KCNQ1OT1 facilitates progression of non-small-cell lung carcinoma via modulating miRNA-27b-3p/HSP90AA1 axis

Long noncoding RNA KCNQ1OT1 participates in the regulation of imprinted genes within the kcnq1 domain. But its roles in carcinogenesis and metastasis remain largely elusive. Herein, we evaluated its potential in non-small-cell lung cancer (NSCLC) progression. We demonstrated that the KCNQ1OT1 level was upregulated in NSCLC tissues and cell lines. High KCNQ1OT1 level correlated with poor overall and progression-free survival in NSCLC patients. KCNQ1OT1 facilitated proliferation, migration, and invasion in H460 cells. Furthermore, knockdown of KCNQ1OT1 reduced the expression of HSP90AA1. KCNQ1OT1 presented a positive correlation with HSP90AA1 which predicted the tumor progression in NSCLC from The Cancer Genome Atlas database. Intriguingly, KCNQ1OT1 modulated HSP90AA1 expression by sponging miR-27b-3p. MiR-27b-3p counteracted the effect of KCNQ1OT1 on HSP90AA1 expression, H460 cell migration, and invasion. These data revealed a role for KCNQ1OT1 as an oncogene through miR-27b-3p/HSP90AA1 axis during NSCLC progression.     

New insights into the central sympathetic hyperactivity post‐myocardial infarction: Roles of METTL3‐mediated m6A methylation

Ventricular arrhythmias (VAs) triggers by sympathetic nerve hyperactivity contribute to sudden cardiac death in myocardial infarction (MI) patients. Microglia‐mediated inflammation in the paraventricular nucleus (PVN) is involved in sympathetic hyperactivity after MI. N6‐methyladenosine (m⁶A), the most prevalent mRNA and epigenetic modification, is critical for mediating cell inflammation. We aimed to explore whether METTL3‐mediated m⁶A modification is involved in microglia‐mediated sympathetic hyperactivity after MI in the PVN. MI model was established by left coronary artery ligation. METTL3‐mediated m⁶A modification was markedly increased in the PVN at 3 days after MI, and METTL3 was primarily located in microglia by immunofluorescence. RNA‐seq, MeRIP‐seq, MeRIP‐qPCR, immunohistochemistry, ELISA, heart rate variability measurements, renal sympathetic nerve activity recording and programmed electrical stimulation confirmed that the elevated toll‐like receptor 4 (TLR4) expression by m⁶A modification on TLR4 mRNA 3''‐UTR region combined with activated NF‐κB signalling led to the overwhelming production of pro‐inflammatory cytokines IL‐1β and TNF‐α in the PVN, thus inducing the sympathetic hyperactivity and increasing the incidence of VAs post‐MI. Targeting METTL3 attenuated the inflammatory response and sympathetic hyperactivity and reduced the incidence of VAs post‐MI.     

长链非编码RNA KCNQ1OT1影响脂多糖诱导的血管内皮细胞凋亡及其炎性因子表达的机制

该研究探讨了长链非编码RNA KCNQ1OT1对脂多糖(LPS)诱导的血管内皮细胞(VEC)凋亡和炎性因子表达的影响以及其可能机制.通过体外培养VEC,分别转染KCNQ1OT1过表达载体、miR-223抑制剂或共转染KCNQ1OT1过表达载体与miR-223模拟物后,用1.0mg/mLLPS干预24h,然后采用RT-q...     

KCNQ1OT1 regulates the retinoblastoma cell proliferation,migration and SIRT1/JNK signaling pathway by targeting miR-124/SP1 axis

Objective: Long non-coding RNA (lncRNA) KCNQ1OT1 was reported to be tightly associated with tumorigenesis and progression of multiple cancers. However, the expression and biological functions of KCNQ1OT1 in retinoblastoma (RB) are still unknown. We aim to elucidate the potential function and underlying mechanism of KCNQ1OT1 in regulating the progression of RB. Methods: The levels of KCNQ1OT1 were assayed by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) analysis. The cell proliferation of RB cells (Y79 and WERI-Rb-1) were evaluated through Cell Counting Kit 8 (CCK-8) assay. Meanwhile, Y79 and WERI-Rb-1 cell apoptosis and cell cycle were assessed by Flow Cytometry analysis. Dual luciferase reporter assay were performed to illustrate the interaction between KCNQ1OT1, miR-124, and SP1. Results: We found that KCNQ1OT1 was up-regulated and miR-124 was down-regulated in RB tissues and cells. Moreover, knockdown of KCNQ1OT1 reduced the proliferation, migration, and cell cycle, as well as promoted cell apoptosis of Y79 and WERI-Rb-1 cells. Western blot analysis consistently proved cell cycle and apoptosis related protein expression levels. More importantly, KCNQ1OT1 was a sponge of microRNA (miR)-124. MiR-124 inhibition strongly reversed the effect on cell proliferation, cycle arrest, and apoptosis by KCNQ1OT1 knockdown mediation. In addition, KCNQ1OT1 regulated expression of SP1, a direct target of miR-124 in RB. On the other hand, miR-124 inhibitor abrogated the active effect of KCNQ1OT1 silencing on silent information regulator 1 (SIRT1)/c-Jun N-terminal kinase (JNK) signaling pathway. The function of KCNQ1OT1 was verified in vivo. Conclusions: These findings implied that KCNQ1OT1 silencing inhibited RB progression and activated SIRT1/JNK signaling pathway partially by modulating the miR-124/SP1 axis.     

Degradation of WTAP blocks antiviral responses by reducing the m6A levels of IRF3 and IFNAR1 mRNA

N ⁶‐methyladenosine (m⁶A) is a chemical modification present in multiple RNA species and is most abundant in mRNAs. Studies on m⁶A reveal its comprehensive roles in almost every aspect of mRNA metabolism, as well as in a variety of physiological processes. Although some recent discoveries indicate that m⁶A can affect the life cycles of numerous viruses as well as the cellular antiviral immune response, the roles of m⁶A modification in type I interferon (IFN‐I) signaling are still largely unknown. Here, we reveal that WT1‐associated protein (WTAP), one of the m⁶A “writers”, is degraded via the ubiquitination‐proteasome pathway upon activation of IFN‐I signaling. With the degradation of WTAP, the m⁶A levels of IFN‐regulatory factor 3 (IRF3) and interferon alpha/beta receptor subunit 1 (IFNAR1) mRNAs are reduced, leading to translational suppression of IRF3 and instability of IFNAR1 mRNA. Thus, the WTAP‐IRF3/IFNAR1 axis may serve as negative feedback pathway to fine‐tune the activation of IFN‐I signaling, which highlights the roles of m⁶A in the antiviral response by dictating the fate of mRNAs associated with IFN‐I signaling.     

Role of miR‐218‐GREM1 axis in epithelial‐mesenchymal transition of oral squamous cell carcinoma: An in vivo and vitro study based on microarray data

Oral squamous cell carcinoma (OSCC) is a prevalent cancer that develops in the head and neck area and has high annual mortality despite optimal treatment. microRNA‐218 (miR‐218) is a tumour inhibiting non‐coding RNA that has been reported to suppress the cell proliferation and invasion in various cancers. Thus, our study aims to determine the mechanism underlying the inhibitory role of miR‐218 in OSCC. We conducted a bioinformatics analysis to screen differentially expressed genes in OSCC and their potential upstream miRNAs. After collection of surgical OSCC tissues, we detected GREM1 expression by immunohistochemistry, RT‐qPCR and Western blot analysis, and miR‐218 expression by RT‐qPCR. The target relationship between miR‐218 and GREM1 was assessed by dual‐luciferase reporter gene assay. After loss‐ and gain‐of‐function experiments, OSCC cell proliferation, migration and invasion were determined by MTT assay, scratch test and Transwell assay, respectively. Expression of TGF‐β1, Smad4, p21, E‐cadherin, Vimentin and Snail was measured by RT‐qPCR and Western blot analysis. Finally, effects of miR‐218 and GREM1 on tumour formation and liver metastasis were evaluated in xenograft tumour‐bearing nude mice. GREM1 was up‐regulated, and miR‐218 was down‐regulated in OSCC tissues, and GREM1 was confirmed to be the target gene of miR‐218. Furthermore, after up‐regulating miR‐218 or silencing GREM1, OSCC cell proliferation, migration and invasion were reduced. In addition, expression of TGF‐β signalling pathway‐related genes was diminished by overexpressing miR‐218 or down‐regulating GREM1. Finally, up‐regulated miR‐218 or down‐regulated GREM1 reduced tumour growth and liver metastasis in vivo. Taken together, our findings suggest that the overexpression of miR‐218 may inhibit OSCC progression by inactivating the GREM1‐dependent TGF‐β signalling pathway.     

LncRNA SNHG1 promotes sepsis‐induced myocardial injury by inhibiting Bcl‐2 expression via DNMT1

Myocardial injury is a frequently occurring complication of sepsis. This study aims to investigate the molecular mechanism of long noncoding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1)‐mediated DNA methyltransferase 1/B‐cell lymphoma‐2 (DNMT1/Bcl‐2) axis in sepsis‐induced myocardial injury. Mice and HL‐1 cells were treated with lipopolysaccharide (LPS) to establish animal and cellular models simulating sepsis and inflammation. LncRNA SNHG1 was screened out as a differentially expressed lncRNA in sepsis samples through microarray profiling, and the upregulated expression of lncRNA SNHG1 was confirmed in myocardial tissues of LPS‐induced septic mice and HL‐1 cells. Further experiments suggested that silencing of lncRNA SNHG1 reduced the inflammation and apoptotic rate of LPS‐induced HL‐1 cells. LncRNA SNHG1 inhibited Bcl‐2 expression by recruiting DNMT1 to Bcl‐2 promoter region to cause methylation. Inhibition of Bcl‐2 promoter methylation reduced the inflammation and apoptotic rate of LPS‐induced HL‐1 cells. In vivo experiments substantiated that lncRNA SNHG1 silencing alleviated sepsis‐induced myocardial injury in mice. Taken together, lncRNA SNHG1 promotes LPS‐induced myocardial injury in septic mice by downregulating Bcl‐2 through DNMT1‐mediated Bcl‐2 methylation.     

METTL3/N6‐methyladenosine/ miR‐21‐5p promotes obstructive renal fibrosis by regulating inflammation through SPRY1/ERK/NF‐κB pathway activation

Renal fibrosis induced by urinary tract obstruction is a common clinical occurrence; however, effective treatment is lacking, and a deeper understanding of the mechanism of renal fibrosis is needed. Previous studies have revealed that miR‐21 impacts liver and lung fibrosis progression by activating the SPRY1/ERK/NF‐kB signalling pathway. However, whether miR‐21 mediates obstructive renal fibrosis through the same signalling pathway has not been determined. Additionally, studies have shown that N6‐methyladenosine (m⁶A) modification‐dependent primary microRNA (pri‐microRNA) processing is essential for maturation of microRNAs, but its role in the maturation of miR‐21 in obstructive renal fibrosis has not yet been investigated in detail. To address these issues, we employed a mouse model of unilateral ureteral obstruction (UUO) in which the left ureters were ligated for 3, 7 and 14 days to simulate the fibrotic process. In vitro, human renal proximal tubular epithelial (HK‐2) cells were transfected with plasmids containing the corresponding sequence of METTL3, miR‐21‐5p mimic or miR‐21‐5p inhibitor. We found that the levels of miR‐21‐5p and m⁶A modification in the UUO model groups increased significantly, and as predicted, the SPRY1/ERK/NF‐kB pathway was activated by miR‐21‐5p, confirming that miR‐21‐5p plays an important role in obstructive renal fibrosis by enhancing inflammation. METTL3 was found to play a major catalytic role in m⁶A modification in UUO mice and drove obstructive renal fibrosis development by promoting miR‐21‐5p maturation. Our research is the first to demonstrate the role of the METTL3‐m⁶A‐miR‐21‐5p‐SPRY1/ERK/NF‐kB axis in obstructive renal fibrosis and provides a deeper understanding of renal fibrosis.     

ALKBH5-m6A-FOXM1 signaling axis promotes proliferation and invasion of lung adenocarcinoma cells under intermittent hypoxia

Obstructive sleep apnea (OSA) is closely associated with cancer progression and cancer-related mortality. N6-methyladenosine (m⁶A) is involved in the process of intermittent hypoxia (IH) promoting tumor progression. However, it is unclear how m⁶A regulates the development of lung adenocarcinoma under IH. In this study, we found that ALKBH5 was elevated in lung adenocarcinoma cells and subcutaneous tumors in mice under IH, which was associated with decreased m⁶A levels in these cells and tissues. Next, we knocked out ALKBH5 in a human lung adenocarcinoma cell line under IH, and we found that the proliferation and invasion of these cells were significantly inhibited. Mechanistic analysis showed that under IH, knockout of ALKBH5 in lung adenocarcinoma cells upregulated the level of m⁶A in Forkhead box M1 (FOXM1) mRNA and decreased the translation efficiency of FOXM1 mRNA, resulting in downregulation of the FOXM1 protein. The FOXM1 protein is elevated in lung adenocarcinoma cells and subcutaneous tumor tissues of mice under IH. By knocking out FOXM1 in lung adenocarcinoma cells under IH, proliferation and invasion of these cells were inhibited, and overexpression of FOXM1 partially restored the inhibition of growth and invasion of lung adenocarcinoma cells due to ALKBH5 knockout. Collectively, our findings demonstrate that the m⁶A demethylase ALKBH5 affects the proliferation and invasion of lung adenocarcinoma cells under IH by downregulating m⁶A modification on FOXM1 mRNA and by promoting FOXM1 expression.     

LncRNA DLX6-AS1 promotes tumor proliferation and metastasis in osteosarcoma through modulating miR-641/HOXA9 signaling pathway

Osteosarcoma (OS) is the most common primary malignant bone tumor. Recently, increasing evidence has shown that the long noncoding RNA (lncRNA) DLX6-AS1 (distal-less homeobox 6 antisense 1) plays significant roles in various types of cancers. However, the functions and underlying mechanisms of DLX6-AS1 have not been explored in OS yet. In this study, we assessed the expression of DLX6-AS1 in OS tissues and cell lines and explored the underlying molecular mechanisms. DLX6-AS1 was found to be significantly upregulated in OS tissues and OS cell lines. High expression of DLX6-AS1 was significantly correlated with advanced TNM stage, high tumor grade, and distant metastasis of patients with OS. Knockdown of DLX6-AS1 suppressed OS cell proliferation, invasion, and migration, and induced cell apoptosis. Knockdown of DLX6-AS1 also suppressed in vivo tumor growth. Bioinformatics and luciferase assay analysis showed that DLX6-AS1 functioned as a competing endogenous RNA (ceRNA) to negatively regulate miR-641 expression. Furthermore, miR-641 was found to target the 3′ untranslated region of homeobox protein Hox-A9 (HOXA9) and suppressed the expression of HOXA9. Mechanistic studies showed that DLX6-AS1 regulated OS cell proliferation, invasion, and migration via regulating HOXA9 by acting as a ceRNA for miR-641. Our results suggested that DLX6-AS1 functions as a ceRNA by targeting miR-641/HOXA9 signal pathway to suppress OS cell proliferation and metastasis. Our study may provide novel insights into understanding pathogenesis and development of OS.     

m6A modification of HSATIII lncRNAs regulates temperature‐dependent splicing

Nuclear stress bodies (nSBs) are nuclear membraneless organelles formed around stress‐inducible HSATIII architectural long noncoding RNAs (lncRNAs). nSBs repress splicing of hundreds of introns during thermal stress recovery, which are partly regulated by CLK1 kinase phosphorylation of temperature‐dependent Ser/Arg‐rich splicing factors (SRSFs). Here, we report a distinct mechanism for this splicing repression through protein sequestration by nSBs. Comprehensive identification of RNA‐binding proteins revealed HSATIII association with proteins related to N⁶‐methyladenosine (m⁶A) RNA modification. 11% of the first adenosine in the repetitive HSATIII sequence were m⁶A‐modified. nSBs sequester the m⁶A writer complex to methylate HSATIII, leading to subsequent sequestration of the nuclear m⁶A reader, YTHDC1. Sequestration of these factors from the nucleoplasm represses m⁶A modification of pre‐mRNAs, leading to repression of m⁶A‐dependent splicing during stress recovery phase. Thus, nSBs serve as a common platform for regulation of temperature‐dependent splicing through dual mechanisms employing two distinct ribonucleoprotein modules with partially m⁶A‐modified architectural lncRNAs.     

Mesenchymal stroma/stem‐like cells of GARP knockdown inhibits cell proliferation and invasion of mouse colon cancer cells (MC38) through exosomes

Mesenchymal stroma/stem‐like cells (MSCs) have antitumour activity, and MSC‐derived exosomes play a role in the growth, metastasis and invasion of tumour cells. Additionally, glycoprotein A repetition predominant (GARP) promotes oncogenesis in breast cancer. Therefore, GARP is speculated to be a target gene for cancer therapy. We aimed to explore the therapy role of MSC‐derived exosomes targeting GARP in mouse colon cancer cell MC38. We successfully established a GARP knockdown system using three kinds of siRNA‐GARP in MSC cells. Exosomes were isolated from MSC and siGARP‐MSC cells, and verified by the exosome surface protein markers CD9, CD63 and CD81. GARP expression was significantly decreased in siGARP‐MSC exosomes compared with that of MSC exosomes. We found that siGARP‐MSC exosomes inhibited cell proliferation, migration and invasion of MC38 cells, using CCK‐8, colony formation, wound‐healing and Transwell invasion assays. Furthermore, siGARP‐MSC exosomes impeded IL‐6 secretion and partly inactivated JAK1/STAT3 pathway, measured using ELISA and RT‐qPCR. In conclusion, MSC‐derived exosomes targeting GARP are a potential strategy for cancer therapy.