Low miR-34a Expression Research Articles

JMJD2A participates in cytoskeletal remodeling to regulate castration-resistant prostate cancer docetaxel resistance

BackgroundTo investigate underlying mechanism of JMJD2A in regulating cytoskeleton remodeling in castration-resistant prostate cancer (CRPC) resistant to docetaxel.MethodsTissue samples from CRPC patients were collected, and the expression of JMJD2A, miR-34a and cytoskeleton remodeling-related proteins were evaluated by qPCR, western blot and immunohistochemistry, and pathological changes were observed by H&E staining. Further, JMJD2A, STMN1 and TUBB3 were knocked down using shRNA in CRPC cell lines, and cell viability, apoptosis and western blot assays were performed. The interaction between miR-34a/STMN1/β3-Tubulin was analyzed with dual-luciferase reporter and co-immunoprecipitation assays.ResultsIn clinical experiment, the CRPC-resistant group showed higher expression of JMJD2A, STMN1, α-Tubulin, β-Tubulin and F-actin, and lower expression of miR-34a and β3-Tubulin compared to the sensitive group. In vitro experiments showed that JMJD2A could regulate cytoskeletal remodeling through the miR-34a/STMN1/β3-Tubulin axis. The expression of miR-34a was elevated after knocking down JMJD2A, and miR-34a targeted STMN1. The overexpression of miR-34a was associated with a decreased expression of STMN1 and elevated expression of β3-Tubulin, which led to the disruption of the microtubule network, decreased cancer cell proliferation, cell cycle arrest in the G0/G1 phase, and increased apoptosis.ConclusionJMJD2A promoted docetaxel resistance in prostate cancer cells by regulating cytoskeleton remodeling through the miR-34a/STMN1/β3-Tubulin axis.

Regulating Function of miR-146a Derived from Bone Marrow Mesenchymal Stem Cell (BMSC) in Acute Lung Injury

This study assesses the mechanism of miR-146a derived from Bone marrow mesenchymal stem cell (BMSC) in acute lung injury. The model of ALI rats was established through endotracheal perfusion of LPS followed by analysis histological changes by HE staining. The source of BMSC was detected through flow cytometry and change of miRNA was detected through Array method. The miR-146a level in lung tissue was detected with RT-PCR and expression of Bcl-2, Bax and Capase-9 was detected with IF and Western Blot. A high expression of CD90 and CD105 was found in BMSC with negative CD11bc and CD34 level. 39 downregulated miRNAs and 20 upregulated miRNAs were found in ALI with miR-146a being the most significant. The apoptotic level induced with LPS could be restrained by miR-146a. In addition, miR-146a could upregulate Bcl-2 and downregulate Bax and Caspase-9. In conclusion, ALI could be restrained by the low expression of miR-146a.

Plasma-Based microRNA Expression Analysis in Advanced Stage NSCLC Patients Treated with Nivolumab.

Simple SummaryNivolumab (anti-PD-1 inhibitor) is the first monoclonal antibody approved for the treatment of NSCLC, with research results showing that patients who had received previous lines of therapy had a better response to this treatment and better overall survival. Tissue-level analyses fail to capture the dynamic tumor-host relationship, in contrast to circulating biomarkers, which can reflect the systemic response of the tumor, allowing for repeated sampling and monitoring. In the context of liquid biopsy, microRNAs are studied as biomarkers of immunotherapy efficacy based on their role in regulating antitumor immunity. The present study suggests that miR-200c and miR-34a plasma expression levels have a prognostic role in patients with advanced NSCLC receiving Nivolumab. It further supports that the expression profile of circulating immunomodulatory microRNAs provides information on the survival of patients with advanced NSCLC receiving Nivolumab and could represent promising circulating biomarkers that may provide information about patients’ responses to immunotherapy.Since circulating microRNAs (miRNAs) are involved in the modulation of the immune response, they are tested as liquid biopsy-based biomarkers in patients with NSCLC treated with immunotherapy. We analyzed the expression levels and examined the clinical significance of immunoregulatory miRNAs involved in immune checkpoint regulation (miR-34a, miR-200b, miR-200c), T-cell activity (miR-155), and the function of myeloid-derived suppressive cells (MDSCs) (miR-223) or regulatory T lymphocytes (Tregs) (miR-146a), in patients with advanced NSCLC (N = 69) treated with anti-PD-1 (Nivolumab) immunotherapy as 2nd or 3rd line of treatment therapy. Plasma levels of circulating miRNAs were analyzed by RT-qPCR before the initiation of immunotherapy. Expression of miR-34a, miR-146a, mir-200c, and miR-223 was found to be associated with response to immunotherapy. High miR-200c expression emerged as an independent prognostic factor for inferior overall survival in all patients with NSCLC (OS, HR: 2.243, 95% CI: 1.208–4.163; p = 0.010) and in patients with non-Squamous (non-SqCC) subtype (N = 38) (HR: 2.809, 95% CI: 1.116–7.074; p = 0.028). Low miR-34a expression independently predicted for shorter OS (HR: 3.189, 95% CI: 1.193–8.527; p = 0.021) in the non-SqCC subgroup. Our findings suggest that alterations in circulating miR-200c and miR-34a expression levels are associated with the response and outcome in patients with advanced NSCLC treated with anti-PD1 immunotherapy.

Expression and role of miR-146a and SMAD4 in placental tissue of pregnant women with preeclampsia.

To investigate the expression of miR-146a in severe preeclampsia (PE) and its effect on trophoblast cell proliferation, invasion and apoptosis, as well as its relationship with SMAD4. Participants were divided into the severe PE group (n=30) and the normal group (n=30). The expression of miR-146a and SMAD4 in placenta tissue was detected by immunohistochemistry, qRT-PCR, and western blot. Trophoblast cell lines HTR-8/SVneo were cultured to detect the expression of miR-146a under the Cobalt chloride (CoCl2 )-simulated hypoxia. The effects of miR-146a transfection on cell proliferation, invasion, apoptosis, and SMAD4 expression were analyzed. Compared with the normal group, miR-146a expression was decreased and the protein and mRNA levels of SMAD4 were increased in placenta tissues of the severe PE group. Our in vitro experiments showed that the expression of miR-146a decreased after CoCl2 treatment. Silencing miR-146a caused increased expression of SMAD4 and decreased expression of VEGF. After transfection with miR-146a inhibitor, compared with the NC group, the invasion and proliferation of HTR-8/Svneo cells were decreased, while the apoptosis was enhanced. The expression of miR-146a decreased in severe PE and was negatively correlated with SMAD4 expression. The expression of miR-146a was inhibited under hypoxia, and the low expression of miR-146a affected the proliferation, invasion, and apoptosis of trophoblast cells.

Abstract 1496: Circulating mir21 and mir125b in women living with human immunodeficiency virus: Utility of biomarkers for monitoring cervical carcinogenesis

Abstract Background: As of 2018, the prevalence of Human immunodeficiency virus (HIV), and cervical cancer (Ca) attributable to HIV was higher in Africa than in other continents. Identifying individuals at a high risk of developing Ca among immunocompromised persons, using less invasive techniques, remains a major challenge. Purpose: The study evaluated HIV infection-associated dysregulation of Ca-linked oncomirs (miR-21, miR-146a, miR-155, miR-182, and miR-200c) and tumor suppressors (miR-let-7b, miR-125b, miR-143, miR-145, and p53 gene), in a bid to identify early indicators of genetic instability, and biomarkers for monitoring of high-risk individuals. Methods: This case-control study included 173 women without abnormal Pap smear; confirmed HIV seropositive women (HIV+ = 103) and HIV seronegative women (HIV- = 70). Relative expressions of miRNAs and p53 gene in blood and cervical cells) were determined following RNA extraction, reverse transcriptase Polymerase Chain Reaction (PCR), and gel electrophoresis. T-test was used to compare the data from HIV+ and HIV- women. Significance was set at p≤ 0.05. Results: Similar pattern of miR-21, miR-146a, miR-182, miR-200c, miR-125b, and miR-145 expression was observed in both samples. Higher expressions of miR-155 and p53 gene were observed in cervical cells of HIV+ women compared with HIV- women (p= 0.046, and 0.033, respectively) whereas lower expressions of miR-155 and p53 gene were observed in the blood of HIV+ women compared with HIV- women (p= 0.539 and 0.049, respectively). In both blood and cervical cells, higher miR-21 expression (p= 0.032 and 0.198, respectively) and lower miR-125b expression (p= 0.050 and 0.004, respectively) were observed in HIV+ women compared with HIV-women. In blood, a lower expression of miR-146a was observed in HIV+ women compared with HIV- women (p= 0.036) whereas in cervical cells, lower expressions of miR-182 and miR-200c were observed in HIV+ women compared with HIV- women (p= 0.035 and 0.045, respectively). The low expression of miR-146a, miR-182, and miR-200c in HIV+ women could be due to immune exhaustion, viral immune evasive mechanism, or an indicator of viral latency while the higher expression of miR-21, and lower expression of miR-125b and p53 could be early indicators of genetic instability prior to epithelial transformation. Conclusion: This study suggests that circulating high expression of miR-21 and low expression of miR-125b and p53 gene could be used in identifying individuals at risk of developing Ca, especially among immunocompromised patients. Keywords: Human immunodeficiency virus, Cervical cancer, Detection, Biomarkers, MicroRNAs Citation Format: Jude Ogechukwu Okoye, Anthony Ajuluchukwu Ngokere, Charles Chinedum Onyenekwe, Samuel Ifedioranma Ogenyi, Olaposi Omotuyi. Circulating mir21 and mir125b in women living with human immunodeficiency virus: Utility of biomarkers for monitoring cervical carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1496.

Long non-coding RNA CHRF accelerates LPS-induced acute lung injury through microRNA-146a/Notch1 axis.

BackgroundThe present study sought to investigate the regulatory role of the long non-coding RNA (lncRNA) cardiac hypertrophy-related factor (CHRF) in a mouse model of acute lung injury (ALI) and in primary mouse pulmonary microvascular endothelial cells (MPVECs) treated with lipopolysaccharide (LPS).MethodsC57BL/6 mice were given adenovirus (Ad) sh-CHRF or negative control (NC) before undergoing cecal ligation and perforation. MPVECs transfected with Adsh-CHRF or NC were treated with LPS. Double luciferase assay was used to detect the binding of miR-146a to CHRF or Notch1. Subsequently, MPVECs were co-transfected with miR-146a inhibitor and sh-CHRF for 24 hours, and then treated with LPS.ResultsHigh expression of CHRF was detected in septic mice. Cecal ligation and perforation induced ALI and apoptosis in mice, whereas, CHRF knockout could inhibit ALI. The protein expression levels of TNF-α, IL-1β and IL-6 in the lung and bronchoalveolar lavage fluid of the CLP group were up-regulated, whereas the expression of IL-4 and IL-10 was down-regulated. CHRF inhibition reduced the production of proinflammatory cytokines in septic mice. The inhibitory effect of CHRF gene knockdown on lung inflammation and apoptosis was confirmed in the septic cell model. Mechanistic investigation showed that CHRF up-regulated the level of Notch1 by sponging miR-146a. Additionally, the low expression of miR-146a reversed the inhibitory effect of CHRF gene knockout on LPS-induced inflammatory response and apoptosis. Together, in vivo and in vitro results demonstrated that CHRF enhanced sepsis-induced ALI by targeting miR-146a and up-regulating Notch1.ConclusionsCHRF can induce inflammation and apoptosis caused by sepsis by miR-146a/Notch1 axis. Therefore, it may serve as a potential drug target for treating sepsis-induced ALI.

Expression and Significance of Serum Mir-146a and Mir-30e in Patients with Secondary Hemophagocytic Lymphohistiocytosis

ObjectiveHemophagocytic lymphohistiocytosis (HLH) is a clinical syndrome which is caused by uncontrolled activation and proliferation of CD8+ T cell , and abundant releasing of in inflammatory cytokines. It has been confirmed that miR-146a is widely participate in the regulation of intensity of inflammatory response, while miR-30e is related to the regulation of NK cell cytotoxicity. In this study, RT-PCR was used to detect the expression of serum miR-146a and miR-30e in patients with secondary HLH (sHLH). Their correlation with clinical data, and diagnosis and prognosis value in sHLH patients were explored. In addition, their relation with the proportion of cytotoxic lymphocyte subsets and the expression of perforin were explored.MethodsA total of 35 patients with sHLH and 10 healthy controls were enrolled in this study. Serum expression levels of miR-146a and miR-30e were detected by using RT-PCR. The expression of perforin protein oncytotoxic lymphocytes and the proportion of cytotoxic lymphocyte subsets were tested by flow cytometry. The clinical data of sHLH patients were collected.Results(1) The serum expression level of miR-146a in the newly diagnosed sHLH group was significantly lower than that of healthy control group (P <0.001); the serum expression level of miR-30e in the newly diagnosed sHLH group was significantly lower than that of healthy control group (P <0.001). (2) The serum expression level of miR-146a and miR-30e in the lymphoma-associated HLH group, the infection- associated HLH group, and the autoimmune-associated HLH group were compared group by group, however no significant difference was found (P >0.05). (3) ROC analysis revealed that the serum expression level of miR-146a can effectively identify sHLH patients from healthy people, with area under the curve (AUC) was 0.931; the serum expression level of miR-30e can effectively identify sHLH patients from healthy people, with area under the curve (AUC) was 0.914. (4) Correlation analysis was applied and no correlation was found between the serum expression level of miR-146a and miR-30e, and Age, WBC, HB, PLT, SF, ALB, ALT, AST, LDH, FIB, TG, ESR, sCD25, CRP level in the newly diagnosed sHLH (P >0.05). (5) According to the median of serum expression level, sHLH patients were divided in to Low expression group and High expression group. OS analysis was applied and revealed that there was no significance difference between the Low miR-146a expression group and the High miR-146a expression group (P >0.05); meanwhile there was no significance difference between the low miR-30e expression group and the high miR-30e expression group (P >0.05). However, sHLH patients were divided in to Low miR-30e/miR-146a group and High miR-30e/miR-146a group according to the median of miR-30e/miR-146a. The OS of Low miR-30e/miR-146a group was significantly longer than High miR-30e /miR-146a group (P=0.022). (6)The proportion of CD8+ T cell in Low miR-146a group was significantly lower than that in High miR-146a group (P = 0.016). The serum exression level of miR-146a was corelated with the proportion of CD8+ T cell (r=0.734, P = 0.007). However, the proportion of cytotoxic lymphocyte subsets in Low miR-146a group has no significant difference with that in Low miR-146a group (P >0.05); the serum exression level of miR-30e has no corelation with the proportion of cytotoxic lymphocyte subsets (P >0.05). (7) The expression of perforin in cytotoxic lymphocyte subsets in Low miR-30e group has no significant difference with that in Low miR-30e group (P >0.05); the serum exression level of miR-30e has no corelation with the expression of perforin in cytotoxic lymphocyte subsets (P >0.05).ConclusionThe serum expression level of miR-146a and miR-30e in the newly diagnosed sHLH group was significantly decreased, which is important in the differential diagnosis of sHLH. Individuals with lower serum expression level of miR-146a and miR-30e were more likely to be sHLH patients. The ratio of miR-30e and miR-146a (miR-30e /miR-146a) can indicate the prognosis of sHLH patients. The prognosis of sHLH patients with lower miR-30e /miR-146a was better than that of sHLH patients with higher miR-30e /miR-146a. The serum exression level of miR-146a was corelated with the proportion of CD8+ T cell. The serum exression level of miR-146a was corelated with the proportion of CD8+ T cell. Therefore, miR-146a may play a role in the deficiency of CD8+ T cell in the pathogenesis of sHLH. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mir-34a as predictor of immunotherapy efficacy in NSCLC patients.

e21191 Background: MicroRNAs are critical modulators of the immune response and regulate the expression of various immune checkpoints during immunotherapy. In the present study we evaluated the predictive significance of immune related miRNAs in the plasma of non-small cell lung cancer (NSCLC) patients treated with immune checkpoint inhibitors. Methods: Plasma was obtained from 62 NSCLC patients before the initiation of immunotherapy, administered as second- or third-line treatment. The expression profile of immune-related miR-34a, miR-146a, miR-155, miR-200b, miR-202 and miR-223 was assessed by RT-qPCR. Patients were classified in high and low expression groups according to the median value of each miRNA. Patients that had achieved partial response (PR) or stable disease (SD) were classified as responders, whereas patients with progressive disease (PD) were classified as non-responders. Results: No statistical correlations were observed among miRNA expression and clinical outcomes in the whole group of patients (N = 62). However, in the non-squamous subgroup (N = 36) lower miR-34a expression levels were observed in non-responders compared to responders (80% vs 20%; p = 0.029). Univariate binary logistic regression analysis revealed that only low miR-34a expression (HR: 8.000, 95% CI: 1.215-52.693; p = 0.031) was correlated with the probability of developing progressive disease as best response to immunotherapy. Patients with low miR-34a expression levels were associated with shorter progression free survival (PFS) and overall survival (OS) compared to patients with high 34a expression (1.97 vs 6.33 months; p = 0.042 and 2.93 vs 9,60 months; p = 0.012, respectively). Furthermore, low miR-34a expression emerged as an independent predictor of shorter PFS and OS (HR: 2.449; p = 0.049 and HR: 3.203; p = 0.016, respectively). No other correlations were observed among the rest of the miRNAs and clinical outcomes. Conclusions: Our findings suggest that circulating miR-34a may serve as a potential predictive biomarker in NSCLC patients treated with immunotherapy. These observations need to be further validated in a larger cohort of patients.

URINARY EXOSOME MIR-146A IS A POTENTIAL MARKER OF ALBUMINURIA IN ESSENTIAL HYPERTENSION

Objective: There is increasing interest in using extracellular vesicle-derived microRNAs (miRNAs) as biomarkers in renal dysfunction and injury. Preliminary evidence indicates that miRNAs regulate the progression of glomerular disease. Indeed, exosomes from the renal system have provided novel evidence in the clinical setting of albuminuria. Thus, the aim of this study was to quantify the urinary miRNAs present in exosome and microvesicles (MVs), and to assess their association with the presence of increased urinary albumin excretion (UAE) in essential hypertension. Design and method: Exosomes were isolated from urine specimens from a cohort of hypertensive patients with (n = 24) or without albuminuria (n = 28), and from 20 healthy volunteers as a control group. Urinary exosomes were phenotyped by Western blot, tunable resistive pulse sensing (TRPS), electronic microscopy and western blot. Expression of exosomal miR-146a and miR-335∗ was analysed by qRT-PCR and any associations between albuminuria and exosomal miRNAs were established. Results: Urinary miRNAs are highly enriched in exosome subpopulations compared to MVs, both in patients with or without increased UAE (p &lt; 0.001), but not in the control group. High albuminuria was associated with 2.5-fold less miR-146a in exosomes (p = 0.017), whereas miR-146a levels in MV did not change. In addition, exosome miR-146a levels were inversely associated with albuminuria (r = 0.65, p &lt; 0.0001), and discriminated the presence of urinary albumin excretion presence [area under the curve (AUC) = 0.80, 95% confidence interval: 0.66–0.95; p = 0.0013). Conclusions: Our results indicate that miRNAs were enriched in the urinary exosome subpopulation in hypertensive patients and that low miR-146a expression in exosomes was associated with the presence of albuminuria. Thus, urinary exosome miR-146a may be a potentially useful tool for studying early renal injury in hypertension.

Prognostic value of low microRNA-34a expression in human gastrointestinal cancer: a systematic review and meta-analysis

BackgroundMounting evidence shows that microRNA-34a (miR-34a) is involved in cancer prognosis. Therefore, we summarize the predictive role of miR-34a for survival in patients with gastrointestinal cancers (GICs).MethodsAll eligible studies were found by searching PubMed, Web of Science and EMBASE, and survival results were extracted. Then, the hazard ratio (HR) with the corresponding 95% confidence interval (CI) was calculated to evaluate the prognostic role of miR-34a in GICs. The association between miR-34a expression and clinicopathological characteristics was estimated by odds ratios (ORs) and 95% CIs.ResultsA total of 20 studies were included in this meta-analysis. For overall survival (OS), lower miR-34a expression could probably predict poorer outcome in GICs, with a pooled HR of 1.86 (95% CI: 1.52–2.28, P < 0.01). For disease-free survival (DFS), progression-free survival (PFS), and recurrence-free survival (RFS), lower miR-34a expression was related to worse DFS/PFS/RFS with a pooled HR of 1.86 (95% CI: 1.31–2.63, P < 0.01). A significant relation of differentiation/TNM stage/lymphatic metastasis and the expression level of miR-34a was identified.ConclusionThis meta-analysis revealed that lower miR-34a expression is significantly connected with worse OS and DFS/PFS/RFS in GIC patients. In addition, the miR-34a expression level is relatively lower in patients with lymph node metastasis than in patients without lymph node metastasis, and decreased miR-34a expression levels are linked to poor tumour differentiation and late TNM stage. MiR-34a may become a new factor for the prognosis prediction and progression of GICs.

Epigallocatechin-3-gallate Enhances the Efficacy of MicroRNA-34a Mimic and MiR-93 Inhibitor Co-transfection in Prostate Cancer Cell Line

The potential role of microRNAs (miRNA) as therapeutic molecules has moved them from basic research to the field of cancer therapy. High expression of miR-93 and low expression of miR-34a have previously been indicated in prostate cancer (PC), which is the second leading cause of cancer-related death in men. Androgen receptor (AR) and prostate-specific antigen (PSA) play key roles in the initiation and progression of this cancer. Therefore, this study aimed to investigate the effects of the transfection and co-transfection of miR-34a mimic and miR-93 inhibitor with or without epigallocatechin-3-gallate (EGCG) on prostate cancer cell line and also to evaluate their effects on the expression of AR, PSA. Human lymph node carcinoma of the prostate (LNCaP) cells were treated with miR-34a mimic or/and miR-93 inhibitor with or without EGCG. Gene or protein expressions were assessed by real-time PCR or western blotting of lysates. The transfection with miR-34a mimics significantly reduced the mRNA expression of AR (p=0.0016), and PSA (p=0.038) compared to the control. Also, the miR-93 inhibitor led to a decrease in the mRNA expression of AR (p=0.0057) and PSA (p&gt;0.05) compared to the control group. Furthermore, the co-transfection, along with EGCG, caused more decrease in both the AR (p&lt;0.001) and the PSA (p=0.003) expression compared with the co-transfection without EGCG. Our study indicates that the reduced expression of AR and PSA in PC cells followed by treatment with miR-34a mimic and miR-93 inhibitor and their combination with EGCG as a natural substance may be a promising therapeutic way for controlling the growth of these malignant cells.

MicroRNA-146a suppresses tumor malignancy via targeting vimentin in esophageal squamous cell carcinoma cells with lower fibronectin membrane assembly

BackgroundEsophageal squamous cell carcinoma (ESCC) is widely prevalent in Taiwan, and high metastatic spread of ESCC leads to poor survival rate. Fibronectin (FN) assembly on the cell membrane may induce ESCC mobility. MicroRNAs (MiRNAs) are abundant in and participate in tumorigenesis in many cancers. However, the role of MiRNA in FN assembly-related ESCC mobility remains unexplored.MethodsWe divided ESCC CE81T cells into high-FN assembly (CE81FN+) and low-FN assembly (CE81FN−) groups by flow cytometry. MiRNA microarray analysis identified miR-146a expression as the most down-regulated miRNA in comparison of CE81FN+ and CE81FN− cells.ResultsCell proliferation and migration were decreased when CE81FN+ cells overexpressed transgenic miR-146a compared to the parental cells, indicating an inverse correlation between low miR-146a expression and high proliferation as well as motility of FN assembly ESCC cells. Furthermore, vimentin is the target gene of miR-146a involved in ESCC tumorigenesis. MiR-146a suppressed cell proliferation, migration and invasion of CE81FN+ cells through the inhibition of vimentin expression, as confirmed by real-time PCR, Western blotting and Transwell™ assay. Analysis of one hundred and thirty-six paired ESCC patient specimens revealed that low miR-146a and high vimentin levels were frequently detected in tumor, and that the former was associated with late tumor stages (III and IV). Notably, either low miR-146a expression or high vimentin level was significantly associated with poor overall survival rate among ESCC patients.ConclusionsThis is the first report to link FN assembly in the cell membrane with miR-146a, vimentin and ESCC tumorigenesis both in vitro and in ESCC patients.

Identification of a Novel miR-146a-POU3F2/SMARCA5 Pathway in Glioblastoma

Rapid tumor growth, widespread brain-invasion, and therapeutic resistance primarily contribute to an inevitable tumor recurrence and dismal patient outcome in glioblastoma. Although GBM stem cells (GSCs) are shown to play key roles in these processes, the molecular pathways driving the GSC phenotype (GBM-stemness) remain poorly defined. A global miRNA expression analysis was performed using FFPE GBM tumor samples, and we found that low expression of miR-146a correlated with poor clinical outcome and tumor recurrence. Based on this finding, the hypothesis is that miR-146a functions as a tumor suppressor and inhibits stemness in GBM. Expression analysis was performed using FFPE GBM tumor samples (n = 268) followed by univariable (UVA) and multivariable (MVA) analyses. Functional studies were conducted to define the role of miR-146a-POU3F2/SMARCA5 pathway in GBM tumorigenesis and therapeutic response. MicroRNA-146a was one of the top miRNAs that correlated with better prognosis in GBM patients by UVA analyses (p = 9.21E-05), which was independent of MGMT promoter methylation by MVA analyses (p<0.001). miR-146a expression was significantly downregulated in recurrent GBM tumors compared with the paired primary GBM tumors (p = 0.003). Overexpression of miR-146a significantly inhibited tumor cell growth and sensitized patient-derived primary GBM cells to temozolomide (TMZ) treatment in vitro, and showed statistically significant smaller tumor size (p<0.01) and prolonged survival (p = 0.001) in vivo. In addition, miR-146a was downregulated in glioma cancer stem cells, and overexpression of miR-146a significantly reduced stemness. Mechanistically, miR-146a directly silenced POU3F2 and SMARCA5, two transcription factors that mutually regulated each other, significantly compromising GBM-stemness and increasing TMZ response. Moreover, overexpression of miR-146a significantly inhibited NF-κB pathways through targeting POU3F2 and SMARCA5 in GBM. Collectively, our data show that miR-146a-POU3F2/SMARCA5 pathway plays a critical role in suppressing GBM-stemness and increasing TMZ-response, suggesting that POU3F2 and SMARCA5 may serve as novel therapeutic targets in GBM. Fundings: R01CA188228 (to A.C., R.B., K.L., and J.S.B-S.), and the T&P Bohnenn Fund for Neuro-Oncology Research (to P.A.R.). R01CA169368, R01CA11522358, R01CA1145128, R01CA108633, 1RC2CA148190, and U10CA180850-01; A Brain Tumor Funders Collaborative Grant; Ohio State University Comprehensive Cancer Center Award (all to A.C.).

Identification of microRNA expression signature for the diagnosis and prognosis of cervical squamous cell carcinoma

Cervical cancer is the fourth leading cause of cancer death among women globally. The prognosis of cervical cancer patients differs considerably, and clinical outcomes are difficult to predict. Given the significant roles of miRNAs in human cancers, identification of novel and reliable miRNA biomarkers is important for targeted cervical cancer therapy. In the present study, we aimed to reveal biological significance of miR-200a, miR-423, miR-34a, miR-193a, and miR-455 for the prognosis and diagnosis of cervical cancer and their association with the clinical outcomes of patients. Distinct expression profiles of miRNAs in formalin-fixed paraffin-embedded tissue samples of patients and healthy controls were evaluated using qRT-PCR. We identified miR-200a, miR-455, and miR-34a were significantly downregulated in cervical squamous cell carcinoma tissues compared to normal cervix tissue from healthy controls. Both miR-455 and miR-34a confer a promising diagnostic factor for the cervical cancer while miR-200a showed no significance in ROC analysis. Notably, low expression of miR-34a was markedly associated with the poor overall survival of cervical cancer patients as revealed by Kaplan-Meier survival analysis. Also, univariate and multivariate analysis indicated miR-34a expression as an independent prognostic factor. Consequently, our results underline the importance of distinct expression miRNAs in cervical squamous cell carcinoma.

Expression Level of MiR-146a in Acute Myeloid Leukemia Patients and Its Clinical Significance

To detect the expression of miR-146a in patients with AML, and to evaluate the relationship between miR-146a expression level and clinical characteristics, treatment response, EFS and OS. 154 patients with newly diagnosed AML were enrolled in AML group, 50 controls (patients with thrombocytopenic purpura or voluntary donor of bone marrow) were enrolled in control group. The miR-146a expression levels in bone marrow mononuclear cells was detected by RT-PCR between 2 group. AML patients were treated with chemotherapeutic drugs, and their clinical response and survivals were assessed. The expression level of MiR-146a in AML group was significantly lower than that in control group. The ROC showed that miR-146a could distinguish the patients in AML and control group better (area under curve 0.819 (95%CI: 0.761-0.877). Meanwhile, the proportion of good and moderate good prognosis (P＜0.001), proportion of WBC count ≤15.2×109/L (P＜0.05), CR rate (P＜0.05), EFS (P＜0.01) and OS (P＜0.01) in patients with high miR-146a expression were higher than those in patients with low miR-146a expression. Cox's model showed that miR-146a expression level positively realated with incressed EFS and OS. MiR-146a is downregulated in AML patients, which might be served as a biomarker for predicting risk and prognosis of AML patients.

The DNMT1/miR-34a/FOXM1 Axis Contributes to Stemness of Liver Cancer Cells.

Background Whether DNA methyltransferase 1 (DNMT1)/miR-34a/FoxM1 signaling promotes the stemness of liver cancer stem cells (LCSCs) remains unclear. This study aimed to assess whether methylation-based silencing of miR-34a by DNMT1 contributes to stemness features via FoxM1 upregulation in LCSCs. Methods The CD133+ subgroup of MHCC97H cells sorted by MACS was used as LCSCs. DNMT1, BMI1, SOX2, and OCT4 mRNA levels, and miR-34a amounts were determined by qRT-PCR. DNMT1, CD44, and FoxM1 proteins were analyzed by immunoblot. Sphere and colony formation abilities were detected by respective assays. CD133+ cell percentages were assessed by flow cytometry. In vivo oncogenicity was evaluated using a tumor xenograft model in mice. The effects of DNMT1/miR-34a signaling on the stemness of LCSCs were examined by knockdown or overexpression of DNMT1 and/or transfection of miR-34a mimic or inhibitor using lentivirus-delivery systems. FoxM1 association with miR-34a was detected by a reporter assay. Results We here showed that LCSCs exhibited elevated DNMT1 activity and expression, lower miR-34a expression with higher promoter methylation, and stronger stemness, compared with the parental liver cancer cells. DNMT1 knockdown repressed DNMT1, increased miR-34a amounts by promoter demethylation, and reduced stemness in LCSCs, whereas DNMT1 overexpression had the opposite effects in liver cancer cells. Transfection with miR-34a mimic repressed the stemness of LCSCs, while miR-34a inhibitor significantly downregulated miR-34a and enhanced stemness, without affecting DNMT1 in liver cancer cells. MiR-34a mimic rescued the effects of DNMT1 overexpression on the stemness of LCSCs, without affecting DNMT1 expression. Finally, FOXM1 was identified as a direct target by miR-34a in LCSCs. Conclusions We revealed that aberrant activation of DNMT1 causes miR-34a promoter methylation and suppression, leading to FoxM1 upregulation by disinhibition and promotion of LCSC stemness. These findings suggest that blockage of DNMT1/miR-34a-mediated FOXM1 upregulation might suppress liver cancer by targeting LCSCs.

Dexmedetomidine improves propofol-induced neuronal injury in rat hippocampus with the involvement of miR-34a and the PI3K/Akt signaling pathway

AimsDexmedetomidine (DEX) is a selective agonist of α2-adrenergic receptors with anesthetic attributes and neuroprotective effects. This study was designed to explore the mechanisms of DEX in the propofol-induced neuronal injury in rat hippocampus. Materials and methodsRat hippocampi were treated with propofol, and then neuronal injury, neuronal apoptosis, PSD95 and apoptosis-related protein expression in CA1 region were measured after DEX administration and/or ant-miR-34a. miR-34a expression was detected using RT-qPCR, while the binding of miR-34a and Sirtuin1 (SIRT1) was identified with dual luciferase reporter gene assay, and the activation of PI3K/Akt signaling pathway was detected. Additionally, hippocampal neurons were cultured in vitro and treated with DEX and propofol. The viability and apoptosis of hippocampal neurons, fluorescence intensity of Ca2+ and neuronal morphology were detected. Key findingsIn vivo experiments, propofol induced obvious neuronal injury in rat hippocampus, while DEX at different doses reduced hippocampal neuronal apoptosis and miR-34a expression but increased PSD95 expression in rat hippocampus. Low expression of miR-34a reduced propofol-induced neuronal injury by targeting SIRT1 and activating the PI3K/Akt pathway. In vitro experiments, propofol induced neuronal injury, which was alleviated by DEX treatment, accompanied with increased neuronal viability, but decreased apoptosis and fluorescence intensity of Ca2+. The attenuation of neuronal injury achieved by DEX was impaired by over-expression of miR-34a. Meanwhile, over-expression of SIRT1 in neurons with overexpressed miR-34a improved p-Akt and p-PI3K expression. SignificanceDEX could inhibit propofol-induced neuronal injury in rat hippocampus by inhibiting miR-34a expression, upregulating SIRT1 and activating the PI3K/Akt pathway.

Expression of miR-34a and Ki67 in nasopharyngeal carcinoma and the relationship with clinicopathological features and prognosis.

Expression levels of miR-34a and Ki67 in nasopharyngeal carcinoma (NPC) and the relationship with clinicopathological features and prognosis were studied. A prospective study was performed on 56 cases of NPC tissues and 56 cases of adjacent tissues collected in Xiangyang No. 1 People's Hospital. The expression levels of miR-34a, Ki67 in NPC and adjacent tissues were detected by RT-qPCR. The association among the expression levels of miR-34a and Ki67, the clinicopathological features and prognosis of patients was analyzed. The relative expression levels of miR-34a in 56 cases of NPC were lower than those of the adjacent tissues. The expression of miR-34a in NPC was significantly associated with bone metastasis and TNM staging (P<0.001). The relative expression of Ki67 in 56 cases of NPC was higher than that of the adjacent tissues. The expression of Ki67 in NPC was significantly associated with lymphatic metastasis and TNM staging (P<0.001). The 5-year survival of patients with low expression of miR-34a was significantly lower than that of patients with high expression, and the survival of patients with high expression of Ki67 was significantly lower than that of patients with low expression. According to Pearson's correlation analysis, Ki67 expression was negatively correlated with miR-34a expression in NPC tissues. In conclusion, the expression of Ki67 in NPC was upregulated, while the expression of miR-34a in NPC was downregulated. miR-34a expression in NPC was significantly associated with bone metastasis and TNM staging, and Ki67 expression in NPC was significantly associated with lymphatic metastasis and TNM staging. In addition, there was a negative correlation between miR-34a and Ki67 expression levels, and the two can be used as predictors of NPC-associated mortality. The expression levels of miR-34a and Ki67, as well as TNM staging were associated with the prognosis of NPC patients.

The DNMT1/miR-34a Axis Is Involved in the Stemness of Human Osteosarcoma Cells and Derived Stem-Like Cells.

The DNA methyltransferase 1 (DNMT1)/miR-34a axis promoted carcinogenesis of various types of cancers. However, no literature reported its contribution to the stemness of osteosarcoma cancer stem-like cells (OSLCs). We sought to determine whether the DNMT1/miR-34a axis facilitates the stemness of OSLCs. We here revealed the higher DNMT1 activity and expression, lower miR-34a expression with high methylation of its promoter, and stronger stemness of OSLCs, as manifested by elevated sphere and colony formation capacities; CD133, CD44, ABCG2, Bmi1, Sox2, and Oct4 protein amounts in vitro; and carcinogenicity in a nude mouse xenograft model, when compared to the parental U2OS cells. 5-Azacytidine (Aza-dC) repressed DNMT1 activation and upregulated miR-34a expression by promoter demethylation and suppressed the stemness of OSLCs in a dose-dependent manner. DNMT1 knockdown increased miR-34a and reduced the stemness of OSLCs. Transfection with a miR-34a mimic repressed the stemness of OSLCs but did not alter DNMT1 activity and expression. Conversely, DNMT1 overexpression declined miR-34a levels, promoting the stemness of U2OS cells. Transfection with a miR-34a inhibitor enhanced the stemness of U2OS cells, without affecting the DNMT1 activity and expression. Importantly, reexpression of miR-34a could rescue the effects of DNMT1 overexpression on miR-34a inhibition as well as the stemness promotion without affecting the activity and expression of DNMT1. Our results revealed that aberrant activation of DNMT1 caused promoter methylation of miR-34a, leading to miR-34a underexpression, and the role of the DNMT1/miR-34a axis in promoting and sustaining the stemness of OSLCs.

MicroRNA‑34a inhibits cell growth and migration in human glioma cells via MMP‑9.

The present study was designed to investigate the function of matrix metalloproteinase-9 (MMP-9) in human glioma cells and the potential regulatory mechanisms. Reverse transcription-quantitative polymerase chain reaction was used to analyze the expression of MMP-9 and microRNA-34a (miR-34a) in the plasma of patients with glioma and healthy volunteers. MTT and Transwell assays were used to assess cell growth and migration, respectively. Annexin-V/propidium iodide staining was used to measure cell apoptosis. In addition, MMP-9 expression was measured using western blot analysis. In patients with glioma, MMP-9 expression was increased, while miR-34a expression was suppressed, compared with the normal group. Overall survival (OS) and disease-free survival (DFS) of patients with high MMP-9 expression were decreased compared with those with low MMP-9 expression. OS and DFS of patients with low miR-34a expression were decreased compared with those with high miR-34a expression. Downregulation of miR-34a promoted cell growth and migration, and inhibited apoptosis in U251-MG glioma cells. However, overexpression of miR-34a inhibited cell growth and migration, and induced apoptosis in glioma cells. Furthermore, downregulation of miR-34a using anti-miR-34a induced MMP-9 protein expression in glioma cells; whereas, overexpression of miR-34a suppressed MMP-9 protein expression in glioma cells. SB-3CT, an inhibitor of MMP-9, attenuated the effects of miR-34a mimic on glioma cells. Together, these results indicated that miR-34a inhibited cell growth and migration in human glioma cells by regulating MMP-9.