Exosomal miR-663b from "M1" macrophages promotes pulmonary artery vascular smooth muscle cell dysfunction through inhibiting the AMPK/Sirt1 axis.

BACKGROUND: Inflammatory mediators from macrophages are proven to be involved in pulmonary vascular remodeling in pulmonary hypertension (PH). Here, this study intends to explore the mechanism of "M1" macrophage-derived exosomal miR-663b in pulmonary artery smooth muscle cells (PASMCs) dysfunctions and pulmonary hypertension. METHODS: Hypoxia-treated PASMCs were utilized for constructing an in-vitro pulmonary hypertension model. THP-1 cells were treated with PMA (320 nM) and LPS (10 µg/mL) + IFN-γ (20 ng/ml) for eliciting macrophage "M1" polarization. Exosomes derived from "M1" macrophages were isolated and added into PASMCs. The proliferation, inflammation, oxidative stress, and migration of PASMCs were evaluated. RT-PCR or Western blot examined the levels of miR-663b and the AMPK/Sirt1 pathway. Dual luciferase activity assay and RNA pull-down assay were carried out for confirming the targeted association between miR-663b and AMPK. An in-vivo PH model was built. Macrophage-derived exosomes with miR-663b inhibition were used for treating the rats, and alterations of pulmonary histopathology were monitored. RESULTS: miR-663b was obviously up-regulated in hypoxia-elicited PASMCs and M1 macrophages. miR-663b overexpression boosted hypoxia-induced proliferation, inflammation, oxidative stress, and migration in PASMCs, whereas miR-663b low expression resulted in the opposite situation. AMPK was identified as a target of miR-663b, and miR-663b overexpression curbed the AMPK/Sirt1 pathway. AMPK activation ameliorated the damaging impact of miR-663b overexpression and "M1" macrophage exosomes on PASMCs. In vivo, "M1" macrophage exosomes with miR-663b low expression alleviated pulmonary vascular remodeling in pulmonary hypertension rats. CONCLUSION: Exosomal miR-663b from "M1" macrophage facilitates PASMC dysfunctions and PH development by dampening the AMPK/Sirt1 axis.

Hsa_circ_0049396 inhibited oral squamous cell carcinoma progression by regulating the miR-663b/ENDOU axis.

BACKGROUND: Circular RNA (circRNAs) play an important role in oral squamous cell carcinoma (OSCC) progression and has been widely reported. In this study, we aimed to investigate the role of a novel circRNA, circ_0049396, and its underlying mechanism in OSCC. METHODS: The expression levels of circ_0049396, miR-663b, and theuridylate-specific endoribonuclease (ENDOU) were assessed by quantitative reverse transcription PCR (qRT-PCR). Cell proliferation and migration were evaluated using CCK-8 and Transwell assays, respectively. Western blotting was performed to measure the levels of the apoptosis-associated proteins (Bcl-2 and Bax). The functional role of circ _0049396 was further validated in a xenograft experiment in vivo. The interactions of miR-663b with circ_0049396/ENDOU were verified using the dual luciferase reporter, RNA pull-down, and RNA immunoprecipitation (RIP) assays. RESULTS: The expression of circ_0049396 and ENDOU was downregulated in OSCC tissues and cells, whereas miR-663b was upregulated. Circ_0049396 overexpression weakened OSCC cell proliferation and migration but enhanced their apoptosis. Circ_0049396 overexpression suppresses tumorigenesis in vivo. The circ_0049396/miR-663b/ENDOU regulatory network predicted through bioinformatic analysis was validated using RNA pull-down, luciferase reporter, and RIP experiments. MiR-663b mimic enhanced the migratory and proliferative abilities of OSCC cells, but suppressed apoptosis. Furthermore, circ_0049396 or ENDOU overexpression partially reversed the malignant behavior of miR-663b-overexpressing OSCC cells. CONCLUSIONS: Our study illustrated that circ_0049396 overexpression inhibited the malignant behavior of OSCC cells by regulating the miR-663b/ENDOU axis. Based on our findings, circ_0049396 can be used as a potential therapeutic agent for OSCC.

Effect of microRNA-663b on migration, invasion and epithelial­mesenchymal transition of oral squamous cell carcinoma cells.

OBJECTIVES: To explore the effect of microRNA-663b (miR-6636) on migration, invasion and epithelial-mesenchymal transition (EMT) of oral squamous cell carcinoma cells (OSCC). METHODS: Use R Studio of gene expression omnibus (GEO) database to analyze expressions of miR-663b in the OSCC and adjacent normal tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-663b in tissues and cells. The transfection efficiency of HN30 cells with miR-663b knockout plasmid was detected. Transwell assay was used to detect the effect of the migration and invasion ability. Bioinformatics method was used to predict the targeted mRNA that may bind to miR-663b and double luciferase assay was used to verify the binding. Western blot assay was used to detect the expression of EMT-related markers. RESULTS: The expression of miR-663b was up-regulated in OSCC tissues and higher in HN30, CAL27 and SCC-9 cells than in HOEC cells (P<0.05). Knockout of miR-663b could inhibit migration and invasion of HN30 cells (P<0.05) and inhibit the occurrence of EMT. Bioinformatics prediction software predicts that SH3BP2 was the target gene of miR-663b, and patients with low SH3BP2 expression had a poor prognosis (P<0.05). MiR-663b could bind to SHBP2 (P<0.05). The expression of SH3BP2 was increased and the occurrence of EMT was inhibited in HN30 cells with miR-663b knocked out. CONCLUSIONS: Knockout of miR-663b can inhibit the migration, invasion and EMT of OSCC by targeting SH3BP2.

Long non-coding RNA HULC regulates TLR4 expression by acting as ceRNA to attract miR-663b in skin fibroblasts of pediatric burns.

OBJECTIVE: The study aims to elucidate the impact of LncRNA HULC in human skin fibroblasts (HSF) after burns in children. HULC might act as endogenous sponges for miR-663b to regulate the gene expression of TLR4. METHODS: This study included 46 children with deep second-degree burns. On the 5th day after the injury, eligible samples from all patients were collected. HSF cells were selected to establish a thermal-injured model. qRT-PCR was applied to detect the expression of HULC, miR-663b, and TLR4 mRNA in burn wound and normal skin tissue. The dual-luciferase reporter and RIP assay were performed to explore a targeted binding relationship between HULC and miR-663b, or miR-663b and TLR4. Cell proliferation and invasion were evaluated through the assay of CCK-8 and transwell assay. The expression levels of α-SMA, Collagen I, MMP-1, and TIMP-1, which are associated with extracellular matrix (ECM) production, were examinated by western blot. RESULTS: HULC and TLR4 mRNA expression were reduced on the 5th day after thermal injury in burn wounds, while miR-663b expression increased significantly (P<0.05), when compared to expression in the normal tissue. HULC and TLR4 mRNA concentration in HSF cells showed a transient increase after thermal injury, and a gradual decline with time was observed subsequently when compared to the control group. An inverse expression of miR-663b with the expression of HULC and TLR4 mRNA was observed simultaneously (P<0.05). A deficiency of HULC promotes the proliferation, invasion, and ECM synthesis of HSF cells with thermal injury; HULC functions as a ceRNA of miR-663b. Inhibitors of miR-663b partially rescued the effects on thermal-injured HSF cells induced by HULC deficiency (P<0.05). TLR4 is a target gene of miR-663b. The up-regulation of TLR4 also partially reversed the effect on the thermal-injury of HSF cells resulting from HULC deficiency (P<0.05). CONCLUSION: LncRNA HULC may function as a molecular sponge to regulate the expression of the miR-663b/TLR4, and thereby inhibit the proliferation, invasion, and ECM synthesis of thermal-injured HSF cells. HULC knockdown might significantly promote wound healing in children after burns.

Exosomal miR­663b exposed to TGF­ß1 promotes cervical cancer metastasis and epithelial­mesenchymal transition by targeting MGAT3.

Transforming growth factor (TGF)­ß1 is a key cytokine affecting the pathogenesis and progression of cervical cancer. Tumor­derived exosomes contain microRNAs (miRNAs/miRs) that interact with cancer and stromal cells, thereby contributing to tissue remodeling in the tumor microenvironment (TME). The present study was designed to clarify how TGF­ß1 affects tumor biological functions through exosomes released by cervical cancer cells. Deep RNA sequencing found that TGF­ß1 stimulated cervical cancer cells to secrete more miR­663b­containing exosomes, which could be transferred into new target cells to promote metastasis. Further studies have shown that miR­663b directly targets the 3'-untranslated regions (3'­UTR) of mannoside acetylglucosaminyltransferase 3 (MGAT3) and is involved in the epithelial­mesenchymal transition (EMT) process. Remarkably, the overexpression of MGAT3 suppressed cervical cancer cell metastasis promoted by exosomal miR­663b, causing increased expression of epithelial differentiation marker E­cadherin and decreased expression of mesenchymal markers N­cadherin and ß­catenin. Throughout our study, online bioinformation tools and dual luciferase reporter assay were applied to identify MGAT3 as a novel direct target of miR­663b. Exosome PKH67­labeling experiment verified that exosomal miR­663b could be endocytosed by cervical cancer cells and subsequently influence its migration and invasion functions which were measured by wound healing and Transwell assays. The expression of miR­663b and MGAT3 and the regulation of the EMT pathway caused by MGAT3 were detected by quantitative real­time transcription­polymerase chain reaction (qPCR) and western blot analysis. These results, thus, provide evidence that cancer cell­derived exosomal miR­663b is endocytosed by cervical cancer cells adjacent or distant after TGF­ß1 exposure and inhibits the expression of MGAT3, thereby accelerating the EMT process and ultimately promoting local and distant metastasis.

Expression of miR-18a-5p, miR-144-3p, and miR-663b in colorectal cancer and their association with cholesterol homeostasis.

OBJECTIVE: Though cholesterol accumulation is an established hallmark of a tumor cell, the relationship between the two is still not clear. Previously, we identified 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGCR), Sterol Regulatory Element BindingTranscription Factor 2 (SREBF2), Nuclear Receptor Subfamily 1 Group H Member 3 (NR1H3), and Nuclear Receptor Subfamily 1 Group H Member 2 (NR1H2) as the key cholesterol homeostasis genes involved in colorectal cancer (CRC). In the present study, we aimed to identify microRNAs regulating these key genes in CRC. METHODS: miR-18a-5p, miR-144-3p, and miR-663b were selected as the miRNAs targeting NR1H2, HMGCR, and SREBF2, respectively, based on the bioinformatic prediction tools and literature review. Their expression was evaluated in the local and The Cancer Genome Atlas (TCGA) cohorts. Receiver Operating Characteristic Curves and Kaplan Meier analysis were performed to elucidate their diagnostic and prognostic potential. Pearson or Spearman's correlations were used to evaluate the relationship between miRNAs and their target genes. Protein-protein interaction networks and Gene Ontology analyses were performed to investigate the potential molecular mechanism of these miRNAs. RESULTS: Deregulated expression of miR-18a-5p, miR-144-3p, and miR-663b was associated with various clinicopathological features. miR-18a-5p exhibited an inverse correlation with NR1H2. miR-18a-5p and miR-144-3p also had a significant direct correlation with miR-33a-5p, an important modulator of cholesterol homeostasis. These miRNAs also exhibited high centrality in the mirna-protein interaction network. miR-144-3p and miR-663b exhibited the potential to be used as diagnostic biomarkers. CONCLUSIONS: miR-18a-5p and miR-144-3p exhibited the potential to modulate cholesterol homeostasis in CRC. miR-663b is an interesting candidate in CRC pathophysiology.

Expression and Role of MicroRNA-663b in Childhood Acute Lymphocytic Leukemia and its Mechanism.

Recent studies have shown that microRNAs (miRNAs) play a key role in various malignant tumors. MiR-663b has been found to have important roles in several cancers, however, the role of miR-663b in T cell acute lymphocytic leukemia (T-ALL) remains unclear. Therefore, we speculated that miR-663b might also play a crucial role in the development and process of T-ALL. In the present study, we found that miR-663b was up-regulated in the blood of children with T-ALL and T-ALL cell lines. TargetScan and dual luciferase reporter assay results showed that CD99 was a direct target of miR-663b. In order to further study the biological function of miR-663b in the development of T-ALL and to clarify its potential molecular mechanism, we detected the changes in proliferation, apoptosis, migration, and invasion of T-ALL cell line Jurkat before and after miR-663b inhibitor transfection. We found that miR-663b inhibitor inhibited Jurkat cell proliferation and induced apoptosis. In addition, miR-663b inhibitor repressed Jurkat cell migration and invasion. All these effects of miR-663b inhibitor on Jurkat cells were eliminated by CD99-silencing. These results have provided a new theoretical basis and strategy for the diagnosis and treatment of T-ALL.

Blockade of miR-663b inhibits cell proliferation and induces apoptosis in osteosarcoma via regulating TP73 expression.

OBJECTIVE: This study aimed to investigate the exact role of miR-663b in osteosarcoma (OS) progression and further explore the underlying molecular mechanisms. MATERIALS AND METHODS: The miR-663b expression in human OS cell lines was determined by qRT-PCR, and the results suggested that miR-663b was highly expressed in human OS cells. TargetScan was used to predict the potential targets of miR-663b, and the prediction was confirmed by dual-luciferase reporter assay. To investigate the role of miR-663b in OS, miR-663b was down-regulated in U2OS cells using miR-663b inhibitor. CCK8 and flow cytometry were preformed to investigate the proliferation and apoptosis of U2OS cells. Moreover, qRT-PCR and western blot analysis were performed to measure the mRNA and protein expression. RESULTS: We found that miR-663b directly targets TP73 and negatively regulates TP73 expression. MiR-663b inhibitor significantly decreased the proliferation ability of U2OS cells, while the percentage of apoptotic cells was markedly increased. The level of Bcl-2 was notably inhibited by miR-663b inhibitor, while Bax expression was significantly enhanced. Moreover, miR-663b down-regulation promoted p53 and p21 expression in U2OS cells. CONCLUSIONS: MiR-663b down-regulation represses proliferation and induces apoptosis in OS by targeting TP73. Therefore, we provide a potential therapeutic target for OS treatment (Fig. 6, Ref. 27).

Epigenetic inhibition of miR-663b by long non-coding RNA HOTAIR promotes pancreatic cancer cell proliferation via up-regulation of insulin-like growth factor 2.

Pancreatic cancer is one of the most deadly cancers with a poor prognosis. Although microRNAs are involving in the carcinogenesis and development of pancreatic cancer, little information is known regarding the role of miR-663b in pancreatic cancer. In this study, the expression of miR-663b in pancreatic cancer cells was down-regulated by hypermethylation in its putative promoter region, and overexpression of miR-663b repressed cell proliferation, invasion and migration, and induced apoptosis in pancreatic cancer cells. Bioinformatics analysis, luciferase report assay and rescue experiments showed that insulin-like growth factor 2 (IGF2) was a direct target of miR-663b. Results from clinical samples showed that the expression level of miR-663b correlated with the pathological grading, and the expression of miR-663b was down-regulated and was inversely correlated with IGF2 expression level in pancreatic cancer tissues. More importantly, the long non-coding RNA, HOX transcript antisense RNA (HOTAIR), was up-regulated in both pancreatic cancer cells and tissues, and HOTAIR suppressed the expression of miR-663b in pancreatic cancer by histone modification on H3K4me3 and H3K27me3 on miR-663b promoter. Further in vivo studies demonstrated that the stable overexpression of miR-663b or knock-down of HOTAIR inhibited tumor growth and was associated with IGF2 expression. In summary, our studies demonstrated that miR-663b is epigenetically repressed by HOTAIR and exerts its tumor-suppressive function via targeting IGF2 in pancreatic cancer.