Restoration of miR-98 relieves the inhibitory effect of nicotine on the differentiation of P19 cells into cardiomyocytes

Objective

To study whether miR-98 participates in the effects of nicotine on myocardial differentiation.

Results

By western blot, MTT and flow cytometry assays, we found that nicotine suppresses P19 cell differentiation into cardiomyocytes and apoptosis, and promotes proliferation, while restoration of miR-98 relieves the inhibitory effect of nicotine on the P19 cell differentiation. By target prediction analysis and luciferase reporter assay, we observed that miR-98 inhibits the protein expression of Wnt1 by directly acting on the 3′-UTR of Wnt1 mRNA. We assumed that the effect of miR-98 on Wnt1 might alter the activity of the Wnt1/β-catenin signaling pathway and be associated with myocardial differentiation. In summary, nicotine restrains differentiation of P19 cells into cardiomyocytes and decreases the level of miR-98.

Conclusions

Restoration of miR-98 relieves the inhibitory effect of nicotine on differentiation of P19 cells via targeting the 3′-UTR of Wnt1, which offers novel insights into our understanding of underlying molecular mechanisms of congenital heart defects.

     

MicroRNA-144-3p inhibits proliferation and induces apoptosis of human salivary adenoid carcinoma cells via targeting of mTOR

Objectives

To investigate the biological functions of microRNA-144-3p with respect to proliferation and apoptosis of human salivary adenoid carcinoma cell lines via mTOR.

Results

After transfection of microRNA-144-3p agomir, cell viability assays confirmed that the salivary adenoid carcinoma cell (SACC) proliferation was inhibited and apoptosis was induced. Dual luciferase reporter assay validated that the mammalian target of rapamycin (mTOR) was a direct target of miR-144-3p. Western blot, immunofluorescent analysis and a xenograft mouse model of adenoid cystic carcinoma indicated that miR-144-3p was a tumor suppressor and repressed mTOR expression and signaling in SACCs.

Conclusions

MicroRNA-144-3p inhibits proliferation and induces apoptosis of human salivary adenoid carcinoma cells by downregulating mTOR expression in vitro and in vivo.

     

Circ_0027599/PHDLA1 suppresses gastric cancer progression by sponging miR-101-3p.1

Background

Pleckstrin homology-like domain family A member 1 (PHLDA1) is a tumor suppressor gene in gastric cancer, but its role regulated by circular RNAs (circRNAs) is not known. CircRNAs are important regulators in cancer growth and progression, however, the molecular roles of circRNAs in gastric cancer are rarely known. The study was aimed to investigate the role of circRNAs in regulating PHLDA1 expression in gastric cancer.

Results

The circRNA expression profile in the gastric cancer tissues by circRNA microarray showed that hsa_circ_0027599 (circ_0027599) was significantly down-regulated in gastric cancer patients and cells when comparing with the controls. Circ_0027599 overexpression suppressed gastric cancer cell proliferation and metastasis. By using bioinformatics tools and luciferase reporter assays, circ_0027599 was verified as a sponge of miR-101-3p.1 (miR-101) and suppressed cancer cell survival and metastasis. It was also verified that PHLDA1 was regulated by circ_0027599 in gastric cancer cells.

Conclusions

The study uncovered that PHLDA1 was regulated by circ_0027599/miR-101, which suppressed gastric cancer survival and metastasis in gastric cancer.

     

Long non-coding RNA H19 regulates E2F1 expression by competitively sponging endogenous miR-29a-3p in clear cell renal cell carcinoma

Background

Numerous recent studies indicate that the long non-coding RNAs (lncRNAs) are frequently abnormal expressed and take critical roles in many cancers. Renal cell carcinoma is the secondary malignant tumors in the urinary system and has high mortality and morbidity. Around 80% of RCCs is clear cell renal cell carcinoma (ccRCC) and is characterized by high metastasis and relapse rate. However, the clinical significances of lncRNAs in ccRCC are still unknown.

Methods

The human cancer lncRNA PCR array (Yingbio) was performed to detect the differentially expressed lncRNAs in human ccRCC samples. Real-time PCR (RT-PCR), dual-luciferase assay, RNA binding protein immunoprecipitation (RIP) assay, transwell assay, CCK-8 assay, and western blot were performed to explore the molecular mechanism of lncRNAs in ccRCC cell migration and invasion.

Results

In this study, lncRNA-H19 was high expressed and negatively correlated with miR-29a-3p in ccRCC. By bioinformatics software, dual-luciferase reporter and RIP assays, we verified that miR-29a-3p was identified as a direct target of lncRNA-H19. RT-PCR and western blot demonstrated that down-regulated lncRNA-H19 could affect the expression of miR-29a-3p targeting E2F1 with competitively binding miR-29a-3p. Furthermore, transwell assays indicated that lncRNA-H19 knockdown inhibited cells migration and invasion, but this effect was attenuated by co-transfection of lncRNA-H19 siRNA and miR-29a-3p inhibitor. Over expression of E2F1 could rescue lncRNA-H19 siRNA induced suppression on cell migration and invasion in ccRCC cells.

Conclusions

These results show a possible competing endogenous RNAs regulatory network involving lncRNA-H19 regulates E2F1 expression by competitively sponging endogenous miR-29a-3p in ccRCC. This mechanism may contribute to a better understanding of ccRCC pathogenesis, and lncRNA-H19 may be further considered as a potential therapeutic target for ccRCC intervention.

     

Intramuscular injection of mechano growth factor E domain peptide regulated expression of memory-related <Emphasis Type="Italic">sod</Emphasis>, miR-134 and miR-125b-3p in rat hippocampus under simulated weightlessness

Objective

To investigate the expression of memory-related antioxidant genes and miRNAs under simulated weightlessness and the regulation of mechano growth factor (MGF) E domain, the peptide preventing nerve damage.

Results

Igf-iea and mgf mRNA levels, expression of antioxidant genes sod1 and sod2 and levels of miR-134 and miR-125b-3p increased in rat hippocampus after 14 days tail suspension to simulate weightlessness which was inhibited with intramuscular injection of E domain peptide. Therefore, administration of MGF E domain peptide could reverse increased expressions of memory-related igf-iea, mgf, sod1, sod2, miR-134 and miR-125b-3p in rat hippocampus under simulated weightlessness.

Conclusions

MGF may regulate the redox state and miRNA-targeted NR-CREB signaling, and intramuscular injection may be the alternative administration because of its safety, convenience and ability to pass through the blood brain barrier.

     

Non-small cell lung cancer: miR-30d suppresses tumor invasion and migration by directly targeting NFIB

Objective

To evaluate the role and the molecular mechanism of miR-30d in non-small cell lung cancer (NSCLC).

Results

qRT-PCR was used to detect miR-30d expression in NSCLC tissues and cell lines. miR-30d was frequently down-regulated in NSCLC and its expression was associated with clinicopathological features of NSCLCC patients. Over-expression of miR-30d notably inhibited cell migration and invasion in NSCLC cell lines. miR-30d could negatively regulate Nuclear factor I B (NFIB) by directly targeting its 3′-UTR, which was confirmed by luciferase assay. NFIB also reversed miR-30d-mediated suppression on the migration and invasion in NSCLC cell lines.

Conclusion

miR-30d suppressed cell migration and invasion by directly targeting NFIB in NSCLC, and NFIB could partially abrogated the inhibition of biological functions by miR-30d.

     

MicroRNA-215 enhances invasion and migration by targeting retinoblastoma tumor suppressor gene 1 in high-grade glioma

Objective

To elucidate the molecular mechanism of microRNA-215 (miR-215) in the migration and invasion of high grade glioma.

Results

42 Patients were analysed for clinicopathological characteristics. qRT-PCR showed that miR-215 was up-regulated in glioma tissues compared with non-neoplastic brain tissues (P < 0.05). The up-regulated miR-215 was closely associated with high grade glioma (P < 0.01) and poor overall survival (P < 0.01). Transwell assay showed that re-expression of miR-215 enhanced migration and invasion of glioma cells. miR-215 also down-regulated retinoblastoma tumor suppressor gene 1 (RB1) expression by targeting its 3′-UTR. Reversely, re-expression of RB1 inhibited partial effect of miR-215 on migration and invasion in vitro.

Conclusions

Re-expression of miR-215 promoted cell migration and invasion of glioma by targeting RB1. miR-215 can thus be used as a biomarker for tumor progression and prognosis in human high grade glioma.

     

Down-regulation of microRNA-126-5p contributes to overexpression of VEGFA in lipopolysaccharide-induced acute lung injury

Objectives

To investigate the role of microRNA-126-5p (miR-126-5p) in acute lung injury induced by bronchial instillation of lipopolysaccharide (LPS), and to explore the potential target(s) of miR-126-5p in acute lung injury.

Results

In the mice with LPS-induced acute lung injury, the level of miR-126-5p in the pulmonary tissues was decreased by 41 % whilst pulmonary vascular endothelial growth factor-A (VEGFA) doubled in its mRNA content and increased threefold in its protein level. Similar results were observed in the alveolar type II (ATII) cells treated with LPS. By using luciferase reporter assay, we found that miR-126-5p inhibited VEGFA expression by targeting its 3′-untranslated region. In addition, overexpression of miR-126-5p attenuated LPS-induced reduction of epithelial sodium channel and aquaporin 1 in ATII cells

Conclusions

MiR-126-5p was down-regulated in LPS-induced acute lung injury in mice. Thus overexpression of miR-126-5p may alleviate acute lung injury by down-regulating VEGFA.

     

miR-93 enhances hepatocellular carcinoma invasion and metastasis by EMT via targeting PDCD4

Objectives

To clarify the potential biological function of miR-93 and its related molecular mechanism underlying metastasis in human hepatocellular carcinoma (HCC).

Results

miR-93 was significantly up-regulated in HCC tissues and was associated with poor 5-year overall survival in HCC patients. Transwell assays showed that over-expression of miR-93 increased HCC cell migration and invasion in vitro. Programmed cell death 4 (PDCD4) was a target gene of miR-93 and miR-93 could down-regulate the expression of PDCD4 by directly targeting its 3′-UTR. The re-expression of PDCD4 could attenuate the HCC cell invasion and migration induced by miR-93, while the knockdown of PDCD4 would promote HCC cell migration and invasion via the epithelial-mesenchymal transition (EMT) pathway.

Conclusions

miR-93 provides new insight into the molecular mechanisms of pathogenesis and progression in HCC and offer a potential therapeutic target for the treatment of HCC patients.

     

Identification of a microRNA (miR-663a) induced by ER stress and its target gene PLOD3 by a combined microRNome and proteome approach

Introduction

MicroRNAs (miRs) regulate gene expression to support important physiological functions. Significant evidences suggest that miRs play a crucial role in many pathological events and in the cell response to various stresses.

Methods

With the aim to identify new miRs induced by perturbation of intracellular calcium homeostasis, we analysed miR expression profiles of thapsigargin (TG)-treated cells by microarray. In order to identify miR-663a-regulated genes, we evaluated proteomic changes in miR-663a-overexpressing cells by two-dimensional differential in-gel electrophoresis coupled to mass spectrometric identification of the differentially represented proteins. Microarray and proteomic analyses were supported by biochemical validation.

Results

Results of microarray revealed 24 differentially expressed miRs; among them, miR-663a turned out to be by ER stress and under the control of the PERK pathway of the unfolded protein response. Proteomic analysis revealed that PLOD3, which is the gene encoding for collagen-modifying lysyl hydroxylase 3 (LH3), is regulated by miR-663a. Luciferase reporter assays demonstrated that miR-663a indeed reduces LH3 expression by targeting to 3′-UTR of PLOD3 mRNA. Interestingly, miR-663a inhibition of LH3 expression generates reduced extracellular accumulation of type IV collagen, thus suggesting the involvement of miR-663a in modulating collagen 4 secretion in physiological conditions and in response to ER stress.

Conclusion

The finding of the ER stress-induced PERK-miR-663a pathway may have important implications in the understanding of the molecular mechanisms underlying the function of this miR in normal and/or pathological conditions.

     

miR-124 promotes proliferation and differentiation of neuronal stem cells through inactivating Notch pathway

Background

Neural stem cells (NSCs) are able to differentiate into neurons and astroglia. miRNAs have been demonstrated to be involved in NSC self-renewal, proliferation and differentiation. However, the exact role of miR-124 in the development of NSCs and its underlying mechanism remain to be explored.

Methods

Primary NSCs were isolated from embryos of Wistar rats. Immunocytochemistry was used to stain purified NSCs. miR-124, Delta-like 4 (DLL4), ki-67, Nestin, β-tubulin III, glial fibrillary acidic protein (GFAP), HES1, HEY2, and cyclin D1 (CCND1) expressions were detected by qRT-PCR and western blot. The interaction between miR-124 and DLL4 was confirmed by luciferase reporter assay. Cell proliferation was assessed by MTT assay.

Results

NSCs could self-proliferate and differentiate into neurons and astrocyte. miR-124 was up-regulated and DLL4 was down-regulated during NSC differentiation. DLL4 was identified as a target of miR-124 in NSCs. Ectopic expression of miR-124 or knockdown of DLL4 promoted the proliferation and the formation of NSCs to neurospheres. Moreover, miR-124 overexpression or DLL4 down-regulation improved β-tubulin III expression but decreased GFAP expression in NSCs. Furthermore, enforced expression of DLL4 partially reversed the effects of miR-124 on NSCs proliferation and differentiation. Elevated expression of miR-124 suppressed the expressions of HES1, HEY2, and CCND1 in NSCs, while these effects were attenuated following the enhancement of DLL4 expression.

Conclusion

miR-124 promoted proliferation and differentiation of NSCs through inactivating Notch pathway.

     

SIRT1 inhibits adipogenesis and promotes myogenic differentiation in C3H10T1/2 pluripotent cells by regulating Wnt signaling

Background

The directed differentiation of mesenchymal stem cells (MSCs) is tightly controlled by a complex network. Wnt signaling pathways have an important function in controlling the fate of MSCs. However, the mechanism through which Wnt/β-catenin signaling is regulated in differentiation of MSCs remains unknown. SIRT1 plays an important role in the regulation of MSCs differentiation.

Results

This study aimed to determine the effect of sirtuin 1 (SIRT1) on adipogenesis and myogenic differentiation of C3H10T1/2 cells. First, the MSC commitment and differentiation model was established by using 5-azacytidine. Using the established model, C3H10T1/2 cells were treated with SIRT1 activator/inhibitor during differentiation. The results showed that resveratrol inhibits adipogenic differentiation and improves myogenic differentiation, whereas nicotinamide promotes adipogenic differentiation. Notably, during commitment, resveratrol blocked adipocyte formation and promoted myotubes differentiation, whereas nicotinamide enhanced adipogenic potential of C3H10T1/2 cells. Furthermore, resveratrol elevated the expression of Cyclin D1 and β-catenin in the early stages. The luciferase assay showed that knockdown SIRT1 inhibits Wnt/β-catenin signaling, while resveratrol treatment or overexpression SIRT1 activates Wnt/β-catenin signaling. SIRT1 suppressed the expression of Wnt signaling antagonists sFRP2 and DACT1. Knockdown SIRT1 promoted adipogenic potential of C3H10T1/2 cells, whereas overexpression SIRT1 inhibited adipogenic differentiation and promoted myogenic differentiation.

Conclusions

Together, our results suggested that SIRT1 inhibits adipogenesis and stimulates myogenic differentiation by activating Wnt signaling.

     

miR-34a attenuates glioma cells progression and chemoresistance via targeting PD-L1

Objective

To investigate the roles of miR-34a in progression and chemoresistance of glioma cells.

Results

Quantitative real-time PCR analysis showed that miR-34a level was lower, but PD-L1 expression level was higher in glioma tissue specimens compared with normal brain tissues and their expression levels were negatively correlated. Ectopic expression of miR-34a inhibited glioma cell proliferation, promoted cell cycle arrest in G1/S phase and cell apoptosis. Additionally, miR-34a/PD-L1 axis was again confirmed and co-expression of PD-L1 with miR-34a mimics attenuated the effects of miR-34a on cell proliferation and apoptosis in glioma cells. Importantly, PD-L1 overexpression resulted in chemoresistance in glioma cells, this effect was attenuated by miR-34a overexpression.

Conclusions

miR-34a inhibits glioma cells progression and chemoresistance via targeting PD-L1.

     

Targeting myomiRs by tocotrienol-rich fraction to promote myoblast differentiation

Background

Several muscle-specific microRNAs (myomiRs) are differentially expressed during cellular senescence. However, the role of dietary compounds on myomiRs remains elusive. This study aimed to elucidate the modulatory role of tocotrienol-rich fraction (TRF) on myomiRs and myogenic genes during differentiation of human myoblasts. Young and senescent human skeletal muscle myoblasts (HSMM) were treated with 50 μg/mL TRF for 24 h before and after inducing differentiation.

Results

The fusion index and myotube surface area were higher (p?<?0.05) on days 3 and 5 than that on day 1 of differentiation. Ageing reduced the differentiation rate, as observed by a decrease in both fusion index and myotube surface area in senescent cells (p?<?0.05). Treatment with TRF significantly increased differentiation at days 1, 3 and 5 of young and senescent myoblasts. In senescent myoblasts, TRF increased the expression of miR-206 and miR-486 and decreased PTEN and PAX7 expression. However, the expression of IGF1R was upregulated during early differentiation and decreased at late differentiation when treated with TRF. In young myoblasts, TRF promoted differentiation by modulating the expression of miR-206, which resulted in the reduction of PAX7 expression and upregulation of IGF1R.

Conclusion

TRF can potentially promote myoblast differentiation by modulating the expression of myomiRs, which regulate the expression of myogenic genes.

     

HuR facilitates cancer stemness of lung cancer cells via regulating miR-873/CDK3 and miR-125a-3p/CDK3 axis

Objectives

To study the roles and mechanisms of HuR in cancer stem cell maintenance of lung cancer.

Results

HuR expression was increased in tumor spheres of lung cancer cells. Knockdown of HuR suppressed spheroid formation and size, inhibited the expression of stemness-related marker, Oct4, Nanog and ALDH in lung cancer cells. Importantly, HuR and CDK3 expressions were increased in lung cancer tissues compared with normal adjacent tissues, and positively correlated. Mechanistically, HuR directly bound to CDK3, and increased CDK3 mRNA stability and expression. Additionally, miR-873 or miR-125a-3p attenuated the promotion of HuR on CDK3 expression and lung cancer stemness. Furthermore, HuR facilitated lung cancer stemness dependent on CDK3 expression. miR-873 or miR-125a-3p level was negatively correlated with HuR and CDK3 expression levels in lung cancer tissues.

Conclusions

HuR facilitates lung cancer stemness via regulating miR-873/CDK3 and miR-125a-3p/CDK3 axis.

     

Downregulation of long non-coding RNA H19 promotes P19CL6 cells proliferation and inhibits apoptosis during late-stage cardiac differentiation via miR-19b-modulated Sox6

Background

Regulating cardiac differentiation to maintain normal heart development and function is very important. At present, biological functions of H19 in cardiac differentiation is not completely clear.

Methods

To explore the functional effect of H19 during cardiac differentiation. Expression levels of early cardiac-specific markers Nkx-2.5 and GATA4, cardiac contractile protein genes α-MHC and MLC-2v were determined by qRT-PCR and western lot. The levels of lncRNA H19 and miR-19b were detected by qRT-PCR. We further predicted the binding sequence of H19 and miR-19b by online softwares starBase v2.0 and TargetScan. The biological functions of H19 and Sox6 were evaluated by CCK-8 kit, cell cycle and apoptosis assay and caspase-3 activity.

Results

The expression levels of α-MHC, MLC-2v and H19 were upregulated, and miR-19b was downregulated significantly in mouse P19CL6 cells at the late stage of cardiac differentiation. Biological function analysis showed that knockdown of H19 promoted cell proliferation and inhibits cell apoptosis. H19 suppressed miR-19b expression and miR-19b targeted Sox6, which inhibited cell proliferation and promoted apoptosis in P19CL6 cells during late-stage cardiac differentiation. Importantly, Sox6 overexpression could reverse the positive effects of H19 knockdown on P19CL6 cells.

Conclusion

Downregulation of H19 promoted cell proliferation and inhibited cell apoptosis during late-stage cardiac differentiation by regulating the negative role of miR-19b in Sox6 expression, which suggested that the manipulation of H19 expression could serve as a potential strategy for heart disease.

     

miR-155-5p modulates malignant behaviors of hepatocellular carcinoma by directly targeting CTHRC1 and indirectly regulating GSK-3β-involved Wnt/β-catenin signaling

Background

Hepatocellular carcinoma (HCC) remains one of the most lethal cancers. MicroRNA-155 (miR-155) and collagen triple helix repeat containing 1 (CTHRC1) were found to be involved in hepatocarcinogenesis, but their detailed functions in HCC are unclear. Here, we aimed to investigate the underlying role of miR-155-5p and CTHRC1 in HCC.

Methods

miR-155-5p and CTHRC1 expression levels were detected by qRT-PCR, IHC and WB in HCC patients and cell lines. Dual-luciferase assay, qRT-PCR and WB were used to validate the target interaction between miR-155-5p and CTHRC1. Biological behaviors, including apoptosis, cell cycle progression, and cell proliferation, invasion and migration, were measured by flow cytometry, CCK-8 assay and Transwell tests. A xenograft model was established to examine the effects of miR-155-5p and CTHRC1 on tumor formation. WB was finally utilized to identify the role of GSK-3β-involved Wnt/β-catenin signaling in HCC growth and metastasis.

Results

Our results showed that miR-155-5p and CTHRC1 were down-regulated and up-regulated, respectively, in HCC patients and cell lines. Dual-luciferase assay verified that CTHRC1 was the direct target of miR-155-5p. Moreover, elevated miR-155-5p expression promoted apoptosis but suppressed cell cycle progression and cell proliferation, invasion and migration in vitro and facilitated tumor formation in vivo; elevated CTHRC1 expression abolished these biological effects. Additionally, miR-155-5p overexpression increased metastasis- and anti-apoptosis-related protein expression and decreased pro-apoptosis-related protein expression, while forced CTHRC1 expression conserved the expression of these proteins.

Conclusion

Altogether, our data suggested that miR-155-5p modulated the malignant behaviors of HCC by targeting CTHRC1 and regulating GSK-3β-involved Wnt/β-catenin signaling; thereby, miR-155-5p and CTHRC1 might be promising therapeutic targets for HCC patients.