Inhibition of miR‐103a‐3p suppresses lipopolysaccharide‐induced sepsis and liver injury by regulating FBXW7 expression

Inflammation, apoptosis, and oxidative stress are involved in septic liver dysfunction. Herein, the role of miR‐103a‐3p/FBXW7 axis in lipopolysaccharides (LPS)‐induced septic liver injury was investigated in mice. Hematoxylin‐eosin staining was used to evaluate LPS‐induced liver injury. Quantitative real‐time polymerase chain reaction was performed to determine the expression of microRNA (miR) and messenger RNA, and western blot analysis was conducted to examine the protein levels. Dual‐luciferase reporter assay was used to confirm the binding between miR‐103a‐3p and FBXW7. Both annexin V‐fluoresceine isothiocyanate/propidium iodide staining and caspase‐3 activity were employed to determine cell apoptosis. First, miR‐103a‐3p was upregulated in the septic serum of mice and patients with sepsis, and miR‐103a‐3p was elevated in the septic liver of LPS‐induced mice. Then, interfering miR‐103a‐3p significantly decreased apoptosis by suppressing Bax expression and upregulating Bcl‐2 levels in LPS‐induced AML12 and LO2 cells, and septic liver of mice. Furthermore, inhibition of miR‐103a‐3p repressed LPS‐induced inflammation by downregulating the expression of tumor necrosis factor, interleukin 1β, and interleukin 6 in vitro and in vivo. Meanwhile, interfering miR‐103a‐3p obviously attenuated LPS‐induced overactivation of oxidation via promoting expression of antioxidative enzymes, including catalase, superoxide dismutase, and glutathione in vitro and in vivo. Moreover, FBXW7 was a target of miR‐103a‐3p, and overexpression of FBXW7 significantly ameliorated LPS‐induced septic liver injury in mice. Finally, knockdown of FBXW7 markedly reversed anti‐miR‐103a‐3p‐mediated suppression of septic liver injury in mice. In conclusion, interfering miR‐103a‐3p or overexpression of FBXW7 improved LPS‐induced septic liver injury by suppressing apoptosis, inflammation, and oxidative reaction.     

Endoplasmic reticulum stress‐induced exosomal miR‐27a‐3p promotes immune escape in breast cancer via regulating PD‐L1 expression in macrophages

Immune escape of breast cancer cells contributes to breast cancer pathogenesis. Tumour microenvironment stresses that disrupt protein homeostasis can produce endoplasmic reticulum (ER) stress. The miRNA‐mediated translational repression of mRNAs has been extensively studied in regulating immune escape and ER stress in human cancers. In this study, we identified a novel microRNA (miR)‐27a‐3p and investigated its mechanistic role in promoting immune evasion. The binding affinity between miR‐27a‐3p and MAGI2 was predicted using bioinformatic analysis and verified by dual‐luciferase reporter assay. Ectopic expression and inhibition of miR‐27a‐3p in breast cancer cells were achieved by transduction with mimics and inhibitors. Besides, artificial modulation of MAGI2 and PTEN was done to explore their function in ER stress and immune escape of cancer cells. Of note, exosomes were derived from cancer cells and co‐cultured with macrophages for mechanistic studies. The experimental data suggested that ER stress biomarkers including GRP78, PERK, ATF6, IRE1α and PD‐L1 were overexpressed in breast cancer tissues relative to paracancerous tissues. Endoplasmic reticulum stress promoted exosome secretion and elevated exosomal miR‐27a‐3p expression. Elevation of miR‐27a‐3p and PD‐L1 levels in macrophages was observed in response to exosomes‐overexpressing miR‐27a‐3p in vivo and in vitro. miR‐27a‐3p could target and negatively regulate MAGI2, while MAGI2 down‐regulated PD‐L1 by up‐regulating PTEN to inactivate PI3K/AKT signalling pathway. Less CD4⁺, CD8⁺ T cells and IL‐2, and T cells apoptosis were observed in response to co‐culture of macrophages and CD3⁺ T cells. Conjointly, exosomal miR‐27a‐3p promotes immune evasion by up‐regulating PD‐L1 via MAGI2/PTEN/PI3K axis in breast cancer.     

Down‐regulated lncRNA SLC25A5‐AS1 facilitates cell growth and inhibits apoptosis via miR‐19a‐3p/PTEN/PI3K/AKT signalling pathway in gastric cancer

Mounting evidence has illustrated the vital roles of long non‐coding RNAs (lncRNAs in gastric cancer (GC). Nevertheless, the majority of their roles and mechanisms in GC are still largely unknown. In this study, we investigate the roles of lncRNA SLC25A5‐AS1 on tumourigenesis and explore its potential mechanisms in GC. The results showed that the expressions of SLC25A5‐AS1 in GC were significantly lower than that of adjacent normal tissues, which were significantly associated with tumour size, TNM stage and lymph node metastasis. Moreover, SLC25A5‐AS1 could inhibit GC cell proliferation, induce G1/G1 cell cycle arrest and cell apoptosis in vitro, as well as GC growth in vivo. Dual‐luciferase reporter assay confirmed the direct interaction between SLC25A5‐AS1 and miR‐19a‐3p, rescue experiment showed that co‐transfection miR‐19a‐3p mimics and pcDNA‐SLC25A5‐AS1 could partially restore the ability of GC cell proliferation and the inhibition of cell apoptosis. The mechanism analyses further found that SLC25A5‐AS1 might act as a competing endogenous RNAs (ceRNA), which was involved in the derepression of PTEN expression, a target gene of miR‐19a‐3p, and regulate malignant phenotype via PI3K/AKT signalling pathway in GC. Taken together, this study indicated that SLC25A5‐AS1 was down‐regulated in GC and functioned as a suppressor in the progression of GC. Moreover, it could act as a ceRNA to regulate cellular behaviours via miR‐19a‐3p/PTEN/PI3K/AKT signalling pathway. Thus, SLC25A5‐AS1 might be served as a potential target for cancer therapeutics in GC.     

MiR‐30‐5p suppresses cell chemoresistance and stemness in colorectal cancer through USP22/Wnt/β‐catenin signaling axis

Colorectal cancer (CRC) remains both common and fatal, and its successful treatment is greatly limited by the development of stem cell‐like characteristics (stemness) and chemoresistance. MiR‐30‐5p has been shown to function as a tumor suppressor by targeting the Wnt/β‐catenin signaling pathway, but its activity in CRC has never been assessed. We hypothesized that miR‐30‐5p exerts anti‐oncogenic effects in CRC by regulating the USP22/Wnt/β‐catenin signaling axis. In the present study, we demonstrate that tissues from CRC patients and human CRC cell lines show significantly decreased miR‐30‐5p family expression. After identifying the 3’UTR of USP22 as a potential binding site of miR‐30‐5p, we constructed a luciferase reporter containing the potential miR‐30‐5p binding site and measured the effects on USP22 expression. Western blot assays showed that miR‐30‐5p decreased USP22 protein expression in HEK293 and Caco2 CRC cells. To evaluate the effects of miR‐30‐5p on CRC cell stemness, we isolated CD133 + CRC cells (Caco2 and HCT15). We then determined that, while miR‐30‐5p is normally decreased in CD133 + CRC cells, miR‐30‐5p overexpression significantly reduces expression of stem cell markers CD133 and Sox2, sphere formation, and cell proliferation. Similarly, we found that miR‐30‐5p expression is normally reduced in 5‐fluorouracil (5‐FU) resistant CRC cells, whereas miR‐30‐5p overexpression in 5‐FU resistant cells reduces sphere formation and cell viability. Inhibition of miR‐30‐5p reversed the process. Finally, we determined that miR‐30‐5p attenuates the expression of Wnt/β‐catenin signaling target genes (Axin2 and MYC), Wnt luciferase activity, and β‐catenin protein levels in CRC stem cells.     

Epigenetic silenced miR‐125a‐5p could be self‐activated through targeting Suv39H1 in gastric cancer

Emerging evidence suggests that microRNAs (miRNAs) serve an important role in tumorigenesis and development. Although the low expression of miR‐125a‐5p in gastric cancer has been reported, the underlying mechanism remains unknown. In the current study, the low expression of miR‐125a‐5p in gastric cancer was verified in paired cancer tissues and adjacent non‐tumour tissues. Furthermore, the GC islands in the miR‐125a‐5p region were hypermethylated in the tumour tissues. And the hypermethylation was negatively correlated with the miR‐125a‐5p expression. Target gene screening showed that the histone methyltransferase Suv39H1 was one of the potential target genes. In vitro studies showed that miR‐125a‐5p could directly suppress the Suv39H1 expression and decrease the H3K9me3 levels. On the other hand, the Suv39H1 could induce demethylation of miR‐125a‐5p, resulting in re‐activation of miR‐125a‐5p. What is more, overexpessing miR‐125a‐5p could also self‐activate the silenced miR‐125a‐5p in gastric cancer cells, which suppressed cell migration, invasion and proliferation in vitro and inhibited cancer progression in vivo. Thus, we uncovered here that the epigenetic silenced miR‐125a‐5p could be self‐activated through targeting Suv39H1 in gastric cancer, suggesting that miR‐125a‐5p might be not only the potential prognostic value as a tumour biomarker but also potential therapeutic targets in gastric cancer.     

Loss of PPM1F expression predicts tumour recurrence and is negatively regulated by miR‐590‐3p in gastric cancer

Objectives

MicroRNAs (miRNAs) as small non‐coding RNA molecules act by negatively regulating their target genes. Recent studies have shown that protein phosphatase Mg2+/Mn2+‐dependent 1F (PPM1F) plays a critical role in cancer metastasis. But, the regulation mechanisms of PPM1F by miRNAs in gastric cancer (GC) remain undefined.

Methods

The correlation of PPM1F or miR‐590‐3p (miR‐590) expression with clinicopathological features and prognosis of the patients with GC was analysed by TCGA RNA‐sequencing data. The miRNAs that target PPM1F gene were identified by bioinformatics and Spearman correlation analysis, and the binding site between miR‐590 and PPM1F 3′UTR was confirmed by dual luciferase assay. MTT and Transwell assays were conducted to evaluate the effects of miR‐590 or (and) PPM1F on cell proliferation and invasion.

Results

We found that PPM1F expression was downregulated in GC tissues and cell lines and was correlated with tumour recurrence in patients with GC. The decreased expression of PPM1F was attributed to the dysregulation of miR‐590 expression rather than its genetic or epigenetic alterations. Overexpression of miR‐590 promoted cell proliferation and invasion capability of GC cells, while knockdown of miR‐590 reversed these effects. Moreover, PPM1F was validated as a direct target of miR‐590 and counteracted the tumour‐promoting effects caused by miR‐590. The expression of miR‐590 presented the negative correlation with PPM1F expression and acted as an independent prognostic factor for tumour recurrence in patients with GC.

Conclusion

PPM1F may function as a suppressive factor and is negatively regulated by miR‐590 in GC.

     

MiR‐129‐5p promotes docetaxel resistance in prostate cancer by down‐regulating CAMK2N1 expression

This study focuses on the effect of miR‐129‐5p on docetaxel‐resistant (DR) prostate cancer (PCa) cells invasion, migration and apoptosis. In our study, the expression of CAMK2N1 was assessed by qRT‐PCR in PCa patient tissues and cell lines including PC‐3 and PC‐3‐DR. Cells transfected with miR‐129‐5p mimics, inhibitor, CAMK2N1 or negative controls (NC) were used to interrogate their effects on DR cell invasions, migrations and apoptosis during docetaxel (DTX) treatments. The apoptosis rate of the PCa cells was validated by flow cytometry. Relationships between miR‐129‐5p and CAMK2N1 levels were identified by qRT‐PCR and dual‐luciferase reporter assay. CAMK2N1 was found to be down‐expressed in DR PCa tissue sample, and low levels of CAMK2N1 were correlated with high docetaxel resistance and clinical prediction of poor survival. CAMK2N1 levels were decreased in DR PCa cells treated with DXT. We further explored that up‐regulation of miR‐129‐5p could promote DR PCa cells viability, invasion and migration but demote apoptosis. Involved molecular mechanism studies revealed that miR‐129‐5p reduced downstream CAMK2N1 expression to further impact on chemoresistance to docetaxel of PCa cells, indicating its vital role in PCa docetaxel resistance. Our findings revealed that miR‐129‐5p contributed to the resistance of PC‐3‐DR cells to docetaxel through suppressing CAMK2N1 expression, and thus targeting miR‐129‐5p may provide a novel therapeutic approach in sensitizing PCa to future docetaxel treatment.     

Downregulation of microRNA‐15a‐3p is correlated with clinical outcome and negatively regulates cancer proliferation and migration in human osteosarcoma

In this study, we investigated the expression, correlation to clinical outcomes and biological functions of microRNA‐15a‐3p (miR‐15a‐3p) in human osteosarcoma. MiR‐15a‐3p expressions in osteosarcoma cell lines and clinical tissues of osteosarcoma patients were measured by qPCR. Relevance of endogenous miR‐15a‐3p to osteosarcoma patients' clinicopathological factors or overall survival was statistically analyzed. In addition, the independence of miR‐15a‐3p predicting cancer patients' overall survival was analyzed by Cox regression method. Furthermore, in osteosarcoma cell lines, Saos‐2 and HOS cells, miR‐15a‐3p was overexpressed through stable lentiviral transduction. The functional regulations of miR‐15a‐3p overexpression on cancer ell proliferation and migration were then analyzed. MiR‐15a‐3p was significantly downregulated in osteosarcoma cell lines and human osteosarcoma tumors. Downregulation of endogenous miR‐15a‐3p in osteosarcoma tumors was significantly associated with cancer patient's poor clinical outcomes and low survival rate. Also, endogenous miR‐15a‐3p was confirmed to be an independent biomarker for predicating cancer patients' survival. In Saos‐2 and HOS cells, lentivirus‐induced miR‐15a‐3p overexpression had significantly tumor suppressing functions, by inhibiting both proliferation and migration. Significant downregulation of miR‐15a‐3p in osteosarcoma may be an independent biomarker to predicting cancer patients' poor prognosis. Overexpression miR‐15a‐3p may be an efficient functional meaning to suppress osteosarcoma development.     

Circular RNA ciRS‐7 promotes tube formation in microvascular endothelial cells through downregulation of miR‐26a‐5p

Atherosclerosis is one of the most common and crucial heart diseases involving the heart and brain. At present, atherosclerosis and its major complications comprise the leading causes of death worldwide. Our purpose was to identify the role of ciRS‐7 in atherosclerosis. Tubulogenesis of HMEC‐1 cell was evaluated utilizing tube formation assay. Cell Counting Kit‐8 assay and flow cytometry were utilized to test viability and apoptosis. Migration assay was utilized to determine the migration capacity of experimental cells. Western blot was applied to examine apoptosis and tube formation‐associated protein expression. In addition, the above experiments were repeated when silencing ciRS‐7, overexpressing ciRS‐7, and upregulating miR‐26a‐5p. HMEC‐1 cells formed tube‐like structures over time. Silencing ciRS‐7 suppressed viability, migration, and tube formation but promoted apoptosis. Oppositely, overexpressing ciRS‐7 reversed the effect in HMEC‐1 cells. miR‐26a‐5p expression was elevated by silencing ciRS‐7 and reduced by overexpressing ciRS‐7. Moreover, overexpressing ciRS‐7 facilitated viability, migration, and tube formation via upregulating miR‐26a‐5p. Conclusively, overexpressing ciRS‐7 mobilized phosphoinositide 3‐kinase/protein kinase B (PI3K/AKT) pathway and suppressed c‐Jun N‐terminal kinase (JNK)/p38 pathway. ciRS‐7 exerted influence on apoptosis, viability, migration, and tube formation through mediating PI3K/AKT and JNK/p38 pathways by miR‐26a‐5p downregulation in HMEC‐1 cells.     

MiR‐485‐3p modulates neural stem cell differentiation and proliferation via regulating TRIP6 expression

Recent references have showed crucial roles of several miRNAs in neural stem cell differentiation and proliferation. However, the expression and role of miR‐485‐3p remains unknown. In our reference, we indicated that miR‐485‐3p expression was down‐regulated during NSCs differentiation to neural and astrocytes cell. In addition, the TRIP6 expression was up‐regulated during NSCs differentiation to neural and astrocytes cell. We carried out the dual‐luciferase reporter and found that overexpression of miR‐485‐3p decreased the luciferase activity of pmirGLO‐TRIP6‐wt but not the pmirGLO‐TRIP6‐mut. Ectopic expression of miR‐485‐3p decreased the expression of TRIP6 in NSC. Ectopic miR‐485‐3p expression suppressed the cell growth of NSCs and inhibited nestin expression of NSCs. Moreover, elevated expression of miR‐485‐3p decreased the ki‐67 and cyclin D1 expression in NSCs. Furthermore, we indicated that miR‐485‐3p reduced proliferation and induced differentiation of NSCs via targeting TRIP6 expression. These data suggested that a crucial role of miR‐485‐3p in self‐proliferation and differentiation of NSCs. Thus, altering miR‐485‐3p and TRIP6 modulation may be one promising therapy for treating with neurodegenerative and neurogenesis diseases.