Cantharidin inhibits osteosarcoma proliferation and metastasis by directly targeting miR-214-3p/DKK3 axis to inactivate β-catenin nuclear translocation and LEF1 translation

Background: As the leading primary bone cancer in adolescents and children, osteosarcoma patients with metastasis show a five-year-survival-rate of 20-30%, without improvement over the past 30 years. Wnt/β-catenin is important in promoting osteosarcoma development. DKK3 is a Wnt/β-catenin antagonist and predicted to have the specific binding site in 3′-UTR with miR-214-3p.Methods: miR-214-3p and DKK3 levels were investigated in human osteosarcoma tissues and cells by RT-qPCR; the prognostic importance of DKK3 level in osteosarcoma patients was determined with Log-rank test; direct binding between DKK3 with miR-214-3p was identified with targetscan; anti-osteosarcoma mechanism of cantharidin was investigated by miR-214-3p silence/over-expression with or without cantharidin treatment, and nuclear/cytoplasmic protein assay in osteosarcoma cells.Results: Down-regulated DKK3 indicated poor prognosis of osteosarcoma patients. Up-regulated miR-214-3p promoted proliferation and migration, while suppressed apoptosis of osteosarcoma cells by increasing β-catenin nuclear translocation and LEF1 translation via degradation of DKK3. Cantharidin suppressed viabilities, migration and invasion, while promoted cell cycle arrest and apoptosis in 143B and U-2 OS cells via down-regulating miR-214-3p to up-regulate DKK3, thus inhibited p-GSK-3β expression, β-catenin nuclear translocation and LEF1 translation. Meanwhile, cantharidin inhibited tumor growth in xenograft-bearing mice with 143B cell injection in tibia.Conclusion: miR-214-3p mediated Wnt/β-catenin/LEF1 signaling activation by targeting DKK3 to promote oncogenesis of osteosarcoma; cantharidin inhibited proliferation and metastasis of osteosarcoma cells via down-regulating miR-214-3p to up-regulate DKK3 and decrease β-catenin nuclear translocation, indicating that cantharidin may be a prospective candidate for osteosarcoma treatment by targeting miR-214-3p/DKK3/β-catenin signaling.     

Pseudogene PTENP1 sponges miR-214 to regulate the expression of PTEN to modulate osteoclast differentiation and attenuate osteoporosis

Background aimsOsteoporosis (OP) is a common bone metabolic disease with a high incidence. Our study aimed to explore the pseudogene PTENP1/miR-214/PTEN axis to modulate the osteoclast differentiation in osteoporosis.MethodsPatients with osteoporosis were recruited in our study, and RANKL-induced osteoclast differentiation and ovariectomy-induced osteoporosis mouse model were established in vitro and in vivo, respectively.ResultsPseudogene PTENP1 and PTEN were significantly down-regulated and miR-214 was up-regulated in osteoporosis patients. In addition, overexpression of PTENP1 or silence of miR-214 inhibited the expression levels of osteoclast specific markers and osteoclast differentiation induced by RANKL. Overexpression of PTENP1 or silence of miR-214 also inhibited the levels of phosphorylation of PI3K and AKT, p65 nuclear translocation, IκBα degradation and the expression level of NFATc1. AlsoSilence of PTENP1 or overexpression of miR-214 induced the osteoclast differentiation under normal physiological condition. Pseudogene PTENP1 sponged miR-214 to regulate the expression of PTEN.ConclusionsIn an ovariectomy-induced osteoporosis mouse model, obvious pathological changes in bone tissues were found, and bone marrow mononuclear cells in this group were more likely to differentiate into osteoclasts. Therefore, pseudogene PTENP1 sponged miR-214 to regulate the expression of PTEN to inhibit osteoclast differentiation and attenuate osteoporosis by suppressing the PI3K/AKT/NF-κB signaling pathway.     

Role of microRNA-214-targeting phosphatase and tensin homolog in advanced glycation end product-induced apoptosis delay in monocytes

Advanced glycation end products (AGEs) delay spontaneous apoptosis of monocytes and contribute to the development of inflammatory responses. However, the mechanism by which AGEs affect monocyte apoptosis is unclear. We studied the role of microRNA-214 (miR-214) and its target gene in AGE-induced monocytic apoptosis delay. Using microRNA (miRNA) microarray and stem-loop, quantitative RT-PCR assay, we studied genome-wide miRNA expression in THP-1 cells treated with or without AGEs. Significant upregulation of miR-214 was consistently observed in THP-1 and human monocytes treated with various AGEs, and AGE-induced monocytic miR-214 upregulation was likely through activation of receptor for AGEs. A striking increase in miR-214 was also detected in monocytes from patients with chronic renal failure. Luciferase reporter assay showed that miR-214 specifically binds to the phosphatase and tensin homolog (PTEN) mRNA 3'-untranslated region, implicating PTEN as a target gene of miR-214. PTEN expression is inversely correlated with miR-214 level in monocytes. Compared with normal monocytes, AGE-treated monocytes and monocytes from chronic renal failure patients exhibited lower PTEN levels and delayed apoptosis. Overexpression of pre-miR-214 led to impaired PTEN expression and delayed apoptosis of THP-1 cells, whereas knockdown of miR-214 level largely abolished AGE-induced cell survival. Our findings define a new role for miR-214-targeting PTEN in AGE-induced monocyte survival.     

Melatonin ameliorates renal fibroblast-myofibroblast transdifferentiation and renal fibrosis through miR-21-5p regulation

Fibroblast-myofibroblast transdifferentiation (FMT) is widely recognized as the major pathological feature of renal fibrosis. Although melatonin has exerted antifibrogenic activity in many diseases, its role in renal FMT remains unclear. In the present study, the aim was to explore the effect of melatonin on renal FMT and the underlying mechanisms. We established the transforming growth factor (TGF)-β1 stimulated rat renal fibroblast cells (NRK-49F) model in vitro and unilateral ureteral obstruction (UUO) mice model in vivo. We assessed levels of α-smooth muscle actin (α-SMA), col1a1 and fibronectin, STAT3 and AP-1, as well as miR-21-5p and its target genes (Spry1, PTEN, Smurf2 and PDCD4). We found that melatonin reduced the expression of α-SMA, col1a1 and fibronectin, as well as the formation of α-SMA filament in TGF-β1-treated NRK-49F cells. Meanwhile, melatonin inhibited STAT3 phosphorylation, down-regulated miR-21-5p expression, and up-regulated Spry1 and PTEN expression. Moreover, miR-21-5p mimics partially antagonized the anti-fibrotic effect of melatonin. For animal experiments, the results revealed that melatonin remarkably ameliorated UUO-induced renal fibrosis, attenuated the expression of miR-21-5p and pro-fibrotic proteins and elevated Spry1 and PTEN expression. Nevertheless, agomir of miR-21-5p blocked the renoprotective effect of melatonin in UUO mice. These results indicated that melatonin could alleviate TGF-β1-induced renal FMT and UUO-induced renal fibrosis through down-regulation of miR-21-5p. Regulation of miR-21-5p/PTEN and/or miR-21-5p/Spry1 signal might be involved in the anti-fibrotic effect of melatonin in the kidneys of UUO mice.     

MiR-181a-5p promotes neural stem cell proliferation and enhances the learning and memory of aged mice

Hippocampal neural stem cell (NSC) proliferation is known to decline with age, which is closely linked to learning and memory impairments. In the current study, we found that the expression level of miR-181a-5p was decreased in the hippocampal NSCs of aged mice and that exogenous overexpression of miR-181a-5p promoted NSC proliferation without affecting NSC differentiation into neurons and astrocytes. The mechanistic study revealed that phosphatase and tensin homolog (PTEN), a negative regulator of the AKT signaling pathway, was the target of miR-181a-5p and knockdown of PTEN could rescue the impairment of NSC proliferation caused by low miR-181a-5p levels. Moreover, overexpression of miR-181a-5p in the dentate gyrus enhanced the proliferation of NSCs and ameliorated learning and memory impairments in aged mice. Taken together, our findings indicated that miR-181a-5p played a functional role in NSC proliferation and aging-related, hippocampus-dependent learning and memory impairments.     

Inhibition of PTEN Gene Expression by Oncogenic miR-23b-3p in Renal Cancer

Background

miR-23b is located on chromosome number 9 and plays different roles in different organs especially with regards to cancer development. However, the functional significance of miR-23b-3p in renal cell carcinoma (RCC) has not been reported.

Methods and Results

We measured miR-23b-3p levels in 29 pairs of renal cell carcinoma and their normal matched tissues using real-time PCR. The expression level of miR-23b-3p was correlated with the 5 year survival rate of renal cancer patients. In 15 cases (52%), miR-23b-3p expression was found to be high. All patients with moderate to low miR-23b-3p expression survived 5 years, while those with high miR-23b-3p expression, only 50% survived. After knocking down miRNA-23b-3p expression in RCC cell lines, there was an induction of apoptosis and reduced invasive capabilities. MiR-23b-3p was shown to directly target PTEN gene through 3′UTR reporter assays. Inhibition of miR-23b-3p induces PTEN gene expression with a concomitant reduction in PI3-kinase, total Akt and IL-32. Immunohistochemistry showed the lack of PTEN protein expression in cancerous regions of tissue samples where the expression of miR-23b-3p was high. We studied the in vitro effects of the dietary chemo preventive agent genistein on miR-23b-3p expression and found that it inhibited expression of miR-23b-3p in RCC cell lines.

Conclusions

The current study shows that miR-23b-3p is an oncogenic miRNA and inhibits PTEN tumor suppressor gene in RCC. Therefore, inhibition of miR-23b-3p may be a useful therapeutic target for the treatment of renal cell carcinoma.     

miR-214-5p targets KLF5 and suppresses proliferation of human hepatocellular carcinoma cells

MicroRNAs (miRNAs) are small endogenous conserved RNAs regulating genes expression through base pairing with the 3′-untranslated region (3′-UTR) of target messenger RNAs. MiR-214-5p is a newly identified miRNA with its biological role largely unknown. In this study, we explored miR-214-5p expression status in 78 paired tumor and nontumor tissues obtained from patients with hepatocellular carcinoma (HCC) by RT-qPCR. The effects of miR-214-5p expression on HCC cell proliferation, cell cycle progression, and cell migration were measured by CCK-8 assay, flow cytometry, and wound-healing assay. A dual-luciferase activity assay was performed to identify whether KLF5 was a target of miR-214-5p. Kaplan-Meier curve and log-rank test were used to investigate the effects of miR-214-5p and KLF5 on overall survival and disease-free survival of patients with HCC. We found miR-214-5p expression was sharply reduced in HCC tissues and cell lines compared with the normal tissues and cell lines. Functional assay revealed that miR-214-5p overexpression could downregulate cell proliferation, cell migration, and arrested cell cycle at G0/G1 phase. Further, we validated Krüppel-like factor 5 (KLF5) as a direct target of miR-214-5p, and was upregulated in HCC and inversely correlated with the expression of miR-214-5p. Moreover, we found the low expression of miR-214-5p and high expression of KLF5 were correlated with tumor size, tumor stage, and poorer 5-year overall survival and disease-free survival of patients with HCC. In conclusion, our results suggested miR-214-5p functions as a tumor suppressor through targeting KLF5 in HCC. Also, miR-214-5p and KLF5 were identified as potential prognostic markers and might be therapeutic targets in HCC.     

Aldose reductase regulates miR-200a-3p/141-3p to coordinate Keap1–Nrf2, Tgfβ1/2, and Zeb1/2 signaling in renal mesangial cells and the renal cortex of diabetic mice

Aberrant regulation in oxidative stress, fibrogenesis, and the epithelial–mesenchymal transition (EMT) in renal cells under hyperglycemic conditions contributes significantly to the onset and progression of diabetic nephropathy. The mechanisms underlying these hyperglycemia-induced dysregulations, however, have not been clearly elucidated. Herein, we report that aldose reductase is capable of regulating the expression of miR-200a-3p/141-3p negatively in renal mesangial cells. MiR-200a-3p/141-3p, in turn, act to target Keap1, Tgfβ2, fibronectin, and Zeb2 directly and regulate Tgfβ1 and Nrf2 indirectly under high-glucose conditions, resulting in profound dysregulations in Keap1–Nrf2, Tgfβ1/2, and Zeb1/2 signaling. In vivo in streptozotocin-induced diabetic mice, we found that aldose reductase deficiency caused significant elevations in miR-200a-3p/141-3p in the renal cortex, which were accompanied by a significant downregulation of Keap1, Tgfβ1/2, and fibronectin but significant upregulation of Nrf2. Moreover, in vivo administration of inhibitors of miR-200a-3p in diabetic animals significantly exacerbated cortical and glomerular fibrogenesis and increased urinary albumin excretion, tightly linking dysregulated miR-200a-3p with the development of diabetic nephropathy. Collectively, our results reveal a novel mechanism whereby hyperglycemia induces aldose reductase to regulate renal expression of miR-200a-3p/141-3p to coordinately control hyperglycemia-induced renal oxidative stress, fibrogenesis, and the EMT. Our novel findings also suggest that inhibition of aldose reductase and in vivo renal cortical restoration of miR-200a-3p/141-3p or their combination are very promising avenues for the development of therapeutic strategies or drugs against diabetic nephropathy.     

Inhibition of miRNA-21 prevents fibrogenic activation in podocytes and tubular cells in IgA nephropathy

Podocytopathy and tubular interstitial fibrosis impact on renal outcomes of IgA nephropathy (IgAN). We found that level of miR-21 was up regulated in both glomerular and tubular–interstitial tissues of patients with IgAN. Enhanced expression of miR-21 mainly located in podocytes and tubular cells. Mesangial cell derived cytokines contributed to the increase of miR-21 in podocytes and HK2 cells. IgA-HMC medium prepared with pIgA from IgAN, lead to obvious fibrogenic activation, evidenced by the loss of Podocin and CD2AP in podocytes, loss of E-cadherin and Megalin in HK2 cells and increase of FN and Col I in both cells. miR-21 targeted PTEN in these cells. Expression of PTEN was decreased and phosphorylation of Akt was increased in podocytes and HK2 cells exposed to the medium prepared with pIgA from IgAN. Inhibition of miR-21 preserved the expression of PTEN, prevented the activation of Akt and inhibited the fibrogenic activation in podocytes and HK2 cells exposed to the IgA-HMC medium prepared with pIgA from IgAN. In conclusion, our study suggests that inhibition of miR-21 prevents fibrogenic activation in podocytes and tubular cells by preventing PTEN/Akt pathway activation in IgAN.     

HIV-1 Nef and KSHV oncogene K1 synergistically promote angiogenesis by inducing cellular miR-718 to regulate the PTEN/AKT/mTOR signaling pathway

Kaposi''s sarcoma (KS) is an AIDS-defining cancer with aberrant neovascularization caused by KS-associated herpesvirus (KSHV). Although the interaction between HIV-1 and KSHV plays a pivotal role in promoting the aggressive manifestations of KS, the pathogenesis underlying AIDS-KS remains largely unknown. Here we examined HIV-1 Nef protein promotion of KSHV oncoprotein K1-induced angiogenesis. We showed that both internalized and ectopic expression of Nef in endothelial cells synergized with K1 to facilitate vascular tube formation and cell proliferation, and enhance angiogenesis in a chicken CAM model. In vivo experiments further indicated that Nef accelerated K1-induced angiogenesis and tumorigenesis in athymic nu/nu mice. Mechanistic studies revealed that Nef and K1 synergistically activated PI3K/AKT/mTOR signaling by downregulating PTEN. Furthermore, Nef and K1 induced cellular miR-718, which inhibited PTEN expression by directly targeting a seed sequence in the 3′ UTR of its mRNA. Inhibition of miR-718 expression increased PTEN synthesis and suppressed the synergistic effect of Nef- and K1-induced angiogenesis and tumorigenesis. These results indicate that, by targeting PTEN, miR-718 mediates Nef- and K1-induced angiogenesis via activation of AKT/mTOR signaling. Our results demonstrate an essential role of miR-718/AKT/mTOR axis in AIDS-KS and thus may represent an attractive therapeutic target.     

Retracted: LncRNA MT1JP functions as a tumor suppressor via regulating miR-214-3p expression in bladder cancer

Growing evidence suggested that the long noncoding RNAs (lncRNAs) regulate several pathophysiological processes in tumorigenesis and may be new biomarkers for tumor therapy. In this study, we studied the expression and role of lncRNA MT1JP in the development of bladder cancer. We demonstrated that the expression of MT1JP was downregulated in bladder tumor samples and cell lines. Ectopic expression of MT1JP suppressed cell proliferation, cycle, and invasion in bladder cancer. In addition, our result suggested that miR-214-3p overexpression decreased the luciferase activity of wild-type MT1JP but not mutated-type MT1JP and elevated expression of MT1JP decreased miR-214-3p expression in the bladder cancer cell. Furthermore, we indicated that the expression of miR-214-3p was upregulated in bladder tumor samples and cell lines. Ectopic expression of miR-214-3p promoted cell proliferation, cycle, and invasion in bladder cancer. MT1JP suppressed cell proliferation, cycle, and invasion via negative modulation of miR-214-3p in bladder cancer. These data suggested that lncRNA MT1JP acts a tumor suppressor gene in bladder cancer progression, considering MT1JP as a new therapeutic target in bladder cancer.     

IL-1β promotes hypoxic vascular endothelial cell proliferation through the miR-24-3p/NKAP/NF-κB axis

Purpose: Our previous data indicated that miR-24-3p is involved in the regulation of vascular endothelial cell (EC) proliferation and migration/invasion. However, whether IL-1β affects hypoxic HUVECs by miR-24-3p is still unclear. Therefore, the present study aimed to investigate the role and underlying mechanism of interleukin 1β (IL-1β) in hypoxic HUVECs.Methods: We assessed the mRNA expression levels of miR-24-3p, hypoxia-inducible factor-1α (HIF1A) and NF-κB-activating protein (NKAP) by quantitative real-time polymerase chain reaction (RT-qPCR). ELISA measured the expression level of IL-1β. Cell counting kit-8 (CCK-8) assays evaluated the effect of miR-24-3p or si-NKAP+miR-24 on cell proliferation (with or without IL-1β). Transwell migration and invasion assays were used to examine the effects of miR-24-3p or si-NKAP+miR-24-3p on cell migration and invasion (with or without IL-1β). Luciferase reporter assays were used to identify the target of miR-24-3p.Results: We demonstrated that in acute myocardial infarction (AMI) patient blood samples, the expression of miR-24-3p is down-regulated, the expression of IL-1β or NKAP is up-regulated, and IL-1β or NKAP is negatively correlated with miR-24-3p. Furthermore, IL-1β promotes hypoxic HUVECs proliferation by down-regulating miR-24-3p. In addition, IL-1β also significantly promotes the migration and invasion of hypoxic HUVECs; overexpression of miR-24-3p can partially rescue hypoxic HUVECs migration and invasion. Furthermore, we discovered that NKAP is a novel target of miR-24-3p in hypoxic HUVECs. Moreover, both the overexpression of miR-24-3p and the suppression of NKAP can inhibit the NF-κB/pro-IL-1β signaling pathway. However, IL-1β mediates suppression of miR-24-3p activity, leading to activation of the NKAP/NF-κB pathway. In conclusion, our results reveal a new function of IL-1β in suppressing miR-24-3p up-regulation of the NKAP/NF-κB pathway.     

miRNA-338-3p通过靶向抑制MC1R基因表达抑制羊驼黑色素细胞黑色素的生成

黑色素皮质素1受体MC1R）是在黑色素细胞内表达的G蛋白耦合受体（G protein coupled receptor, GPCR）家族成员,参与黑色素细胞中黑色素的生成。微RNAs（miRNAs）是一类非编码RNA,通过与靶基因3′-UTR结合抑制基因表达。已有研究证明,miR-338-3p 在多种人类肿瘤细胞中（过）表达,可通过下调靶基因表达抑制肿瘤细胞的侵袭迁移能力。然而,有关miR-338-3p对羊驼皮肤黑色素细胞的黑色素合成影响却罕见报道。本研究证明,miRNA-338-3p通过靶向抑制MC1R基因表达,抑制羊驼黑色素细胞黑色素的生成。采用生物信息学预测MC1R基因是miRNA-338-3p的靶基因,其基因表达抑制羊驼黑色素细胞黑色素合成。随后构建miR-338-3p真核表达载体。其基因转染结合qPT-PCR和Western印迹结果揭示,与对照细胞比较,过表达miRNA-338-3p的羊驼黑色素细胞的MC1R基因,及其下游与黑色素生成相关的小眼相关性转录因子（MITF)、酪氨酸酶(TYR)、酪氨酸酶相关蛋白1（TYRP1）、酪氨酸酶相关蛋白2（TYRP2）编码基因mRNA及蛋白质表达水平明显下调。酶联免疫吸附分析显示,过表达miRNA-338-3p的羊驼皮肤黑色素细胞的黑色素产量,较对照细胞显著下降（P＜0.01）。综上结果,miR-338-3p可通过抑制靶基因MC1R表达,下调其下游基因MITF、TYR、TYRP1和TYRP2基因的表达,从而抑制羊驼皮肤黑色素细胞黑色素的合成。miRNA-338-3p在羊驼生长发育过程中,是否参与调控体内皮肤黑色素细胞的黑色素生成尚待进一步研究。     

HOXC13-AS promotes breast cancer cell growth through regulating miR-497-5p/PTEN axis

Dysregulated long noncoding RNAs (lncRNAs) remains to be explored in tumorigenesis. LncRNA HOXC13 antisense RNA (HOXC13-AS) has been found as an oncogene in many cancers; however, the role of HOXC13-AS in breast cancer still elusive. In this study, the HOXC13-AS levels and its role in cell proliferation was first measured by real-time quantitative polymerase chain reaction, Cell Counting Kit-8 assay, and colony formation assay. It showed that HOXC13-AS was increased in breast cancer tissues compared with the adjacent normal tissues and upregulated HOXC13-AS promoted the growth of breast cancer cells. Then, we found that the miR-497-5p levels were downregulated in cancer tissues compared with the adjacent tissues and miR-497-5p suppressed breast cancer cell proliferation. Further study showed that HOXC13-AS could function as a “sponge” for miR-497-5p then suppress miR-497-5p expression. Moreover, we next identified that Phosphatase and Tensin homolog (PTEN) is the target of miR-497-5p. Overexpression of miR-497-5p by chemical mimics decreased the expression of PTEN, while downregulation of miR-497-5p by HOXC13-AS rescued the expression of PTEN. Finally, we showed that HOXC13-AS promoted the proliferation of breast cancer cells and tumor growth through miR-497-5p/PTEN axis in vitro and in vivo. Hence, we conclude that HOXC13-AS, which is significantly upregulated in breast cancers, promoted cell proliferation through the suppressed miR-497-5p and further upregulated PTEN.     

MicroRNA-382 induced by HIF-1α is an angiogenic miR targeting the tumor suppressor phosphatase and tensin homolog

Recent studies have revealed that microRNAs (miRs) play important roles in the regulation of angiogenesis. In this study, we have characterized miR-382 upregulation by hypoxia and the functional relevance of miR-382 in tumor angiogenesis. miRs induced by hypoxia in MKN1 human gastric cancer cells were investigated using miRNA microarrays. We selected miR-382 and found that the expression of miR-382 was regulated by HIF-1α. Conditioned media (CM) from MKN1 cells transfected with a miR-382 inhibitor (antagomiR-382) under hypoxic conditions significantly decreased vascular endothelial cell (EC) proliferation, migration and tube formation. Algorithmic programs (Target Scan, miRanda and cbio) predicted that phosphatase and tensin homolog (PTEN) is a target gene of miR-382. Deletion of miR382-binding sequences in the PTEN mRNA 3′-untranslated region (UTR) diminished the luciferase reporter activity. Subsequent study showed that the overexpression of miR-382 or antagomiR-382 down- or upregulated PTEN and its downstream target AKT/mTOR signaling pathway, indicating that PTEN is a functional target gene of miR-382. In addition, PTEN inhibited miR-382-induced in vitro and in vivo angiogenesis as well as VEGF secretion, and the inhibition of miR-382 expression reduced xenograft tumor growth and microvessel density in tumors. Taken together, these results suggest that miR-382 induced by hypoxia promotes angiogenesis and acts as an angiogenic oncogene by repressing PTEN.