Long noncoding RNA NEAT1 decreases polycystic ovary syndrome progression via the modulation of the microRNA-324-3p and BRD3 axis.

Long noncoding RNA nuclear-enriched abundant transcript 1 (NEAT1) is believed to be involved in many gynecological and obstetrics disorders. Nevertheless, the role of NEAT1 in polycystic ovary syndrome (PCOS) is scarcely investigated. Our study aimed to investigate the role of NEAT1, microRNA (miR)-324-3p, and bromodomain containing 3 (BRD3) in PCOS. First, 80 women with PCOS and 80 healthy (non-PCOS) women were included, and their serum hormone levels were tested. Next, the PCOS mouse model was established by dehydroepiandrosterone injection, and then NEAT1, miR-324-3p, and BRD3 expression levels were detected in the PCOS mice. Lentivirus carrying short hairpin-NEAT1 or miR-324-3p agomir was injected into the PCOS mice to determine the change in biochemical indices and pathology. Moreover, a rescue experiment was conducted, after which, the binding relationships among NEAT1, miR-324-3p, and BRD3 were analyzed. NEAT1 and BRD3 were expressed at a high level while miR-324-3p was expressed at a low level in women with PCOS and PCOS mice. Reduced levels of NEAT1 or elevated levels of miR-324-3p mitigated metabolic disorders and alleviated ovarian pathological changes in PCOS mice. Mechanistically, NEAT1 sponged miR-324-3p and miR-324-3p targeted BRD3. In the rescue experiment, elevated miR-324-3p or reduced BRD3 level reversed the effects of the enhanced NEAT1 on metabolic disorders and ovarian pathological changes in PCOS mice. NEAT1 exacerbates metabolic disorders and ovarian pathological changes in PCOS mice by downregulating miR-324-3p and upregulating BRD3. This study gives a novel direction in PCOS treatment.

[Significance of microRNA 216a, 324-5p and 29a expression in peripheral blood in patients with acute pancreatitis and their correlation with liver injury].

Objective: To investigate the significance of microRNA (miR)-216a, miR-324-5p, miR-29a expression in peripheral blood in patients with acute pancreatitis (AP) and their correlation with liver injury. Methods: It was a case-control study design. To select 130 AP patients admitted from June 2017 to May 2019 in the First People's Hospital of Shangqiu, and the patients were divided into mild AP group (MAP group) and moderately severe AP group (SAP group) according to the disease severity, or 54 patients in the liver injury group (20 were MAP and 34 were SAP) and 76 in the non-liver injury group(all were MAP) according to liver injury. And another 40 healthy volunteers were selected as the healthy group. The expressions of miR-216a, miR-324-5p and miR-29a in peripheral blood of MAP group, SAP group, healthy group and liver injury group, non-liver injury group were compared, and the correlation between the miRNA levels and clinical indexes was analyzed. The predictive value of miRNA levels in peripheral blood for AP complicated with liver injury was analyzed by receiver operating characteristic (ROC) curve. Results: The levels of miR-216a and miR-29a in MAP group and SAP group were higher than those in healthy group, and the level of miR-324-5p was lower than that in healthy group (all P<0.01). The levels of miR-216a and miR-29a in SAP group were higher than those in MAP group, and the level of miR-324-5p was lower than that in healthy group (all P<0.01). Balthazar CT Score, acute physiology and chronic health evaluations (APACHE Ⅱ) score, C-reactive protein level, length of hospital stay were positively correlated with the levels of miR-216a and miR-29a in peripheral blood (all P<0.05), and negatively correlated with the levels of miR-324-5p (P<0.05). The levels of miR-216a and miR-29a in the peripheral blood in the liver injury group were higher than those in the non-liver injury group, and they were higher inSAP patients than those in MAP patients in the liver injury group (all P<0.05). The level of miR-324-5p in the peripheral blood in the liver injury group was lower than that in the non-liver injury group, and it was lower in SAP patients than that in MAP patientsin the liver injury group (all P<0.05). The area under ROC curve of miR-216a, miR-324-5p, and miR-29a in peripheral blood to predicate the AP complicated with liver damage was 0.694, 0.750 and 0.814, respectively. Conclusions: The levels of miR-216a and miR-29a increase in peripheral blood and the level of miR-324-5p decreases in patients with AP, and they are closely related to Balthazar CT score, APACHEⅡ score, C-reactive protein and length of hospital stay. The levels of miR-216a, miR-324-5p, miR-29a has certain predictive value for AP with liver injury, of which miR-29a has the highest predictive value.

Clinical Value Evaluation of microRNA-324-3p and Other Available Biomarkers in Patients With HBV Infection-Related Hepatocellular Carcinoma.

BACKGROUND: Patients with hepatitis B virus (HBV) infection are at high risk of hepatocellular carcinoma (HCC). This study aimed to evaluate the expression of microRNA-324-3p (miR-324-3p) in HBV-related HCC and explore the clinical significance of serum miR-324-3p and other available biomarkers in the diagnosis and prognosis of HBV-related HCC. METHODS: Expression of miR-324-3p in HBV infection-related cells and patients was estimated using quantitative real-time polymerase chain reaction (PCR). Receiver operating characteristic (ROC) curves were constructed to evaluate the diagnostic performance of serum miR-324-3p, alpha-fetoprotein (AFP), and protein induced by vitamin K absence/antagonist II (PIVKA-II) in the differentiation of HBV-related HCC from healthy controls and chronic hepatitis B patients (CHB). The relationship between serum miR-324-3p and patients' clinical features was assessed using the chi-square test, and the value of miR-324-3p to predict overall survival prognosis was evaluated using Kaplan-Meier methods and Cox regression assay in patients with HBV-related HCC. RESULTS: HBV-related HCC cells had significantly increased miR-324-3p compared with normal and HBV-unrelated HCC cells, and serum miR-324-3p in HCC patients with HBV infection was also higher than that in healthy controls and CHB. Serum miR-324-3p had relatively high diagnostic accuracy for the screening of HCC cases with HBV infection, and the combination of miR-324-3p, AFP, and PIVKA-II showed improved diagnostic performance. Additionally, high-serum miR-324-2p in HBV-related HCC patients was associated with cirrhosis, tumor size, clinical stage, and poor overall survival prognosis. CONCLUSIONS: High-serum miR-324-3p may be involved in the progression of HBV-related hepatitis to HCC and may serve as a candidate biomarker for the diagnosis and prognosis of HBV-related HCC.

High miR-324-5p expression predicts unfavorable prognosis of gastric cancer and facilitates tumor progression in tumor cells.

BACKGROUND: Gastric cancer (GCa) is one of the six major malignancies in the world with low survival rate. Although there are advances in therapeutic approaches, the prognosis of patients with GCa remains not optimistic. Therefore, this study aimed to evaluate the prognostic value of miR-324-5p, as well as its functional role in GCa progression. METHODS: The expression of miR-324-5p in tumor tissues and cell lines was examined using real-time quantitative PCR. The prognostic value of miR-324-5p in patients with GCa was evaluated by Kaplan-Meier survival curve and Cox regression analysis. Gain- and loss-of-function experiments were performed to evaluate the biological function of miR-324-5p during the progression of GCa, and a target gene of miR-324-5p was proposed. RESULTS: The expression of miR-324-5p was up-regulated in GCa tissues and cell lines. Patients with high expression of miR-324-5p had more cases with positive lymph node metastasis, advanced TNM stage, and worse overall survival compared with patients with low expression. The elevated miR-324-5p was an independent prognostic indicator of GCa. In addition, the inhibition of miR-324-5p could suppress GCa cell proliferation, migration and invasion and promote cell apoptosis, and PTEN was demonstrated to serve as a direct target of miR-324-5p in GCa progression. CONCLUSION: The present study indicates that miR-324-5p overexpression predicts poor prognosis in GCa patients, and the reduction of miR-324-5p can inhibit GCa biological processes. PTEN is a target gene of GCa, which may mediate the biological function of miR-324-5p in GCa progression.

Long non-coding RNA TPT1-AS1 alleviates cell injury and promotes the production of extracellular matrix by targeting the microRNA-324-5p/CDK16 axis in human dermal fibroblasts after thermal injury.

Long non-coding RNAs (lncRNAs) are associated with the healing of burn wounds in the dermis. The present study aimed to probe the role and regulatory network of the lncRNA TPT1 antisense RNA 1 (TPT1-AS1) in human dermal fibroblasts (HDFs) following thermal injury. A model of thermally injured cells was constructed with HDFs. The levels of TPT1-AS1, microRNA (miR)-324-5p and cyclin-dependent kinase (CDK)16 were determined through reverse transcription-quantitative PCR. Cell viability, cell cycle distribution, cell apoptosis rate and extracellular matrix (ECM) synthesis were assessed with a series of in vitro gain-of-function experiments and MTT, flow cytometry and western blot analyses. The binding ability of miR-324-5p and TPT1-AS1 (or the 3' untranslated region of CDK16) was identified via bioinformatics analysis and luciferase reporter assay. It was found that TPT1-AS1 and CDK16 were downregulated, but miR-324-5p was upregulated, in the HDFs after thermal injury. TPT1-AS1 elevation induced cell viability and ECM synthesis but attenuated cell cycle arrest at the G0/G1 stage and decreased the cell apoptosis rate of thermally injured HDFs. In addition, TPT1-AS1 sponged miR-324-5p to modulate CDK16 expression. Moreover, silencing CDK16 weakened the impacts of TPT1-AS1 upregulation on cell function and ECM synthesis in heat-treated HDFs. In summary, TPT1-AS1 relieved cell injury and induced ECM synthesis by sponging miR-324-5p and targeting CDK16 in the HDFs after thermal injury, implying a protective role for TPT1-AS1 in the burn wound healing process.

Silencing of miR-324-5p alleviates rat spinal cord injury by Sirt1.

MicroRNAs (miRNAs) are implicated in the pathogenesis of spinal cord injury (SCI) as primary regulators. Previous studies have reported that miR-324-5p is involved in the modulation of neural injury, while the underlying mechanisms of miR-324-5p in SCI remain unclear. In a SCI rat model, miR-324-5p was significantly upregulated in the spinal cord tissues after SCI. Downregulation of miR-324-5p via injection of adeno-associated viruses (AAV) expressing miR-324-5p inhibitor relieved animal motor deficits and pathological changes in the tissues. Furthermore, downregulation of miR-324-5p significantly altered the expression of genes regulating neural growth, apoptosis, and the inflammatory and antioxidant response, which are implicated in SCI pathogenesis. In a H2O2-induced cell injury model, miR-324-5p silencing rescued the elevated apoptosis of PC12 cells. Finally, miR-324-5p directly targeted the 3'-untranslated region of NAD-dependent protein deacetylase sirtuin-1 (Sirt1) and negatively regulated the levels of Sirt1, an anti-inflammatory protein involved in SCI. Silencing of Sirt1 aggravated SCI and rescued the effects of miR-324-5p downregulation in rats. Overall, our findings indicated that silencing of miR-324-5p alleviates the loss of animal locomotion and concurrently mediates several degenerative processes relevant to the pathogenesis of SCI by Sirt1, which may provide clues for SCI treatment.

Long Non-coding RNA ENST00000453774.1 Confers an Inhibitory Effect on Renal Fibrosis by Inhibiting miR-324-3p to Promote NRG1 Expression.

Progressive or chronic renal diseases arise from a process of destructive renal fibrosis. Therefore, the molecular basis of renal fibrosis has attracted increasing attention. In this investigation, we set out to elucidate the potential interaction among long non-coding RNA ENST00000453774.1 (lncRNA 74.1), microRNA-324-3p (miR-324-3p), and NRG1, and to investigate their roles in the context of cellular autophagy and renal fibrosis. We collected 30 renal fibrosis tissue samples for analysis. In other studies, HK-2 cells were stimulated with TGF-ß1 to induce a cell model of renal fibrosis, followed by alteration on the expression of lncRNA 74.1, miR-324-3p, or NRG1, or by the addition of AKT activator SC79 in the HK-2 cells. The expression levels of lncRNA 74.1, miR-324-3p, NRG1, autophagy-related proteins (ATG5, ATG7, LC3II/I, and P62), and the corresponding fibrosis markers (Collagen I, Fibronectin, and α-SMA) were subsequently determined using various assay methods. In addition, the proportion of LC3 positive cells and number of autophagosomes were recorded. Results revealed that lncRNA 74.1 and NRG1 were poorly expressed and miR-324-3p was highly expressed in renal fibrosis tissues and modeled cells. LncRNA 74.1 could bind to miR-324-3p, which led to upregulated NRG1 expression and inhibition of the PI3K/AKT signaling pathway. Meanwhile, overexpression of lncRNA 74.1 or down-regulation of miR-324-3p increased the levels of ATG5, ATG7, LC3II, and LC3I, and decreased levels of P62, Collagen I, Fibronectin, and α-SMA, accompanied by elevated proportions of LC3 positive cells and autophagosomes. Findings concur in showing that lncRNA 74.1 could induce cellular autophagy and alleviate renal fibrosis by regulating the miR-324-3p-mediated NRG1/PI3K/AKT axis. This axis may thus present a potential molecular target in renal fibrosis treatment.

Long non­coding RNA NEAT1 regulates glioma cell proliferation and apoptosis by competitively binding to microRNA­324­5p and upregulating KCTD20 expression.

Previous studies have demonstrated that long non­coding RNAs (lncRNAs) serve a key role in the development and progression of several types of cancer, including glioma. The lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) contributes to cancer growth through its effects on cell proliferation, migration, invasion and drug resistance. However, the exact regulatory mechanisms via which NEAT1 acts in glioma are unclear. In the present study, the expression levels and function of NEAT1 in glioma tissues and cell lines were examined in vitro and in vivo. By reverse transcription­quantitative PCR and fluorescence in situ hybridization analysis, NEAT1 expression was upregulated in glioma tissues compared with in adjacent normal brain tissues, and elevated NEAT1 levels were associated with poor prognosis. Cell Counting Kit­8, colony formation, ethynyldeoxyuridine, flow cytometry and western blotting assays were performed to detect the effects of NEAT1 on cell biological behavior. Knockdown of NEAT1 in glioma cell lines was associated with cell cycle arrest at the G0/G1 phase, decreased proliferation and elevated apoptosis in vitro, and resulted in reduced tumor growth and increased survival in a mouse xenograft model of glioma. Using bioinformatics analysis, RNA immunoprecipitation experiments and luciferase reporter assays, it was demonstrated that NEAT1 may competitively bind to microRNA (miR)­324­5p, thus blocking its interaction with target mRNAs. Potassium channel tetramerization protein domain containing 20 (KCTD20) was identified as a specific miR­324­5p target. Accordingly, the inhibition of NEAT1 resulted in the downregulation of KCTD20 through competitive binding with miR­324­5p, decreased cell proliferation and increased apoptosis. Concomitant NEAT1 knockdown and inhibition of miR­324­5p partially reversed the effects of NEAT1 knockdown on cell proliferation and apoptosis, and further regulated KCTD20 expression. Collectively, the present findings demonstrated that NEAT1 acted as a competing endogenous RNA for miR­324­5p, and identified the NEAT1/miR­324­5p/KCTD20 axis as a novel regulatory axis and a potential therapeutic target for human glioma.

Comprehensive identification of microRNA arm selection preference in lung cancer: miR-324-5p and -3p serve oncogenic functions in lung cancer.

MicroRNA (miRNA/miR) dysfunction is a hallmark of lung cancer, and results in the dysregulation of tumor suppressors and oncogenes during lung cancer progression. Selection of the 5p and 3p arms of miRNA is a mechanism that improves the modulation of miRNA biological functions and complicates the regulatory network in human types of cancer. However, the involvement of arm selection preference of miRNA in lung cancer remains unclear. In the present study, changes in miRNA arm selection preference were comprehensively identified in lung cancer and corresponding adjacent normal tissues by analyzing The Cancer Genome Atlas. Arm selection was revealed to be consistent in the majority of miRNAs in lung cancer. Only a few miRNAs had significantly altered arm selection preference in lung cancer. Among these, the biological functions of the individual arms of miR-324 were investigated further. The data revealed that miR-324-5p and -3p were significantly overexpressed in lung cancer cells. Ectopic expression of miR-324-5p significantly promoted cell proliferation and invasion in lung cancer cells, while miR-324-3p overexpression significantly increased cell proliferation but did not alter the invasion of lung cancer cells. In conclusion, the arm selection preference of miRNA may be an additional mechanism through which biological functions are modulated. The results of the present study provide a novel insight into the underlying mechanisms of lung cancer and may direct research into future therapies.

MicroRNA-324-5p suppresses the migration and invasion of MM cells by inhibiting the SCFß-TrCP E3 ligase.

Multiple myeloma (MM) is a cytogenetically heterogeneous malignancy of plasma cells in bone marrow. Among the cytogenetic abnormalities of MM, del(17p) is a well-recognized high-risk genetic lesion associated with the late stage and progression of the disease. MicroRNA (miR)-324-5p, located at 17p13.1, was identified to be involved in the dysregulation of a number of types of malignant disease. However, whether miR-324-5p is associated with the development and progression of MM remains unknown. In the present study, the expression status of miR-324-5p in MM, and its effect on the migratory and invasive ability of MM cells were investigated. Using ubiquitination pathway polymerase chain reaction array, the inhibitory effect of miR-324-5p on the ubiquitinated proteins was investigated. It was identified that miR-324-5p levels were decreased in samples from patients with MM and MM cell lines. Increased expression of miR-324-5p by transfection of miR-324-5p mimic suppressed the proliferative, migratory and invasive abilities of MM.1R cells. Furthermore, increased expression of miR-324-5p in MM.1R cells inhibited the ubiquitination pathway and decreased the levels of ubiquitination-associated proteins, particularly the Skp1-Cullin1-F-box ß-transducin repeat-containing protein (SCFß-TrCP) E3 ligase. In addition, the results of the present study demonstrated that the SCFß-TrCP E3 ligase may contribute to the suppression of MM cell motility by inhibiting the expression of metastasis-associated genes, including metastasis suppressor 1. In conclusion, the results of the present study suggested that miR-324-5p may act as a tumor suppressor by impairing the motility of MM cells by suppressing the ubiquitination pathway.