PGM5-AS1 Promotes Progression of Diffuse Large B-Cell Lymphoma and Immune Escape by Regulating miR-503-5p.

Purpose: Diffuse large B-cell lymphoma (DLBCL) is a prevalent malignant condition with a dismal prognosis. LncRNA PGM5 antisense RNA 1 (PGM5-AS1) appears to be intricately involved in the progression of DLBCL, yet the modulatory mechanism remains unclear. The purpose of this study was to explore the expression of lncRNA PGM5-AS1 in DLBCL and its effect on the disease progression of DLBCL, as well as to explore its mechanisms. Patients and Methods: A total of 35 patients were included in the study. The expression levels of PGM5-AS1 and miR-503-5p in DLBCL tumor tissues and cell lines were detected by RT-qPCR. Cell proliferation was assessed using CCK8. Apoptosis rate was determined by flow cytometry. Cell invasion was examined by transwell assays. The specific interaction between PGM5-AS1 and miR-503-5p was verified through dual luciferase reporter gene assays. The immune related factors were detected by ELASA kits. The CD8+ T cells cytotoxicity was evaluated by LDH cytotoxicity kit. Results: In DLBCL tumor tissues and cells, upregulated PGM5-AS1 expression, downregulated miR-503-5p expression, and elevated PD-L1 expression were observed. PGM5-AS1 functioned as a regulator in controlling DLBCL cell proliferation, apoptosis, and invasion by downregulating miR-503-5p expression. When CD8+ T cells were co-cultured with cells transfected with si-PGM5-AS1, the secretion of immunoregulatory factors increased, and the cytotoxicity of CD8+ T cells increased. These effects were mitigated by miR-503-5p inhibitors. Conclusion: PGM5-AS1 accelerated DLBCL development and facilitated tumor immune escape through the miR-503-5p. Our discoveries offered an insight into lncRNA PGM5-AS1 serving as a prospective therapeutic target for DLBCL.

LncRNA CYTOR knockdown inhibits tumor development via regulating miR-503-5p/PCSK9 in lung adenocarcinoma.

BACKGROUND: The intricate biological mechanism underlying lung adenocarcinoma (LUAD), characterized by a deficiency of distinctive biomarkers, remain elusive. The presence of Long non-coding RNAs (lncRNAs) have been established to play a role in carcinogenesis. Nevertheless, the regulatory effects and mechanisms of lncRNA CYTOR in LUAD have yet to be elucidated. METHODS: In this study, RT-qPCR and Western blot were adopted to examine gene mRNA and protein expression, respectively. Cell proliferation was evaluated by CCK-8 assays. Transwell was performed to assay cell migration and invasion. The function of CYTOR in vivo was investigated through a xenograft animal model. RESULTS: We observed an apparent upregulation of CYTOR in LUAD. Silencing CYTOR significantly reduced proliferation, migration, and invasion capabilities of LUAD cells. Mechanism analysis indicated that CYTOR targeted the miR-503-5p/PCSK9 axis. Additionally, inhibiting of miR-503-5p partially reversed the inhibitory effects of CYTOR silencing on the malignant progression of LUAD cells. Animal experiments revealed that CYTOR/miR-503-5p/PCSK9 curbed tumor formation of nude mice in vivo. CONCLUSION: These findings demonstrated that lncRNA CYTOR acted as an oncogene in LUAD, regulating tumor malignant progression through the miR-503-5p/PCSK9 axis. This study unveiled a new regulation mechanism of LUAD progression, offering potential therapeutic targets for LUAD.

Upregulation of circ-IGF1R increased therapeutic effect of hypoxia-pretreated ADSC-derived extracellular vesicle by regulating miR-503-5p/HK2/VEGFA axis.

Diabetes mellitus is a major cause of blindness and chronic ulcers in the working-age population worldwide. Wound healing is deeply dependent on neovascularization to restore blood flow. Former research has found that differentially expressed circular RNAs (circRNAs) are associated with hyperglycaemia-induced endothelial cell damage, and hypoxia-pretreated adipose-derived stem cells (ADSCs)-extracellular vesicle (HEV) transplants have a more therapeutic effect to enhance wound healing in diabetic mice by delivery circRNA. The current investigation employed high-throughput sequencing to identify circRNAs that are abnormally expressed between EV and HEV. The regulatory mechanism and predicted targets of one differentially expressed circRNA, circ-IGF1R, were investigated utilizing bioinformatics analyses, luciferase reporter assays, angiogenic differentiation assays, flow cytometric apoptosis analysis and RT-qPCR. Circ-IGF1R expression increased in HEV, and downregulation of circ-IGF1R suppressed and reversed the promotion effect of HEV on angiogenesis in ulcerated tissue. Bioinformatics analyses and luciferase reporter assays confirmed that miR-503-5p was the downstream target of circ-IGF1R, and inhibiting miR-503-5p restored the promotion effect of HEV on angiogenesis after circ-IGF1R silence. The study also found that miR-503-5p can interact with 3'-UTR of both HK2 and VEGFA. Overexpression of HK2 or VEGFA restored the promotion effect of HExo on angiogenesis after circ-IGF1R silence. Overexpression miR-503-5p or silence HK2/VEGFA reversed the protective effect of circ-IGF1R to MLMECs angiogenic differentiation. Overexpression of circ-IGF1R increased the protective effect of HEV on the promotion of wound healing in mice with diabetes. Circ-IGF1R promotes HIF-1α expression through miR-503-5p sponging. Our data demonstrate that circ-IGF1R overexpression EVs from ADSCs suppress high glucose-induced endothelial cell damage by regulating miR-503-5p/HK2/VEGFA axis.

MiR-503-5p alleviates peripheral neuropathy-induced neuropathic pain in T2DM mice by regulating SEPT9 to inhibit astrocyte activation.

Diabetic peripheral neuropathy (DPN) is a common complication of type 2 diabetes mellitus (T2DM) that causes peripheral and autonomic nervous system dysfunction. Dysregulation of miRNAs plays a crucial role in DPN development. However, the role of miR-503-5p in DPN remains unknown. Herein, T2DM mice (db/db) were used as a DPN model in vivo, and astrocytes isolated from db/db mice were induced with high glucose levels as a DPN model in vitro. MiR-503-5p expression was analyzed using qRT-PCR. GFAP, MCP-1, and SEPT9 protein levels were analyzed using western blotting and immunofluorescence. Luciferase assays were performed to investigate the interaction between miR-503-5p and SEPT9. We found that miR-503-5p expression decreased in the spinal cord of DPN model mice and astrocytes treated with high glucose (HG). The db/db mice displayed higher body weight and blood glucose, lower mechanical withdrawal threshold and thermal withdrawal latency, and higher GFAP and MCP-1 protein levels than db/m mice. However, tail vein injection of agomiR-503-5p remarkably reversed these parameters, whereas antigomiR-503-5p enhanced them. HG markedly facilitated GFAP and MCP-1 protein expression in astrocytes, whereas miR-503-5p mimic or inhibitor transfection markedly blocked or elevated GFAP and MCP-1 protein expression, respectively, in astrocytes with HG. SEPT9 was a target of miR-503-5p. In addition, SEPT9 protein levels were found to be elevated in db/db mice and astrocytes treated with HG. Treatment with agomiR-503-5p and miR-503-5p mimic was able to reduce SEPT9 protein levels, whereas treatment with antigomiR-503-5p and miR-503-5p inhibitor led to inhibition of the protein. Furthermore, SEPT9 overexpression suppressed the depressing effect of miR-503-5p overexpression in astrocytes subjected to HG doses. In conclusion, miR-503-5p was found to alleviate peripheral neuropathy-induced neuropathic pain in T2DM mice by regulating SEPT9 expression.

Exosome-delivered circRNA circSYT15 contributes to cisplatin resistance in cervical cancer cells through the miR-503-5p/RSF1 axis.

The development of chemotherapy resistance is a major obstacle for cervical cancer (CC) patients. Exosome-mediated transfer of circular RNAs (circRNAs) was found to have relevance to the CC. This study is designed to explore the role and mechanism of exosomal circRNA synaptotagmin 15 (circSYT15) on cisplatin (DDP) resistance in CC. Cell proliferation ability and apoptosis rate were detected by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), colony formation, and flow cytometry assays. CircSYT15, microRNA-503-5p (miR-503-5p), Remodeling spacing factor 1 (RSF1) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Exosomes were analyzed by a transmission electron microscope and nanoparticle tracking analysis. CD63, CD81, TSC101, Bcl-2, Bax, C-caspase 3, and RSF1 protein levels were examined by western blot assay. The binding between miR-503-5p and circSYT15 or RSF1 was predicted by circBank or Starbase and then verified by a dual-luciferase reporter and RNA Immunoprecipitation (RIP). The biological role of exosomal circSYT15 in DDP resistance of CC in vivo. CircSYT15 was upregulated in the DDP-resistant CC cells and exosomes isolated from DDP-resistant CC cells. CircSYT15 knockdown repressed the proliferation and drug resistance of CC and induced apoptosis in CC cells. Exosomes shuttled circSYT15 act as a sponge to affect RSF1 expression, thereby promoting proliferation and drug resistance and repressing apoptosis of sensitive CC cells. Exosomal circSYT15 boost DDP resistance of cervical cancer in vivo. Exosome-mediated transfer of circSYT15 enhanced DDP resistance in CC partly by targeting the miR-503-5p/RSF1 axis, providing a foundation for future clinical applications of CC drug resistance.

Long Non-Coding RNA ZSCAN16-AS1 Promotes the Malignant Progression of Melanoma Through Regulating the miR-503-5p/ARL2 Axis.

Background: LncRNA zinc finger and SCAN domain containing 16 antisense RNA 1 (ZSCAN16-AS1), a newly identified lncRNA, has been proven to accelerate hepatocellular carcinoma progression. However, the function and molecular mechanism of ZSCAN16-AS1 in melanoma are still unknown. Methods: The level of ZSCAN16-AS1 in melanoma tissues was detected and reported in The Cancer Genome Atlas (TCGA) and GEO#GSE15605. CCK-8, Transwell and flow cytometry assays were used to explore the role of ZSCAN16-AS1 in melanoma cells. Luciferase reporter assays and RNA pull-down assays were used to verify the molecular mechanism of ZSCAN16-AS1. Results: Here, we found that ZSCAN16-AS1 expression was increased in melanoma. We confirmed that ZSCAN16-AS1 promotes the growth and metastasis of melanoma. ZSCAN16-AS1 exerts its pro-tumour role through sponging of miR-503-5p to liberate ADP-ribosylation factor-like protein 2 (ARL2) mRNA transcripts. Conclusion: These results demonstrated the role and molecular mechanism of ZSCAN16-AS1 in the occurrence and development of melanoma. Therefore, ZSCAN16-AS1 may be used as a specific biomarker in the diagnosis and treatment of melanoma patients.

Hsa-miR-503-5p regulates CTDSPL to accelerate cisplatin resistance and angiogenesis of lung adenocarcinoma cells.

The study aimed to assess the role of hsa-miR-503-5p in cisplatin resistance and angiogenesis in LUAD and its underlying mechanisms. Hsa-miR-503-5p expression in LUAD and the target gene downstream of hsa-miR-503-5p was predicted by bioinformatics analysis. Binding relationship between the two genes was verified by dual-luciferase reporter assay. qRT-PCR was conducted for detecting gene expression in cells, CCK-8 for IC50 value, angiogenesis assay for human umbilical vein endothelial cell (HUVEC) angiogenic ability, flow cytometry for apoptosis ability, transwell assay for migration ability, and western blot for detecting the protein expression of vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, and CTD small phosphatase like (CTDSPL). The results showed that hsa-miR-503-5p showed high expression, while its target gene CTDSPL presented decreased expression in LUAD. Hsa-miR-503-5p also had high expression in cisplatin-resistant LUAD cells. Knockdown of hsa-miR-503-5p resensitized LUAD cells to cisplatin, inhibited angiogenesis of drug-resistant cells, and reduced the protein expression of VEGFR1, VEGFR2, and EMT-related targets in cisplatin-resistant LUAD cells, but promoted the apoptosis ability. Hsa-miR-503-5p bound to CTDSPL gene and promoted cisplatin resistance and malignant progression of LUAD cells by negatively regulating CTDSPL. Our results revealed that hsa-miR-503-5p and CTDSPL may be novel targets for overcoming cisplatin resistance in LUAD.

Diabetic macrophage small extracellular vesicles-associated miR-503/IGF1R axis regulates endothelial cell function and affects wound healing.

Diabetic foot ulcer (DFU) is a break in the skin of the foot caused by diabetes. It is one of the most serious and debilitating complications of diabetes. The previous study suggested that dominant M1 polarization during DFU could be the leading reason behind impaired wound healing. This study concluded that macrophage M1 polarization predominates in DFU skin tissue. iNOS was increased in HG-induced M1-polarized macrophages; conversely, Arg-1 was decreased. Macrophage pellets after HG stimulation can impair endothelial cell (EC) function by inhibiting cell viability, tube formation and cell migration, indicating M1 macrophage-derived small extracellular vesicles (sEVs) -mediated HUVEC dysfunction. sEVs miR-503 was significantly upregulated in response to HG stimulation, but inhibition of miR-503 in HG-stimulated macrophages attenuated M1 macrophage-induced HUVEC dysfunction. ACO1 interacted with miR-503 and mediated the miR-503 package into sEVs. Under HG stimulation, sEVs miR-503 taken in by HUVECs targeted IGF1R in HUVECs and inhibited IGF1R expression. In HUVECs, miR-503 inhibition improved HG-caused HUVEC dysfunction, whereas IGF1R knockdown aggravated HUVEC dysfunction; IGF1R knockdown partially attenuated miR-503 inhibition effects on HUVECs. In the skin wound model in control or STZ-induced diabetic mice, miR-503-inhibited sEVs improved, whereas IGF1R knockdown further hindered wound healing. Therefore, it can be inferred from the results that the M1 macrophage-derived sEVs miR-503 targets IGF1R in HUVECs, inhibits IGF1R expression, leads to HUVEC dysfunction, and impedes wound healing in diabetic patients, while packaging miR-503 as an M1 macrophage-derived sEVs may be mediated by ACO1.

MiR-503 pleiotropically regulates epithelial-mesenchymal transition and targets PTK7 to control lung cancer metastasis.

OBJECTIVE: In lung cancer patients, most deaths are caused by the distant dissemination of cancer cells. Epithelial-mesenchymal transition (EMT) and collective cell migration are distinct and important mechanisms involved in cancer invasion and metastasis. Additionally, microRNA dysregulation contributes significantly to cancer progression. In this study, we aimed to explore the function of miR-503 in cancer metastasis. METHODS: Molecular manipulations (silencing or overexpression) were performed to investigate the biological functions of miR-503 including migration and invasion. Reorganization of cytoskeleton was assessed using immunofluorescence and the relationship between miR-503 and downstream protein tyrosine kinase 7 (PTK7) was assessed using quantitative real-time PCR, immunoblotting, and reporter assays. The tail vein metastatic animal experiments were performed. RESULTS: Herein, we demonstrated that the downregulation of miR-503 confers an invasive phenotype in lung cancer cells and provided in vivo evidence that miR-503 significantly inhibits metastasis. We found that miR-503 inversely regulates EMT, identified PTK7 as a novel miR-503 target, and showed the functional effects of miR-503 on cell migration and invasion were restored upon reconstitution of PTK7 expression. As PTK7 is a Wnt/planar cell polarity protein crucial for collective cell movement, these results implicated miR-503 in both EMT and collective migration. However, the expression of PTK7 did not influence EMT induction, suggesting that miR-503 regulates EMT through mechanisms other than PTK7 inhibition. Furthermore, we discovered that PTK7 mechanistically activates focal adhesion kinase (FAK) and paxillin, thereby controlling the reorganization of the cortical actin cytoskeleton. CONCLUSION: Collectively, miR-503 is capable of governing EMT and PTK7/FAK signaling independently to control the invasion and dissemination of lung cancer cells, indicating that miR-503 represents a pleiotropic regulator of cancer metastasis and hence a potential therapeutic target for lung cancer.

Experimental study of miR-503 regulating the activity as well as the function of degenerated human nucleus pulposus cells of the intervertebral disc through inhibiting Wnt pathway.

OBJECTIVES: To preliminarily explore miR-503 in human degenerative disc nucleus pulposus cell effects as well as mechanisms. METHODS: We utilized bioinformatics analysis to determine the miRNA differential expression as well as key signal pathways existing in human nucleus pulposus cells of the degenerative intervertebral discs. Human degenerative disc nucleus pulposus cell model was cultured and established in vitro. miR-503 and TNIK-related genes are knocked down and overexpressed by lentiviral infection, then we added Wnt signaling pathway agonists; CCK-8, ELISA, RT-PCR, Western blot were used to detect proliferation, apoptosis, and activity of cells. RESULTS: Bioinformatics results demonstrated that miR-503 was significantly down-regulated in human nucleus pulposus cells of the degenerated intervertebral discs. The targeted differentially expressed genes were mainly enriched in Wnt signaling pathway. However, after screening differential genes in the Wnt pathway, it was demonstrated that miR-503 mainly regulates TNIK to achieve Wnt pathway regulation. Cell experiments in vitro showed that cell activity and function were decreased while apoptosis was increased in the degenerative cell model. CONCLUSIONS: miR-503 can improve the function and activity of human nucleus pulposus cells of degenerated intervertebral disc by inhibiting Wnt expression. miR-503 mainly regulates the Wnt pathway through targeted binding with TNIK.

MIR503HG: A potential diagnostic and therapeutic target in human diseases.

LncRNAs are involved in many physiological and pathological processes, including chromatin remodeling, transcription, posttranscriptional gene expression, mRNA stability, translation, and posttranslational modification, and their functions depend on subcellular localization. MIR503HG is a lncRNA as well as a host gene for the miRNAs miR-503 and miR-424. MIR503HG functions independently or synergistically with miR-503. MIR503HG affects cell proliferation, invasion, metastasis, apoptosis, angiogenesis, and other biological behaviors. The mechanism of MIR503HG in disease includes interaction with protein, sponging miRNA to regulate downstream target gene, and participation in NF-κB, TGF-ß, ERK/MAPK, and PI3K/AKT signaling pathways. In this review, we summarize the molecular mechanisms of MIR503HG in disease and its potential applications in diagnosis, prognosis, and treatment. We also raise some unanswered questions in this area, providing insights for future research.

MIR503HG silencing promotes endometrial stromal cell progression and metastasis and suppresses apoptosis in adenomyosis by activating the Wnt/ß­catenin pathway via targeting miR­191.

MIR503HG is a 786 bp long lncRNA located on chromosome Xq26.3, and it can regulate diverse cellular processes. The pathogenesis of adenomyosis (AD) is associated with endometrial stromal cells (ESCs). The present study investigated the specific role of MIR503HG in AD pathogenesis and progression using ESCs derived from the endometrium of patients with AD as a model. Expression of MIR503HG and microRNA (miR)-191 were assessed using reverse transcription-quantitative PCR. An immunocytochemistry assay was used to detect cytokeratin- or vimentin-positive ESCs. Transfections of ESCs with MIR503HG overexpression plasmid, short hairpin-MIR503HG and miR-191 inhibitor were performed. ESC viability, migration, invasion and apoptosis were evaluated using Cell Counting Kit-8, Transwell and flow cytometry assays. The association between MIR503HG and miR-191 was predicted by StarBase and confirmed using a dual-luciferase reporter assay. Expression of epithelial-mesenchymal transition-related markers (E-cadherin and N-cadherin) and Wnt/ß-catenin pathway-related molecules (ß-catenin) in ESCs were analyzed by western blotting. The isolated ESCs were vimentin-positive and cytokeratin-negative. MIR503HG was lowly expressed in the endometrial tissues derived from patients with AD. MIR503HG overexpression hindered ESC viability, migration and invasion while enhancing the apoptosis and downregulating miR-191 expression. MIR503HG knockdown induced the opposite effects, accompanied by downregulation of the E-cadherin expression and upregulation of N-cadherin and ß-catenin levels. MIR503HG directly targeted miR-191 that was highly expressed in endometrial tissues derived from patients with AD. In ESCs, downregulation of miR-191 inhibited the viability, migration and invasion and the expression of N-cadherin and ß-catenin levels while enhancing the apoptosis and E-cadherin expression in ESCs. Moreover, downregulation of miR-191 partially reversed the effect of MIR503HG knockdown. Collectively, overexpressed MIR503HG impeded the proliferation and migration of ESCs derived from endometrium of patients with AD, while promoting apoptosis via inhibition of the Wnt/ß-catenin pathway via targeting miR-191.

miR-503 targets MafK to inhibit subcutaneous preadipocyte adipogenesis causing a decrease of backfat thickness in Guanzhong Black pigs.

Reducing backfat thickness (BFT), determined by subcutaneous fat deposition, is vital in Chinese developed pig breeds. The level of miR-503 in the backfat of Guanzhong Black pigs was found to be lower than that in Large White pigs, implying that miR-503 may be related to BFT. However, the effect and mechanism of miR-503 on adipogenic differentiation in subcutaneous preadipocytes remain unknown. Compared with Large White pigs, the BFT and body fat content of Guanzhong Black pigs were greater, but the level of miR-503 was lower in subcutaneous adipose tissue (SAT) at 180 days of age. Furthermore, miR-503 promoted preadipocyte proliferation by increasing the proportion of S-phase and EdU-positive cells. However, miR-503 inhibited preadipocyte differentiation by downregulating adipogenic gene expression. Mechanistically, miR-503 directly targeted musculoaponeurotic fibrosarcoma oncogene homolog K (MafK) in both proliferating and differentiating preadipocytes to repress adipogenesis. Our findings provide a novel miRNA biomarker for reducing pig BFT levels to improve carcass quality.

Exosomal LINC00460/miR-503-5p/ANLN positive feedback loop aggravates pancreatic cancer progression through regulating T cell-mediated cytotoxicity and PD-1 checkpoint.

BACKGROUND: Long non-coding RNA (lncRNA) LINC00460 is an onco-lncRNA in a variety of cancers, including pancreatic cancer (PC). This study is aimed to investigate the regulatory mechanisms of LINC00460 in PC. METHODS: The tumor and adjacent normal tissues were collected from 73 PC patients. The expression of LINC00460, miR-503-5p, and ANLN was detected using qRT-PCR. We then analyzed the proliferation, migration, invasion, and apoptosis/cell cycle of PC cells by performing the MTT/EdU, transwell, and flow cytometry assays, respectively. The xenograft tumor model were utilized to confirm the effect of LINC00460 knockdown on PC through anti-PD-1 therapy in vivo, and the sensitivity of PANC-1 cells to the cytotoxicity of CD8+ T cells in vitro. Western blotting was used to determine the protein levels. A co-culture model was utilized to explore the effects of exosomes on macrophages. RESULTS: LINC00460 was up-regulated in PC tissues and cells. LINC00460 knockdown suppressed cell proliferation, migration, and invasion, facilitated cell apoptosis and G0/G1 phase arrest, and inhibited the tumor growth through anti-PD-1 therapy. Both miR-503-5p down-regulation and ANLN up-regulation reversed the effects of LINC00460 knockdown on inhibiting the proliferation, migration and invasion, and on promoting the apoptosis, G0/G1 phase arrest, and the sensitivity of PC cells to the cytotoxicity of CD8+ T cells. Exosomes were uptaken by the ambient PC cells. PANC-1 cells-derived exosomal LINC00460-induced M2 macrophage polarization accelerates the cell migration and invasion. CONCLUSIONS: LINC00460 silencing attenuates the development of PC by regulating the miR-503-5p/ANLN axis and exosomal LINC00460-induced M2 macrophage polarization accelerates the migration and invasion of PANC-1 cells, thus LINC00460 may act as a possible therapeutic target for treating PC.

Tumor Suppressor miRNA-503 Inhibits Cell Invasion in Head and Neck Cancer through the Wnt Signaling Pathway via the WNT3A/MMP Molecular Axis.

Head and neck cancer (HNC) is the fifth most common cancer worldwide, and its incidence and death rates have been consistently high throughout the past decades. MicroRNAs (miRNAs) have recently gained significant attention because of their role in the regulation of a variety of biological processes via post-transcriptional silencing mechanisms. Previously, we determined a specific profile of miRNAs associated with HNC using a miRNA microarray analysis. Of the 23 miRNAs with highly altered expression in HNC cells, miR-503 was the most significantly downregulated miRNA. In this study, we confirmed that miR-503 acts as a tumor suppressor, as our results showed decreased levels of miR-503 in cancer cells and patients with HNC. We further characterized the role of miR-503 in the malignant functions of HNC. Although there was a minimal effect on cell growth, miR-503 was found to inhibit cellular invasion significantly. Algorithm-based studies identified multiple potential target genes and pathways associated with oncogenic mechanisms. The candidate target gene, WNT3A, was confirmed to be downregulated by miR-503 at both the mRNA and protein levels and validated by a reporter assay. Furthermore, miR-503 modulated multiple invasion-associated genes, including matrix metalloproteinases (MMPs), through the Wnt downstream signaling pathway. Overall, this study demonstrates that miR-503 suppresses HNC malignancy by inhibiting cell invasion through the Wnt signaling pathway via the WNT3A/MMP molecular axis. The modulation of miR-503 may be a novel therapeutic approach to intervene in cancer invasion.

[MIR503HG promotes esophageal squamous cell carcinoma cell proliferation, invasion and migration via hsa-miR-503 pathway].

Objective: To explore the function and mechanism of long non-coding RNA MIR503HG in esophageal squamous cell carcinoma (ESCC). Methods: The MIR503HG expression data in 60, 119 and 23 cases of ESCC and their paired adjacent tissues were chosen from three ESCC datasets GSE53622, GSE53624 and GSE130078, respectively. The expression data of MIR503HG in 81 ESCC tissues and 271 unpaired normal esophageal tissues were screened from the combined dataset of Cancer Genome Atlas and Genotype-Tissue Expression Database (TCGA+ GTEx). The MIR503HG knockdown plasmid was constructed, packaged into lentivirus. The lentivirus was used to infect with esophageal squamous cell carcinoma cell lines KYSE30 and KYSE510 to screen out the stable MIR503HG knockdown cell lines. ESCC cell line KYSE30 was transiently transfected with miRNA mimics to overexpress hsa-miR-503-3p and hsa-miR-503-5p.The expression levels of MIR503HG, hsa-miR-503-3p and hsa-miR-503-5p were detected by quantitative real-time polymerase chain reaction. The proliferation ability of the cells was detected by cell counting kit 8 and clone formation assay. The invasion and migration ability of the cells were detected by Transwell assay. Cell cycle was detected by flow cytometry. The effect of MIR503HG on the proliferation of ESCC was detected by xenograft experiment in BALB/c-nu/nu mice. Results: Both GEO and TCGA+ GTEx databases showed that the expression of MIR503HG in ESCC tissues was higher than that in adjacent tissues and normal esophageal tissues (P<0.01). Compared with shNC group, the proliferation rates of KYSE30 and KYSE510 cells after knockdown of MIR503HGwere significantly inhibited (P<0.001). The colony formation numbers of KYSE30 cells in shMIR503HG1 group and shMIR503HG2 group were (2.00±1.41) and (1.33±0.47), respectively, significantly lower than that of the shNC group (P=0.002). The clone formation numbers of KYSE510 cells in shMIR503HG1 group and shMIR503HG2 group were (174.67±15.97) and (80.33±6.34), respectively, significantly lower than that of the shNC group (P<0.001). The invasive numbers of KYSE30 cells in shMIR503HG1 group and shMIR503HG2 group were 75.33±6.02 and 45.67±7.59, significantly lower than that of the shNC group(P<0.001). The migrating number of KYSE30 cells in shMIR503HG1 group and shMIR503HG2 group were 244.00±10.23 and 210.67±13.52, significantly lower than that of the shNC group(P<0.001), and the cell cycle was arrested in G(0)/G(1) phase. The xenograft experiment showed that the subcutaneous tumor in shMIR503HG group was significantly smaller than that in shNC group, and the tumor weight in shMIR503HG group was (0.097±0.026) g, which was lower than (0.166±0.021) g in shNC group (P<0.001). After knockdown of MIR503HG, the relative expression levels of hsa-miR-503-3p in KYSE30 cells of shMIR503HG1 group and shMIR503HG2 group were 0.66±0.02 and 0.58±0.00, respectively, the relative expression levels of hsa-miR-503-5p were 0.64±0.00 and 0.68±0.03, respectively, which were all lower than those in shNC group (P<0.01). After knockdown of MIR503HG, overexpression of hsa-miR-503-3p and hsa-miR-503-5p attenuated the inhibitory effects of knockdown of MIR503HG on proliferation (P<0.001), invasion (P<0.01) and migration (P<0.001) of KYSE30 cells. Conclusions: MIR503HG promotes the proliferation, invasion and migration of ESCC cells by regulating hsa-miR-503 pathway and can be used as a new potential target for targeted therapy of ESCC.

ALKBH5-mediated m6A modification of circCCDC134 facilitates cervical cancer metastasis by enhancing HIF1A transcription.

BACKGROUND: Metastasis is the main cause of mortality in cervical cancer (CC). Circular RNAs (circRNAs) have been demonstrated to play a crucial role in carcinoma biology. However, the expression and function of circRNAs in cervical cancer metastasis are still unclear. METHODS: In the present study, we identified a circRNA with an N6-methyladenosine (m6A) modification, circCCDC134, whose expression was increased in CC tissues by circRNA-Seq and qPCR. CircCCDC134 upregulation in CC was fine-tuned by ALKBH5-mediated m6A modification, which enhanced its stability in a YTHDF2-dependent manner. The functional experiments illustrated that circCCDC134 enhanced tumour proliferation and metastasis in vitro and in vivo. For the comprehensive identification of RNA-binding proteins, circRNA pull-down and mass spectrometry (ChIRP-MS), chromatin immunoprecipitation-seq (Chip-seq), RNA binding protein immunoprecipitation (RIP) and luciferase reporter assays were used to perform mechanistic investigations. RESULTS: The results revealed that circCCDC134 recruited p65 in the nucleus and acted as a miR-503-5p sponge to regulate the expression of MYB in the cytoplasm, ultimately stimulating HIF1A transcription and facilitating CC growth and metastasis. CONCLUSION: These findings indicate that circCCDC134 is an important therapeutic target and provide new regulatory model insights for exploring the carcinogenic mechanism of circCCDC134 in CC.

Neutrophil Extracellular Traps (NETs) Promote Non-Small Cell Lung Cancer Metastasis by Suppressing lncRNA MIR503HG to Activate the NF-κB/NLRP3 Inflammasome Pathway.

Neutrophil extracellular traps (NETs) that are produced in the tumour microenvironment (TME) have been suggested to play an essential role in the dissemination of metastatic cancer under multiple infectious and inflammatory conditions. However, the functions of NETs in promoting non-small cell lung cancer (NSCLC) metastasis and the underlying mechanisms remain incompletely understood. Here, we found that NETs promoted NSCLC cell invasion and migration by inducing epithelial to mesenchymal transition (EMT). To explore how NETs contribute to NSCLC metastasis, microarrays were performed to identify substantial numbers of long noncoding RNAs (lncRNAs) and mRNAs that were differentially expressed in NSCLC cells after stimulation with NETs. Interestingly, we observed that the expression of lncRNA MIR503HG was downregulated after NETs stimulation, and ectopic MIR503HG expression reversed the metastasis-promoting effect of NETs in vitro and in vivo. Notably, bioinformatics analysis revealed that differentially expressed genes were involved in the NOD-like receptor and NF-κB signalling pathways that are associated with inflammation. NETs facilitated EMT and thereby contributed to NSCLC metastasis by activating the NF-κB/NOD-like receptor protein 3 (NLRP3) signalling pathway. Further studies revealed that MIR503HG inhibited NETs-triggered NSCLC cell metastasis in an NF-κB/NLRP3-dependent manner, as overexpression of NF-κB or NLRP3 impaired the suppressive effect of MIR503HG on NETs-induced cancer cell metastasis. Together, these results show that NETs activate the NF-κB/NLRP3 pathway by downregulating MIR503HG expression to promote EMT and NSCLC metastasis. Targeting the formation of NETs may be a novel therapeutic strategy for treating NSCLC metastasis.

MIR503HG impeded ovarian cancer progression by interacting with SPI1 and preventing TMEFF1 transcription.

MIR503 host gene (MIR503HG) acts as an important tumor suppressor in many human cancers, but its role and regulatory mechanism in ovarian cancer need to be further studied. In this study, lower expressed MIR503HG was observed in ovarian tumor tissues and cells than in adjacent normal tissues and normal human ovarian epithelial cells. MIR503HG overexpression impaired the proliferative, invasive and EMT properties, and facilitated cell apoptosis in ovarian cancer cells. Nuclear and cytoplasmic separation test suggested that MIR503HG was mainly expressed in the nucleus. RNA immunoprecipitation and RNA pull-down assays confirmed that MIR503HG could bind to transcription factor SPI1 (Spi-1 proto-oncogene), and dual luciferase reporter gene and Chromatin immunoprecipitation assays verified that SPI1 could bind to TMEFF1 (Transmembrane protein with EGF like and two follistatin like domains 1) promoter, suggesting that MIR503HG suppressed TMEFF1 expression by competitively binding SPI1 and blocking transcriptional activation of TMEFF1. Moreover, interference with TMEFF1 reversed the promotion effect of MIR503HG silence on the malignant behaviors of ovarian cancer cells. Moreover, MIR503HG knockdown activated the MAPK and PI3K/AKT pathways by increasing the expression of TMEFF1. In addition, overexpression of MIR503HG in vivo suppressed the tumorigenic ability in nude mice. In conclusion, MIR503HG acted as a tumor suppressor lncRNA in ovarian cancer by suppressing transcription factor SPI1-mediated transcriptional activation of TMEFF1.

Ultrasound microbubble-mediated miR-503-5p downregulation suppressed in vitro CRC progression via promoting SALL1 expression.

BACKGROUND: This study compared the effect of ultrasound microbubble-mediated miR-503-5p downregulation with that of pure liposome-mediated miR-503-5p downregulation on colorectal cancer (CRC) progression and explored the downstream mechanism. METHODS: Bioinformatics tools were utilized to predict miR-503-5p-targeted genes and CRC progression-associated genes. MiR-503-5p and sal-like 1 (SALL1) expressions in CRC cells and tissues were analyzed by qRT-PCR and/or bioinformatics tools; their correlations with overall survival and clinicopathological features of CRC patients were presented, and their interaction was validated by dual-luciferase reporter assay. CRC cells received ultrasound microbubble-mediated miR-503-5p downregulation and/or liposome-mediated miR-503-5p downregulation or SALL1 silencing. Cell phenotype changes were evaluated by flow cytometry, as well as MTT, Wound healing, Transwell and tube formation assays. E-cadherin, N-cadherin, Vimentin, B-cell lymphoma (Bcl)- 2, Cleaved caspase-3, and SALL1 expressions in cells were analyzed by Western blot. RESULTS: Upregulated miR-503-5p in CRC tissues and cells was detected, associated with poorer cell differentiation, easier lymph node metastasis and higher TNM stages, and related to poorer prognoses of CRC patients. Ultrasound microbubble-mediated miR-503-5p downregulation relative to pure liposome-mediated miR-503-5p downregulation better decreased viability, inhibited migration, invasion and tube formation, enhanced apoptosis, upregulated SALL1, E-cadherin and Cleaved caspase-3, and downregulated miR-503-5p, N-cadherin, Vimentin and Bcl-2 in CRC cells. SALL1 was targeted by miR-503-5p, low-expressed in CRC tissues and cells and positively related to CRC patients' survival. Silencing SALL1 exerted the opposite effects, which reversed the effects of ultrasound microbubble-mediated miR-503-5p downregulation and vice versa. CONCLUSION: Ultrasound microbubble-mediated miR-503-5p downregulation suppressed in vitro CRC progression via promoting SALL1 expression.