Pharmacological p38 MAPK inhibitor SB203580 enhances AML stem cell line KG1a chemosensitivity to daunorubicin by promoting late apoptosis, cell growth arrest in S-phase, and miR-328-3p upregulation.

Acute myeloid leukaemia (AML) is characterized by uncontrolled proliferation of myeloid progenitor cells and impaired maturation, leading to immature cell accumulation in the bone marrow and bloodstream, resulting in hematopoietic dysfunction. Chemoresistance, hyperactivity of survival pathways, and miRNA alteration are major factors contributing to treatment failure and poor outcomes in AML patients. This study aimed to investigate the impact of the pharmacological p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 on the chemoresistance potential of AML stem cell line KG1a to the therapeutic drug daunorubicin (DNR). KG1a and chemosensitive leukemic HL60 cells were treated with increasing concentrations of DNR. Cell Titer-Glo®, flow cytometry, phosphokinase and protein arrays, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were employed for assessment of cell viability, half-maximal inhibitory concentration (IC50) determination, apoptotic status detection, cell cycle analysis, apoptosis-related protein and gene expression monitoring. Confocal microscopy was used to visualize caspase and mitochondrial permeability transition pore (mPTP) activities. Exposed at various incubation times, higher DNR IC50 values were determined for KG1a cells than for HL60 cells, confirming KG1a cell chemoresistance potential. Exposed to DNR, late apoptosis induction in KG1a cells was enhanced after SB203580 pretreatment, defined as the combination treatment. This enhancement was confirmed by increased cleavage of poly(ADP-ribose) polymerase, caspase-9, caspase-3, and augmented caspase-3/-7 and mPTP activities in KG1a cells upon combination treatment, compared to DNR. Using phosphokinase and apoptosis protein arrays, the combination treatment decreased survival Akt phosphorylation and anti-apoptotic Bcl-2 expression levels in KG1a cells while increasing the expression levels of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21, compared to DNR. Cell cycle analysis revealed KG1a cell growth arrest in G2/M-phase caused by DNR, while combined treatment led to cell growth arrest in S-phase, mainly associated with cyclin B1 expression levels. Remarkably, the enhanced KG1a cell sensitivity to DNR after SB203580 pretreatment was associated with an increased upregulation of miR-328-3p and slight downregulation of miR-26b-5p, compared to DNR effect. Altogether, these findings could contribute to the development of a new therapeutic strategy by targeting the p38 MAPK pathway to improve treatment outcomes in patients with refractory or relapsed AML.

MiR-328-3p promotes TGF-ß1-induced proliferation, migration, and inflammation of airway smooth muscle cells by regulating the PTEN/Akt pathway.

BACKGROUND: Recent studies have shown that the up-regulation of microRNA miR-328-3p expression increases seasonal allergy and asthma symptoms in children, but the specific mechanism remains unclear. Therefore, the aim of this study was to explore the role and mechanism of -miR-328-3p in transforming growth factor (TGF)-ß1-induced airway smooth muscle cells (ASMCs). METHODS: The effect of TGF-ß1 on the expression of miR-328-3p in ASMCs was examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cells proliferation, migration, and inflammatory factors in TGF-ß1-induced ASMCs were measured by cell counting kit-8 (CCK-8), transwell, and enzyme-linked immunosorbent assay (ELISA), respectively. Besides, TargetScan was used to predict phosphatase and tensin homolog (PTEN), the downstream target of miR-328-3p; double-luciferase reporter assay, western blot, and qRT-PCR were used to verify the targeting relationship between miR-328-3p and PTEN; western blot was also used to examine the effects of PTEN and miR-328-3p knockdown on the expression levels of PTEN, Akt, and p-Akt proteins. RESULTS: The expression of miR-328-3p was up-regulated in TGF-ß1-induced ASMCs. Knockdown of miR-328-3p significantly inhibited proliferation, migration, and inflammation of ASMCs induced by TGF-ß1 and decreased levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß. The dual--luciferase reporter assay results confirmed that PTEN was a target gene of miR-328-3p. Moreover, inhibition of PTEN expression reversed the inhibitory effect of low miR-328-3p expression on -TGF-ß1-induced ASMC's proliferation, migration, and inflammation. In comparison to the knockdown of miR-328-3p alone, the simultaneous knockdown of miR-328-3p with PTEN decreased PTEN protein expression levels and increased p-Akt/Akt ratio in TGF-ß1-induced ASMCs. CONCLUSION: Through regulating the expression of PTEN and the activity of Akt signaling pathway, miR-328-3p promotes TGF-ß1-induced proliferation, migration, and inflammation of ASMCs.

Decrease in LINC00963 attenuates the progression of pulmonary arterial hypertension via microRNA-328-3p/profilin 1 axis.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a severe cardiopulmonary disease characterized by vascular hyperplasia and remodeling. Long noncoding RNA LINC00963 can regulate cell proliferation and metastasis in nonsmall cell lung cancer. However, the function of LINC00963 on PAH progression is rarely reported. METHODS: Quantitative real-time PCR was used to determine the expression levels of LINC00963, microRNA (miRNA)-328-3p, and profilin 1 (PFN1), as well as vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), and hypoxia-inducible factor (HIF)-α. The protein level of PFN1 was measured by western blotting. The viability and migration of hypoxia-induced pulmonary arterial smooth muscle cells (PASMCs) were assessed by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-h-tetrazolium bromide, and transwell assays, respectively. The target relationships between miR-328-3p and LINC00963/PFN1 were confirmed by dual-luciferase reporter assay. A PAH mouse model was conducted to explore the effects of hypoxia on cardiopulmonary functions. RESULTS: In hypoxia-induced PASMCs and PAH mouse model, high expression levels of LINC00963 and PFN1, and low expression of miR-328-3p, were determined. The viability, migration of hypoxia-induced PASMCs, the expression of VEGF, FGF-2, and HIF-α were significantly repressed by transfection of si-LINC00963 or miR-328-3p mimics. The inhibitory effects of LINC00963 silencing on cell viability, migration, and the levels of VEGF, FGF-2, and HIF-α were partly eliminated by miR-328-3p inhibitor or increasing the expression of PFN1. Hypoxia treatment increased the levels of RVSP, mPAP, and RV/(LV+S), as well as the thickness of pulmonary artery wall. CONCLUSIONS: Silencing of LINC00963 ameliorates PAH via modulating miR-328-3p/PFN1.

MiR-328-3p inhibits lung adenocarcinoma-genesis by downregulation PYCR1.

BACKGROUND: A vast majority of patients with NSCLC (non-small cell lung cancer) have lung adenocarcinoma (LA), and the survival rate of LA varies from 5% to 75% depending on the severity of this adenocarcinoma. PYCR1 (abnormal pyrroline-5-carboxylate reductase 1) gene and miR-328-3p have been found to be associated with cancer development. However, the underlying mechanism of interaction between miR-328-3p and PYCR1 in LA needs further investigation. METHODS: The expressions of miR-328-3p and PYCR1 in samples with LA were identified by RT-qPCR. Next, we investigated the targeting relationship between these two biological factors using luciferase assay. CCK-8, BrdU, transwell-migration, and flow cytometry assays were performed to detect cell viability, cell proliferation, cell migration and cell apoptosis in LA cells. RESULTS: We noticed that miR-328-3p expression was downregulated in LA samples. MiR-328-3p mimic restricted cell proliferation and cell migration, while it enhanced cell apoptosis. Furthermore, the overexpression of PYCR1 promoted the proliferation and migration of LA cells, but it repressed cell apoptosis. Moreover, PYCR1 directly interacted with miR-328-3p in the LA cells, and miR-328-3p restrained the expression of PYCR1, thus suppressing LA tumorigenesis. CONCLUSION: In summary, our study revealed that miR-328-3p targeting to PYCR1 suppressed the malignancy of LA cells by impeding cell proliferation and migration, while effectively promoting cell apoptosis.

Circ_0004104 knockdown alleviates oxidized low-density lipoprotein-induced dysfunction in vascular endothelial cells through targeting miR-328-3p/TRIM14 axis in atherosclerosis.

BACKGROUND: Circular RNAs have shown important regulatory roles in cardiovascular diseases, containing atherosclerosis (AS). We intended to explore the role of circ_0004104 in AS using oxidized low-density lipoprotein (ox-LDL)-induced vascular endothelial cells and its associated mechanism. METHODS: Real-time quantitative polymerase chain reaction and Western blot assay were conducted to analyze RNA levels and protein levels, respectively. Cell viability, apoptosis, angiogenic ability and inflammatory response were assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, flow cytometry, capillary-like network formation assay and enzyme-linked immunosorbent assay, respectively. Cell oxidative stress was assessed using commercial kits. Dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA-pull down assay were performed to verify the intermolecular interaction. RESULTS: ox-LDL exposure up-regulated the level of circ_0004104 in HUVECs. ox-LDL exposure suppressed cell viability and angiogenic ability whereas promoted the apoptosis, inflammation and oxidative stress of HUVECs partly through up-regulating circ_0004104. MicroRNA-328-3p (miR-328-3p) was confirmed as a target of circ_0004104. MiR-328-3p interference largely reversed circ_0004104 silencing-mediated effects in HUVECs upon ox-LDL exposure. MiR-328-3p interacted with the 3' untranslated region of tripartite motif 14, and circ_0004104 positively regulated TRIM14 expression by sponging miR-328-3p. TRIM14 overexpression largely overturned miR-328-3p accumulation-induced influences in HUVECs upon ox-LDL exposure. CONCLUSION: Circ_0004104 knockdown attenuated ox-LDL-induced dysfunction in HUVECs via miR-328-3p-mediated regulation of TRIM14.

MiR-328-3p inhibits cell proliferation and metastasis in colorectal cancer by targeting Girdin and inhibiting the PI3K/Akt signaling pathway.

MiR-328-3p has been reported to be downregulated and serve as a tumor suppressor in several cancers. Previous studies only have reported the downregulation of miR-328-3p in CRC. However, the roles of miR-328-3p in CRC growth and metastasis were unknown. In this study, we demonstrated that miR-328-3p overexpression inhibited cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). The PI3K/Akt signaling pathway was also inactivated by miR-328-3p overexpression. MiR-328-3p knockdown showed the opposite effects. In addition, we confirmed that miR-328-3p directly bound to 3'UTR of Girdin and negatively regulated its expression. Girdin knockdown or treatment with PI3K inhibitor LY294002 blocked the effects of miR-328-3p inhibitor on cell proliferation, metastasis, and the PI3K/Akt signaling pathway. Moreover, pre-miR-328 decreased numbers of liver metastatic nodules, and reduced the levels of p-Akt, p-Girdin, and Girdin in metastatic tissues in liver. In conclusion, miR-328-3p may inhibit proliferation and metastasis of CRC cells by targeting Girdin and inactivating the PI3K/Akt signaling pathway. MiR-328-3p may be a novel target in cancer therapy.

Upregulation of microRNA-328-3p by hepatitis B virus contributes to THLE-2 cell injury by downregulating FOXO4.

BACKGROUND: Hepatitis B virus (HBV) remains a major cause of chronic hepatitis and hepatocellular carcinoma, and miRNAs play important roles in HBV pathogenesis. Our previous study has shown that miR-328-3p is upregulated in HBV-infected patients and serves as a potent predictor for the prognosis of HBV-related liver failure. METHODS: Here, the role of miR-328-3p in modulating cell injury in HBV-infected liver cells THLE-2 was investigated in detail. MiR-328-3p expression was examined using qRT-PCR. The levels of pro-inflammatory cytokines were measured using ELISA. HBV RNA and HBV DNA levels were quantified. The interactions between STAT3 and miR-328-3p promoter as well as miR-328-3p and FOXO4 were analyzed using chromatin immunoprecipitation (CHIP) assay and luciferase reporter assay, respectively. THLE-2 cell injury was evaluated by examining cell viability and apoptosis. RESULTS: HBV promoted expression of miR-328-3p through the STAT3 signal pathway and that increasingly expressed miR-328-3p downregulated its target FOXO4, leading to the promotion of cell injury in HBV-infected liver cells THLE-2. CONCLUSION: These data demonstrate that HBV-STAT3-miR-328-3p-FOXO4 regulation pathway may play an important role in the pathogenesis of HBV infection.

Glutamate metabotropic receptor 4 (GRM4) inhibits cell proliferation, migration and invasion in breast cancer and is regulated by miR-328-3p and miR-370-3p.

BACKGROUND: Glutamate metabotropic receptors (GRM) play a variety of roles in neuronal cells. However, their clinical significance and biological functions in breast cancer remain unknown. METHODS: RNA sequencing data of breast cancer was obtained from the TCGA dataset (v2) and mined for the expression profiles of GRM family according to cancer subtypes. mRNA expression of GRM family in breast cancer tissues and para-cancerous tissue samples as well as breast cancer cell lines were measured by qPCR. The effects of over- and under-expression of GRM4 on cell capabilities to survive, migrate and invade were determined by colony formation, transwell migration and invasion assays. To explore the upstream regulation pattern of GRM4, miRNAs that target GRM4 were predicted and validated by dual luciferase reporter assay. In addition, the mRNA and protein expression of GRM4 regulated by these miRNAs were further measured by qPCR and western blot assay. RESULTS: GRM4 was the only GRM member that expressed in breast cancer tissues. Ectopic expression of GRM4 was correlated with better prognosis of breast cancer patients. Overexpression of GRM4 could significantly inhibit cell proliferation, migration and invasion capacity in MDA-MB-231, while knockdown of GRM4 could promote these processes. miR-328-3p and miR-370-3p were predicted to regulate the expression of GRM4 and dual luciferase reporter assay demonstrated that miR-328-3p and miR-370-3p directly bound to the 3' UTR of GRM4 and mutations on the binding regions on GRM4 significantly decreased the luciferase activity. qPCR demonstrated that expression of miR-328-3p and miR-370-3p was significantly decreased in breast cancer tissues and cells compared with that in control samples. However, there were no correlations between the expression of miR-328-3p and GRM4, as well as the expression of miR-370-3p and GRM4. Moreover, overexpression of miR-328-3p and miR-370-3p counteracted the inhibitory effect of GRM4-induced cell proliferation, migration and invasion. CONCLUSIONS: Our results suggest that GRM4 might be a tumor suppressor gene in breast cancer under the direct regulation of miR-328-3p and miR-370-3p.

LncRNA XIST modulates miR-328-3p ectopic expression in lung injury induced by tobacco-specific lung carcinogen NNK both in vitro and in vivo.

BACKGROUND AND PURPOSE: It is well acknowledged that tobacco-derived lung carcinogens can induce lung injury and even lung cancer through a complex mechanism. MicroRNAs (MiRNAs) are differentially expressed in tobacco-derived carcinogen nicotine-derived nitrosamine ketone (NNK)-treated A/J mice. EXPERIMENTAL APPROACH: RNA sequencing was used to detect the level of long non-coding RNAs (lncRNAs). Murine and human lung normal and cancer cells were used to evaluate the function of lncRNA XIST and miR-328-3p in vitro, and NNK-treated A/J mice were used to test their function in vivo. In vivo levels of miR-328-3p and lncRNA XIST were analysed, using in situ hybridization. miR-328-3p agomir and lncRNA XIST-specific siRNA were used to manipulate in vivo levels of miR-328-3p and lncRNA XIST in A/J mice. KEY RESULTS: LncRNA XIST was up-regulated in NNK-induced lung injury and dominated the NNK-induced ectopic miRNA expression in NNK-induced lung injury both in vitro and in vivo. Either lncRNA XIST silencing or miR-328-3p overexpression exerted opposing effects in lung normal and cancer cells regarding cell migration. LncRNA XIST down-regulated miR-328-3p levels as a miRNA sponge, and miR-328-3p targeted the 3'-UTR of FZD7 mRNA, which is ectopically overexpressed in lung cancer patients. Both in vivo lncRNA XIST silencing and miR-328 overexpression could rescue NNK-induced lung injury and aberrant overexpression of the lung cancer biomarker CK19 in NNK-treated A/J mice. CONCLUSIONS AND IMPLICATIONS: Our results highlight the promotive effect of lncRNA XIST in NNK-induced lung injury and elucidate its post-transcriptional mechanisms, indicating that targeting lncRNA XIST/miR-328-3p could be a potential therapeutic strategy to prevent tobacco carcinogen-induced lung injury in vivo.

18ß-glycyrrhetinic acid promotes gastric cancer cell autophagy and inhibits proliferation by regulating miR-328-3p/signal transducer and activator of transcription 3.

BACKGROUND: Gastric cancer (GC) is one of the most common cancer types worldwide, and its prevention and treatment methods have garnered much attention. As the active ingredient of licorice, 18ß-glycyrrhetinic acid (18ß-GRA) has a variety of pharmacological effects. The aim of this study was to explore the effective target of 18ß-GRA in the treatment of GC, in order to provide effective ideas for the clinical prevention and treatment of GC. AIM: To investigate the mechanism of 18ß-GRA in inhibiting cell proliferation and promoting autophagy flux in GC cells. METHODS: Whole transcriptomic analyses were used to analyze and screen differentially expressed microRNAs (miRNAs) in GC cells after 18ß-GRA intervention. Lentivirus-transfected GC cells and the Cell Counting Kit-8 were used to detect cell proliferation ability, cell colony formation ability was detected by the clone formation assay, and flow cytometry was used to detect the cell cycle and apoptosis. A nude mouse transplantation tumor model of GC cells was constructed to verify the effect of miR-328-3p overexpression on the tumorigenicity of GC cells. Tumor tissue morphology was observed by hematoxylin and eosin staining, and microtubule-associated protein light chain 3 (LC3) expression was detected by immunohistochemistry. TransmiR, STRING, and miRWalk databases were used to predict the relationship between miR-328-3p and signal transducer and activator of transcription 3 (STAT3)-related information. Expression of STAT3 mRNA and miR-328-3p was detected by quantitative polymerase chain reaction (qPCR) and the expression levels of STAT3, phosphorylated STAT3 (p-STAT3), and LC3 were detected by western blot analysis. The targeted relationship between miR-328-3p and STAT3 was detected using the dual-luciferase reporter gene system. AGS cells were infected with monomeric red fluorescent protein-green fluorescent protein-LC3 adenovirus double label. LC3 was labeled and autophagy flow was observed under a confocal laser microscope. RESULTS: The expression of miR-328-3p was significantly upregulated after 18ß-GRA intervention in AGS cells (P = 4.51E-06). Overexpression of miR-328-3p inhibited GC cell proliferation and colony formation ability, arrested the cell cycle in the G0/G1 phase, promoted cell apoptosis, and inhibited the growth of subcutaneous tumors in BALB/c nude mice (P < 0.01). No obvious necrosis was observed in the tumor tissue in the negative control group (no drug intervention or lentivirus transfection) and vector group (the blank vector for lentivirus transfection), and more cells were loose and necrotic in the miR-328-3p group. Bioinformatics tools predicted that miR-328-3p has a targeting relationship with STAT3, and STAT3 was closely related to autophagy markers such as p62. After overexpressing miR-328-3p, the expression level of STAT3 mRNA was significantly decreased (P < 0.01) and p-STAT3 was downregulated (P < 0.05). The dual-luciferase reporter gene assay showed that the luciferase activity of miR-328-3p and STAT3 3' untranslated regions of the wild-type reporter vector group was significantly decreased (P < 0.001). Overexpressed miR-328-3p combined with bafilomycin A1 (Baf A1) was used to detect the expression of LC3 II. Compared with the vector group, the expression level of LC3 II in the overexpressed miR-328-3p group was downregulated (P < 0.05), and compared with the Baf A1 group, the expression level of LC3 II in the overexpressed miR-328-3p + Baf A1 group was upregulated (P < 0.01). The expression of LC3 II was detected after intervention of 18ß-GRA in GC cells, and the results were consistent with the results of miR-328-3p overexpression (P < 0.05). Additional studies showed that 18ß-GRA promoted autophagy flow by promoting autophagosome synthesis (P < 0.001). qPCR showed that the expression of STAT3 mRNA was downregulated after drug intervention (P < 0.05). Western blot analysis showed that the expression levels of STAT3 and p-STAT3 were significantly downregulated after drug intervention (P < 0.05). CONCLUSION: 18ß-GRA promotes the synthesis of autophagosomes and inhibits GC cell proliferation by regulating the miR-328-3p/STAT3 signaling pathway.

Yanghepingchuan granule improves airway inflammation by inhibiting autophagy via miRNA328-3p/high mobility group box 1/Toll-like receptor 4 targeting of the pathway of signaling in rat models of asthma.

Background: As a type of traditional Chinese medicine, Yanghepingchuan granules (YHPCG) are used to treat inflammatory diseases of the lungs, including asthma. However, the underlying molecular mechanism of the ability of YHPCG to reduce airway inflammation remains unknown. Methods: By sensitizing rats to aluminum hydroxide and ovalbumin, an asthma model was established. During the 14-day treatment period, the rats received YHPCG, TAK242 (TLR4 inhibitor), and a combination of the two treatments. Histopathology and goblet cell hyperplasia were observed in rats with ovalbumin-induced asthma by using hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining. Immunohistochemical, autophagy-related immunofluorescence, and western blotting analyses were performed to determine autophagic activity. The effects of YHPCG on high mobility group box 1 (HMGB1)-mediated Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) pathway-related proteins and inflammatory factors in rats were evaluated via western blotting, PCR analysis, and enzyme-linked immunosorbent assay. A dual luciferase method was used to detect the interaction between miRNA328-3p and HMGB1. Results: YHPCG inhibit the HMGB1/TLR4/NF-κB pathway by upregulating miR-328-3p, reducing autophagosome production, inhibiting autophagy, and effectively preventing the progression of lung inflammation. Conclusions: Asthma airway inflammation can be treated with YHPCG by inhibiting autophagy via miRNA328-3p/HMGB1/TLR4/NF-κB signaling pathways.

Puerarin Inhibits the Progression of Bladder Cancer by Regulating circ_0020394/miR-328-3p/NRBP1 Axis.

Background: Previous studies have shown puerarin to be a potential therapeutic drug for treatment of bladder cancer. But the role and possible molecular mechanism of puerarin remain unknown. Methods: Cell viability, apoptosis, migration, and invasion were assessed by Cell Counting Kit-8 (CCK-8), flow cytometry, and transwell assays, respectively. Western blot was used to measure the levels of all protein. Glucose consumption and lactate production were detected using a glucose and lactate assay kit. Circular RNA_0020394 (circ_0020394), microRNA-328-3p (miR-328-3p), and nuclear receptor binding protein 1 (NRBP1) levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between miRNA and circRNA or mRNA was confirmed using dual-luciferase reporter assay. In vivo experiments were performed to examine the effect of puerarin on tumor growth. Results: Puerarin suppressed cell viability, migration, invasion, and glycolysis, and induced apoptosis in bladder cancer. circ_0020394 was downregulated in puerarin-treated bladder cancer cells, and circ_0020394 overexpression attenuated the inhibitory effect of puerarin on cell progression. Moreover, circ_0020394 could bind to miR-328-3p, and miR-328-3p directly targeted NRBP1. Functionally, miR-328-3p could reverse the promotion effect of circ_0020394 overexpression on the progression of puerarin-treated cells, and silencing NRBP1 counteracted the effects of anti-miR-328-3p on puerarin-treated cells. Mechanically, circ_0020394 could increase NRBP1 expression by acting as miR-328-3p sponge in puerarin-treated bladder cancer cells. Besides, puerarin inhibited tumorigenesis in vivo by increasing miR-328-3p and decreasing the levels of circ_0020394 and NRBP1. Conclusions: Puerarin impedes cell viability, migration, invasion, and glycolysis, and promoted apoptosis in bladder cancer by regulating circ_0020394/miR-328-3p/NRBP1 axis.

lncRNA SNHG1 regulates odontogenic differentiation of human dental pulp stem cells via miR-328-3p/Wnt/ß-catenin pathway.

BACKGROUND: Elucidating the mechanism of odontogenic differentiation of human dental pulp stem cells (hDPSCs) is the key to in-depth mastery and development of regenerative endodontic procedures (REPs). In odontogenic differentiation, lncRNAs have a regulatory role. The goal of this research is to determine the involvement of short nucleolar RNA host gene 1 (SNHG1) in hDPSCs' odontogenic differentiation and the mechanism that underpins it. METHODS: hDPSCs were isolated from the dental pulp tissue of healthy immature permanent teeth. Follow-up experiments were performed when the third generation of primary cells were transfected. The proliferation ability was measured by CCK-8. The biological effects of SNHG1 and miR-328-3p were determined by real-time quantitative polymerase chain reaction (qRT-PCR), western blot (WB), alkaline phosphatase (ALP) staining and activity, alizarin red S staining (ARS) and quantification, and immunofluorescence staining. The binding of SNHG1 and miR-328-3p was confirmed using a dual-luciferase reporter assay. qRT-PCR and WB were used to determine whether the canonical Wnt/ß-catenin pathway was activated. RESULTS: On the 0th, 3rd, and 7th days of odontogenic differentiation of hDPSCs, SNHG1 showed a gradual up-regulation trend. SNHG1 overexpression enhanced the mRNA and protein expression of dentin sialophosphoprotein (DSPP), dentine matrix protein 1 (DMP-1) and ALP. We found that SNHG1 could bind to miR-328-3p. miR-328-3p inhibited the odontogenic differentiation of hDPSCs. Therefore, miR-328-3p mimics rescued the effect of SNHG1 overexpression on promoting odontogenic differentiation. In addition, SNHG1 inhibited Wnt/ß-catenin pathway via miR-328-3p in odontogenic differentiation of hDPSCs. CONCLUSION: lncRNA SNHG1 inhibits Wnt/ß-catenin pathway through miR-328-3p and then promotes the odontogenic differentiation of hDPSCs.

miR-328-3p, a Predictor of Stroke, Aggravates the Cerebral Ischemia-Reperfusion Injury.

BACKGROUND: In the present study, we aimed to identify microRNAs (miRNAs) that affected the prognosis of stroke and assess their biological effects. MATERIALS AND METHODS: A high-throughput sequencing (HTS) analysis was performed to screen distinctive miRNAs in serum exosomes of stroke patients, and these miRNAs were subsequently validated using individual quantitative real-time polymerase chain reaction (qRT-PCR) in a cohort consisting of 39 stroke patients and 20 normal controls. Briefly, miR-328-3p agomir or agomir NC was injected into rats before ischemia and reperfusion (I/R) injury. Zea-Longa score, neurological severity score (mNSS), triphenyltetrazolium chloride (TTC) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, transmission electron microscopy, and hematoxylin and eosin (H&E) staining were used to examine the brain injury. Immunohistochemistry was utilized to determine the expressions of TNF-α and IL-6. RESULTS: The expression of serum exosomal miR-328-3p was significantly reduced in patients with an infarct volume ≥10 cm3 (P=0.01). Serum exosomal miR-328-3p was associated with the short-term prognosis (P=0.02), and the level of miR-328-3p was an independent relative factor for short-term prognosis (OR 5.276, P=0.02). The sensitivity of miR-328-3p level higher than 1.24 to predict the severity of the patient's 1-week prognosis was 70%, and the specificity was 83% (AUC=0.74, P=0.02). The mNSS was higher in the miR-328-3p agomir group compared with the agomir NC group (P=0.03). Neutrophil infiltration was more serious in the miR-328-3p agomir group. CONCLUSION: Our study indicated that miR-328-3p played a critical predictive role in the short-term prognosis of stroke, and up-regulation of miR-328-3p aggravated cerebral I/R injury.

Myocardial infarction cardiomyocytes-derived exosomal miR-328-3p promote apoptosis via Caspase signaling.

Exosomal miRNAs are used as novel non-invasive biomarkers for detection strategies of human disease. Here, we aimed to investigate the potential clinical value of exosomal miRNAs for myocardial infarction (MI) diagnosis and treatment. Differentially expressed miRNAs were obtained from normal cardiomyocytes, MI cardiomyocytes and adjacent normal cardiomyocytes using miRNA microarray analysis. Exosomes were isolated by centrifugation and identified by transmission electron microscopy (TEM) and western blot. The expression of miR-328-3p in exosomes was then verified by qRT-PCR. Cell apoptosis was measured using flow cytometry and TUNEL analysis. The MI severity was confirmed by masson's trichrome staining and echocardiography. MiR-328-3p was significantly increased in the MI cardiomyocytes and adjacent normal cardiomyocytes. We further confirmed miR-328-3p increasing in the exosomes from MI cardiomyocytes, which can be taken into normal cardiomyocytes. Furthermore, exogenous exosomal miR-328-3p increased apoptosis of cardiomyocytes and promoted MI. Genes regulated by miR-328-3p are mainly enriched in Caspase signaling, which is an important apoptosis regulating signaling pathway. Additionally, Caspase-3 inhibitor, Z-DEVD-FMK, reversed apoptosis and MI promoting function of miR-328-3p. Exosomal miR-328-3p is a potential novel diagnostic biomarker and therapeutic target for MI, and Z-DEVD-FMK could reverse the apoptosis progression induced by miR-328-3p.

MicroRNA-328-3p facilitates the progression of gastric cancer via KEAP1/NRF2 axis.

Gastric cancer is a common lethal malignancy and causes great cancer-related mortality worldwide. MicroRNA (miR)-328-3p is implicated in the progression of various human cancers; however, its role and mechanism in the progression of gastric cancer remain unclear.Human gastric cancer cells were incubated with miR-328-3p mimic, inhibitor or the matched negative control. Cell viability, colony formation, migrative and invasive capacity, cell apoptosis and oxidative stress were measured. To clarify the involvement of nuclear factor-E2-related factor 2 (NRF2) and kelch-like ECH-associated protein 1 (KEAP1), small interfering RNA was used. miR-328-3p was upregulated in human gastric cancer cells and tissues, and its level positively correlated with the progression of gastric cancer. miR-328-3p promoted cell viability, colony formation, migration and invasion, thereby facilitating the progression of gastric cancer. miR-328-3p mimic reduced, while miR-328-3p inhibitor increased apoptosis and oxidative stress of human gastric cancer cells. Mechanistically, miR-328-3p upregulated NRF2 via targeting KEAP1to attenuate excessive free radical production and cell apoptosis. miR-328-3p functions as an oncogenic gene and inhibiting miR-328-3p may help to develop novel therapeutic strategies of human gastric cancer.

Circular RNA-HIPK3 regulates human pulmonary artery endothelial cells function and vessel growth by regulating microRNA-328-3p/STAT3 axis.

The proliferation and migration of pulmonary artery endothelial cells are the pathological basis of pulmonary vascular remodeling with pulmonary hypertension. Recent studies have shown that circular RNA (circRNA) regulates biological processes in various vascular diseases, including pulmonary arterial hypertension. It has been reported that circRNA regulates the vascular endothelial cells' function. Therefore, circRNA may have crucial roles in human pulmonary artery endothelial cells (hPAECs) proliferation, migration, and tube formation in pulmonary arterial hypertension. In this study, we aimed to discover the role and mechanism of circular RNA HIPK3 (circHIPK3) in the proliferation and migration of pulmonary hypertension hPAECs. First, we used platelet-derived growth factor-stimulated hPAECs as a cellular model of pulmonary arterial hypertension. The results showed that platelet-derived growth factor promoted hPAECs proliferation, migration, and tube formation. Notably, platelet-derived growth factor upregulated the expression of circHIPK3 in hPAECs and regulated their proliferation, migration, and angiogenesis. Mechanistically, we confirmed miR-328-3p was copiously pulled down by circHIPK3 in hPAECs. Luciferase reporter and RNA immunoprecipitation assays further indicated the cytoplasmic interactions between circHIPK3 and miR-328-3p. Subsequently, we found that circHIPK3 might increase the expression of STAT3 by sponging miR-328-3p. Collectively, our results demonstrated that the circHIPK3-miR-328-3p-STAT3 axis contributed to the pathogenesis of pulmonary arterial hypertension by stimulating hPAECs proliferation, migration, and angiogenesis. The circHIPK3 has an accelerated role in pulmonary arterial hypertension development, implicating the potential values of circHIPK3 in pulmonary arterial hypertension therapy.

KDM4C contributes to cytarabine resistance in acute myeloid leukemia via regulating the miR-328-3p/CCND2 axis through MALAT1.

AIMS: Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm, in which relapse due to drug resistance is the main cause for treatment failure and the disease progression. In this study, we aimed to investigate the molecular mechanism of KDM4C-dependent MALAT1/miR-328-3p/CCND2 axis in cytarabine (Ara-C) resistance in the context of AML. METHODS: Bioinformatics analysis was performed to predict the targeting relationships among KDM4C, MALAT1, miR-328-3p, and CCND2 in AML, which were validated with chromatin immunoprecipitation and dual-luciferase reporter assay. Methylation-specific polymerase chain reaction was conducted to detect the methylation of MALAT1 promoter. After conducting gain- and loss-of-function assays, we investigated the effect of KDM4C on cell Ara-C resistance. A NOD/SCID mouse model was established to further investigate the roles of KDM4C/MALAT1/miR-328-3p/CCND2 in Ara-C resistant AML cells. RESULTS: KDM4C expression was upregulated in AML. KDM4C upregulation promoted the demethylation in the promoter region of MALAT1 to increase its expression, MALAT1 targeted and inhibited miR-328-3p expression, enhancing the Ara-C resistance of HL-60/A. miR-328-3p targeted and suppressed the expression of CCND2 in HL-60/A to inhibit the Ara-C resistance. Mechanistically, KDM4C regulated miR-328-3p/CCND2 through MALAT1, resulting in Ara-C resistance in AML. Findings in an in vivo xenograft NOD/SCID mouse model further confirmed the contribution of KDM4C/MALAT1/miR-328-3p/CCND2 in the Ara-C resistant AML. CONCLUSION: Our study demonstrated that KDM4C may up-regulate MALAT1 expression, which decreases the expression of miR-328-3p. The downregulation of miR-328-3p increased the level of CCND2, which induced the Ara-C resistance in AML.

The ceRNA PVT1 inhibits proliferation of ccRCC cells by sponging miR-328-3p to elevate FAM193B expression.

Clear cell renal cell carcinoma (ccRCC) is a common and fatal malignancy. Long noncoding RNAs (lncRNAs) have emerged as crucial biomarkers and regulators in many cancers, warranting the detailed investigation of their biological functions and molecular mechanisms. In this study, we explored the role and mechanism of plasmacytoma variant translocation 1 (PVT1), a competitive endogenous RNA (ceRNA) in ccRCC tissues in vitro and in vivo. We found that PVT1 is upregulated in ccRCC cells and promoted cell proliferation. Bioinformatic analysis, dual-luciferase reporter assays, argonaute 2-RNA immunoprecipitation (AGO2-RIP), quantitative PCR arrays, western blot assay, and rescue experiments were conducted to explore the underlying mechanisms of PVT1. Our analyses revealed that miR-328-3p was a direct target of PVT1 and that FAM193B was a direct target of miR-328-3p. FAM193B is upregulated in ccRCC tissues and promotes cell proliferation by activating the MAPK/ERK and PI3K/AKT pathways. Our results indicated that PVT1 promotes ccRCC cells proliferation by sponging miR-328-3p to upregulate FAM193B and activate the MAPK/ERK and PI3K/AKT pathways. Collectively, these results suggest that PVT1- miR-328-3p-FAM193B loop could serve as a potential biomarker and therapeutic target for ccRCC.

LncRNA SNHG17 promotes tumor progression and predicts poor survival in human renal cell carcinoma via sponging miR-328-3p.

Accumulating data shows that dysregulation of long non-coding RNAs (lncRNAs) are involved in human tumors' occurrence and progression. Small nucleolar RNA host genes (SNHGs) are recently revealed to play a carcinogenic role in various human neoplasms. However, the functions and underlying mechanisms of lncRNA SNHG17 in renal cell carcinoma (RCC) are still elusive. We analyzed the relationship between SNHG17 expression levels and clinicopathologic characteristics and prognosis in patients with RCC according to TCGA RNA-sequencing data and our cohort data. Loss-of-function and gain-of-function experiments were conducted to examine the biological behaviors of SNHG17 on RCC cell proliferation, migration, invasion, apoptosis, and tumor growth in vivo. The interaction between SNHG17, miR-328-3p, and Histone'sH2Avariant (H2AX) was verified by bioinformatics, dual-luciferase reporter gene, and RNA immunoprecipitation (RIP). Highly expressed SNHG17 was evident in RCC tissue samples and cell lines, and SNHG17 overexpression was related to advanced TNM stage and reduced relapse-free and overall survival of patients with RCC. Knockdown of SNHG17 prohibited malignant phenotypes, whereas ectopic SNHG17 expression showed the opposite effects. More importantly, SNHG17 could upregulate the expression of H2AX by acting as a miR-328-3p sponge. In vivo experiments confirmed that SNHG17 promoted the growth of RCC tumors. SNHG17/miR-328-3p/H2AXaxis might be involved in RCC progression, which provided a potential therapeutic target for RCC.