MEG3-Mediated Oral Squamous-Cell-Carcinoma-Derived Exosomal miR-421 Activates Angiogenesis by Targeting HS2ST1 in Vascular Endothelial Cells.

Exosomal microRNAs (miRNAs) from cancer cells play a key role in mediating the oral squamous cell carcinoma (OSCC) microenvironment. The objective of this study was to investigate how the long non-coding RNA (lncRNA) MEG3 affects OSCC angiogenesis through exosomal miR-421. Global miRNA microarray analysis and quantitative real-time PCR (qRT-PCR) were performed to determine the level of miRNAs in OSCC cell-derived exosomes. Cell migration, invasion, tube formation, immunohistochemistry, and hemoglobin concentrations were used to study the effects of exosomal miR-421 in angiogenesis. Western blotting was used to determine the expression level of HS2ST1 and VEGFR2-related downstream proteins. MiRNA array and qRT-PCR identified the upregulation of miR-421 in OSCC cell-derived exosomes. Furthermore, exosomal miR-421 can be taken up by human umbilical vein endothelial cells (HUVECs) and then target HS2ST1 through VEGF-mediated ERK and AKT phosphorylation, thereby promoting HUVEC migration, invasion, and tube formation. Additionally, forced expression of the lncRNA MEG3 in OSCC cells reduced exosomal miR-421 levels and then increased HS2ST1 expression, thereby reducing the VEGF/VEGFR2 pathway in HUVECs. Our results demonstrate a novel mechanism by which lncRNA MEG3 can act as a tumor suppressor and regulate endothelial angiogenesis through the exosomal miR-421/HS2ST1 axis, which provides a potential therapeutic strategy for OSCC angiogenesis.

MicroRNA 421 induces the formation of high-invasive cell subsets of ovarian cancer from low-invasive cell subsets mediated by exosomes by activating the PI3K/AKT pathway.

Intratumoral heterogeneity (ITH) results in treatment failure in ovarian cancer (OC). Exosomes are related to the formation of a heterogeneous tumor microenvironment, and microRNAs play a crucial role in the progression of OC. Therefore, we aimed to explore the effect of exosomes and microRNA 421 (miR-421), which is mediated by exosomes, on ITH and the diagnosis of OC. Exosomes derived from A2780 cells with the highest (AHC) or lowest (ALC) invasive/migratory capacity cells (AHE/ALE) were extracted by differential centrifugation. We conducted a series of experiments to verify the role of AHE and miR-421 in promoting the transformation of low-invasive cells to high-invasive cells by regulating the PI3K/AKT pathway, and we also measured the levels of CA125 in serum exosomes. The results of assays showed that the AHE and miR-421, mediated by exosomes, significantly increased the malignancy of ALC cells by activating the PI3K/AKT pathway. The expression of miR-421 was significantly increased in the serum exosomes derived from high-grade serous ovarian cancer (HGSOC) patients. Our findings indicate that MiR-421, mediated by exosomes, could induce the transformation of highly invasive cell subpopulations from subpopulations of OC cells with low invasive potential by activating the PI3K/AKT signaling pathway.

Autophagy activated by GR/miR-421-3p/mTOR pathway as a compensatory mechanism participates in chondrodysplasia induced by prenatal caffeine exposure in male fetal rats.

Autophagy has been implicated in the developmental toxicity of multiple organs in offspring caused by adverse environmental conditions during pregnancy. We have previously found that prenatal caffeine exposure (PCE) can cause fetal overexposure to maternal glucocorticoids, leading to chondrodysplasia. However, whether autophagy is involved and what role it plays has not been reported. In this study, a PCE rat model was established by gavage of caffeine (120 mg/kg.d) on gestational day 9-20. The results showed that reduced cartilage matrix synthesis in male fetal rats in the PCE group was accompanied by increased autophagy compared to the control group. Furthermore, the expression of mTOR, miR-421-3p, and glucocorticoid receptor (GR) in male fetal rat cartilage of PCE group was increased. At the cellular level, we confirmed that corticosterone inhibited matrix synthesis in fetal chondrocytes while increasing autophagic flux. However, administration of autophagy enhancer (rapamycin) or inhibitor (bafilomycin A1 or 3-methyladenine) partially increased or further decreased aggrecan expression respectively. At the same time, we found that corticosterone could increase the expression of miR-421-3p through GR and target to inhibit the expression of mTOR, thereby enhancing autophagy. In conclusion, PCE can cause chondrodysplasia and autophagy enhancement in male fetal rats. Intrauterine high corticosterone activates GR/miR-421-3p signaling and down-regulates mTOR signaling in fetal chondrocytes, resulting in enhanced autophagy, which can partially compensate for corticosterone-induced fetal chondrodysplasia. This study confirmed the compensatory protective effect of autophagy on the developmental toxicity of fetal cartilage induced by PCE and its epigenetic mechanism, providing novel insights for exploring the early intervention and therapeutic target of fetal-originated osteoarthritis.

Exosomes regulate SIRT3-related autophagy by delivering miR-421 to regulate macrophage polarization and participate in OSA-related NAFLD.

PURPOSE: To analyze the role of and mechanism underlying obstructive sleep apnea (OSA)-derived exosomes in inducing non-alcoholic fatty liver (NAFLD). METHODS: The role of OSA-derived exosomes was analyzed in inducing hepatocyte fat accumulation in mice models both in vivo and in vitro. RESULTS: OSA-derived exosomes caused fat accumulation and macrophage activation in the liver tissue. These exosomes promoted fat accumulation; steatosis was more noticeable in the presence of macrophages. Macrophages could internalize OSA-derived exosomes, which promoted macrophage polarization to the M1 type. Moreover, it inhibited sirtuin-3 (SIRT3)/AMP-activated protein kinase (AMPK) and autophagy and promoted the activation of nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasomes. The use of 3-methyladenine (3-MA) to inhibit autophagy blocked NLRP3 inflammasome activation and inhibited the M1 polarization of macrophages. miR-421 targeting inhibited SIRT3 protein expression in the macrophages. miR-421 was significantly increased in OSA-derived exosomes. Additionally, miR-421 levels were increased in OSA + NAFLD mice- and patient-derived exosomes. In the liver tissues of OSA and OSA + NAFLD mice, miR-421 displayed similar co-localization with the macrophages. Intermittent hypoxia-induced hepatocytes deliver miR-421 to the macrophages via exosomes to inhibit SIRT3, thereby participating in macrophage M1 polarization. After OSA and NAFLD modeling in miR-421-/- mice, liver steatosis and M1 polarization were significantly reduced. Additionally, in the case of miR-421 knockout, the inhibitory effects of OSA-derived exosomes on SIRT3 and autophagy were significantly alleviated. Furthermore, their effects on liver steatosis and macrophage M1 polarization were significantly reduced. CONCLUSIONS: OSA promotes the delivery of miR-421 from the hepatocytes to macrophages. Additionally, it promotes M1 polarization by regulating the SIRT3/AMPK-autophagy pathway, thereby causing NAFLD.

MiR-421 mediates PM2.5-induced endothelial dysfunction via crosstalk between bronchial epithelial and endothelial cells.

OBJECTIVE: PM2.5 is closely linked to vascular endothelial injury and has emerged as a major threat to human health. Our previous research indicated that exposure to PM2.5 induced an increased release of miR-421 from the bronchial epithelium. However, the role of miR-421 in PM2.5-induced endothelial injury remains elusive. MATERIALS AND METHODS: We utilized a subacute PM2.5-exposure model in mice in vivo and an acute injury cell model in vitro to simulate PM2.5-associated endothelial injury. We also used quantitative real-time polymerase chain reaction, western blot, enzyme-linked immunosorbent assay, and immunohistochemistry to investigate the role of miR-421 in PM2.5-induced endothelial injury. RESULTS: Our findings reveal that inhibition of miR-421 attenuated PM2.5-induced endothelial injury and hypertension. Mechanistically, miR-421 inhibited the expression of angiotensin-converting enzyme 2 (ACE2) in human umbilical vein endothelial cells and upregulated the expression of the downstream molecule inducible nitric oxide synthase (iNOS), thereby exacerbating PM2.5-induced endothelial injury. CONCLUSIONS: Our results indicate that PM2.5 exposure facilitates crosstalk between bronchial epithelial and endothelial cells via miR-421/ACE2/iNOS signaling pathway, mediating endothelial damage and hypertension. MiR-421 inhibition may offer a new strategy for the prevention and treatment of PM2.5-induced vascular endothelial injury.

CircNRD1 elevates THAP domain containing 11 through sequestering microRNA-421 to inhibit gastric cancer growth and tumorigenesis.

We explored the role and mechanism of circular RNAcircNRD1 in gastric cancer (GC) progression, aiming to identify new bio-markers for the treatment and prognosis of GC patients. The RNA expression was examined by reverse transcription-quantitative polymerase chain reaction. Cell proliferation, migration and invasion were analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, scratch assay and transwell assay. Western blot assay was conducted for protein expression measurement. Dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays were conducted to verify the interaction between microRNA-421 (miR-421) and circNRD1 or THAP domain containing 11 (THAP11). Xenograft tumor model was established to perform in vivo experiments. CircNRD1 was notably downregulated in GC tissues and cell lines. Additionally, decreased circNRD1 level was closely associated with advanced tumor stage and dismal prognosis in GC patients. CircNRD1 overexpression suppressed the proliferation and metastasis of GC cells. CircNRD1 acted as a molecular sponge for miR-421 in GC cells, and the antitumor impacts of circNRD1 overexpression in GC cells could be alleviated by miR-421 overexpression. miR-421 directly targeted THAP11, and circNRD1 could up-regulate THAP11 expression in GC cells through sponging miR-421. THAP11 knockdown reversed circNRD1 overexpression-induced tumor suppressing effects in GC cells. CircNRD1 overexpression significantly blocked tumor growth in vivo. CircNRD1 suppressed the proliferation and metastasis of GC cells in vitro and blocked tumor growth in vivo via modulating miR-421/THAP11 axis.

Evaluation of angiotensin converting enzyme 2 (ACE2), angiotensin II (Ang II), miR-141-3p, and miR-421 levels in SARS-CoV-2 patients: a case-control study.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly contagious virus that uses angiotensin converting enzyme 2 (ACE2), a pivotal member of the renin-angiotensin system (RAS), as its cell-entry receptor. Another member of the RAS, angiotensin II (Ang II), is the major biologically active component in this system. There is growing evidence suggesting that serum miRNAs could serve as prognostic biomarkers for SARS-CoV-2 infection and regulate ACE2 expression. Therefore, the aim of this study is to evaluate the changes in the serum levels of sACE2 and Ang II, as well as the expression level of miR-141-3p and miR-421 in SARS-CoV-2 positive and negative subjects. METHODS: In the present study, the serum levels of sACE2 and Ang II were measured in 94 SARS-CoV-2 positive patients and 94 SARS-CoV-2 negative subjects with some symptoms similar to those of SARS-CoV-2 positive patients using the ELISA method. In addition, the expression level of miR-141-3p and miR-421 as ACE2 regulators and biomarkers was evaluated using quantitative real-time PCR (qRT-PCR) method. RESULTS: The mean serum sACE2 concentration in the SARS-CoV-2-positive group was 3.268 ± 0.410 ng/ml, whereas in the SARS-CoV-2 negative group, it was 3.564 ± 0.437 ng/ml. Additionally, the mean serum Ang II level in the SARS-CoV-2 positive and negative groups were 60.67 ± 6.192 ng/L and 67.97 ± 6.837 ng/L, respectively. However, there was no significant difference in the serum levels of sACE2 (P value: 0.516) and Ang II (P value: 0.134) between the SARS-CoV-2 positive and negative groups. Meanwhile, our findings indicated that the expression levels of miR-141-3p and miR-421 in SARS-CoV-2 positive group were significantly lower and higher than SARS-CoV-2 negative group, respectively (P value < 0.001). CONCLUSIONS: Taken together, the results of this study showed that the serum levels of sACE2 and Ang II in SARS-CoV-2 positive and negative subjects were not significantly different, but the expression levels of miR-141-3p and miR-421 were altered in SARS-CoV-2 positive patients which need more investigation to be used as biomarkers for COVID-19 diagnosis.

Downregulated antisense lncRNA ENTPD3-AS1 contributes to the development of lung adenocarcinoma.

The poor outcome of patients with lung adenocarcinoma (LUAD) highlights the importance to identify novel effective prognostic markers and therapeutic targets. Long noncoding RNAs (lncRNAs) have generally been considered to serve important roles in tumorigenesis and the development of various types of cancer, including LUAD. Here, we aimed to investigate the role of ENTPD3-AS1 (ENTPD3 Antisense RNA 1) in LUAD and to explore its potential mechanisms by performing comprehensive bioinformatic analyses. The regulatory effect of ENTPD3-AS1 on the expression of NR3C1 was validated by siRNA-based silencing. The effect of miR-421 on the modulation of NR3C1 was determined by miRNA mimics and inhibitors transfection. ENTPD3-AS1 was expressed at lower levels in tumor parts and negatively correlated with unfavorable prognosis in LUAD patients. It exerted functions as a tumor suppressor gene by competitively binding to oncomir, miR-421, thereby attenuating NR3C1 expression. Transfection of lung cancer A549 cells with miR-421 mimics decreased the expression of NR3C1. Transfection of lung cancer A549 cells with miR-421 inhibitors increased the expression of NR3C1 with lower cellular functions as proliferation and migration via epithelial-mesenchymal transition. In addition, inhibition of ENTPD3-AS1 by siRNA transfection decreased the levels of NR3C1, supporting the ENTPD3-AS1/miR-421/NR3C1 cascade. Moreover, the bioinformatic analysis also showed that ENTPD3-AS1 could interact with the RNA-binding proteins (RBPs), CELF2 and QKI, consequently regulating RNA expression and processing. Taken together, we identified that ENTPD3-AS1 and its indirect target NR3C1 can act as novel biomarkers for determining the prognosis of patients with LUAD, and further study is required.

Overexpression of circZNF720 Inhibits Hepatocellular Carcinoma Progression by Regulating miR-421/MAPK9.

Hepatocellular carcinoma (HCC) is characterized by high incidence, high death rate and poor long term prognosis. Abnormal expression of circular RNA (circRNA) has been demonstrated to be closely associated with malignant progression of HCC. However, the effect of circZNF720 on HCC is largely incompletely understood. This study sought to explore the effect of circZNF720 on the progression of HCC by CCK-8, transwell, and flow cytometry experiments. The prognosis of circZNF720 on patients was analyzed by Kaplan-Meier curve. Then, the regulatory mechanisms among circZNF720, miR-421, and MAPK9 were identified by qRT-PCR, dual-luciferase reporter assay, and RIP analysis, respectively. The outcomes of this study disclosed that circZNF720 was lowly expressed in HCC, and this low expression was strongly linked with a poor prognosis for HCC patients. Overexpression of circZNF720 inhibited the proliferation, migration, and invasion of cells, and increased the apoptosis of cells. Mechanistic studies revealed that circZNF720 targeted miR-421 and regulated the development of HCC by acting as a sponge for miRNAs. In addition, MAPK9 was a downstream target of miR-421, and circZNF720 introduction hampered HCC progression by upregulating MAPK9 expression through sponging miR-421. In conclusion, circZNF720 restrained HCC progression by targeting miR-421/MAPK9. This study provides a novel target for the treatment of HCC.

Regulation of PM2.5 on mitochondrial damage in H9c2 cells through miR-421/SIRT3 pathway and protective effect of miR-421 inhibitor and resveratrol.

Fine particulate matter (PM2.5) exposure is associated with cardiovascular disease (CVD) morbidity and mortality. Mitochondria are sensitive targets of PM2.5, and mitochondrial dysfunction is closely related to the occurrence of CVD. The epigenetic mechanism of PM2.5-triggered mitochondrial injury of cardiomyocytes is unclear. This study focused on the miR-421/SIRT3 signaling pathway to investigate the regulatory mechanism in cardiac mitochondrial dynamics imbalance in rat H9c2 cells induced by PM2.5. Results illustrated that PM2.5 impaired mitochondrial function and caused dynamics homeostasis imbalance. Besides, PM2.5 up-regulated miR-421 and down-regulated SIRT3 gene expression, along with decreasing p-FOXO3a (SIRT3 downstream target gene) and p-Parkin expression and triggering abnormal expression of fusion gene OPA1 and fission gene Drp1. Further, miR-421 inhibitor (miR-421i) and resveratrol significantly elevated the SIRT3 levels in H9c2 cells after PM2.5 exposure and mediated the expression of SOD2, OPA1 and Drp1, restoring the mitochondrial morphology and function. It suggests that miR-421/SIRT3 pathway plays an epigenetic regulatory role in mitochondrial damage induced by PM2.5 and that miR-421i and resveratrol exert protective effects against PM2.5-incurred cardiotoxicity.

Circ_MACF1 targets miR-421 to upregulate FMO2 to suppress paclitaxel resistance and malignant cellular behaviors in lung adenocarcinoma.

BACKGROUND: Chemoresistance remains an enormous challenge in the treatment of lung adenocarcinoma (LADC). Circular RNAs (circRNAs) exhibit important regulation in tumor progression and chemoresistance. This research focused on exploring the regulatory function and mechanism of circ_MACF1 (has_circ_0011780) in paclitaxel (PTX) resistance in LADC. METHODS: Circ_MACF1, miR-421 and flavin-containing monooxygenase 2 (FMO2) were determined by RT-qPCR. MTT was applied to detect IC50 of PTX. The proliferation analysis was performed using EdU and colony formation assay. Cell apoptosis and motility were examined using flow cytometry and transwell assay, respectively. Western blot was administered for protein detection. A dual-luciferase reporter assay was performed for confirming target interaction. PTX sensitivity in vivo was researched via xenograft tumor assay. RESULTS: Expression of circ_MACF1 was decreased in PTX-resistant LADC tissues and cells. Circ_MACF1 overexpression reduced chemoresistance, proliferation, motility and accelerated apoptosis in PTX-resistant LADC cells. Circ_MACF1 targeted miR-421 and miR-421 upregulation reverted circ_MACF1-evoked effects. FMO2 served as a downstream target of miR-421 and circ_MACF1 sponged miR-421 to elevate the expression of FMO2. MiR-421 enhanced PTX resistance and LADC progression via targeting FMO2. FMO2 knockdown enhanced IC50 of PTX and cell proliferation. In vivo, circ_MACF1 elevated PTX sensitivity of LADC by mediating miR-421/FMO2 axis. CONCLUSION: These findings elucidated that circ_MACF1 inhibited PTX resistance by absorbing miR-421 to upregulate FMO2 in LADC.

MEG3 Regulates CSE-Induced Apoptosis by Regulating miR-421/DFFB Signal Axis.

Introduction: Chronic obstructive pulmonary disease (COPD) is a common respiratory disease with irreversible and progressive obstruction of airflow. Currently, there are no clinically available treatments to prevent COPD progression. Apoptosis of human lung microvascular endothelial cells (HPMECs) and bronchial epithelial cells (HBECs) is often observed in COPD, but its pathogenesis has not been fully elucidated. LncRNA maternally expressed gene 3 (MEG3) is closely related to CSE-induced apoptosis, but the specific mechanism of MEG3 in COPD is still unknown. Methods: In the present study, cigarette smoke extract (CSE) is used to treat HPMECs and HBECs. Flow cytometry assay is used to detect the apoptosis of these cells. The expression of MEG3 in CSE-treated HPMECs and HBECs is detected by qRT-PCR. LncBase v.2 is used to predict miRNAs binding to MEG3, and miR-421 is found to bind to MEG3. Dual luciferase report analysis and RNA immunoprecipitation experiment jointly clarified the binding relationship between MEG3 and miR-421. Results: MiR-421 was downregulated in CSE-treated HPMECs/HBECs, and miR-421 overexpression mitigated CSE-induced apoptosis in these cells. Subsequently, DFFB was found to be directly targeted by miR-421. The overexpression of miR-421 dramatically reduced the expression level of DNA fragmentation factor subunit beta (DFFB). DFFB was found downregulated in CSE-treated HPMECs and HBECs. MEG3 contributed to the apoptosis of HPMECs and HBECs induced by CSE by regulating the miR-421/DFFB axis. Conclusion: This study presents a new perspective on the diagnosis and treatment of COPD caused by CSE.

Mir-421 and mir-550a-1 are potential prognostic markers in esophageal adenocarcinoma.

OBJECTIVE: To identify the prognostic indicators of esophageal adenocarcinoma (EAC) for future EAC diagnosis and treatment. METHODS: The EAC dataset from The Cancer Genome Atlas was screened for differentially expressed microRNAs (miRNAs) and mRNAs associated with EAC. Weighted gene coexpression network analysis was performed to cluster miRNAs or mRNA with similar expression patterns to identify the miRNAs or mRNA that are highly associated with EAC. Prognostic miRNAs for overall survival (OS) were identified using Cox proportional-hazards regression analysis and least absolute shrinkage and selection operator based on survival duration and status. Two types of miRNAs were selected to develop a prognostic signature model for EAC using multiple Cox regression analysis. Furthermore, the signature was validated using internal validation sets 1 and 2. The receiver operating characteristic curve and concordance index were used to evaluate the accuracy of the signature and validation sets. The expression of miR-421, miR-550a-3p, and miR-550a-5p was assessed using quantitative polymerase chain reaction (qPCR). The proliferation, invasion, and migration of EAC cells were assessed using CCK8 and transwell assays. The OS of target mRNAs was assessed using Kaplan-Meier analysis. Functional enrichment analysis of the target mRNAs was performed using Metascape. RESULTS: The prognostic signature and validation sets comprising mir-421 and mir-550a-1 had favorable predictive power in OS. Compared with the patients with EAC in the high-expression group, those assigned to the low-expression group displayed increased OS according to survival analysis. Differential and qPCR analysis showed that miR-421, miR-550a-3p, and miR-550a-5p were highly expressed in the EAC tissues and cell lines. Moreover, the downregulation of miR-421 and miR-550a-3p with inhibitor markedly suppressed the proliferation, invasion, and migration in OE33 cells compared with the negative control. A total of 20 target mRNAs of three miRNAs were predicted, among which seven target mRNAs-ASAP3, BCL2L2, LMF1, PPM1L, PTPN21, SLC18A2, and NR3C2-had prognostic value; PRKACB, PDCD4, RPS6KA5, and BCL2L2 were enriched in the miRNA cancer pathway. CONCLUSION: Prognostic indicators of EAC may be useful in future EAC diagnosis and treatment.

CircSCNN1A is a tumor suppressor in renal cell carcinoma via inducing the upregulation of MPP7 by the sponge effect on miR-421.

BACKGROUND: Circular RNAs (circRNAs) function as oncogenic factors or tumor repressors in variety of human malignancies. CircRNA Sodium Channel Epithelial 1 Subunit Alpha (circSCNN1A, hsa_circ_0025135) was downregulated in renal cell carcinoma (RCC). This study performed further research of circSCNN1A in RCC. METHODS: The level detection for circSCNN1A, microRNA-421 (miR-421) or Membrane Palmitoylated Protein 7 (MPP7) was conducted by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell behaviors were analyzed by Cell counting kit-8 (CCK-8) assay for cell viability, EDU assay for cell proliferation, flow cytometry for cell apoptosis, transwell assay for cell invasion and tube formation assay for angiogenesis. The protein expression was determined using western blot. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and pull-down assay were applied to validate the interaction between targets. In vivo research was performed by xenograft tumor assay. RESULTS: The level of circSCNN1A was significantly downregulated in RCC tissues and cells. RCC cell proliferation, invasion and angiogenesis were reduced but apoptosis was promoted by circSCNN1A overexpression. Interestingly, circSCNN1A could interact with miR-421. Overexpression of miR-421 has reversed the anti-tumor function of circSCNN1A in RCC cells. MPP7 served as a target of miR-421, and MPP7 inhibited the malignant phenotypes of RCC cells. In addition, miR-421 downregulation induced the inhibitory effect on the RCC development via elevating the MPP7 level. Moreover, RCC tumorigenesis was suppressed by circSCNN1A through the miR-421/MPP7 axis in vivo. CONCLUSION: The experimental data revealed that circSCNN1A upregulated the expression of MPP7 via sponging miR-421, then inhibiting the progression of RCC.

Constructing a novel competing Endogenous RNAs network based on NR3C1 and X-linked inhibitor of apoptosis protein genes reveals potential prognostic biomarkers in colorectal cancer.

Background: Long noncoding RNAs (lncRNAs) have been recognized as the main modulatory molecules in various cancers and perform as competing endogenous RNAs (ceRNAs). The nuclear hormone receptor superfamily of ligand-activated transcription factors (NR3C1) regulates numerous proliferative and metabolic processes such as tumorigenesis and metabolic diseases. Furthermore, X-linked inhibitor of apoptosis protein (XIAP) belongs to a family of the inhibitors of apoptosis proteins, is located downstream of the glucocorticoid receptor (GR or NR3C1) pathway, and cooperates with GR to suppress apoptosis. However, the underlying mechanisms of NR3C1 and XIAP in colorectal cancer (CRC) remain mainly unclear. This research aims to clarify the potential RNA biomarkers and to construct a novel ceRNA network in CRC. Materials and Methods: Multistep bioinformatics methods such as Lnc2cancer and miRDB databases were applied to identify candidate lncRNAs and miRNAs. The interaction energy between lncRNAs, NR3C1, and XIAP genes was analyzed by the LncRRIsearch database. Plus, microRNAs and lncRNA were evaluated via the Diana tools database to select microRNAs with the most binding scores. Quantitative reverse transcription-polymerase chain reaction (QRT-PCR) was applied to verify RNA molecules' expression levels and their association with the clinicopathological factors in 30 CRC tissues compared to 30 adjacent tissues. Results: QRT-PCR showed upregulation of KCNQ1OT1, NR3C1, and XIAP and downregulation of miR-421. The ceRNA network was constructed with 17 lncRNAs, 2 mRNAs, and 42 miRNAs. Thus, we explained the potential interactions between KCNQ1OT1 and miR-421 with NR3C1 and XIAP genes. Conclusion: Our study represents potential prognostic biomarkers and a new ceRNA network for further study in CRC.

Cancer-associated fibroblast-secreted miR-421 promotes pancreatic cancer by regulating the SIRT3/H3K9Ac/HIF-1α axis.

This study was designed to explore the effects of exosomal miR-421 secreted by cancer-associated fibroblasts (CAFs) on pancreatic cancer (PC) progression and the mechanisms involved. CAFs and exosomes (exos) were isolated and identified. PC cells were treated with CAF-derived exos (CAF-exos). Western blotting and quantitative polymerase chain reaction (qPCR) were used to measure miR-421, sirtuin-3 (SIRT3), and hypoxia duciblefactors-1 alpha (HIF-1α) levels. Cell counting kit-8 (CCK-8), wound-healing, and transwell migration assays were used to measure proliferation, migration, and invasion abilities of the cells. Dual-luciferase assay and RNA immunoprecipitation (RIP) experiment analyzed the relationship between miR-421 and SIRT3. Chromatin immunoprecipitation (f)-verified H3K9Ac enrichment in the HIF-1α promoter region. In vivo tumorigenesis experiments were performed to further explore the effects of exosomal miR-421 from CAFs on PC. CAFs and exos were successfully isolated. CAF-exo-treated PC cells highly expressed miR-421 and had increased cell proliferation, migration, and invasion abilities. Knocking down miR-421 increased the expression of SIRT3. SIRT3 is a target of miR-421, and inhibiting the expression of SIRT3 reversed the negative effects of miR-421 knockdown on PC cell. Knocking down miR-421 in CAF-exo inhibited the expression of HIF-1α in PC cells. Moreover, SIRT3-mediated HIF-1α expression by regulating H3K9Ac. HIF-1α overexpression reversed the inhibiting effects of SIRT3 overexpression on PC progression and counteracted the inhibiting effects of miR-421 knockdown on glycolysis. Moreover, in vivo tumorigenesis experiments showed that knocking down miR-421 attenuated CAF-exo induced tumor growth. Exosomal miR-421 from CAFs promoted PC progression by regulating the SIRT3/H3K9Ac/HIF-1α axis. This study provided insights into the molecular mechanism of PC.

The protective effects of lncRNA ZFAS1/miR-421-3p/MEF2C axis on cerebral ischemia-reperfusion injury.

LncRNA ZNFX1 antisense RNA 1 (ZFAS1) could improve neuronal damage and inhibit inflammation and apoptosis. We conducted an in-depth exploration on the protective mechanism of ZFAS1 in cerebral ischemia-reperfusion injury. Overexpressed or silenced plasmids of ZFAS1 were transfected into the cells to analyze the effects of oxygen-glucose deprivation/reperfusion (OGD/R) treatment on the viability, apoptosis and related gene expressions of Neuro-2a cell by performing MTT assay, flow cytometry, qRT-PCR, and Western blot. Bioinformatic analysis, qRT-PCR, dual-luciferase reporter assay and RNA immunoprecipitation were used to screen and verify the miRNA(s) which could competitively bind with ZFAS1 and downstream mRNA(s) targeted by the miRNA(s). The effects of ZFAS1 and the above target miRNA(s) or gene(s) on the apoptosis of OGD/R-injured cells, apoptosis-related proteins, inflammatory factors and p65/IκBα pathway were further verified via the rescue test. The results from the middle cerebral artery occlusion (MCAO) mouse model in vivo were consistent with those from the cellular experiments. The expression of lncRNA ZFAS1 in OGD/R-injured cells was inhibited, and the up-regulation of ZFAS1 protected Neuro-2a cells. MiR-421-3p was predicted to be the target miRNA of ZFAS1 and could offset the protective effect of ZFAS1 overexpression on OGD/R-injured cells following its up-regulation. MEF2C, which was the downstream target gene of miR-421-3p, reversed the OGD/R-induced enhanced cell damage caused by miR-421-3p mimic when MEF2C was overexpressed. In in vivo studies, ZFAS1 overexpression reduced brain tissue infarction, apoptosis and gene regulation caused by MCAO, while miR-421-3p mimic had the opposite effect. Collectively, the regulation of lncRNA ZFAS1/miR-421-3p/MEF2C axis showed protective effects on cerebral ischemia-reperfusion injury.

Extracellular vesicles enclosed-miR-421 suppresses air pollution (PM2.5 )-induced cardiac dysfunction via ACE2 signalling.

Air pollution, via ambient PM2.5, is a big threat to public health since it associates with increased hospitalisation, incidence rate and  mortality of cardiopulmonary injury. However, the potential mediators of pulmonary injury in PM2.5 -induced cardiovascular disorder are not fully understood. To investigate a potential cross talk between lung and heart upon PM2.5 exposure, intratracheal instillation in vivo, organ culture ex vivo and human bronchial epithelial cells (Beas-2B) culture in vitro experiments were performed respectively. The exposed supernatants of Beas-2B were collected to treat primary neonatal rat cardiomyocytes (NRCMs). Upon intratracheal instillation, subacute PM2.5 exposure caused cardiac dysfunction, which was time-dependent secondary to lung injury in mice, thereby demonstrating a cross-talk between lungs and heart potentially mediated via small extracellular vesicles (sEV). We isolated sEV from PM2.5 -exposed mice serum and Beas-2B supernatants to analyse the change of sEV subpopulations in response to PM2.5 . Single particle interferometric reflectance imaging sensing analysis (SP-IRIS) demonstrated that PM2.5 increased CD63/CD81/CD9 positive particles. Our results indicated that respiratory system-derived sEV containing miR-421 contributed to cardiac dysfunction post-PM2.5 exposure. Inhibition of miR-421 by AAV9-miR421-sponge could significantly reverse PM2.5 -induced cardiac dysfunction in mice. We identified that cardiac angiotensin converting enzyme 2 (ACE2) was a downstream target of sEV-miR421, and induced myocardial cell apoptosis and cardiac dysfunction. In addition, we observed that GW4869 (an inhibitor of sEV release) or diminazene aceturate (DIZE, an activator of ACE2) treatment could attenuate PM2.5 -induced cardiac dysfunction in vivo. Taken together, our results suggest that PM2.5 exposure promotes sEV-linked miR421 release after lung injury and hereby contributes to PM2.5 -induced cardiac dysfunction via suppressing ACE2.

CircSEC24A (hsa_circ_0003528) interference suppresses epithelial-mesenchymal transition of hepatocellular carcinoma cells via miR-421/MMP3 axis.

Accumulating evidence indicates that circular RNAs (circRNAs) function as conclusive modulators in diverse tumors, including in hepatocellular carcinoma (HCC). Nonetheless, knowledge of the latent mechanisms involving circRNAs in HCC development is insufficient. circSEC24A (hsa_circ_0003528) was discovered by microarray analysis of patients with HCC. Binding sites between circSEC24A, miR-421, miR-421 and matrix metalloproteinase 3 (MMP3) were predicted using online bioinformatics tools. Interactions involving miRNA and target genes or circRNAs were verified by luciferase reporter-gene and RNA pull-down assays. Two HCC cell lines (HCCLM3 and Hep3B) and normal THLE-2 liver cells were used for in vitro experiments. miRNA and mRNA expression levels were detected by RT-qPCR, and protein expression was measured by western blotting. Cell proliferation was evaluated using Cell Counting Kit 8 (CCK-8) assays along with colony formation assays. Cell invasion and migration were determined using the Transwell and wound healing migration assays. A xenograft model was used to evaluate the role of circSEC24A in vivo. circSEC24A expression was significantly upregulated in HCCLM3 and Hep3B cells. Silencing circSEC24A mitigated the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of HCC cells, which was abrogated by downregulation of miR-421. Meanwhile, MMP3 could bind to miR-421 to decrease the functional effects of miR-421 and induce tumor metastasis. Knockdown of cicSEC24A suppressed tumor growth in vivo. circSEC24A interference suppressed HCC cell EMT by sponging miR-421, further regulating MMP3, and inhibiting tumor growth in vivo. Therefore, circSEC24A could represent a potential target for HCC patient treatment.