miR-29b-3p’s Effects on Prostate Cancer

Objection: Our research wanted to discuss miR-29b-3p in PCa occurrence and development and relative mechanisms. Methods: Collecting adjacent and cancer tissues from prostate cancer patients and measuring miR-29b-3p expressions by RT-qPCR and ISH assay. Using DU145 and PC3 cell lines which the miR-29b-3p were high expression in our study. Using miR inhibitor to knockdown miR-29b-3p in DU145 and PC3. Using CCK-8 and flow cytometry to measure cell proliferation and cell apoptosis, invasion cell number by transwell and wound healing rate by wound healing assay. The relative proteins expressions were measured using WB assay. p-AKT nuclear levels were evaluated using Cell immunofluorescence test. Using dual-luciferase reporter gene assay to analysis correlation miR-29b-3p and PTEN. Results: miR-29b-3p gene significantly increased. miR-29b-3p knockdown had effects to depress cell proliferation, increase cell apoptosis, depress invasion cells number and wound healing rates. PTEN proteins were significantly up-regulation and p-AKT and MMP-9 proteins expressions were significantly down-regulation (P < 0.001, respectively). And p-AKT nuclear volume were significantly depressed. And miR-29b-3p could target PTEN. Conclusion: miR-29b-3p played an oncology gene in prostate cancer via regulation PTEN/AKT pathway in vitro study.

MicroRNA-637 Relieves Oxidative Damage in Human Melanocytes Through Down-Regulating Transient Receptor Potential Melastatin 2 Expression

Background: Vitiligo, a chronic, autoimmune destruction of melanocytes, caused by the disappearance of epidermal melanocytes, but the mechanism is not fully understood. Although emerging evidence demonstrated that abnormal regulation of microRNAs (miRNAs) were associated with the pathogenesis of diseases, the functions of miR-637 in vitiligo remain unclear. Objective: This research was designed to explore the potential roles of miR-637 in hydrogen peroxide (H2O2)-induced human primary melanocytes in vitiligo. Methods: Human primary melanocytes were induced by 250 μmol/L H2O2 for 4 h to establish oxidative injury of melanocytes model. Cell viability and apoptosis analyzed by MTT and flow cytometry assay, respectively. The relevance between miR-637 and transient receptor potential melastatin 2 (TRPM2) was checked using TargetScan and dual luciferase reporter gene assay. The expression of miR-637 and TRPM2 was evaluated using qRT-PCR and/or Western blot analysis. Reactive oxygen species (ROS) accumulation, superoxide dismutase (SOD) and catalase (CAT) activities were measured using specific assay kits. In addition, the expression of Bcl-2 and Bax were evaluated using Western blot assay. Results: TRPM2 was up-regulated, while miR-637 was down-regulated in H2O2-stimulated human primary melanocytes. TRPM2 directly interacted with miR-637. Up-regulation of miR-637 memorably increased miR-637 level and inhibited TRPM2 expression. Furthermore, miR-637 mimic fortified cell viability, reduced apoptotic cells, enhanced Bcl-2 expression, reduced Bax level, as well as inhibited the ratio of Bax/Bcl-2 in H2O2-induced melanocytes. Meanwhile, miR-637 mimic obviously suppressed the accumulation of ROS and increased SOD and CAT activity. Nevertheless, all these findings were inverted by TRPM2-plasmid. Likewise, TRPM2-siRNA led to increased cell viability, reduced apoptotic cells, enhanced Bcl-2 expression, reduced Bax level, inhibited Bax/Bcl-2 ratio, inhibited ROS production, but increased SOD and CAT activity in H2O2-induced melanocytes. Conclusion: Our findings suggested that TRPM2 was up-regulated, while miR-637 was down-regulated in injurious melanocytes of vitiligo. Up-regulation of miR-637 relieved oxidative stress-stimulated melanocyte injury via down-regulating TRPM2 expression. Our results provide new insights into the functions of miR-637 in the development of vitiligo, indicating that miR-637 may be a latent target for vitiligo therapy.

miR-30a-5p inhibits the proliferation and collagen formation of cardiac fibroblasts in diabetic cardiomyopathy

Cardiac fibrosis is one of the major pathological characteristics of diabetic cardiomyopathy (DCM). MicroRNAs (miRNAs, miRs) have been identified as key regulators in the progression of cardiac fibrosis. This study aimed to investigate the role of miR-30a-5p in DCM and the underlying mechanism. The rat model of diabetes mellitus (DM) was established by streptozotocin injection, and the rat primary cardiac fibroblasts (CFs) were isolated from cardiac tissue and then treated with high glucose (HG). MTT assay was performed to assess the viability of CFs. Dual-luciferase reporter gene assay was conducted to verify the interaction between miR-30a-5p and Smad2. The expression of miR-30a-5p was downregulated in the myocardial tissues of DM rats and HG-stimulated CFs. Overexpression of miR-30a-5p reduced Smad2 levels and inhibited collagen formation in HG-stimulated CFs and DM rats, as well as decreased the proliferation of CFs induced by HG. Smad2 was a target of miR-30a-5p and its expression was inhibited by miR-30a-5p. Furthermore, the simultaneous overexpression of Smad2 and miR-30a-5p reversed the effect of miR-30a-5p overexpression alone in CFs. Our results indicated that miR-30a-5p reduced Smad2 expression and also induced a decrease in proliferation and collagen formation in DCM.

Hsa_circ_0060927 Is a Novel Tumor Biomarker by Sponging miR-195-5p in the Malignant Transformation of OLK to OSCC

ObjectiveTo investigate the clinical significance of differentially expressed circRNAs and candidate circRNAs in the transformation of oral leukoplakia (OLK) to oral squamous cell carcinoma (OSCC).MethodsWe performed high-throughput circRNA sequencing in six cases of normal oral mucosal (NOM) tissues, six cases of OLK tissues, and six cases of OSCC tissues. Ten circRNAs with significant differential expression were verified by qRT-PCR. Enzyme tolerance assay and Sanger sequencing were performed on the screened target circRNA hsa_circ_0060927, and a qRT-PCR assay of hsa_circ_0060927 was performed in three tissues (24 cases in each group); this was followed by an ROC analysis. The ceRNA network was predicted using TargetScan and miRanda. MiR-195-5p and TRIM14 were selected as the downstream research objects of hsa_circ_0060927. The sponge mechanism of hsa_circ_0060927 was detected by AGO2 RIP. The interaction between hsa_circ_0060927 and miR-195-5p was verified by RNA pull-down assay and dual luciferase reporter gene assay. The expressions of hsa_circ_0060927, miR-195-5p, and TRIM14 were verified by normal oral epithelial primary cells and cell lines of LEUK1, SCC9, and SCC25. The hsa_circ_0060927 overexpressed plasmid and miR-195-5p mimics were constructed to transfection LEUK1 to detect the changes in cell proliferation, apoptosis, and migration.ResultsThe results of qRT-PCR validation were consistent with the sequencing results. Hsa_circ_0060927 is a true circRNA with trans-splicing sites. The expression of hsa_circ_0060927 increased in NOM, OLK, and OSCC. Overexpression of hsa_circ_0060927 enhanced the ability of cell proliferation and migration, and decreased cell apoptosis capacity. The prediction of ceRNA network suggested that hsa_circ_0060927 could regulate the target gene TRIM14 through sponging miR-195-5p. AGO2 RIP indicated that hsa_circ_0060927 had a sponge mechanism. RNA pull-down and dual luciferase reporter gene assay suggested that hsa_circ_0060927 interacted with miR-195-5p. Hsa_circ_0060927 was positively correlated with the expression of TRIM14, and could relieve the inhibition of miR-195-5p on TRIM14 to regulate cell proliferation, apoptosis, and migration of LEUK1 cells.ConclusionHsa_circ_0060927 acted as a potential key ceRNA to sponge downstream miR-195-5p and promote OLK carcinogenesis by upregulating TRIM14. Hsa_circ_0060927 was expected to be a molecular marker for the prevention and treatment of OLK carcinogenesis through the hsa_circ_0060927/miR-195-5p/TRIM14 axis.

MicroRNA-409-3p Targeting at ATXN3 Reduces the Apoptosis of Dopamine Neurons Based on the Profile of miRNAs in the Cerebrospinal Fluid of Early Parkinson’s Disease

Precise recognition of early Parkinson’s disease (PD) has always been a challenging task requiring more feasible biomarkers to be integrated to improve diagnostic accuracy. MicroRNAs (miRNAs) of cerebrospinal fluid (CSF) are believed to be potential and promising candidate biomarkers for PD. However, the role of altered miRNAs of CSF play in PD is unclear. Here, we recruited patients with early stages of PD and controls to analyze the expression of miRNA in CSF by the Next Generation Sequencing (NGS). Furthermore, we tested the levels of these miRNA in SH-SY5Y cells treated with MPP+ using real-time quantitative PCR. We found 21 miRNAs were upregulated in CSF of early PD patients and miR-409-3p, one of the identified 21 miRNAs, was further confirmed in SH-SY5Y cells treated with MPP+. Also, more cells survived in the overexpression of the miR-409-3p group when SH-SY5Y cells and mice were treated with MPP+ and MPTP, respectively. Mechanistically, we demonstrated the binding of miR-409-3p and 3’UTR of ATXN3 through a dual luciferase reporter gene assay. Moreover, miR-409-3p mimic reduced the aggregation of polyglutamine-expanded mutant of ATXN3 and apoptosis. Our results provide experimental evidence for miR-409-3p in CSF as a diagnostic marker of PD.

MicroRNA-149 suppresses osteogenic differentiation of mesenchymal stem cells via inhibition of AKT1-dependent Twist1 phosphorylation

Osteogenic differentiation is a vital process for growth, repair, and remodeling of bones. Accumulating evidence have suggested that microRNAs (miRNAs or miRs) play a crucial role in osteogenic differentiation of mesenchymal stem cells (MSCs). Hence, the current study set out to elucidate the role of miR-149 in osteogenic differentiation of MSCs and the underlying mechanism. First, rat models of bone differentiation were established using the Masquelet-induced membrane technique, and MSCs were isolated. The expression of miR-149 and AKT1 in the rats and cells was detected with RT-qPCR and western blot analysis. The relationships among miR-149, AKT1, and Twist1 were further predicted by online bioinformatics prediction and verified using dual luciferase reporter gene assay. Alteration of miR-149, AKT1, or Twist1 was performed to further explore their effect on osteogenic differentiation of MSCs. miR-149 was poorly expressed in the process of osteogenic differentiation of MSCs, while AKT1 was highly expressed. miR-149 negatively regulated the expression of AKT1, which in turn diminished the protein levels of Twist1 and promoted the phosphorylation levels of Twist1. Lastly, miR-149 acted as an inhibitor of osteogenic differentiation of MSCs, which could be reversed by AKT1. To sum up, miR-149 silencing promoted osteogenic differentiation of MSCs by enhancing Twist1 degradation through AKT1 upregulation, representing a new method for bone repair treatment.

Targeting ZFP64/GAL-1 axis promotes therapeutic effect of nab-paclitaxel and reverses immunosuppressive microenvironment in gastric cancer

Background Chemoresistance is a main obstacle in gastric cancer (GC) treatment, but its molecular mechanism still needs to be elucidated. Here, we aim to reveal the underlying mechanisms of nanoparticle albumin-bound paclitaxel (nab-paclitaxel) resistance in GC. Methods We performed RNA sequencing (RNA-seq) on samples from patients who were resistant or sensitive to nab-paclitaxel, and identified Zinc Finger Protein 64 (ZFP64) as critical for nab-paclitaxel resistance in GC. CCK8, flow cytometry, TUNEL staining, sphere formation assays were performed to investigate the effects of ZFP64 in vitro, while subcutaneous tumor formation models were established in nude mice or humanized mice to evaluate the biological roles of ZFP64 in vivo. Chromatin immunoprecipitation sequencing (CHIP-seq) and double-luciferase reporter gene assay were conducted to reveal the underlying mechanism of ZFP64. Results ZFP64 overexpression was linked with aggressive phenotypes, nab-paclitaxel resistance and served as an independent prognostic factor in GC. As a transcription factor, ZFP64 directly binds to Galectin-1 (GAL-1) promoter and promoted GAL-1 transcription, thus inducing stem-cell like phenotypes and immunosuppressive microenvironment in GC. Importantly, compared to treatment with nab-paclitaxel alone, nab-paclitaxel plus GAL-1 blockade significantly enhanced the anti-tumor effect in mouse models, particularly in humanized mice. Conclusions Our data support a pivotal role for ZFP64 in GC progression by simultaneously promoting cellular chemotherapy resistance and tumor immunosuppression. Treatment with the combination of nab-paclitaxel and a GAL-1 inhibitor might benefit a subgroup of GC patients.

Regulation of NSCLC Cell Proliferation By MARCH7 Via the NF-κB and Wnt/β-Catenin Signaling Pathways

The aim of the study was to explore the role of the E3 ubiquitin ligase MARCH7 in the development of non-small-cell lung cancer (NSCLC) and to explore the underlying molecular mechanism.Western blot and immunohistochemistry results showed that the expression of MARCH7 in NSCLC cancer tissues was higher than that in paracancerous tissues. Tissue microarray staining results and clinicopathological parameters of NSCLC patients revealed that MARCH7 expression was closely related to TNM stage, degree of tumor differentiation and lymph node metastasis of NSCLC patients. Furthermore, univariate and multivariate analyses and survival curve analysis showed that high expression of MARCH7 was associated with poor prognosis.In vitro, siRNA was constructed and transfected into A549 cells to inhibit the expression of MARCH7. The CCK-8 assay indicated that the growth rate of tumor cells in the interference group was reduced. The number of colonies and cells in the interference group decreased in the plate clone formation experiment. Flow cytometry showed that G0/G1 phase cells were predominantly increased after blocking endogenous MARCH7 expression, and G0/G1 phase arrest occurred in A549 cells. The reporter gene activity of the NF-κB signaling pathway and Wnt/β-catenin signaling pathway was reduced, as validated by a double luciferase reporter gene assay. Western blot analysis showed that the expression of NF-κB P50, NF-κB P65 and β-catenin was decreased, while the expression of E-cadherin was elevated.In vivo, MARCH7-overexpressing virus was constructed and transfected into A549 cells and then subcutaneously injected into nude mice. It was demonstrated that the tumor volume was significantly larger in the MARCH7 overexpression group than in the control nude mice during the same period. Elevated expression of PCNA and Ki-67 was observed in the tumor mass of the MARCH7 overexpression group, as measured by immunohistochemical analysis, accompanied by enhanced levels of NF-κB P50, NF-κB P65 and β-catenin, as detected by Western blot. These results provide a new idea for the experimental basis for the treatment of NSCLC in the future.

Lnc027912 Reduces Lipid Accumulation Through Akt/MTOR/SREBP1C Axis in Hepatic Cells

Background: Various metabolism diseases are closely related to lipid metabolism disorder, but long noncoding-RNAs (lncRNA) involve in regulating function of lipid was limited elucidated. Previous our work have found that lnc027912 involve in cholesterol metabolism. Here, we further explore the role of lipid metabolism-associated lncRNA-lnc027912 in oleic acid- (OA) and palmitic acid (PA)-induced hepatic cells. Methods: The overexpression of lnc027912 cell model was constructed by using virus particles transfection, and the level of lnc027912 in AML12 cells were detected by RT-qPCR. High fat cell model was established by treating AML12 cells with OA and PA, and the level of lipid drops was detected by Oil red O staining and triglyceride analyze Kit. The lipid metabolism related-genes, such as SREBP1C, FAS, PPARγ, MTTP, ApoE and ApoC3 level, was detected using RT-qPCR and Western blot. The role of SREBP1C in lipid metabolism was further analyzed using double luciferase reporter gene assay and Immunofluorescence. The Akt/mTOR signal pathway related genes was detected by Western blot. Results: We found that TG level was inhibited in overexpression of lnc027912 cell. Upregulated lnc027912 of AML12 cells treated with OA and PA showed a significant decrease in lipid accumulation and TG levels. Furthermore, overexpression of lnc027912, the lipid biosynthesis genes of SREBP1C, FAS and PPARγ was significantly decreased and a significant increase in expression of MTTP and ApoE. Interestingly, lnc027912 inhibited Akt/mTOR signaling axis and decreased SREBP1C transit into nucleus and the promoter activity of SREBP1C and regulated expression of its targets. Conclusions: Our study revealed a new insights into the molecular function of lnc027912 in lipid metabolism by Akt/mTOR/SREBP1C signaling axis and highlights the potential of lnc027912 as a new therapeutic target for lipid disorder diseases (such as, NAFLD).

MiR-218 Promotes Adriamycin-Induced H9C2 Apoptosis by Inhibiting Stress-Associated Endoplasmic Reticulum Protein 1

Objective. Adriamycin is a clinically important chemotherapeutic drug, but its use is restricted due to its myocardial toxicity. Therefore, it is especially important to explore the toxicity mechanism of Adriamycin (ADR) to cardiomyocytes. Methods. The myocardial toxicity model of ADR was constructed in vitro, and the effect of miR-218 inhibitor and sh-Serp1 on the activity of H9C2 cells induced by ADR was detected by MTT method. Also, flow cytometry, real-time polymerase chain reaction (RT-PCR), and TUNEL staining were used to detect the cell apoptosis. The activity of LDH was detected by colorimetry, and the interaction of miR-218 with Serp1 was detected by double-luciferase reporter gene assay. Western blotting technique was used to detect the expression level of caspase3 and p38 MAPK signal pathway. Results. miR-218 inhibitor can obviously inhibit ADR-induced decrease in cell activity of H9C2 cells, inhibit cell apoptosis, and inhibit p38 MAPK signaling pathway activation. Conversely, sh-Serp1 aggravated the decrease in H9C2 cell activity and promoted cell apoptosis. Conclusion. Upregulation of miR-218 expression will promote ADR-induced apoptosis of H9C2 cells. At the same time, we confirmed that the mechanism by which miR-218 promotes myocardial apoptosis was through the Serp1/p38 MAPK/caspase-3 signaling pathway.