miR-149 promotes human osteocarcinoma progression via targeting bone morphogenetic protein 9 (BMP9)

Objectives

To investigate the roles of miR-149 in the progression of human osteosarcoma (OS).

Results

miR-149 level was upregulated in tissues from OS patients more than in normal subjects. Cell proliferation and apoptosis assays revealed that miR-149 increased cell proliferation and inhibited cell apoptosis in OS cell line (MG63). An increase of Bcl-2 gene expression and a decrease of cleaved-caspase-3, and cleaved-PARP expression were observed in MG63 cells with transfection of miR-149. Additionally, bone morphogenetic protein 9 (BMP9) was identified as a target of miR-149 in MG63 cells, and BMP9 expression was negatively correlated with miR149 level in OS clinical samples. Co-overexpression of BMP9 with miR-149 in MG63 cells prohibited miR-149-mediated promotive effects on OS progression. Importantly, overexpression of miR-149 conferred chemoresistance in MG63 cells.

Conclusions

miR-149 promotes OS progression via targeting BMP9.

     

MicroRNA-190b confers radio-sensitivity through negative regulation of Bcl-2 in gastric cancer cells

Objectives

To determine the role of miR-190b in radio-sensitivity of gastric cancer (GC).

Results

In radio-resistant GC cells, down-regulation of miR-190b and up-regulation of Bcl-2 were observed. The protein expression of Bcl-2 was negatively regulated by miR-190b. Overexpression of miR-190b significantly decreased cell viability and enhanced radio-sensitivity of GC cells. Of note, these effects of miR-190b on GC cells radio-sensitivity were abolished by Bcl-2.

Conclusion

miR-190b confers radio-sensitivity of GC cells, possibly via negative regulation of Bcl-2.

     

Bioinformatics prediction of miR-30a targets and its inhibition of cell proliferation of osteosarcoma by up-regulating the expression of PTEN

Background

MiRNAs are frequently abnormally expressed in the progression of human osteosarcoma. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is one of the tumor suppressors in various types of human cancer. In the present study, we detected how hsa-miR-30a-3p regulated PTEN and further tested the role of hsa-miR-30a-3p in the cell proliferation of osteosarcoma cells.

Methods

The levels of miR-30a were determined by real time PCR. The expression of PTEN was tested by western blotting analysis. Cell distribution of PTEN was observed with confocal laser scanning microscope. Cell viability was determined by MTT assay.

Results

The expression of miR-30a and PTEN was obviously decreased in MG-63, 143B and Saos-2 cells compared with primary osteoblasts. TargetScan analysis data showed miR-30a might bind with position 30-57 of 3’UTR of PTEN. Transfection with miR-30a-3p increased the level of PTEN in MG-63 cells, while transfection with miR-30a-3p inhibitor significantly decreased the expression of PTEN in osteosarcoma cells. Transfection with miR-30a-3p significantly inhibited cell proliferation of osteosarcoma cells, while miR-30a inhibitor obviously promoted cell viability of MG63 cells and Saos-2 cells. Inhibition of PTEN eliminated the proliferation inhibitory effect of miR-30a-3p.

Conclusion

Thus, all these findings revealed the anti-tumor effects of miR-30a in human osteosarcoma cells, which could be mediated by regulating the level of PTEN.

     

miR-1260b promotes cell migration and invasion of hepatocellular carcinoma by targeting the regulator of G-protein signaling 22

Objectives

To investigate whether miR-1260b can regulate migration and invasion of hepatocellular carcinoma (HCC) by targeting RGS22.

Results

miR-1260b was up-regulated in HCC tissues compared with their corresponding non-cancerous tissues. Over-expression of miR-1260b increased migration and invasion of HepG2 and SMMC-7721 cells associated with HCC. Regulator of G-protein signaling 22 (RGS22) was identified as a directly target of miR-1260b and was inhibited by miR-1260b. Knockdown of RGS22 increased proliferation of HCC cells.

Conclusions

The new identified miR-1260b/RGS22 axis provides useful therapeutic methods for treatment of HCC deepening on our understanding of underlying mechanisms of HCC tumorigenesis.

     

MicroRNA-15b silencing inhibits IL-1β-induced extracellular matrix degradation by targeting SMAD3 in human nucleus pulposus cells

Objectives

To determine the role of microRNA-15b (miR-15b) in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation in the nucleus pulposus (NP).

Results

MiR-15b was up-regulated in degenerative NP tissues and in IL-1β-stimulated NP cells, as compared to the levels in normal controls (normal tissue specimens from patients with idiopathic scoliosis). Bioinformatics and luciferase activity analyses showed that mothers against decapentaplegic homolog 3 (SMAD3), a key mediator of the transforming growth factor-β signaling pathway, was directly targeted by miR-15b. Functional analysis demonstrated that miR-15b overexpression aggravated IL-1β-induced ECM degradation in NP cells, while miR-15b inhibition had the opposite effects. Prevention of IL-1β-induced NP ECM degeneration by the miR-15b inhibitor was attenuated by small-interfering-RNA-mediated knockdown of SMAD3. In addition, activation of MAP kinase and nuclear factor-κB up-regulated miR-15b expression and down-regulated SMAD3 expression in IL-1β-stimulated NP cells.

Conclusions

MiR-15b contributes to ECM degradation in intervertebral disc degeneration (IDD) via targeting of SMAD3, thus providing a novel therapeutic target for IDD treatment.

     

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.

     

Over-expression of miR-146b and its regulatory role in intestinal epithelial cell viability,proliferation, and apoptosis in piglets

Background

Weaning stress affects the small intestine of piglets. MiR-146b is differentially expressed in suckling and weaned piglets. In this study, we evaluated the effects of miR-146b on cell viability, proliferation, and apoptosis in IPEC-J2 cells.

Results

Transfection with miR-146b mimics successfully increased miR-146b levels by 1000× (P?<?0.001). The over-expression of miR-146b significantly promoted the apoptosis (P?<?0.01) of IPEC-J2 cells, with no significant effects on cell viability or proliferation. MiR-146b suppressed the luciferase activity of the miR-TLR4-wt by 57% compared with the negative control, while mutation of the miR-146b binding site significantly blocked the suppressive effect (P?<?0.05). Western blot results showed that TLR4 levels decreased in IPEC-J2 cells transfected with miR-146b mimics (P?<?0.05).

Conclusions

The over-expression of miR-146b promotes IPEC-J2 cell apoptosis. TLR4 is a direct target of miR-146b in IPEC-J2 cells.

Reviewers

This article was reviewed by Eugene Berezikov and Jan B Hoek.

     

MiRNA-145 suppresses lung adenocarcinoma cell invasion and migration by targeting N-cadherin

Objective

To investigate the roles of miR-145 in lung adenocarcinoma (LAC) and to clarify the regulation of N-cadherin by miR-145.

Results

In 57 paired clinical LAC tissues, diminished miR-145 was significantly correlated with the lymph node metastasis and was negatively correlated with N-cadherin mRNA level expression. Wound healing and transwell assays revealed a reduced capability of tumor metastasis induced by miR-145 in LAC. miR-145 negatively regulated the invasion of cell lines through targeting N-cadherin by directly binding to its 3′-untranslated region. Silencing of N-cadherin inhibited invasion and migration of LAC cell lines similar to miR-145 overexpression.

Conclusions

MiR-145 could inhibit invasion and migration of lung adenocarcinoma cell lines by directly targeting N-cadherin.

     

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.

     

MiR-9 enhances the sensitivity of A549 cells to cisplatin by inhibiting autophagy

Objectives

To demonstrate that miR-9 inhibits autophagy by down-regulating Beclin1 and thus enhances the sensitivity of A549 cells to cisplatin.

Results

MiR-9 inhibited Beclin1 expression by binding to its 3′UTR. The inhibition decreased the cisplatin-induced autophagy in A549 cells, evidenced by the decreased expression of LC3II and GFP-LC3 puncta and the increased expression of P62. Upregulation of miR-9 level enhanced the sensibility of A549 cells to cisplatin and increased the cisplatin-induced apoptosis. Overexpression of Beclin1 reversed above effects of miR-9 mimics, cisplatin-induced autophagy was increased and apoptosis was decreased.

Conclusions

MiR-9 inhibits autophagy via targeting Beclin1 3′UTR and thus enhances cisplatin sensitivity in A549 cells.

     

miR-130b-3p/301b-3p negatively regulated Rb1cc1 expression on myogenic differentiation of chicken primary myoblasts

Objectives

To explore the roles of miR-130b-3p and miR-301b-3p which may regulate Rb1-inducible coiled-coil 1 (Rb1cc1) expression during myogenic differentiation of chicken primary myoblasts.

Results

After 4 days of myogenic differentiation, myotubes appeared and after 6 days the cells fused to each other and expression of MyHC could be detected by immunofluorescence staining. TargetScan and RNAhybrid 2.2 showed miR-130b-3p and miR-301b-3p were well complementary with the target site of Rb1cc1 3′-untranslated region (3′-UTR). Using the dual-luciferase assay, we found miR-130b-3p and miR-301b-3p could inhibit Rb1cc1 expression by binding to its 3′-UTR. Real-time PCR showed Rb1cc1 mRNA expression level was almost reciprocal to that of miR-130b-3p or miR-301b-3p during myogenic differentiation. Furthermore, over-expression of miR-130b-3p or miR-301b-3p down-regulated the expression levels of Rb1cc1, myoblast determination protein, myogenin and myosin heavy chain.

Conclusions

miR-130b-3p or miR-301b-3p negatively regulate Rb1cc1 expression to affect myogenic differentiation.

     

miR-215 functions as an oncogene in high-grade glioma by regulating retinoblastoma 1

Objectives

To investigate the roles of miR-215 in high-grade glioma and to clarify the regulation of retinoblastoma 1 (RB1) by miR-215.

Results

miR-215 is frequently up-regulated in high-grade glioma tissues. Increased miR-215 expression is significantly associated with World Health Organization grade (P < 0.01) tumor size (P < 0.05) and poor prognosis (P < 0.01). Over-expression of miR-215 promoted cell proliferation and knockdown of miR-215 inhibited cell proliferation in vitro. RB1 was identified as a direct and functional target of miR-215. RB1 is generally down-regulated in glioma tissues and its expression inversely correlated with miR-215, which is up-regulated in high-grade glioma tissues, and its expression was negatively correlated with miR-215.

Conclusions

The new miR-215/RB1 axis provides new insights into the molecular mechanism and treatment for glioma.

     

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.

     

RNA binding protein Lin28A promotes osteocarcinoma cells progression by associating with the long noncoding RNA MALAT1

Objectives

To explore the effects of Lin28A on progression of osteocarcinoma (OS) cells.

Results

Lin28A mRNA and protein expressions were significantly increased in OS tissues compared with that in normal adjacent tissues. Expressions of Lin28A and long noncoding RNA MALAT1 were positively correlated. Patients with higher Lin28A expression had shorter overall survival. Moreover, Lin28A knockdown inhibited OS cells proliferation, migration, invasion and promoted cell apoptosis; Lin28A was found to harbor binding sites on MALAT1 sequences and associated with MALAT1, and increased MALAT1 stability and expression. Notably, the inhibition of Lin28A knockdown was attenuated or even reversed by MALAT1 overexpression.

Conclusions

RNA binding protein Lin28A could facilitate OS cells progression by associating with the long noncoding RNA MALAT1.

     

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.

     

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.

     

MiR-320 regulates cardiomyocyte apoptosis induced by ischemia–reperfusion injury by targeting AKIP1

Background

MicroRNAs play important roles in regulation of the cardiovascular system. The purpose of this study was to investigate microRNA-320 (miR-320) expression in myocardial ischemia-reperfusion (I/R) injury and the roles of miR-320 in cardiomyocyte apoptosis by targeting AKIP1 (A kinase interacting protein 1).

Methods

The level of miR-320 was detected using quantitative real-time polymerase chain reaction (qRT-PCR), and cardiomyocyte apoptosis was detected via terminal dUTP nick end-labeling assay. Cardiomyocyte apoptosis and the mitochondrial membrane potential were evaluated via flow cytometry. Bioinformatics tools were used to identify the target gene of miR-320. The expression levels of AKIP1 mRNA and protein were detected via qRT-PCR and Western blot, respectively.

Results

Both the level of miR-320 and the rate of cardiomyocyte apoptosis were substantially higher in the I/R group and H9c2 cells subjected to H/R than in the corresponding controls. Overexpression of miR-320 significantly promoted cardiomyocyte apoptosis and increased the loss of the mitochondrial membrane potential, whereas downregulation of miR-320 had an opposite effect. Luciferase reporter assay showed that miR-320 directly targets AKIP1. Moreover, knock down and overexpression of AKIP1 had similar effects on the H9c2 cells subjected to H/R.

Conclusions

miR-320 plays an important role in regulating cardiomyocyte apoptosis induced by I/R injury by targeting AKIP1 and inducing the mitochondrial apoptotic pathway.