Mimic Transfection Research Articles

MicroRNA Let-7 targets AMPK and impairs hepatic lipid metabolism in offspring of maternal obese pregnancies

Nutritional status during gestation may lead to a phenomenon known as metabolic programming, which can be triggered by epigenetic mechanisms. The Let-7 family of microRNAs were one of the first to be discovered, and are closely related to metabolic processes. Bioinformatic analysis revealed that Prkaa2, the gene that encodes AMPK α2, is a predicted target of Let-7. Here we aimed to investigate whether Let-7 has a role in AMPKα2 levels in the NAFLD development in the offspring programmed by maternal obesity. Let-7 levels were upregulated in the liver of newborn mice from obese dams, while the levels of Prkaa2 were downregulated. Let-7 levels strongly correlated with serum glucose, insulin and NEFA, and in vitro treatment of AML12 with glucose and NEFA lead to higher Let-7 expression. Transfection of Let-7a mimic lead to downregulation of AMPKα2 levels, while the transfection with Let-7a inhibitor impaired both NEFA-mediated reduction of Prkaa2 levels and the fat accumulation driven by NEFA. The transfection of Let-7a inhibitor in ex-vivo liver slices from the offspring of obese dams restored phospho-AMPKα2 levels. In summary, Let-7a appears to regulate hepatic AMPKα2 protein levels and lead to the early hepatic metabolic disturbances in the offspring of obese dams.

The regulatory role of miR-20a in bovine cumulus cells and its contribution to oocyte maturation.

Dynamic changes in microRNAs in oocyte and cumulus cells before and after maturation may explain the spatiotemporal post-transcriptional gene regulation within bovine follicular cells during the oocyte maturation process. miR-20a has been previously shown to regulate proliferation and differentiation as well as progesterone levels in cultured bovine granulosa cells. In the present study, we aimed to demonstrate the function of miR-20a during the bovine oocyte maturation process. Maturation of cumulus-oocyte complexes (COCs) was performed at 39°C in an humidified atmosphere with 5% CO2 in air. The expression of miR-20a was investigated in the cumulus cells and oocytes at 22 h post culture. The functional role of miR-20a was examined by modulating the expression of miR-20a in COCs during in vitro maturation (IVM). We found that the miR-20a expression was increased in cumulus cells but decreased in oocytes after IVM. Overexpression of miR-20a increased the oocyte maturation rate. Even though not statistically significant, miR-20a overexpression during IVM increased progesterone levels in the spent medium. This was further supported by the expression of STAR and CYP11A1 genes in cumulus cells. The phenotypes observed due to overexpression of miR-20a were validated by BMP15 supplementation during IVM and subsequent transfection of BMP15-treated COCs using miR-20a mimic or BMPR2 siRNA. We found that miR-20a mimic or BMPR2 siRNA transfection rescued BMP15-reduced oocyte maturation and progesterone levels. We concluded that miR-20a regulates oocyte maturation by increasing cumulus cell progesterone synthesis by simultaneous suppression of BMPR2 expression.

Differential miRNA Expression Profiling Reveals Correlation of miR125b-5p with Persistent Infection of Japanese Encephalitis Virus.

MicroRNAs (miRNAs) play versatile roles in multiple biological processes. However, little is known about miRNA’s involvement in flavivirus persistent infection. Here, we used an miRNA array analysis of Japanese encephalitis virus (JEV)-infected cells to search for persistent infection-associated miRNAs in comparison to acute infection. Among all differentially expressed miRNAs, the miR-125b-5p is the most significantly increased one. The high level of miR-125b-5p in persistently JEV-infected cells was confirmed by Northern analysis and real-time quantitative polymerase chain reaction. As soon as the cells established a persistent infection, a significantly high expression of miR-125b-5p was readily observed. Transfecting excess quantities of a miR-125b-5p mimic into acutely infected cells reduced genome replication and virus titers. Host targets of miR125b-5p were analyzed by target prediction algorithms, and six candidates were confirmed by a dual-luciferase reporter assay. These genes were upregulated in the acutely infected cells and sharply declined in the persistently infected cells. The transfection of the miR125b-5p mimic reduced the expression levels of Stat3, Map2k7, and Triap1. Our studies indicated that miR-125b-5p targets both viral and host sequences, suggesting its role in coordinating viral replication and host antiviral responses. This is the first report to characterize the potential roles of miR-125b-5p in persistent JEV infections.

Identification of a cartilage specific novel miRNA which directly targets PRMT3 in rats

ObjectiveThrough experiments to testify a candidate novel miRNA previously discovered by us is a real miRNA and involved in cartilage development. Design: The miR-novel and the newly hairpin miRNA transcribed sequence (pre-miR-novel) was verified as a genuinely existing miRNA by northern blotting. The predicted secondary structure, sequence alignment and targets of pre-miR-novel were performed by “RNAstructure 5.3” program, LASTN2.8.0+/miRbase22 program and RNA hybird program, respective. GO/KEGG pathway analysis also were performed. The miR-novel expression in cartilage tissue during development was detected by RT-qPCR and dot blotting. The chondrocyte differentiation model was established to examine whether miR-novel is involved in cartilage development. The regulation of PRMT3 expression by novel miRNA was determined with the luciferase reporter gene assay and Western blotting after novel miRNA mimic or inhibitor transfection. Results: It’s potential role in specifically regulating rodent cartilage development and associated cellular processes. Furthermore, the expression of protein arginine N-methyltransferase 3 (PRMT3), as a predicted target of the novel miRNA, was found consistently downregulated at rat cartilage during developmental stages and RCJ3.1C5.18 (C5.18) cells during the proliferating and hypertrophic phases of the cartilage development, where the miR-novel expression was significantly up-regulated. Both the dual-luciferase reporter gene assay and the up- or down-regulation of miR-novel suggest that the later can specifically bind with the Prmt3 3′-UTR. Conclusion: Overall, this study provides the first comprehensive evidence that a genuine cartilage-specific novel miRNA directly targets PRMT3 and may regulate multitudinous cellular processes and signal transduction during cartilage development.

LncRNA XIST/miR-137 axis strengthens chemo-resistance and glycolysis of colorectal cancer cells by hindering transformation from PKM2 to PKM1.

Glycolysis was an essential driver of chemo-resistance in colorectal cancer (CRC), albeit with limited molecular explanations. We strived to elucidate the involvement of lncRNA XIST/miR-137/PKM axis in chemo-tolerance and glycolysis of CRC. Altogether 212 pairs of tumor tissues and adjacent normal tissues were collected from CRC patients. Moreover, human CRC epithelial cell lines, including HT29, SW480, SW620 and LoVo, were purchased in advance, and their activity was estimated after transfection of si-XIST or miR-137 mimic. Furthermore, 5-FU/cisplatin-resistance of CRC cells was determined through MTT assay, and glycolytic potential of CRC cells was appraised based on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Highly-expressed XIST were predictive of severe symptoms and unfavorable 3-year survival of CRC patients (P< 0.05). Besides, silencing of XIST not only diminished proliferative, migratory and invasive power of CRC cells (P< 0.05), but also enhanced sensitivity of CRC cells responding to 5-FU/cisplatin (P< 0.05). Glycolytic potency of CRC cells was also undermined by si-XIST, with decreased maximal respiration and maximal glycolytic capacity in the si-XIST group as relative to NC group (P< 0.05). Nevertheless, miR-137 mimic attenuated the facilitating effect of pcDNA3.1-XIST on proliferation, migration, invasion, 5-FU/cisplatin-resistance and glycolysis of CRC cells (P< 0.05). Ultimately, ratio of PKM2 mRNA and PKM1 mRNA, despite being up-regulated by pcDNA3.1-XIST, was markedly lowered when miR-137 mimic was co-transfected (P< 0.05). LncRNA XIST/miR-137 axis reinforced glycolysis and chemo-tolerance of CRC by elevating PKM2/PKM1 ratio, providing an alternative to boost chemo-therapeutic efficacy of CRC patients.

LncRNA DANCR regulates lymphatic metastasis of bladder cancer via the miR-335/VEGF-C axis.

BackgroundSubstantial evidence indicate that long non-coding RNA (lncRNA) and microRNA (miRNA) act as key role in bladder cancer. Differentiation antagonistic ncRNA (DANCR) could be used as a biomarker in the occurrence and development of cancer. This study aims to explore the mechanism of DANCR/miR-335/VEGF-C axis affecting lymphatic metastasis of bladder cancer.MethodsqRT-PCR detects the expression of DANCR in bladder cancer cell lines (SW780, 5637, T24, UM-UC-3) and normal bladder cell lines (SV-HUC-1), and selects T24 cell lines for subsequent experiments. The expression levels of DANCR, miR-335 and VEGF were measured by qRT-PCR, and the dual luciferase reporter gene verified the targeted regulation of DANCR on miR-335 and miR-335 on VEGF. CCK-8, Transwell and Wound healing assay detect the proliferation, invasion and migration ability of bladder cancer cells, Endothelial cell adhesion assay and Western blot further prove the lymphatic metastasis of bladder cancer.ResultsIn this study, DANCR was highly expressed in bladder cancer cell lines. Transfection of si-DANCR significantly inhibits the proliferation, migration, invasion and lymphatic metastasis of bladder cancer cells. Dual luciferase assay confirmed that DANCR targets miR-335/VEGF-C. Transfection of miR-335 mimic promotes the proliferation, migration, invasion and lymphatic metastasis of bladder cancer cells, overexpression of DANCR eliminates the promotion of miR-335 mimic on bladder cancer cells. Further experiments proved that inhibition of miR-335 and overexpression of VEGF-C can reverse the inhibitory effect of silencing DANCR on bladder cancer cells.ConclusionsIn bladder cancer, DARCR plays an important role, which regulates the proliferation, migration, invasion and lymphatic metastasis of bladder cancer cells through the miR-335/VEGF-C molecular axis.

MicroRNA-145-5p Protects Human Melanocytes Against Oxidative Damage by Targeting Transient Receptor Potential Melastatin 2 (TRPM2)

Background: Oxidative stress was reported to be involved in the progression of vitiligo. microRNAs (miRNAs) have been confirmed to display critical roles in vitiligo. In this study, we conjectured that miR-145-5p might be related to the development of vitiligo by regulating the key genes expression in melanocytes. Methods: H2O2 was used to induce the dysfunction of melanocytes. The levels of TRPM2 and miR-145-5p in H2O2-induced human primary melanocytes were assessed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). TargetScan and Dual luciferase reporter gene assay were conducted to confirm the correlation between miR-145-5p and TRPM2. Cell viability and apoptosis were determined using MTT and Flow cytometry analysis. Reactive oxygen species (ROS), antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were determined using specific assay kits. The levels of cleaved caspase-3 and pro-Caspase3 were measure by western blotting. Results: TRPM2 was upregulated while miR-145-5p was downregulated in H2O2-induced human primary melanocytes. Dual luciferase reporter assay confirmed that TRPM2 was a target gene of miR-145-5p. miR-145-5p mimic transfection significantly increased cell viability and inhibited cell apoptosis in H2O2-treated melanocytes. In addition, overexpression of miR-145-5p enhanced the antioxidant activity of SOD and CAT, and decreased intracellular ROS accumulation. Notably, these findings were abolished by TRPM2-plasmid. Conclusions: Taken together, our study demonstrated that oxidative stress induced up-regulation of TRPM2 and down-regulation of miR-145-5p in melanocytes. In addition, overexpression of miR-145-5p alleviated melanocytes destruction via targeting TRPM2. These results indicated that miR-145-5p might serve as a potential target for anti-oxidative therapy in vitiligo.

MiR-144-3p ameliorates the progression of osteoarthritis by targeting IL-1β: Potential therapeutic implications.

The pro-inflammatory cytokine interleukin 1 beta (IL-1β) plays a critical role in osteoarthritis (OA) disease pathogenesis. MicroRNA (miRNA) activity is related to inflammation in OA and some miRNAs specifically regulate IL-mediated degradation of cartilage type II collagen. Previous studies have indicated that miR-144-3p is a useful target in the regulation of pro-inflammatory cytokines in different diseases. However, the role of miR-144-3p in OA is unclear. In this study, we observed a negative correlation between miR-144-3p and IL-1β expression in OA. miR-144-3p mimic transfection of OA synovial fibroblasts downregulated levels of IL-1β expression, while blocking the MAPK, PI3K/Akt, and NF-κB signaling pathways relating to IL-1β production, and effectively increased miR-144-3p expression in OASFs. Findings from an anterior cruciate ligament transection rat model revealed that administration of miR-144-3p mimic effectively ameliorated OA progression and reduced the numbers of IL-1β-positive cells in synovial tissue. This study suggests that miR-144-3p is a useful therapeutic target in OA disease.

MiR-34a mimic or pre-mir-34a, which is the better option for cancer therapy? KatoIII as a model to study miRNA action in human gastric cancer cells

BackgroundAberrantly expressed microRNAs play important roles in gastric tumorigenesis. However, use of miRNAs as a therapeutic option in gastric cancer still remains as a challenging problem.MethodsWe performed transient transfection of miR-34a-5p mimic and stable transfection of pre-mir-34a into KatoIII cells. Then, we evaluated the effect of transfected miRNAs on numerous cellular and molecular processes.ResultsFollowing transient transfection of miR-34a-5p mimic at 25 nM—a commonly used concentration—into KatoIII cells, inhibition of two target genes expression, namely Notch1 and β-catenin, was not observed, but a non-significant marginal increase of these genes was detected. No changes were detected in the percentage of apoptotic cells as well as in CD44 + and EpCAM + cells after 25 nM miR-34a-5p mimic transfection. Interestingly, stable transfection of pre-mir-34a into KatoIII cells (named as KatoIII-pGFPC1-34a cells) caused a significant repression in β-catenin protein and Notch1 mRNA levels (p < 0.05 and p < 0.01, respectively) relative to equivalent control (KatoIII- pGFPC1-empty cells). The percentage of CD44 + cells in the KatoIII-pGFPC1-34a cells (< 40%) was significantly lower than that in control cells (~ 95%) (p < 0.05). An increase of ~ 3.5% in apoptotic cells and a slower proliferation rate were detected in KatoIII-pGFPC1-34a cells.ConclusionsOur study revealed that the effect of miR mimic in target gene repression can be dependent to its concentration as well as to the cell type. Meanwhile, our findings further support a regulatory function for pre-miRNAs in target repression and will help to develop effective therapeutic strategies in cancer treatment.

Effects of metal nanoparticles on tight junction-associated proteins via HIF-1\u03b1/miR-29b/MMPs pathway in human epidermal keratinocytes

BackgroundThe increasing use of metal nanoparticles in industry and biomedicine raises the risk for unintentional exposure. The ability of metal nanoparticles to penetrate the skin ranges from stopping at the stratum corneum to passing below the dermis and entering the systemic circulation. Despite the potential health risks associated with skin exposure to metal nanoparticles, the mechanisms underlying the toxicity of metal nanoparticles on skin keratinocytes remain unclear. In this study, we proposed that exposure of human epidermal keratinocytes (HaCaT) to metal nanoparticles, such as nickel nanoparticles, dysregulates tight-junction associated proteins by interacting with the HIF-1α/miR-29b/MMPs axis.MethodsWe performed dose-response and time-response studies in HaCaT cells to observe the effects of Nano-Ni or Nano-TiO2 on the expression and activity of MMP-2 and MMP-9, and on the expression of tight junction-associated proteins, TIMP-1, TIMP-2, miR-29b, and HIF-1α. In the dose-response studies, cells were exposed to 0, 10, or 20 μg/mL of Nano-Ni or Nano-TiO2 for 24 h. In the time-response studies, cells were exposed to 20 μg/mL of Nano-Ni for 12, 24, 48, or 72 h. After treatment, cells were collected to either assess the expression of mRNAs and miR-29b by real-time PCR or to determine the expression of tight junction-associated proteins and HIF-1α nuclear accumulation by Western blot and/or immunofluorescent staining; the conditioned media were collected to evaluate the MMP-2 and MMP-9 activities by gelatin zymography assay. To further investigate the mechanisms underlying Nano-Ni-induced dysregulation of tight junction-associated proteins, we employed a HIF-1α inhibitor, CAY10585, to perturb HIF-1α accumulation in one experiment, and transfected a miR-29b-3p mimic into the HaCaT cells before Nano-Ni exposure in another experiment. Cells and conditioned media were collected, and the expression and activities of MMPs and the expression of tight junction-associated proteins were determined as described above.ResultsExposure of HaCaT cells to Nano-Ni resulted in a dose-dependent increase in the expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 and the activities of MMP-2 and MMP-9. However, exposure of cells to Nano-TiO2 did not cause these effects. Nano-Ni caused a dose-dependent decrease in the expression of miR-29b and tight junction-associated proteins, such as ZO-1, occludin, and claudin-1, while Nano-TiO2 did not. Nano-Ni also caused a dose-dependent increase in HIF-1α nuclear accumulation. The time-response studies showed that Nano-Ni caused significantly increased expressions of MMP-2 at 24 h, MMP-9 at 12, 24, and 48 h, TIMP-1 from 24 to 72 h, and TIMP-2 from 12 to 72 h post-exposure. The expression of miR-29b and tight junction-associated proteins such as ZO-1, occludin, and claudin-1 decreased as early as 12 h post-exposure, and their levels declined gradually over time. Pretreatment of cells with a HIF-1α inhibitor, CAY10585, abolished Nano-Ni-induced miR-29b down-regulation and MMP-2/9 up-regulation. Introduction of a miR-29b-3p mimic into HaCaT cells by transfection before Nano-Ni exposure ameliorated Nano-Ni-induced increased expression and activity of MMP-2 and MMP-9 and restored Nano-Ni-induced down-regulation of tight junction-associated proteins.ConclusionOur study herein demonstrated that exposure of human epidermal keratinocytes to Nano-Ni caused increased HIF-1α nuclear accumulation and increased transcription and activity of MMP-2 and MMP-9 and down-regulation of miR-29b and tight junction-associated proteins. Nano-Ni-induced miR-29b down-regulation was through Nano-Ni-induced HIF-1α nuclear accumulation. Restoration of miR-29b level by miR-29b-3p mimic transfection abolished Nano-Ni-induced MMP-2 and MMP-9 activation and down-regulation of tight junction-associated proteins. In summary, our results demonstrated that Nano-Ni-induced dysregulation of tight junction-associated proteins in skin keratinocytes was via HIF-1α/miR-29b/MMPs pathway.

MiR-489-3p modulates cell proliferation and apoptosis in cerulein-induced acute pancreatitis by targeting X-linked inhibitor of apoptosis protein

IntroductionThis study aims to explore the effect and mechanism of miR�489-3p on the proliferation and apoptosis of pancreatic acinar cells in acute pancreatitis (AP).Material and methodsX-linked inhibitor of apoptosis protein (XIAP) and miR-489-3p expression in serum of AP patients, pancreatic AR42J cells, and rat AP tissues were measured using quantitative reverse transcription poly�merase chain reaction and western blot. The effect of miR-489-3p on pro�liferation and apoptosis was determined by MTT assay and flow cytometry. The relationship between XIAP and miR-489-3p was verified using luciferase assay RNA immunoprecipitation assay. Histological changes in rat pancreatic tissues were observed via haematoxylin and eosin staining.ResultsMeasurement of miR-489-3p and XIAP expressions in AP patients revealed a negative correlation between miR-489-3p and XIAP. Increased miR-489-3p expression in AP patients indicated a poor prognosis. Cerulein was used to induce AP in AR42J cells and standard deviation rats. Exper�iments in cells and AP rat models showed that miR-489-3p can increase cell apoptosis and inhibit cell proliferation by regulating XIAP, as shown by elevated expressions of pro-apoptotic proteins (p53 and Bax) and decreased expression of proliferation indicator (Ki-67) after transfection of miR-489- 3p mimics. Meanwhile, knockdown of miR-489-3p abrogated the inhibitory effects of miR-489-3p on cell proliferation and the promotion on cell apop�tosis. Luciferase assay and RNA immunoprecipitation assay confirmed that XIAP can directly bind miR-489-3p.ConclusionsWe concluded that miR-489-3p modulates cell proliferation and apoptosis in AP by targeting XIAP. Given that high expression of miR�489-3p in AP indicated poor prognosis, it raises the possibility that miR�489-3p might be a novel and valuable therapeutic target and a prognosis indicator for AP.

Long noncoding RNA CASC7 inhibits the proliferation and migration of papillary thyroid cancer cells by inhibiting miR-34a-5p

Long noncoding RNAs (lncRNAs) play an essential role in the progression of papillary thyroid cancer (PTC). However, the expression and function of lncRNA cancer susceptibility candidate 7 (CASC7) in PTC remain unknown. The purpose of this study was to investigate the role and molecular mechanism of CASC7 in regulating PTC cell behavior. The expression of CASC7, miR-34a-5p, and tumor protein P73 (TP73) was determined by qRT-PCR and western blot. Cell proliferation was examined by MTT assay. Cell apoptosis was assessed by flow cytometry following Annexin V and PI staining. Cell migration was determined by Transwell migration assay. The interaction between miR-34a-5p and CASC7 or TP73 was examined by luciferase reporter assay. CASC7 and TP73 expression were significantly lower, whereas miR-34a-5p expression was higher in PTC tissues than the adjacent normal tissues. Furthermore, CASC7 overexpression inhibited cell proliferation and migration, whereas facilitated cell apoptosis in human PTC cell lines (K1 and TPC-1). Mechanistically, CASC7 acted as a sponge of miR-34a-5p to upregulate TP73 expression. Moreover, miR-34a-5p mimic transfection could abate the CASC7-regulated PTC cell proliferation, migration, and apoptosis. Collectively, CASC7 inhibited the proliferation and migration of PTC cells by sponging miR-34a-5p to upregulate TP73 expression.

Regulation of connective tissue growth factor expression by miR-133b for the treatment of renal interstitial fibrosis in aged mice with unilateral ureteral obstruction

IntroductionRenal interstitial fibrosis, an important pathological feature of kidney aging and chronic renal failure, is regulated by mesenchymal stem cells (MSCs). We have previously demonstrated low expression of miR-133b in MSC-derived extracellular vesicles (MSC-EVs) in aged rats. However, miR-133b can mediate the inhibition of epithelial-mesenchymal transition (EMT) of renal tubules induced by transforming growth factor-β1 (TGF-β1). We investigated the effect of miR-133b for the treatment of geriatric renal interstitial fibrosis and evaluated its target genes.MethodsWe performed real-time polymerase chain reaction to detect miR-133b expression induced during EMT of HK2 cells by TGF-β1 at different concentrations (0, 6, 8, and 10 ng/mL) and at different time points (0, 24, 48, and 72 h). The target genes of miR-133b were validated using the dual-luciferase reporter assay. In vitro experiments were performed to evaluate mRNA and protein expression of miR-133b targets, E-cadherin, α-smooth muscle actin (SMA), fibronectin, and collagen 3A1 (Col3A1), in HK2 cells transfected with miR-133b under TGF-β1 stimulation. A 24-month-old unilateral ureteral obstruction (UUO) mouse model was established and injected with transfection reagent and miR-133b into the caudal vein. The target gene of miR-133b and other parameters mentioned above such as mRNA and protein expression levels and renal interstitial fibrosis were detected at 7 and 14 days.ResultsmiR-133b expression gradually decreased with an increase in TGF-β1 concentration and treatment time, and the miR-133b mimic downregulated connective tissue growth factor (CTGF) expression. The dual-luciferase reporter assay confirmed CTGF as a direct target of miR-133b. Transfection of the miR-133b mimic inhibited TGF-β1-induced EMT of HK2 cells; this effect was reversed by CTGF overexpression. miRNA-133b expression significantly increased (approximately 70–100 times) in mouse kidney tissues after injection of the miRNA-133b overexpression complex, which significantly alleviated renal interstitial fibrosis in mice with UUO.ConclusionmiR-133b exerted targeted inhibitory effects on CTGF expression, which consequently reduced TGF-β1-induced EMT of HK2 cells and renal interstitial fibrosis in aged mice with UUO.

Punica granatum Extract Inhibits Bladder Cancer Cell Viability, Invasion and Migration through Down-Regulation of HOXD10 Signalling Pathway.

The present study investigated Punica granatum extract (PGE) as potential proliferation inhibitory agent for bladder cancer cells and elucidated the possible mechanism. PGE reduced viabilities of HT-1197 and RT4 cells in concentration-based manner at 72 h. Colony forming potential of HT-1197 and RT4 cells was also significantly (p < 0.05) inhibited on exposure to 2 and 12 mg/mL PGE. Exposure to 12 mg/mL PGE for 72 h significantly (p < 0.05) decreased miR‑10b expression and suppressed migration potential of HT-1197 and RT4 cells. In PGE exposed HT-1197 and RT4 cells, invasiveness was reduced to 30.25 and 33.47%, respectively. PGE treatment of HT-1197 and RT4 cells caused a significant (p < 0.05) elevation in HOXD10 protein and mRNA levels compared to control. The miR‑10b mimic transfection in HT-1197 and RT4 cells reversed inhibitory effect of PGE on cell viability. Thus, PGE exhibited cytotoxicity and anti-invasive effect on HT-1197 and RT4 cells through targeting miR‑10b and up-regulation of HOXD10 expression. Thus, PGE may be developed as therapeutic agent for treatment of bladder cancer.

MiRNA-200a Regulating Proliferation, Migration, and Infiltration of Tongue Squamous Cell Carcinoma Cells by Targeting DEK Proto-Oncogene

Tongue squamous cell carcinoma (TSCC) is the most frequently occurring oral cancer and is characterized by high proliferation and metastasis rates. Incomplete understanding of the pathogenesis of TSCC coupled with frequent tongue movement increases the difficulty of therapy. Additionally, TSCC is prone to recurrence and metastasis after treatment. Thus, exploring mechanisms of proliferation, migration, and infiltration of TSCC cancer cells is essential for reducing morbidity and mortality. Transfection of miRNA-200a mimics into SCC15 cells showed that miRNA-200a expression decreased significantly, and DEK expression significantly increased. Transfection of miRNA-200a mimics (miRNA-200a group), negative control mimics (miRNA-NC group), empty vector (miRNA-200a + pcDNA3.1 group), and miRNA-200a mimics and DEK overexpression vector (miRNA-200a + DEK group) into SCC15 cells respectively indicates that overexpression of miRNA-200a substantially inhibits SCC15 cell proliferation, infiltration and migration, decreases PCNA and Vimentin expression, and promotes E-cadherin expression. miRNA-200a + DEK transfection induced greater cell proliferation, infiltration and migration, much higher PCNA and Vimentin expression, and significantly lower E-cadherin expression. Luciferase reporter gene detection of overexpressed DEK or DEK expression after inhibiting miRNA-200a expression indicated a targeting association between miRNA-200a and DEK. miRNA-200a inhibits proliferation, infiltration and migration ability of TSCC by targeting DEK and may represent a novel means for clinical intervention in TSCC. miRNA-200a inhibits proliferation, invasion, and migration of TSCC by targeting DEK.

MiR-467b alleviates lipopolysaccharide-induced inflammation through targeting STAT1 in chondrogenic ATDC5 cells.

Osteoarthritis (OA) is one of the most common degenerative joint diseases worldwide. Chondrocytes are activated in OA patients, accompanied by excessive chondrogenic proliferation and production of inflammatory cytokines. MiR-467b is implicated in the regulation of artherosclerosis and pro-inflammatory cytokine secretion. However, the precise role of miR-467b in OA remains unclear. In the present study, we induced inflammation in chondrogenic ATDC5 cells using lipopolysaccharide (LPS). LPS treatment significantly elevated the production of interleukin-6 (IL-6), IL-1β and tumour necrosis factor-α (TNF-α) in ATDC5 cells, accompanied by decreased miR-467 level. Then, we over-expressed miR-467b using its specific mimics in ATDC5 cells, and LPS-induced inflammation was significantly inhibited as evidenced by decreased IL-6, IL-1β and TNF-α levels. MiR-467b agomir also alleviated inflammation in rat knee osteoarthritis (KOA) model. In addition, we validated that signal transducer and activator of transcription 1 (STAT1) was a downstream target of miR-467b. LPS treatment significantly increased the STAT1 expression while miR-467b mimic transfection partially reversed this effect. Moreover, STAT1 knockout reversed the increased contents of IL-6, IL-1β and TNF-α. Furthermore, miR-467b over-expression significantly decreased the production of IL-6, IL-1β and TNF-α induced by LPS treatment, which was partially reversed by further STAT1 over-expression. In summary, our findings demonstrated that miR-467b alleviated LPS-induced inflammation through targeting STAT1, and this miR-467b/STAT1 regulation axis may provide a new therapeutic target for OA clinical management.

Silica nanoparticles inducing the apoptosis via microRNA-450b-3p targeting MTCH2 in mice and spermatocyte cell.

Silica nanoparticles (SiNPs) could cause reproductive toxicity. The role of miRNAs in reproductive toxicity induced by SiNPs is still ambiguous. The present study was designed to investigate the role of miRNA-450b-3p. Invivo, 40 male mice were randomly divided into control, and 20mg/kg SiNPs groups. The mice were administrated by tracheal perfusion for 35 days. Invitro, spermatocyte cells (GC-2spd cells) were divided into 6 groups: 0μg/mL SiNPs groups, 5μg/mL SiNPs groups, 5μg/mL SiNPs+miRNA-450b-3p mimic transfection group, 5μg/mL SiNPs+miRNA-450b-3p mimic negative control group, 5μg/mL SiNPs+miRNA-450b-3p inhibitor transfection group, and 5μg/mL SiNPs+miRNA-450b-3p inhibitor negative control group. The results showed that SiNPs induced the apoptosis of spermatogenic cells, decreased the quantity and quality of the sperm, reduced the expressions of miR-450b-3p, and increased the protein expressions of the MTCH2, BID, BAX, Cytochrome C, Caspase-9, and Caspase-3 in the testis. Invitro, the mimic of miRNA-450b-3p reversed the decrease of viability and the increase of apoptosis rate and significantly antagonized the expression enhancements of the MTCH2, BID, BAX, Cytochrome C, Caspase-9, Caspase-3 induced by SiNPs, while inhibitor of miRNA-450b-3p further promoted the effects induced by SiNPs. The result suggested that SiNPs could inhibit the miR-450b-3p expression resulting in activation of the mitochondrial apoptosis signaling pathways by regulating the MTCH2 in the spermatocyte cells and, thus, induce the reproductive toxicity.

Potential Target miR-455 Delaying Arterial Stenosis Progression Through PTEN.

Background: Vascular smooth muscle cells (VSMC) underwent phenotypic switching upon stimulation signals, and this is the prerequisite for their proliferation and migration. Previous work revealed that miR-455 may be involved in vascular stenosis. Thus, this study aimed to explore potential targets and mechanisms underlying the dynamics of miR-455 in vascular stenosis.Methods: miR-455 and PTEN expression levels were studied in normal and stenosis tissue, as well as in VSMC in proliferation model. Manipulating miR-455 expression levels was achieved by transfection of either miR-455 mimic or inhibitor, and its effect on cell proliferation was studied by CCK-8 assay. Its effect on gene expression was studied by RT-qPCR and western blot. The expression regulation mechanism was studied by luciferase reporter system. Finally, the effect of miR-455 on regulating vascular stenosis was studied using a rat balloon-injured carotid artery stenosis model.Results: High expression levels of miR-455 were detected in both stenosis arterial tissues and VSMC proliferation models. In contrast, the expression levels of PTEN were downregulated in these systems. miR-455 transfected VSMC showed higher levels of proliferation and decreased levels of PTEN. Potential binding sites between miR-455 and PTEN 3′UTR were predicted and confirmed. NF-kB p65 was found to bind directly on miR-455 promoter region and regulate its transcription. The progression of arterial stenosis could be delayed by introducing miR-455 antagomir.Conclusions: The p65/miR-455/PTEN signaling pathway plays a crucial role in regulating VSMC proliferation and vascular stenosis. This indicated that miR-455 is a novel target that would help improve treatment outcomes in patients suffering from vascular stenosis.

MicroRNA-30d can inhibit the proliferation, migration and invasion of human mesothelial cell MSTO-211H

Objective: To investigate the inhibitory effect and molecular mechanism of microRNA-30d (miR-30d) in the process of proliferation, migration and invasion of malignant mesothelioma cell line MSTO-211H. Methods: In April 2017, the human MSTO-211H cells was used to establish miR-30d overexpressed MSTO-211H cell model by transfection of miR-30d mimics. The qRT-PCR was performed to detect the expression level of miR-30d in the cells transfected miR-30d mimics. The effects of miR-30d on the proliferation, apoptosis, migration and invasion of MSTO-211H cells were analyzed by CCK-8 experiment, flow cytometry, cell scratch experiment and Transwell method. Results: After transfection of miR-30d, the expression level of miR-30d in the MSTO-211H+miR-30d cells group was significantly higher than MSTO-211H+miR NC cells group (P<0.01) . The cell activity of MSTO-211H+miR-30d group (105.13%±2.35%) was significantly lower than MSTO-211H+miR NC cells group (115.40%±1.35%) , and the level of apoptosis (3.97%±0.36%) was significantly higher than MSTO-211H+miR NC cells group (1.47%±0.10%) (P<0.01) . The relative migration areas at 12 and 24 h of MSTO-211H+miR-30d cells group (9.35±3.16 μm(2) and 58.19±1.82 μm(2)) were significantly lower than MSTO-211H+miR NC cells group (54.42±5.26 μm(2) and 88.32±1.96 μm(2)) (P<0.01) . Compared with the MSTO-211H+miR NC cells group, the numbers of cell migration and cell invasion were reduced in the MSTO-211H+miR-30d cells group (P<0.01) . Conclusion: miR-30d can regulate the progression of malignant pleural mesothelioma by inhibiting the proliferation, apoptosis, migration and invasion of MSTO-211H cells.

MicroRNA-301a Promotes Cell Proliferation and Resistance to Apoptosis through PTEN/PI3K/Akt Signaling Pathway in Human Ovarian Cancer

Background: MicroRNAs are endogenous small noncoding RNAs, which play a critical role in regulating various biological and pathologic processes. Furthermore, miR-301a has been detected to be overly expressed in tumorigenic progression of ovarian cancer. However, the effects of miR-301a on ovarian cancer are still unclear. Objective: The objective of this study is to investigate the molecular mechanisms of miR-301a in epithelial ovarian cancer cells. Methods: The miR-301a expression in ovarian cancer cells was detected. Then, cell proliferation, cell cycle, and apoptosis of the miR-301a-mimic-transfected ovarian cancer cells were determined, as well as the effects of the miR-301a mimic on the PTEN/phosphoinositide 3-kinase (PI3K) signaling pathway were explored. Results: We found that the miR-301a expression levels were markedly upregulated in ovarian cancer tissues and cells, and upregulation of miR-301a-promoted cell viability and proliferation. Our results also showed that the miR-301a-mimic accelerated cell cycle progression of ovarian cancer cells by targeting the CDK4/Cyclin-D1 pathway but not the CDK2/Cyclin-E pathway. Moreover, transfection of the miR-301a mimic into ovarian cancer cells could decrease the PTEN expression while increasing the PI3K and Akt phosphorylation, as compared with the miR-301a inhibitor group and the negative control group. Conclusion: Therefore, miR-301a should be an oncogene in ovarian cancer, and overexpression of miR-301a promoted proliferation of ovarian cancer cells by modulating the PTEN/PI3K/Akt signaling pathway.