MiR-339-5p inhibits replication of porcine reproductive and respiratory syndrome virus by targeting viral gene regions.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a variable virus, whose spread cannot be totally stopped by vaccination. PRRSV infection results in abortion and respiratory symptoms in pregnant pigs. One crucial component of the anti-viral infection strategy is microRNA (miRNA), a class of multifunctional small molecules. It is unknown whether miR-339-5p can specifically target the PRRSV gene and prevent the virus from replicating, despite the fact that miR-339-5p is markedly up-regulated during the PRRSV infection. In this pursuit, the present study revealed that the two PRRSV areas targeted by miR-339-5p were PRRSV nsp2-3378 to 3403 and PRRSV nsp2-3112 to 3133 using the miRanda program. Dual luciferase reporter assays showed that the miR-339-5p target region of the PRRSV gene sequence exhibited 100% homology and was highly conserved. Furthermore, the ability of miR-339-5p to target PRRSV gene areas was verified. It was found that the overexpression of miR-339-5p markedly reduced the PRRSV replication through PRRSV infection trials. The precursor sequence of ssc-miR-339-5p was amplified using the DNA of pig lung tissue as a template in order to create a fragment of 402 bp of porcine-derived miR-339-5p precursor sequence, which was then used to produce the eukaryotic expression plasmid of miR-339-5p. In conclusion, miR-339-5p can target the specific PRRSV gene areas and prevent PRRSV replication, offering fresh perspectives for the creation of medications that combat the PRRSV infection.

Nuclear lncRNA NORSF reduces E2 release in granulosa cells by sponging the endogenous small activating RNA miR-339.

BACKGROUND: Functioning as a competing endogenous RNA (ceRNA) is the main action mechanism of most cytoplasmic lncRNAs. However, it is not known whether this mechanism of action also exists in the nucleus. RESULTS: We identified four nuclear lncRNAs that are presented in granulosa cells (GCs) and were differentially expressed during sow follicular atresia. Notably, similar to cytoplasmic lncRNAs, these nuclear lncRNAs also sponge miRNAs in the nucleus of GCs through direct interactions. Furthermore, NORSF (non-coding RNA involved in sow fertility), one of the nuclear lncRNA acts as a ceRNA of miR-339. Thereby, it relieves the regulatory effect of miR-339 on CYP19A1 encoding P450arom, a rate-limiting enzyme for E2 synthesis in GCs. Interestingly, miR-339 acts as a saRNA that activates CYP19A1 transcription and enhances E2 release by GCs through altering histone modifications in the promoter by directly binding to the CYP19A1 promoter. Functionally, NORSF inhibited E2 release by GCs via the miR-339 and CYP19A1 axis. CONCLUSIONS: Our findings highlight an unappreciated mechanism of nuclear lncRNAs and show it acts as a ceRNA, which may be a common lncRNA function in the cytoplasm and nucleus. We also identified a potential endogenous saRNA for improving female fertility and treating female infertility.

miR-339-3p promotes AT1-AA-induced vascular inflammation by upregulating NFATc3 protein expression in vascular smooth muscle cells.

Vascular inflammation induced by angiotensin II-1 receptor autoantibody (AT1-AA) is involved in the occurrence and development of various cardiovascular diseases. miR-339-3p is closely related to the degree of vasodilation of aortic aneurysm and is also involved in the occurrence and development of acute pancreatitis. However, it is still unclear whether miR-339-3p influences AT1-AA-induced vascular inflammation. In this study, the role and mechanism of miR-339-3p in AT1-AA-induced vascular inflammation are studied. RT-PCR detection shows that the miR-339-3p levels in the thoracic aorta and serum exosomes of AT1-AA-positive rats are significantly increased. The miRwalk database predicts the mRNAs that miR-339-3p can bind to their 5'UTR. Subsequently, it is found that the number of genes contained in the T cell receptor pathway is high through KEGG analysis, and NFATc3 among them can promote the secretion of various inflammatory cytokines. AT1-AA-induced upregulation of miR-339-3p expression in vascular smooth muscle cells (VSMCs) can lead to a significant increase in NFATc3 protein level and promote vascular inflammation. Inhibition of miR-339-3p with antagomir-339-3p can significantly reverse AT1-AA-induced high expressions of IL-6, IL-1ß and TNF-α proteins in rat thoracic aorta and VSMCs. That is, AT1-AA can upregulate the expression of miR-339-3p in VSMCs, and the increased miR-339-3p targets the 5'UTR of NFATc3 mRNA to increase the protein level of NFATc3, thereby aggravating the occurrence of vascular inflammation. These findings provide new experimental evidence for the involvement of miRNAs in regulating vascular inflammatory diseases.

Circ_0001821 Potentiates Cell Growth, Metastasis, and Stemness in Colorectal Cancer by Regulating miR-339-3p/CST1.

Dysregulation of circRNAs is associated with human cancer progression, but its expression pattern and function in cancer are not fully understood. The aim of our study is to investigate the effects of circ_0001821 on colorectal cancer (CRC) proliferation, migration, invasion, and stemness. The expression level of circ_0001821 in CRC tissues and cells was detected by quantitative real-time PCR. The effects of circ_0001821 silencing on the proliferation, migration, invasion, and stemness of CRC cells were examined by cell counting kit-8, 5-Ethynyl-2'-deoxyuridine, transwell, sphere formation, and western blot assays. Bioinformatics and dual-luciferase reporter gene assay were used to verify the relationship between circ_0001821 and miR-339-3p, miR-339-3p and CST1 in CRC cells. Circ_0001821 was highly expressed in CRC tissues and cells. Circ_0001821 silencing inhibited the proliferation, migration, invasion, and stemness of CRC cells, and transfection of miR-339-3p inhibitor partly attenuated this effect. In addition, circ_0001821 can bind miR-339-3p to regulate CST1 expression. Circ_0001821 silencing also curbed tumor growth in vivo. Circ_0001821 promoted CRC cell proliferation, migration, invasion, and stemness by regulating the miR-339-3p/CST1 axis.

KCNQ1OT1 promotes retinoblastoma progression by targeting miR-339-3p that suppresses KIF23.

BACKGROUND: Long noncoding RNAs (lncRNAs) are involved in tumor formation and development. KCNQ1OT1 regulates the malignant proliferation of retinoblastoma (RB), but the specific mechanism remains to be further investigated. METHODS: The KCNQ1OT1, miR-339-3p and KIF23 expression levels in RB were detected by qRT-PCR and western blotting. The cell viability, proliferation, migration ability and caspase-3 activity of RB cells were evaluated by CCK-8, BrdU, transwell and caspase-3 activity analysis. Western blot was used to detect the Bax and Bcl-2 protein expression in RB cells. The binding relationship between KCNQ1OT1, miR-339-3p and KIF23 was detected by luciferase, RIP and RNA pull-down assay. RESULTS: KCNQ1OT1 and KIF23 were up-regulated frequently in RB, and miR-339-3p was down-regulated. Functional studies showed that downregulation of KCNQ1OT1 or KIF23 inhibited the survival and migration of RB cells, and facilitated apoptosis. Interference with miR-339-3p showed the opposite effect. Mechanisms suggested that KCNQ1OT1 exited its oncogenic activity by positively regulating the expression of KIF23 and sponging miR-339-3p. CONCLUSION: KCNQ1OT1/miR-339-3p/KIF23 may be a new biomarker for the diagnosis and treatment of RB.

MiRNA-339-5p promotes isoproterenol-induced cardiomyocyte hypertrophy by targeting VCP to activate the mTOR signaling.

MicroRNAs (miRNAs) regulate multiple biological processes and participate in various cardiovascular diseases. This study aims to investigate the role of miR-339-5p in cardiomyocyte hypertrophy and the involved mechanism. Neonatal rat cardiomyocytes (NRCMs) were cultured and stimulated with isoproterenol (ISO). The hypertrophic responses were monitored by measuring the cell surface area and expression of hypertrophic markers including ß-myosin heavy chain (ß-MHC) and atrial natriuretic factor (ANF). Bioinformatic prediction tools and dual-luciferase reporter assay were performed to identify the target gene of miR-339-5p. Quantitative real-time polymerase chain reaction and western blot analysis were used to determine the levels of miR-339-5p and its downstream effectors. Our data showed that miR-339-5p was upregulated during cardiomyocyte hypertrophy triggered by ISO. MiR-339-5p overexpression resulted in enlargement of cell size and increased the levels of hypertrophic markers. In contrast, inhibition of miR-339-5p significantly attenuated ISO-induced hypertrophic responses of NRCMs. Valosin-containing protein (VCP), a suppressor of cardiac hypertrophy via inhibiting mechanistic target of rapamycin (mTOR) signaling, was validated as a target of miR-339-5p. MiR-339-5p suppressed VCP protein expression, leading to elevated phosphorylation of mTOR and ribosomal protein S6 kinase (S6K). VCP depletion activated the mTOR/S6K cascade and could compromise the anti-hypertrophic effects of miR-339-5p inhibitor. Additionally, the hypertrophic responses caused by miR-339-5p was alleviated in the presence of mTOR inhibitor rapamycin. In conclusion, our research revealed that miR-339-5p promoted ISO-induced cardiomyocyte hypertrophy by targeting VCP to activate the mTOR signaling, suggesting a promising therapeutic intervention by interfering miR-339-5p.

miR-339-3p inhibits cell growth and epithelial-mesenchymal transition in nasopharyngeal carcinoma by modulating the KAT6A/TRIM24 axis.

OBJECTIVE: To explore the effect and mechanism of the miR-339-3p/KAT6A/TRIM24 axis in nasopharyngeal carcinoma (NPC) cell growth and epithelial-mesenchymal transition (EMT) progression. METHODS: CNE2 and 5-8F NPC cell lines were transfected with miR-339-3p-mimic or sh-KAT6A alone or co-transfected with miR-339-3p-mimic and oe-KAT6A. The expression levels of miR-339-3p, KAT6A, TRIM24, and EMT-related proteins were assessed, in addition to cell biological behaviors. Then, the relationship between miR-339-3p and KAT6A was predicted and validated. The correlations between miR-339-3p and KAT6A or between KAT6A and TRIM24 were analyzed by Pearson coefficient and the enrichment of H3K23ac in TRIM24 promoter region was measured by chromatin immunoprecipitation. RESULTS: miR-339-3p was downregulated, but KAT6A and TRIM24 were highly expressed in NPC cells and tissues. Upregulated miR-339-3p or downregulated KAT6A could inhibit the growth and EMT of NPC cells. Further experiments showed that miR-339-3p regulated NPC cell growth and EMT by mediating KAT6A in a targeted fashion. KAT6A was positively correlated with TRIM24, and the enrichment of H3K23ac was much higher in NPC tissues. miR-339-3p suppressed the growth and EMT of NPC cells by the KAT6A/TRIM24 axis. In a xenograft study, miR-339-3p overexpression inhibited NPC tumor growth in vivo. CONCLUSION: Conclusively, miR-339-3p inhibited the growth and EMT of NPC cells via the KAT6A/TRIM24 axis.

Silencing LINC00665 inhibits cutaneous melanoma in vitro progression and induces apoptosis via the miR-339-3p/TUBB.

BACKGROUND: LncRNAs are closely related to cutaneous melanoma (CM) tumorigenesis and metastasis, and it can affect the progression of CM by regulating cell proliferation, migration, invasion, apoptosis, and other cellular mechanisms. This study investigated the role of LINC00665 in CM. METHODS: Expressions of LINC00665, miR-339-3p, and tubulin beta chain (TUBB) in CM cells were analyzed by qRT-PCR and/or Western blot. The LINC00665/miR-339-3p/TUBB targeting network was predicted by bioinformatics tools, screened out by Venn diagrams and analyzed by Pearson's correlation coefficients, followed by validation via dual-luciferase reporter assay and/or pull-down assay. Transfection of siLINC00665 or miR-339-3p inhibitor/mimic was conducted with CM cells whose viability, proliferation, migration, invasion, cell cycle progression, and apoptosis were measured by CCK-8 assay, colony formation assay, wound healing assay, Transwell assay, and flow cytometry. The associations of TUBB with tumor biological characteristics and other proteins were analyzed by CanserSEA and String, respectively. RESULTS: High-expressed LINC00665 was detected in CM cells. Silencing LINC00665 decreased CM cell viability; inhibited colony formation, cell cycle progression, migration and invasion; enhanced apoptosis; and upregulated miR-339-3p. LINC00665 targeted miR-339-3p which targeted TUBB. MiR-339-3p upregulation induced effects similar to the LINC00665-silencing-induced effects and could downregulate TUBB, which was associated with malignant behaviors and related to other five proteins. MiR-339-3p downregulation induced the opposite effects of what miR-339-3p upregulation induced, and the miR-339-3p downregulation-induced effects could be reversed by LINC00665 silencing. CONCLUSION: Silencing LINC00665 inhibits in vitro CM progression and induces apoptosis via the miR-339-3p/TUBB axis.

CircDOCK1 promotes the tumorigenesis and cisplatin resistance of osteogenic sarcoma via the miR-339-3p/IGF1R axis.

BACKGROUND: Circular RNAs (circRNAs), a class of noncoding RNAs (ncRNAs), may modulate gene expression by binding to miRNAs. Additionally, recent studies show that circRNAs participate in some pathological processes. However, there is a large gap in the knowledge about circDOCK1 expression and its biological functions in osteogenic sarcoma (OS). METHODS: Differentially expressed circRNAs in OS cell lines and tissues were identified by circRNA microarray analysis and quantitative real-time PCR (qRT-PCR). To explore the actions of circDOCK1 in vivo and in vitro, circDOCK1 was knocked down or overexpressed. To assess the binding and regulatory associations among miR-339-3p, circDOCK1 and IGF1R, we performed rescue experiments, RNA immunoprecipitation (RIP), RNA pulldown assays and dual-luciferase assays. Moreover, we performed apoptosis assays to reveal the regulatory effects of the circDOCK1/miR-339-3p/IGF1R axis on cisplatin sensitivity. RESULTS: CircDOCK1 expression remained stable in the cytoplasm and was higher in OS tissues and cells than in the corresponding controls. Overexpression of circDOCK1 increased oncogenicity in vivo and malignant transformation in vitro. In the U2OS and MG63 cell lines, circDOCK1 modulated tumor progression by regulating IGF1R through sponging of miR-339-3p. Additionally, in the U2OS/DDP and MG63/DDP cell lines, cisplatin sensitivity was regulated by circDOCK1 via the miR-339-3p/IGF1R axis. CONCLUSIONS: CircDOCK1 can promote progression and regulate cisplatin sensitivity in OS via the miR-339-3p/IGF1R axis. Thus, the circDOCK1/miR-339-3p/IGF1R axis may be a key mechanism and therapeutic target in OS.

MiR-339 and galectin-3: diagnostic value in patients with airway obstruction after lung transplantation.

Respiratory complications can be the cause of graft dysfunction after lung transplantation (LTx). MicroRNAs are small regulatory molecules-potential biomarkers of respiratory diseases and post-transplant complications. Galectin-3 is highly expressed in fibrosis of transplanted solid organs. The aim was to evaluate the expression of plasma miR-339 and galectin-3 concentrations in lung recipients including with airway obstruction after LTx. The study included 57 lung recipients (34 men and 23 women aged 10 to 74 years) were followed up to 5 years after LTx. The plasma microRNAs were detected by real-time PCR; galectin-3 levels were measured by ELISA. During follow-up in 30 recipients, post-transplant complications were detected: 12 (40.0%) cases of airway obstruction. The levels of miR-339 and galectin-3 were significantly higher in recipients with airway obstruction compare with 27 (47.3%) recipients without any complications (P = 0.036 and P = 0.014, resp.). Increasing miR-339 (above the 0.02 fold change) and galectin-3 (above the 11.7 ng/ml) threshold plasma levels in lung recipients is associated with high risk (RR = 7.14 ± 0.97 [95% CI 1.05-48.60], P = 0.045) of airway obstruction after LTx. A measurement of miR-339 expression in combination with galectin-3 level might be perspective to identify recipients at risk of airway obstruction after LTx.

[Hsa_circ_0005221 promotes prostate cancer progression through the miR-339-5p/STAT5a pathway].

OBJECTIVE: To investigate the molecular mechanism of hsa_circ_0005221 regulating the progression of PCa through the miR-339-5p/STAT5a pathway. METHODS: Localizations of hsa_circ_0005221 and miR-339-5p in cells were detected by nuclear-cytoplasmic isolation. MiRNA-339-5p was selected as the target miRNA bound to hsa_circ_0005221 by RNA pull-down assay. The binding site of the luciferase reporter gene was predicted by software and the binding capability of miR-339-5p validated by luciferase assay. The expression of hsa_circ_0005221 in the prostatic epithelial and PCa cells was determined by qPCR. The hsa_circ_0005221-overexpressed plasmid and siRNA were transfected into the PCa cells for measurement of their proliferation, invasion and migration abilities and the levels of epithelial-mesenchymal transformation (EMT) and apoptosis. After knockdown of hsa_circ_0005221 and transfection of miR-339-5p mimics and miR-339-5p inhibitor, the proliferation, invasion and migration abilities of the DU145 and LNCaP cells were detected, and so were the levels of the EMT signature protein, STAT5a and cell apoptosis. RESULTS: The expression of hsa_circ_0005221 was significantly higher in the PCa than in the prostatic epithelial cells. Nuclear-cytoplasmic isolation experiments showed that hsa_circ_0005221 and miR-339-5p were mainly located in the cytoplasm. The proliferation, invasion and migration abilities and EMT were decreased and the apoptosis increased in the DU145 and LNCaP cells with knockdown of hsa_circ_0005221, which was just the reverse in those with overexpressed hsa_circ_0005221. Among the top 5 miRNAs predicted by software, miR-339-5p, miR-17 and miR-520h were shown by pull-down assay to be bound to hsa_circ_0005221, with most obvious changes in miR-339-5p when hsa_circ_0005221 knocked down or overexpressed. Luciferase reporter gene assay showed the binding of hsa_circ_0005221 to miR-339-5p. Knockdown of hsa_circ_0005221 and transfection of miR-339-5p mimics into the DU145 and LNCaP cells significantly reduced the proliferation, invasion and migration abilities of the cells and the N-cad level, increased their apoptosis and E-cad level, and up-regulated the expression of STAT5a, while overexpression of hsa_circ_0005221 and transfection of miR-339-5p mimics induced just the opposite effects. CONCLUSIONS: Hsa_circ_0005221 enhances the progression of prostate cancer through the miR-339-5p/STAT5a pathway.

NEAT1 induces osteosarcoma development by modulating the miR-339-5p/TGF-ß1 pathway.

The long noncoding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) has been recognized as a tumor oncogene involved in the development of multiple cancers. However, the function of NEAT1 and its molecular mechanism in osteosarcoma (OS) remain unclear. First, we detected the NEAT1 expression in OS cell lines by performing quantitative reverse-transcription polymerase chain reaction. Next, the effects of NEAT1 on OS cell growth, apoptosis, migration, and invasion were tested by lentivirus-mediated downregulation. We observed that inhibition of NEAT1 restrained OS cell progression greatly. Interestingly, in the last few years, increasing studies have shown that some lncRNAs can act as miRNA sponges and reduce the amount of the same. Here, we found that NEAT1 can modulate OS development via sponging miR-339-5p. MiR-339-5p was significantly decreased in OS cells, and its overexpression can remarkably repress the OS proliferation. These results indicated that NEAT1 could function as a tumor oncogene in OS by inhibiting miR-339-5p in vitro. Then, the following assays validated that transforming growth factor ß1 (TGF-ß1) can act as a functional target of miR-339-5p in OS cells. Finally, we indicated that NEAT1 could mediate TGF-ß1 expression by competitively sponging miR-339-5p. NEAT1 induced OS cell proliferation and cell mobility by binding to miR-339-5p and increasing TGF-ß1 in OS. It was demonstrated in our study that lncRNA NEAT1 could impede miR-339-5p expression to maintain the expression of TGF-ß1, which led to the development of OS. Our findings implied that the novel identified NEAT1/miR-339-5p/TGF-ß1 axis might be a new molecular pathway or therapeutic target for OS diagnosis and treatment.

LncRNA MAFG-AS1 facilitates the migration and invasion of NSCLC cell via sponging miR-339-5p from MMP15.

Non-small-cell carcinoma (NSCLC) is the most common cancer along with high mortality rate worldwide. In the present study, our data showed that lncRNA MAF BZIP Transcription Factor G Antisense RNA 1 (MAFG-AS1) was over-expressed in NSCLC tissues and cell lines. Overexpression of MAFG-AS1 promoted the migration, invasion and enhanced epithelial-mesenchymal transition (EMT) of NSCLC cell. In addition, miR-339-5p was predicted to be a target of MAFG-AS1 and the level of miR-339-5p was down-regulated in NSCLC. Over-expression of MAFG-AS1 significantly decreased the level of miR-339-5p in NSCLC cell. Moreover, the matrix metalloproteinase 15 (MMP15) was identified to be a target of miR-339-5p. The level of MMP15 was negatively regulated by miR-339-5p whereas positively controlled by MAFG-AS1. In addition, up-regulation of miR-339-5p neutralized the promoting impact of MAFG-AS1 on the migration, invasion and EMT of NSCLC cell. Finally, the xenograft model suggested that MAFG-AS1 promoted the metastasis of NSCLC cell in vivo. Altogether, we proved that MAFG-AS1-miR-339-5p-MMP15 axis might be a promising therapeutic target for the treatment of patients with NSCLC.

miR-339-5p downregulation contributes to Taxol resistance in small-cell lung cancer by targeting α1,2-fucosyltransferase 1.

Lung cancer is a leading cause of cancer-related mortality, and non-small-cell lung carcinoma is responsible for almost 80% of lung cancer-related deaths. In recent years, lung cancer has shown increasing incidence but poor prognosis, and many studies have demonstrated that microRNAs play crucial roles in the development of lung carcinoma and chemoresistance. This study investigated the role of miR-339-5p involvement in lung carcinoma cell lines and chemoresistance to Taxol. We observed that miR-339-5p was significantly downregulated in Taxol-A549 cells compared with A549 cells. In vitro studies further indicated that miR-339-5p could promote colony formation and attenuate apoptosis of lung carcinoma cell lines through targeting α1,2-fucosyltransferase 1 and regulation of the downstream protein Lewis y. Furthermore, miR-339-5p was found to enhance the proliferation inhibition ability of Taxol in lung carcinoma cell lines as well as in the Taxol-A549 subclone. An in vivo study indicated that both miR-339-5p and Taxol could attenuate the growth of lung carcinoma; moreover, miR-339-5p could synergistically promote this inhibitory function of Taxol. In summary, our results suggest a miR-339-5p molecular network that is involved in controlling lung carcinoma progression. © 2017 The Authors IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 69(11):841-849, 2017.

miR-339-5p inhibits migration and invasion in ovarian cancer cell lines by targeting NACC1 and BCL6.

This study aimed to explore the role of miR-339-5p in ovarian cancer. The expression of miR-339-5p in seven ovarian cancer cell lines (Hey, SKOV3, OVCAR5, SKOV3-IP, A2780, CAOV3, and OVCA433) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The miR-339-5p mimic and inhibitor were used to regulate its expression. Migration, invasion, and proliferation were examined. A bioinformatics analysis was used to predict targets, and a dual-luciferase reporter system was applied for validation, along with Western blot verification. Additionally, the association of miR-339-5p and its target genes with ovarian cancer was analyzed based on The Cancer Genome Atlas (TCGA) database. OVCAR5 and SKOV3 had the highest and lowest miR-339-5p expression, respectively. Inhibition of miR-339-5p expression increased the migration and invasion of OVCAR5 cells, while in SKOV3 cells, upregulated miR-339-5p attenuated the migration and invasion ability. Modulation of miR-339-5p had no effect on proliferation. The genes nucleus accumbens associated 1(BEN and BTB (POZ) domain containing) (NACC1) and B cell lymphoma-6 (bcl6) were validated to be targets of miR-339-5p. Clinically, patients with a high expression of NACC1 had a high risk in the survival analysis. miR-339-5p inhibits migration and invasion in ovarian cancer by targeting NACC1 and BCL6. miR-339-5p may be a biomarker of metastasis in ovarian cancer; NACC1 had a predictive value for ovarian cancer progression.

miR-339-5p inhibits alcohol-induced brain inflammation through regulating NF-κB pathway.

Alcohol-induced neuroinflammation is mediated by the innate immunesystem. Pro-inflammatory responses to alcohol are modulated by miRNAs. The miRNA miR-339-5p has previously been found to be upregulated in alcohol-induced neuroinflammation. However, little has been elucidated on the regulatory functions of this miRNA in alcohol-induced neuroinflammation. We investigated the function of miR-339-5p in alcohol exposed brain tissue and isolated microglial cells using ex vivo and in vitro techniques. Our results show that alcohol induces transcription of miR 339-5p, IL-6, IL-1ß and TNF-α in mouse brain tissue and isolated microglial cells by activating NF-κB. Alcohol activation of NF-κB allows for nuclear translocation of the NF-κB subunit p65 and expression of pro-inflammatory mediators. miR-339-5p inhibited expression of these pro-inflammatory factors through the NF-κB pathway by abolishing IKK-ß and IKK-ε activity.

Adenosine as a probing tool for the mechanistic study of acupuncture treatment.

To date, acupuncture has been widely used despite a lack of solid clinical evidence in the East and West. However, there are few validated in vitro models for the mechanistic studies of acupuncture. We hypothesized that adenosine could be used as a probing tool in the mechanistic studies of acupuncture because of its critical role in the action of acupuncture. Subsequently, we tested this hypothesis using both in vitro and in vivo experiments. First, we found that adenosine stimulation mimicked the effect of acupuncture on microRNA profiling (including miR-339, miR-145 and miR-451) and protein level (including Sirt2) in nerve growth factor-induced differentiated PC12 cells. These miRNA and proteins have been found to be regulated by acupuncture treatment in the brain of spontaneously hypertensive rats. Next, we found that adenosine stimulation downregulated miR-339 expression through adenosine A1 receptor-mediated pathway. Finally, we showed that the concentration of adenosine was actually decreased in the brain of spontaneously hypertensive rats after acupuncture treatment at Taichong acupoint. Taken together, these findings suggest that adenosine could be used as a useful probing tool for acupuncture mechanistic studies, while more validation studies are certainly warranted.

MiR-339-5p Inhibits Ferroptosis by Promoting Autophagic Degradation of FTH1 Through Targeting ATG7 in Liver Cancer Cells.

Background: Liver cancer has a high incidence and mortality rate worldwide, and there is an urgent need to identify new therapeutic strategies and predictive targets to improve the clinical outcomes of advanced liver cancer. Ferroptosis holds promise as a novel strategy for cancer therapy. Epigenetic dysregulation is a hallmark of cancer, and noncoding RNAs are tightly involved in cell fate determination. Therefore, we aimed to identify a novel ferroptosis regulator from aberrantly expressed microRNAs that may serve as a novel biomarker and therapeutic target for liver cancer. Methods: The expression signature and prognostic value of miR-339 was assessed using TCGA data set. The role of miR-339/ATG7/FTH1 axis in liver cancer cells were evaluated through growth curve, colony formation, 7-AAD staining. The role of miR-339 in regulation of ferroptosis was determined by immunofluorescence staining, flow cytometry, and Elisa kits. Results: Here, we showed that miR-339 is aberrantly overexpressed in patients with liver cancer. In addition, miR-339 inhibition dramatically suppresses liver cancer progression. Furthermore, miR-339 silencing drives cell death and inhibits liver cancer progression, indicating that miR-339 may serve as a novel ferroptosis suppressor. Mechanistically, we demonstrated that miR-339 targets ATG7 to facilitate the autophagic degradation of FTH1 and prevent ferroptosis in liver cancer cells. Conclusions: We provide important evidence that the miR-339 inhibition activates of the autophagy pathway to promote ferroptosis by degrading FTH1 in liver cancer cells. We found that miR-339 regulates the balance between ferroptosis and autophagy in liver cancer cells.

Implications for miR-339-5p regulation of trophoblast proliferation and migration in placentas associated with porcine intrauterine growth retardation using integrated transcriptome sequencing analysis.

Placental dysfunction is considered as one of the main etiologies of fetal intrauterine growth retardation (IUGR). MicroRNAs (miRNAs) have been demonstrated to be a vital epigenetic modification involved in regulating the placental function and pregnancy outcomes in mammals. However, the mechanisms underlying placenta-specific miRNAs involved in the occurrence and development of pig IUGR remain unclear. In this work, we compared the placental morphologies of piglets with IUGR and normal birth weight (NBW) by using histomorphological analysis and performed a miRNA-mRNA integrative analysis of the gene expression profiles of IUGR and NBW placentas through RNA sequencing. We also investigated the role of differentially expressed ssc-miR-339-5p/GRIK3 through an in vitro experiment on porcine trophoblast cells (PTr2). IUGR piglets had significantly lower birth weight, placental weight, placental efficiency, and placental villus and capillary densities compared with the NBW piglets (P < 0.05). A total of 81 differentially expressed miRNAs and 726 differentially expressed genes in the placentas were screened out between the IUGR and NBW groups. The miRNA-mRNA interaction networks revealed the key core miRNA (ssc-miR-339-5p) and its corresponding target genes. Subsequently, we found that upregulation of ssc-miR-339-5p significantly inhibited the migration and proliferation of PTr2 cells (P < 0.05). The dual-luciferase reporter system showed that GRIK3 was the target gene of ssc-miR-339-5p, and the transcription level of GRIK3 may be negatively regulated by ssc-miR-339-5p. Additionally, overexpression of ssc-miR-339-5p significantly increased (P < 0.05) the mRNA expression levels of genes involved in the cytokine-cytokine receptor interaction pathway. These results indicate that ssc-miR-339-5p may affect the migration and proliferation of trophoblast cells by regulating the expression of GRIK3 and altering the placental inflammatory response, resulting in a suboptimal morphology and function of the placenta and the development of pig IUGR.

Follicular fluid exosome-derived miR-339-5p enhances in vitro maturation of porcine oocytes via targeting SFPQ, a regulator of the ERK1/2 pathway.

In this study, we aimed to investigate cytoplasmic maturation and miRNA expression of mature oocytes cultured in porcine follicular fluid exosomes. We also examined the effect of miR-339-5p on oocyte maturation. Twenty eight differentially expressed miRNAs were detected using miRNA-seq. We then transfected cumulus oocyte complexes with miR-339-5p mimics and inhibitor during culture. The results showed that exosomes increased endoplasmic reticulum levels and the amount of lipid droplets, and decreased ROS levels, lipid droplet size, and percentage of oocytes with abnormal cortical granule distribution. Overexpressing miR-339-5p significantly decreased cumulus expansion genes, oocyte maturation-related genes, target gene proline/glutamine-rich splicing factor (SFPQ), ERK1/2 phosphorylation levels, oocyte maturation rate, blastocyst rate, and lipid droplet number, but increased lipid droplet size and the ratio of oocytes with abnormal cortical granule distribution. Inhibiting miR-339-5p reversed the decrease observed during overexpression. Mitochondrial membrane potential and ROS levels did not differ significantly between groups. In summary, exosomes promote oocyte cytoplasmic maturation and miR-339-5p regulating ERK1/2 activity through SFPQ expression, thereby elevating oocyte maturation and blastocyst formation rate in vitro.