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.

     

Propofol alleviates hypoxia-induced nerve injury in PC-12 cells by up-regulation of microRNA-153

Background

Although the neuroprotective role of propofol has been identified recently, the regulatory mechanism associated with microRNAs (miRNAs/miRs) in neuronal cells remains to be poorly understood. We aimed to explore the regulatory mechanism of propofol in hypoxia-injured rat pheochromocytoma (PC-12) cells.

Methods

PC-12 cells were exposed to hypoxia, and cell viability and apoptosis were assessed by CCK-8 assay and flow cytometry assay/Western blot analysis, respectively. Effects of propofol on hypoxia-injured cells were measured, and the expression of miR-153 was determined by stem-loop RT-PCR. After that, whether propofol affected PC-12 cells under hypoxia via miR-153 was verified, and the downstream protein of miR-153 as well as the involved signaling cascade was finally explored.

Results

Hypoxia-induced decrease of cell viability and increase of apoptosis were attenuated by propofol. Then, we found hypoxia exposure up-regulated miR-153 expression, and the level of miR-153 was further elevated by propofol in hypoxia-injured PC-12 cells. Following experiments showed miR-153 inhibition reversed the effects of propofol on hypoxia-treated PC-12 cells. Afterwards, we found BTG3 expression was negatively regulated by miR-153 expression, and BTG3 overexpression inhibited the mTOR pathway and AMPK activation. Besides, hypoxia inhibited the mTOR pathway and AMPK, and these inhibitory effects could be attenuated by propofol.

Conclusion

Propofol protected hypoxia-injured PC-12 cells through miR-153-mediataed down-regulation of BTG3. BTG3 could inhibit the mTOR pathway and AMPK activation.

     

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.

     

ERK-mediated autophagy promotes inactivated Sendai virus (HVJ-E)-induced apoptosis in HeLa cells in an Atg3-dependent manner

Background

Apoptosis and autophagy are known to play important roles in cancer development. It has been reported that HVJ-E induces apoptosis in cancer cells, thereby inhibiting the development of tumors. To define the mechanism by which HVJ-E induces cell death, we examined whether HVJ-E activates autophagic and apoptotic signaling pathways in HeLa cells.

Methods

Cells were treated with chloroquine (CQ) and rapamycin to determine whether autophagy is involved in HVJ-E-induced apoptosis. Treatment with the ERK inhibitor, U0126, was used to determine whether autophagy and apoptosis are mediated by the ERK pathway. Activators of the PI3K/Akt/mTOR/p70S6K pathway, 740 Y-P and SC79, were used to characterize its role in HVJ-E-induced autophagy. siRNA against Atg3 was used to knock down the protein and determine whether it plays a role in HVJ-E-induced apoptosis in HeLa cells.

Results

We found that HVJ-E infection inhibited cell viability and induced apoptosis through the mitochondrial pathway, as evidenced by the expression of caspase proteins. This process was promoted by rapamycin treatment and inhibited by CQ treatment. HVJ-E-induced autophagy was further blocked by 740 Y-P, SC79, and U0126, indicating that both the ERK- and the PI3K/Akt/mTOR/p70S6K-pathways were involved. Finally, autophagy-mediated apoptosis induced by HVJ-E was inhibited by siRNA-mediated Atg3 knockdown.

Conclusion

In HeLa cells, HVJ-E infection triggered autophagy through the PI3K/Akt/mTOR/p70S6K pathway in an ERK1/2-dependent manner, and the induction of autophagy promoted apoptosis in an Atg3-dependent manner.

     

MicroRNA-1284 inhibits proliferation and induces apoptosis in SGC-7901 human gastric cancer cells

Objectives

To explore the functional effects of miR-1284 on gastric cancer cells.

Results

Overexpression of miR-1284 significantly reduced SGC-7901 cell proliferation, but improved apoptosis. However, miR-1284 suppression displayed the inversed impacts. Furthermore, the protein levels of p27, Bax, procaspase-3 and active caspase-3 were up-regulated by miR-1284 overexpression, but were down-regulated by miR-1284 suppression. The level of Bcl-2 was down-regulated by miR-1284 overexpression, while it was up-regulated by miR-1284 suppression. The level of p21 was unaffected.

Conclusion

These results suggest that miR-1284 overexpression might be a suppressor for gastric cancer via controlling of cell proliferation and apoptosis.

     

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.

     

MicroRNA-145-based differentiation of human mesenchymal stem cells to smooth muscle cells

Objectives

To investigate the role of microRNA-145, that regulates gene expression of genes related to differentiation, proliferation and the phenotype of smooth muscle cells (SMCs), in the differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) to SMCs.

Results

Real-time PCR analysis indicated significant upregulation of SMC markers, including SM-α-actin, calponin, caldesmon and SMMHC, in SMCs compared to hBM-MSCs. Conversely, Krüppel-like factor 4, the direct target of microRNA-145 and the suppressor of smooth muscle differentiation, was suppressed in hBM-MSC-derived SMCs. Western blot analysis and immunocytochemistry also confirmed that the introduction of microRNA-145 into hBM-MSCs induced mature contractile SMCs. The functionality of hBM-MSC-derived SMCs was assessed by proliferation assay using PDGF-BB and contractility assay using carbachol. The results showed that the produced SMCs contracted in response to carbachol stimulation.

Conclusion

Overexpression of microRNA-145 in undifferentiated hBM-MSCs results in functionally mature contractile SMCs that can be used in drug discovery and cell therapy in SMC disorders such as vascular disease.

     

Knockdown of microRNA-17-5p ameliorates atherosclerotic lesions in ApoE<Superscript>−/−</Superscript> mice and restores the expression of very low density lipoprotein receptor

Objective

To propose and verify a hypothesis that miR-17-5p knockdown may mitigate atherosclerotic lesions using atherosclerotic ApoE^?/? mice as serum microRNA-17-5p (miR-17-5p) is elevated in patients with atherosclerosis.

Results

The level of miR-17-5p was higher while the level of very low density lipoprotein receptor (VLDLR), a predicted target of miR-17-5p, was lower in the peripheral blood lymphocytes (PBLs) of atherosclerosis patients as compared with control PBLs. ApoE^?/? mice fed with a high-cholesterol diet displayed marked atherosclerotic vascular lesions, which were ameliorated after treatment with antagomiR-17-5p. Moreover, the decreased VLDLR in atherosclerotic mice was partly restored when miR-17-5p was antagonized. Further, luciferase assay confirmed VLDLR as a direct target of miR-17-5p in vascular smooth muscle cells (VSMCs). In addition, the elevated expression of proprotein convertase subtilisin kexin 9 (PCSK9), a secreted protease that binds to and promotes VLDLR degradation, in the atherosclerotic mice was suppressed by antagomiR-17-5p.

Conclusions

A novel interaction between miR-17-5p and VLDLR is revealed and suggests that miR-17-5p may be a potential therapeutic target for AS.

     

MiR-338-3p inhibits TNF-α-induced lipogenesis in human sebocytes

Objective

To suppress TNF-α-induced lipogenesis in sebocytes (associated with acne development) with microRNA-338-3p (miR-338-3p) and to explore the underlying mechanisms.

Results

TNF-α increased lipid droplet formation in sebocytes which were used as in vitro model of inflammation-induced acne. Flow cytometry and TLC assays validated that miR-338-3p could suppress TNF-α-induced lipid droplet formation, down-regulate the expression of PREX2a, and inactivate AKT signaling in sebocytes. In addition, suppression of AKT activity by the PI3 K and AKT inhibitors diminished TNF-α-induced lipogenesis. PREX2a siRNA mimics the effects of miR-338-3p on AKT phosphorylation and lipogenesis. PREX2a overexpression consistently restored lipogenesis and AKT phosphorylation attenuated by miR-338-3p.

Conclusions

MiR-338-3p suppresses the TNF-α-induced lipogenesis in sebocytes by targeting PREX2a and down-regulating PI3K/AKT signaling.

     

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-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.

     

Effect of experimental gingivitis induction and erythritol on the salivary metabolome and functional biochemistry of systemically healthy young adults

Introduction

Understanding the changes occurring in the oral ecosystem during development of gingivitis could help improve prevention and treatment strategies for oral health. Erythritol is a non-caloric polyol proposed to have beneficial effects on oral health.

Objectives

To examine the effect of experimental gingivitis and the effect of erythritol on the salivary metabolome and salivary functional biochemistry.

Methods

In a two-week experimental gingivitis challenge intervention study, non-targeted, mass spectrometry-based metabolomic profiling was performed on saliva samples from 61 healthy adults, collected at five time-points. The effect of erythritol was studied in a randomized, controlled trial setting. Fourteen salivary biochemistry variables were measured with antibody- or enzymatic activity-based assays.

Results

Bacterial amino acid catabolites (cadaverine, N-acetylcadaverine, and α-hydroxyisovalerate) and end-products of bacterial alkali-producing pathways (N-α-acetylornithine and γ-aminobutyrate) increased significantly during the experimental gingivitis. Significant changes were found in a set of 13 salivary metabolite ratios composed of host cell membrane lipids involved in cell signaling, host responses to bacteria, and defense against free radicals. An increase in mevalonate was also observed. There were no significant effects of erythritol. No significant changes were found in functional salivary biochemistry.

Conclusions

The findings underline a dynamic interaction between the host and the oral microbial biofilm during an experimental induction of gingivitis.

     

Potential action of androstenedione on the proliferation and apoptosis of stromal endometrial cells

Background

Hyperandrogenic conditions have been associated with a high prevalence of endometrial pathologies related to cell survival. However, the action of androgens on proliferation and apoptosis in endometrial cells is poorly understood. Therefore, the aim of the present study was to evaluate the effect of androstenedione on cell proliferation, cell death and expression of estrogen receptor (ER) isoforms and proteins related to apoptosis in endometrial cells using two in vitro experimental approaches.

Methods

The endometrial tissue was obtained from 20 eumenorrheic women [28.7 (25 – 35) years] during the early secretory phase. We analyzed cell proliferation (immunohistochemistry of Ki-67 and spectrophotometric assay); apoptosis (DNA fragmentation (TUNEL) and Annexin V-FITC binding); ER-alpha, ER-beta bcl-2 and bax mRNA abundance (RT-PCR) in explants and isolated endometrial epithelial (EEC) and stromal cells (ESC) incubated with androstenedione 1 micro mol/l (A4) or A4 plus hydroxyflutamide 10 micro mol/l (F) for 24 h.

Results

In explants, A4 induced an increase of cell proliferation and a decrease on apoptosis in the stromal compartment (p < 0.05). In isolated ESC, proliferation augmented with A4 (p < 0.05), whereas, no significant modifications in the expression of ER-alpha, ER-beta bcl-2 and bax nor in the apoptotic index were observed. In EEC, A4 increase the ER-beta mRNA abundance (p < 0.05) and a decrease of the bcl-2/bax ratio (p < 0.05), without an increase in the apoptotic index. Hydroxyflutamide reverted the effect of androstenedione on the parameters described.

Conclusions

These results indicate that androstenedione may modulate cell survival, expression of ER-beta and proteins related to apoptosis, suggesting a potential mechanism that associates the effect of hyperandrogenemia on the endometrial tissue.

     

Alpha-synuclein contributes to malignant progression of human meningioma via the Akt/mTOR pathway

Background

The aim of this study is to explore the expression of alpha-synuclein (α-synuclein) in benign, atypical, and anaplastic meningiomas and determine its role in the malignant progression of meningiomas.

Methods

Expression of α-synuclein was measured in 44 meningioma samples by real-time PCR analysis. The effects of overexpression or knockdown of α-synuclein on meningioma cell growth, invasiveness, and tumorigenicity were determined.

Results

Atypical and anaplastic meningiomas displayed significantly greater levels of α-synuclein mRNA, relative to benign tumors. Depletion of α-synuclein decreased cell proliferation and colony formation and promoted apoptosis in IOMM-Lee meningioma cells, whereas overexpression of α-synuclein facilitated cell proliferation and colony formation in CH-157MN meningioma cells. Silencing of α-synuclein attenuated IOMM-Lee cell migration and invasion. In contrast, ectopic expression of α-synuclein increased the invasiveness of CH-157MN cells. In vivo studies further demonstrated that downregulation of α-synuclein significantly retarded meningioma growth in nude mice. At the molecular level, the phosphorylation levels of Akt, mTOR, p70S6K and 4EBP were significantly decreased in α-synuclein-depleted IOMM-Lee cells.

Conclusions

In conclusion, α-synuclein upregulation contributes to aggressive phenotypes of meningiomas via the Akt/mTOR pathway and thus represents a potential therapeutic target for malignant meningiomas.

     

Parthenolide induces apoptosis and autophagy through the suppression of PI3K/Akt signaling pathway in cervical cancer

Objective

To investigate the effect of parthenolide on apoptosis and autophagy and to study the role of the PI3K/Akt signaling pathway in cervical cancer.

Results

Parthenolide inhibits HeLa cell viability in a dose dependent-manner and was confirmed by MTT assay. Parthenolide (6 µM) induces mitochondrial-mediated apoptosis and autophagy by activation of caspase-3, upregulation of Bax, Beclin-1, ATG5, ATG3 and down-regulation of Bcl-2 and mTOR. Parthenolide also inhibits PI3K and Akt expression through activation of PTEN expression. Moreover, parthenolide induces generation of reactive oxygen species that leads to the loss of mitochondrial membrane potential.

Conclusion

Parthenolide induces apoptosis and autophagy-mediated growth inhibition in HeLa cells by suppressing the PI3K/Akt signaling pathway and mitochondrial membrane depolarization and ROS generation. Parthenolide may be a potential therapeutic agent for the treatment of cervical cancer.

     

The lncRNA MEG3 downregulation leads to osteoarthritis progression via miR-16/SMAD7 axis

Background

Osteoarthritis (OA) is a chronic joint disease and there is no a definitive cure at present. Long non-coding RNAs (lncRNAs) have been confirmed to play important roles in the development of OA. However, the underlying mechanism of lncRNA maternally expressed gene 3 (MEG3) in OA has not been well elucidated.

Methods

The rat OA model and interleukin-1β (IL-1β)-induced rat chondrocytes were constructed. The expression pattern of lncRNA MEG3 and miR-16 was detected by RT-qPCR assay in cartilage tissues of rat OA model. The effect of MEG3 and miR-16 on IL-1β-induced chondrocytes was evaluated on the basis of cell viability and apoptosis. Then, the interaction among MEG3, miR-16 SMAD7 was explored by dual-luciferase reporter assay and RIP assay.

Results

It is found that lncRNA MEG3 was down-regulated and miR-16 was up-regulated in rat OA cartilage tissues. MEG3 knockdown promoted proliferation and inhibited apoptosis, while miR-16 knockdown suppressed proliferation and promoted apoptosis in IL-1β-induced rat chondrocytes. Moreover, MEG3 was involved in miR-16 pathway and MEG3 suppressed miR-16 expression. Additionally, SMAD7 was a target gene of miR-16 and miR-16 suppressed SMAD7 expression in IL-1β-induced chondrocytes. Moreover, the expression of SMAD7 induced by MEG3 or si-MEG3 was markedly reversed by the introduction of miR-16 or anti-miR-16. Furthermore, MEG3 exerted its anti-proliferation and pro-apoptosis by regulating miR-16 and SMAD7.

Conclusion

MEG3 was down-regulated and miR-16 was up-regulated in cartilage tissues of rat OA model. MEG3 knockdown might lead to the progression of OA through miR-16/SMAD7 axis.