CircSEMA4B targets miR-431 modulating IL-1β-induced degradative changes in nucleus pulposus cells in intervertebral disc degeneration via Wnt pathway

Intervertebral disc (IVD) degeneration (IDD), characterized by elevated levels of proinflammatory mediators, increased Aggrecan and collagen degradation, and increased degradation of extracellular matrix (ECM), has been widely regarded as a significant contributor to low back pain. Genetics are significant factors contribute to IDD. Based on previous data, circular RNA SEMA4B (circSEMA4B) is down-regulated in IDD specimens; herein, we demonstrated circSEMA4B overexpression could attenuate the effect of IL-1β on nucleus pulposus cell (NPC) proliferation, senescence, and ECM and Aggrecan degradation in IDD via Wnt signaling. Moreover, miR-431, a direct target of circSEMA4B, could bind to the 3′UTR of SFRP1 or GSK-3β, two inhibitory regulators of Wnt signaling, to inhibit their expression thus playing a role similar to the activator of Wnt signaling in NPCs. The effect of circSEMA4B knockdown on NPCs was partially reversed by miR-431 inhibition; circSEMA4B serves as a miR-431 sponge to compete with SFRP1 or GSK-3β for miR-431 binding, thus inhibiting IL-1β-induced degenerative process in NPCs through Wnt signaling. Rescuing circSEMA4B expression in NPCs in IDD might present a potential strategy for IDD improvement.     

SKI knockdown suppresses apoptosis and extracellular matrix degradation of nucleus pulposus cells via inhibition of the Wnt/β-catenin pathway and ameliorates disc degeneration

This study aimed to determine the effects of SKI on interleukin (IL)-1β-induced apoptosis of nucleus pulposus (NP) cells, intervertebral disc degeneration (IDD), and the Wnt signaling pathway. NP tissue specimens of different Pfirrmann grades (II–V) were collected from patients with different grades of IDD. Real-time polymerase chain reaction and western blotting were used to compare SKI mRNA and protein expression in NP tissues from patients. Using the IL-1β-induced IDD model, NP cells were infected with lentivirus-coated si-SKI to downregulate the expression of SKI and treated with LiCl to evaluate the involvement of the Wnt/β-catenin signaling pathway. Western blotting, immunofluorescence, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to detect NP cell apoptosis, extracellular matrix (ECM) metabolism, and related protein expression changes in the Wnt/β-catenin signaling pathway. To investigate the role of SKI in vivo, a rat IDD model was established by needle puncture of the intervertebral disc. Rats were injected with lentivirus-coated si-SKI and evaluated by magnetic resonance imaging (MRI), and hematoxylin and eosin (HE) and safranin O staining. SKI expression positively correlated with the severity of human IDD. In the IL-1β-induced NP cell degeneration model, SKI expression increased significantly and reached a peak at 24 h. SKI knockdown protected against IL-1β-induced NP cell apoptosis and ECM degradation. LiCl treatment reversed the protective effects of si-SKI on NP cells. Furthermore, lentivirus-coated si-SKI injection partially reversed the NP tissue damage in the IDD model in vivo. SKI knockdown reduced NP cell apoptosis and ECM degradation by inhibiting the Wnt/β-catenin signaling pathway, ultimately protecting against IDD. Therefore, SKI may be an effective target for IDD treatment.

     

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.

     

PTEN promotes intervertebral disc degeneration by regulating nucleus pulposus cell behaviors

Intervertebral disc degeneration (IDD) is induced by multiple factors including increased apoptosis, decreased survival, and reduced extracellular matrix (ECM) synthesis in the nucleus pulposus (NP) cells. The tumor suppressor phosphatase and tensin homolog deleted from chromosome 10 (PTEN) is the only known lipid phosphatase counteracting the PI3K/AKT pathway. Loss of PTEN leads to activated PI3K/AKT signaling, which plays a key role in a variety of cancers. However, the role of PTEN/PI3K/AKT signaling nexus in IDD remains unknown. Here, we report that PTEN is overexpressed in degenerative NP, which correlates with inactivated AKT. Using the PTEN knockdown approach by lentivirus‐mediated short interfering RNA gene transfer technique, we report that PTEN decreases survival but induces apoptosis and senescence of NP cells. PTEN also inhibits expression and production of ECM components including collagen II, aggrecan, and proteoglycan. Furthermore, PTEN modulates the expression of ECM regulatory molecules SOX‐9 and matrix metalloproteinase‐3 (MMP‐3). Using small‐molecule AKT inhibitor GDC‐0068, we confirm that PTEN regulates NP cell behaviors through its direct targeting of PI3K/AKT. These findings demonstrate for the first time that PTEN/PI3K/AKT signaling axis plays an important role in the pathogenesis of IDD. Targeting PTEN using gene therapy may represent a promising therapeutic approach against disc degenerative diseases.     

Downregulation of adenomatous polyposis coli by microRNA-663 promotes odontogenic differentiation through activation of Wnt/beta-catenin signaling

MicroRNAs (miRNAs) regulate cell differentiation by inhibiting mRNA translation or by inducing its degradation. However, the role of miRNAs in odontogenic differentiation is largely unknown. In this present study, we observed that the expression of miR-663 increased significantly during differentiation of MDPC-23 cells to odontoblasts. Furthermore, up-regulation of miR-663 expression promoted odontogenic differentiation and accelerated mineralization without proliferation in MDPC-23 cells. In addition, target gene prediction for miR-663 revealed that the mRNA of the adenomatous polyposis coli (APC) gene, which is associated with the Wnt/β-catenin signaling pathway, has a miR-663 binding site in its 3′-untranslated region (3′UTR). Furthermore, APC expressional was suppressed significantly by miR-663, and this down-regulation of APC expression triggered activation of Wnt/β-catenin signaling through accumulation of β-catenin in the nucleus. Taken together, these findings suggest that miR-663 promotes differentiation of MDPC-23 cells to odontoblasts by targeting APC-mediated activation of Wnt/β-catenin signaling. Therefore, miR-663 can be considered a critical regulator of odontoblast differentiation and can be utilized for developing miRNA-based therapeutic agents.     

lncRNA HOTAIR upregulates autophagy to promote apoptosis and senescence of nucleus pulposus cells

Intervertebral disc degeneration (IDD) is a complex and chronic disease that involves disc cell senescence, death, and extracellular matrix (ECM) degradation. HOTAIR, a long non-coding RNA (lncRNA) is reportedly associated with autophagy, whereas autophagy is shown to promote IDD. However, how it affects nucleus pulposus (NP) cells, the primary component of intervertebral discs is still unclear. We hypothesized that HOTAIR promotes NP cell apoptosis and senescence through upregulating autophagy. Thus, silencing HOTAIR should inhibit autophagy and exert a therapeutic effect on IDD. Our in vitro experiments in human NP cells revealed that HOTAIR expression positively correlated with IDD grade, and overexpression enhanced autophagy. Autophagy inhibition via 3-methyladenine reversed HOTAIR stimulatory effects on apoptosis, senescence, and ECM catabolism, while the AMP-activated protein kinase (AMPK) inhibitor Compound C suppressed HOTAIR-induced autophagy through regulating AMPK/mTOR/ULK1 pathways. Our in vivo experiment then illustrated that silencing HOTAIR ameliorates IDD in rats. Collectively, we demonstrated that HOTAIR stimulates autophagy to promote NP cell apoptosis, senescence, and ECM catabolism. Therefore, silencing HOTAIR has the potential to become a treatment option for IDD.     

Retracted: HO-1 overexpression alleviates senescence by inducing autophagy via the mitochondrial route in human nucleus pulposus cells

Intervertebral disc degeneration (IDD) is closely associated with aging. Our previous studies have confirmed that heme oxygenase-1 (HO-1) can inhibit nucleus pulposus (NP) cell apoptosis. However, whether or not HO-1 is involved in NP cell senescence and autophagy is unclear. Our results indicated that HO-1 expression was reduced in IDD tissues and replicative senescent NP cells. HO-1 overexpression using a lentiviral vector reduced the NP cell senescence level, protected mitochondrial function, and promoted NP cell autophagy through the mitochondrial pathway. Autophagy inhibitor 3-MA pretreatment reversed the anti-senescent and protective effects on the mitochondrial function of HO-1, which promoted the degradation of the extracellular matrix (ECM) in the intervertebral disc. In vivo, HO-1 overexpression inhibited IDD and enhanced autophagy. In summary, these results suggested that HO-1 overexpression alleviates NP cell senescence by inducing autophagy via the mitochondrial route.     

LINC00261 suppresses human colon cancer progression via sponging miR-324-3p and inactivating the Wnt/β-catenin pathway

Growing evidence indicates long noncoding RNAs (lncRNAs) are significant regulators in the progression of various malignant tumors including colon cancer. Dysregulation of lncRNA LINC00261 has been identified in many cancers. Investigations on LINC00261 function have revealed that LINC00261 could act as a crucial tumor suppressor in various cancers. But, the biological involvement of LINC00261 in colon cancer is still barely known. Here, we found LINC00261 was reduced in colon cancer cells. Meanwhile, overexpressed LINC00261 repressed colon cancer cell viability and proliferation capacity. In addition, colony cancer cell colony formation was inhibited and apoptosis was enhanced by upregulation of LINC00261. Also, colon cancer cell migration and invasion both were restrained by LINC00261. miR-324-3p can exert important functions in several carcinomas, but its role in colon cancer is uninvestigated. In the current study, miR-324-3p was examined and miR-324-3p was greatly increased in colon cancer cells. Moreover, the association between miR-324-3p and LINC00261 was confirmed via performing RNA immunoprecipitation and RNA-pull-down experiments. In cancer biology, aberrant modulation of the Wnt signaling pathway remains a prevalent theme. Overexpression of LINC00261 obviously impaired colon cancer progression via inactivating the Wnt pathway. Furthermore, in the xenograft model assay, an increase of LINC00261 could suppress colon tumor growth via sponging miR-324-3p and inactivating the Wnt pathway. Overall, our results showed that LINC00261 repressed colon cancer progression via regulating miR-324-3p and the Wnt pathway. LINC00261 could be established as a novel therapeutic target for colon cancer.     

MicroRNA-27 promotes the differentiation of odontoblastic cell by targeting APC and activating Wnt/β-catenin signaling

MicroRNAs (miRNAs) play an essential role in regulating cell differentiation either by inhibiting mRNA translation or by inducing its degradation. However, the role of miRNAs in odontoblastic cell differentaion is largely unknown. In the present study, we demonstrate that the expression of miR-27 was significantly increased during MDPC-23 odontoblastic cell differentiation. Furthermore, the up-regulation of miR-27 promotes the differentiation of MDPC-23 odontoblastic cells and accelerates mineralization without cell proliferation. In addition, our results of target gene prediction revealed that the mRNA of adenomatous polyposis coli (APC) associated with Wnt/β-catenin signaling pathway has miR-27 binding site in the its 3′ UTR and is suppressed by miR-27. Subsequentially, the down-regulated APC by miR-27 triggered the activation of Wnt/β-catenin signaling through accumulation of β-catenin in the nucleus. Our data suggest that miR-27 promotes MDPC-23 odontoblastic cell differentiation by targeting APC and activating Wnt/β-catenin signaling. Therefore, miR-27 might be considered a critical candidate as an odontoblastic differentiation molecular target for the development of miRNA based therapeutic agents in the dental medicine.     

circLRP6 regulates high glucose-induced proliferation,oxidative stress,ECM accumulation,and inflammation in mesangial cells

Aberrant regulation in mesangial cell proliferation, extracellular matrix (ECM) accumulation, oxidative stress, and inflammation under hyperglycemic condition contributes significantly to the occurrence and development of diabetic nephropathy (DN). However, the mechanisms underlying the hyperglycemia-induced dysregulations have not been clearly elucidated. Here, we reported that high mobility group box 1 (HMGB1) was highly elevated in high glucose (HG)-treated mesangial cells, and induced the phosphorylation, nuclear translocation, and DNA binding activity of NF-κB via toll-like receptor 4 (TLR4). Function assays showed that inhibition of HMGB1 mitigated HG-induced proliferation, oxidative stress, ECM accumulation, and inflammation in mesangial cells via TLR4/NF-κB pathway. Increasing evidence has shown that circRNA, a large class of noncoding RNAs, functions by binding with miRNAs and terminating regulation of their target genes. We further investigated whether HMGB1 is involved in circRNA–miRNA–mRNA regulatory network. First, HMGB1 was identified and confirmed to be the target of miR-205, and miR-205 played a protective role against HG-induced cell injure via targeting HMGB1. Then circLRP6 was found to be upregulated in HG-treated mesangial cells, and regulate HG-induced mesangial cell injure via sponging miR-205. Besides, overexpression of miR-205 or knockdown of circLRP6 inhibited the NF-κB signaling pathway. Collectively, these data suggest that circLRP6 regulates HG-induced proliferation, oxidative stress, ECM accumulation, and inflammation in mesangial cells via sponging miR-205, upregulating HMGB1 and activating TLR4/NF-κB pathway. These findings provide a better understanding for the pathogenesis of DN.     

Overexpression of miR-623 suppresses progression of hepatocellular carcinoma via regulating the PI3K/Akt signaling pathway by targeting XRCC5

It has been reported that miR-623 is deregulated in lung adenocarcinoma and inhibits tumor growth and invasion. However, it is unclear whether miR-623 has a role in the progression of hepatocellular carcinoma (HCC). Herein, we found that miR-623 was significantly downregulated in HCC, and that its expression was related to poor clinical outcomes of patients with HCC. Upregulation of miR-623 decreased cell proliferation, viability, migration, and invasion and further promoted apoptosis in 7721, Huh7, and Bel-7402 cells. Moreover, we also observed that miR-623 regulated the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), Wnt/β-catenin, and extracellular regulated protein kinases/c-Jun N-terminal kinase (ERK/JNK) signaling pathways as well as the expression level of related proteins. Further, X-ray repair cross complementing 5 (XRCC5) was a direct target for miR-623, and the suppression of PI3K/Akt, Wnt/β-catenin, and ERK/JNK signaling pathways and cell proliferation and invasion abilities caused by miR-623 in HCC cells was significantly reversed by the upregulation of XRCC5. Collectively, our data suggested that miR-623 suppressed the progression of HCC by regulating the PI3K/Akt, Wnt/β-catenin, and ERK/JNK pathways by targeting XRCC5 in HCC in vitro, indicating that miR-623 may be a target for the therapy of HCC.     

Curcumin inhibits oral squamous cell carcinoma SCC-9 cells proliferation by regulating miR-9 expression

Curcumin, a phytochemical derived from the rhizome of Curcuma longa, has shown anticancer effects against a variety of tumors. In the present study, we investigated the effects of curcumin on the miR-9 expression in oral squamous cell carcinoma (OSCC) and explored the potential relationships between miR-9 and Wnt/β-catenin pathway in curcumin-mediated OSCC inhibition in vitro. As the results shown, the expression levels of miR-9 were significantly lower in clinical OSCC specimens than those in the adjacent non-tumor tissues. Furthermore, our results indicated that curcumin inhibited OSCC cells (SCC-9 cells) proliferation through up-regulating miR-9 expression, and suppressing Wnt/β-catenin signaling by increasing the expression levels of the GSK-3β, phosphorylated GSK-3β and β-catenin, and decreasing the cyclin D1 level. Additionally, the up-regulation of miR-9 by curcumin in SCC-9 cells was significantly inhibited by delivering anti-miR-9 but not control oligonucleotides. Downregulation of miR-9 by anti-miR-9 not only attenuated the growth-suppressive effects of curcumin on SCC-9 cells, but also re-activated Wnt/β-catenin signaling that was inhibited by curcumin. Therefore, our findings would provide a new insight into the use of curcumin against OSCC in future.     

Retracted: Suppression of microRNA-342-3p increases glutamate transporters and prevents dopaminergic neuron loss through activating the Wnt signaling pathway via p21-activated kinase 1 in mice with Parkinson's disease

Development of effective therapeutic drugs for Parkinson's disease (PD) is of great importance. Aberrant microRNA (miRNA) expression has been identified in postmortem human PD brain samples, in vitro and in vivo PD models. However, the role of miR-342-3p in PD has been understudied. The study explores the effects of miR-342-3p on expression of glutamate (Glu) transporter, and dopaminergic neuron apoptosis and proliferation by targeting p21-activated kinase 1 (PAK1) through the Wnt signaling pathway in PD mice. After establishment of PD mouse models, gain- or loss-of-function assay was performed to explore the functional role of miR-342-3p in PD. Number of apoptotic neurons and Glu concentration was then determined. Subsequently, PC12 cells were treated with miR-342-3p mimic, miR-342-3p inhibitor, dickkopf-1 (DKK1), and miR-342-3p inhibitor + DKK1. The expression of miR-342-3p, PAK1, the Wnt signaling pathway-related and apoptosis-related genes, Glutamate transporter subtype 1 (GLT-1), l -glutamate/ l -aspartate transporter (GLAST), tyrosine hydroxylase (TH) was measured. Also, cell viability and apoptosis were evaluated. PD mice exhibited increased miR-342-3p, while decreased expression of PAK1, GLT-1, GLAST, TH, and the Wnt signaling pathway-related and antiapoptosis genes. miR-342-3p downregulation could promote expression of PAK1, the Wnt signaling pathway-related and antiapoptosis genes. GLT-1, GLAST, and TH as well as cell viability, but reduce cell apoptosis rate. The results indicated that suppression of miR-342-3p improves expression of Glu transporter and promotes dopaminergic neuron proliferation while suppressing apoptosis through the Wnt signaling pathway by targeting PAK1 in mice with PD.     

Silencing of circ_0000205 mitigates interleukin-1β-induced apoptosis and extracellular matrix degradation in chondrocytes via targeting miR-766-3p/ADAMTS5 axis

The aim of this study was to explore the role of hsa_circRNA_0000205 (circ_0000205) in chondrocyte injury in osteoarthritis (OA) and the underlying mechanism. Expression of circ_0000205, microRNA (miR)-766-3p and a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS)-5 was detected by quantitative real time (qRT)-polymerase chain reaction (PCR) and Western blot assays. Cell proliferation, apoptosis, and extracellular matrix (ECM) synthesis were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 5-ethynyl-2-deoxyuridine assays, flow cytometry, and qRT-PCR and Western blot assays. The target relationship between miR-766-3p and circ_0000205 or ADAMTS5 was confirmed by luciferase reporter assay and RNA immunoprecipitation. IL-1β treatment could attenuate cell viability of primary chondrocytes and proliferating cell nuclear antigen (PCNA) and collagen II type alpha-1 (COL2A1) levels, and elevate apoptosis rate and cleaved caspase-3, ADAMTS5 and matrix metalloproteinase-13 (MMP13) levels, suggesting that IL-1β induced chondrocyte apoptosis and ECM degradation. Expression of circ_0000205 was up-regulated in OA tissues and IL-1β-induced primary chondrocytes, accompanied with miR-766-3p down-regulation and ADAMTS5 up-regulation. Knockdown of circ_0000205 could mitigate IL-1β-induced above effects and improve cell proliferation. Moreover, both depleting miR-766-3p and promoting ADAMTS5 could partially counteract circ_0000205 knockdown roles in IL-1β-cultured primary chondrocytes. Notably, circ_0000205 was verified as a sponge for miR-766-3p via targeting, and ADAMTS5 was a direct target for miR-766-3p. Silencing circ_0000205 could protect chondrocytes from IL-1β-induced proliferation reduction, apoptosis, and ECM degradation by targeting miR-766-3p/ADAMTS5 axis.     

LncRNA H19/miR-194/PFTK1 axis modulates the cell proliferation and migration of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) remains a huge challenge due to its high mortality and morbidity; gene therapy might be a promising treatment for PDAC. The critical role of Wnt-signaling pathway in cancer pathogenesis has been widely recognized; cyclin-dependent kinase 14 (CDK14, PFTK1)-induced low-density lipoprotein receptor-related proteins 5/6 (LRP5/6) phosphorylation is an important issue in Wnt-signaling activation. Long noncoding RNA (LncRNA)-microRNA (miRNA)-messenger RNA (mRNA) modulating the pathogenesis of cancers has been regarded as a major mechanism. In the current study, upregulated lncRNAs positively correlated with PFTK1 were analyzed and selected using The Cancer Genome Atlas (TCGA) database. Of them, lncRNA H19 can activate Wnt signaling in cancers. In PDAC tissues, the expression of H19 and PFTK1 were upregulated; H19 knockdown suppressed the cell proliferation and migration of PDAC, while PFTK1 overexpression partially attenuated the suppressive effect of H19 knockdown. As analyzed by TCGA and predicted by online tools, miR-194 was negatively correlated with PFTK1 and might bind to both H19 and PFTK1, which was further confirmed by luciferase reporter and RNA immunoprecipitation assays. Moreover, the effect of H19 knockdown on PFTK1 protein and the cell proliferation and migration could be partially reversed by miR-194 inhibition; H19/miR-194 axis modulated PDAC cell proliferation and migration through PFTK1 downstream Wnt signaling. Results suggested that rescuing miR-194 expression in PDAC can inhibit lncRNA H19 and PFTK1 expression, subsequently suppressing PDAC cell proliferation and migration. Due to the complexity of the lncRNA-miRNA-mRNA network, further in vivo experiments examining potential side effects are needed in future study to explore the clinical application of these findings.