and invasion of LUAD cells were detected by CCK-8 assay, scratch healing assay, and Transwell assay. Bioinformatics predicted the target miRNA of CDCA8, and dual-luciferase reporter gene assay was used to verify the binding relationship between miR-133b and CDCA8. ResultsData from TCGA-LUAD showed that CDCA8 was significantly overexpressed in LUAD tissue, while its upstream miRNA (miR-133b) was significantly lowly expressed. The result of dual-luciferase test showed that miR-133b targeted CDCA8. The results of in vitro functional experiments showed that overexpression of CDCA8 could promote the proliferation, invasion, and migration of LUAD cells, and miR-133b could reverse this promotion by targeting CDCA8. ConclusionThis study found that CDCA8 was a carcinogenic factor in LUAD cells and it was regulated by upstream miR-133b. miR-133b could inhibit proliferation, invasion, and migration of LUAD cells by targeting CDCA8.

Cyclophosphamide suppresses spermatogenesis in the testis of mice through downregulation of miR-34b and miR-34c.

Cyclophosphamide (CP) is commonly used as an anticancer agent but has been associated with high toxicity in several organs, including the testes. In this study, we aimed to evaluate the effects of CP-induced testicular toxicity, using glial cell line-derived neurotrophic factor (GDNF), occludin and transforming growth factor beta 3 (TGF-β3) primary antibodies, and miR-34b and miR-34c expressions. Eighteen young Balb/c male mice were divided into three groups. The control group received no treatment. The mice of CP group were injected 100mgkg-1 day-1 CP for 5days, and the same amount of saline was injected in the sham group. The animals were sacrificed 24hr after the last injection. Immunohistochemical analysis of testicular tissues showed a decrease in both spermatogenic germ cell count and also GDNF, occludin expressions, but an increase in TGF-β3 expression in the CP group compared to the others group. The expressions of miR-34b and miR-34c were examined by qPCR technique, a significant decrease was observed in tissue samples in the CP-treated group. The expression of GDNF, occludin and TGF-β3 plays an important role in testicular injury caused by CP, and the decrease in the expression of miR-34b/c in tissue samples may be an important marker for the detection of testicular damage.

Long non-coding RNA EGFR-AS1 in colorectal cancer: potential role in tumorigenesis and survival via miRNA-133b sponge and EGFR/STAT3 axis regulation

ABSTRACT Background Colorectal cancer is one of the most common cancers worldwide and a major cause of cancer-related death. Thus molecular biomarkers for colorectal cancer have been proposed. The role of long non-coding RNA EGFR-AS1 in colorectal cancer is still unclear. We aimed to evaluate its expression in different stages of colorectal cancer and determine any possible role in regulating the miR‑133b/EGFR/STAT3 signalling pathway. Materials and Methods The relative expression of EGFR-AS1 and miR‑133b were evaluated by quantitative real-time RT-transcription PCR in 130 colorectal cancer samples and 30 normal tissues. EGFR expression was assessed using immunohistochemistry. Furthermore, levels of p-EGFR, p-STAT3, and apoptotic proteins were determined by ELISA. Results Both EGFR-AS1 and EGFR overexpression were positively linked with colorectal cancer status (both p < 0.01), grade (both p < 0.01), and metastasis (P < 0.01 and p = 0.019 respectively). EGFR-AS1 and miR-133b were significantly inversely correlated (P < 0.01). Low expression of miR-133b was inversely associated with overexpressed EGFR and increased p-STAT3 levels. EGFR-AS1 was an independent prognostic factor for survival of colorectal cancer patients (P < 0.01, HR 2.06; 95% CI 1.32–3.19) where low EGFR-AS1 expression was associated with higher survival rate (p = 0.003). Conclusion EGFR-AS1 may have a role in colorectal cancer by regulation of miR‑133b/EGFR/STAT3 signalling. It may be a potential biomarker for early diagnosis and predicting the survival rate of colorectal cancer.

HuR, TTP, and miR-133b expression in NSCLC and their association with prognosis.

The article "HuR, TTP, and miR-133b expression in NSCLC and their association with prognosis, by L. Qian, A.-H. Ji, W.-J. Zhang, N. Zhao, published in Eur Rev Med Pharmacol Sci 2018; 22 (2): 430-442-DOI: 10.26355/eurrev_201801_14192-PMID: 29424924" has been withdrawn from the authors due to different opinions on the interpretation of the relevant results. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/14192.

Regulation of connective tissue growth factor expression by miR-133b for the treatment of renal interstitial fibrosis in aged mice with unilateral ureteral obstruction

IntroductionRenal interstitial fibrosis, an important pathological feature of kidney aging and chronic renal failure, is regulated by mesenchymal stem cells (MSCs). We have previously demonstrated low expression of miR-133b in MSC-derived extracellular vesicles (MSC-EVs) in aged rats. However, miR-133b can mediate 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.MethodsWe performed real-time polymerase chain reaction to detect miR-133b expression induced during EMT of HK2 cells by TGF-β1 at different concentrations (0, 6, 8, and 10 ng/mL) and at different time points (0, 24, 48, and 72 h). The target genes of miR-133b were validated using the dual-luciferase reporter assay. In vitro experiments were performed to evaluate 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-month-old unilateral ureteral obstruction (UUO) mouse model was established and injected with transfection reagent and miR-133b into the caudal vein. The target gene of miR-133b and other parameters mentioned above such as mRNA and protein expression levels and renal interstitial fibrosis were detected at 7 and 14 days.ResultsmiR-133b expression gradually decreased with an increase in TGF-β1 concentration and treatment time, and the miR-133b mimic downregulated connective tissue growth factor (CTGF) expression. The dual-luciferase reporter assay confirmed CTGF as a direct target of miR-133b. Transfection of the miR-133b mimic inhibited 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 kidney tissues after injection of the miRNA-133b overexpression complex, which significantly alleviated renal interstitial fibrosis in mice with UUO.ConclusionmiR-133b exerted targeted inhibitory effects on CTGF expression, which consequently reduced TGF-β1-induced EMT of HK2 cells and renal interstitial fibrosis in aged mice with UUO.

MiR-133b Modulates the Osteoblast Differentiation to Prevent Osteoporosis Via Targeting GNB4.

MiR-133b is considered to be lowly expressed in osteoporosis patients. This study aimed to probe the role and in-depth mechanism of miR-133b in modulating osteoblast biological behavior and differentiation. The differential expressions of miR-133b and GNB4 in patients with osteoporosis and healthy control were analyzed based on the GEO database. Osteoblastic differentiation of hFOB 1.19 cells was induced in the culture medium containing 10mM β-glycerophosphate, 50nm dexamethasone, and 100μg/ml ascorbic acid. The level of GNB4 was detected using quantitative real-time PCR (qRT-PCR) and Western blot. Cell viability and apoptosis were measured by Cell Counting Kit-8 (CCK-8) and flow cytometry assays, respectively. Western blot was also utilized to measure the levels of osteoblast-related proteins, including ALP, Runx2, Osterix, and OPN. GNB4 was identified and confirmed as a downstream target gene of miR-133b. The expression of miR-133b was declined while the expression of GNB4 was increased in osteoporosis patients. Importantly, up-regulation of miR-133b caused the increase of cell viability and the decrease of apoptosis, which could be blocked by overexpression of GNB4. Also, up-regulation of miR-133b promoted osteoblasts differentiation, as shown by the increase in the expression of ALP, Runx2, Osterix, and OPN. Similarly, this promoting impact resulted from miR-133b overexpression can be reversed via up-regulation of GNB4. These findings revealed that miR-133b can promote the viability and differentiation of osteoblasts by targeting GNB4, hoping to lay a feasible theoretical foundation for the clinical treatment of osteoporosis.