WTAP-Mediated m(6)A Modification of circSMOC1 Accelerates the Tumorigenesis of Non-Small Cell Lung Cancer by Regulating miR-612/CCL28 Axis.

Accumulating evidence reveals that deregulated N6-methyladenosine (m6A) RNA methylation and circular RNAs (circRNAs) are required for the tumorigenesis of non-small cell lung cancer (NSCLC). We aimed to uncover the underlying mechanisms by which WTAP-mediated m6A modification of circRNA contributes to NSCLC. The differentially-expressed circRNAs were identified by a circRNA profiling microarray. The association of circSMOC1 with clinicopathological features and prognosis in patients with NSCLC was estimated by fluorescence in situ hybridization. WTAP-mediated m6A modification of circRNA was validated by RNA immunoprecipitation (RIP) and methylated RIP (MeRIP) assays. The role of circSMOC1 in NSCLC cells was validated by in vitro functional experiments and in vivo tumorigenesis models. CircSMOC1-specific binding with miR-612 was verified by RIP, luciferase gene report and RT-qPCR assays. The effect of circSMOC1 and/or miR-612 on CCL28 expression was detected by RT-qPCR and Western blotting analysis. We found that the expression levels of circSMOC1 were elevated in NSCLC tissues and associated with TNM stage and poor survival in patients with NSCLC. Knockdown of circSMOC1 impaired the tumorigenesis of NSCLC in vitro and in vivo, whereas restored expression of circSMOC1 displayed the opposite effect. Furthermore, WTAP was upregulated in NSCLC and mediated m6A modification of circSMOC1 and circSMOC1 abolished WTAP knockdown-caused tumour-suppressive effects. Then, circSMOC1 acted as a sponge of miR-612 to upregulate CCL28 and miR-612 inhibitors abrogated circSMOC1 knockdown-caused anti-proliferation effects and CCL28 downregulation in NSCLC cells. Knockdown of CCL28 inhibited cell proliferation and invasion and counteracted miR-612 inhibitor-caused tumour-promoting effects. Our findings unveil that WTAP-mediated m6A modification of circSMOC1 facilitates the tumorigenesis of NSCLC by regulating the miR-612/CCL28 axis.

Zhu XX ，Chen XY ，Zhao LT ，Zhang XL ，Li YO ，Shen XY ... -  《-》

RNA binding protein RBM22 suppresses non-small cell lung cancer tumorigenesis by stabilizing LATS1 mRNA.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related mortality worldwide. Despite advancements in diagnostics and therapeutics, the prognosis for NSCLC remains poor, highlighting the urgent need for novel treatment options. RNA binding proteins, particularly RBM22, have emerged as significant contributors to cancer progression by influencing RNA splicing and gene expression. This study investigates the role of RBM22 in NSCLC and its potential as a therapeutic target. We focus on the effects of RBM22 on cell proliferation, invasion, stemness, and its interaction with LATS1 mRNA. RBM22 expression was assessed in samples and cell lines of NSCLC through techniques such as real-time PCR and western blot analysis. To modify RBM22 levels, overexpression and knockdown methods were employed utilizing vectors and siRNAs. We conducted assays for cell proliferation, invasion, and stemness to evaluate the effects of altering RBM22. The interaction between RBM22 and LATS1 mRNA was investigated using RNA immunoprecipitation. In addition, in vivo studies involving subdermal tumor and lung metastasis models in athymic mice were carried out to evaluate how changes in RBM22 influence the tumorigenic and metastatic characteristics of NSCLC. Our analysis revealed a significant underexpression of RBM22 in NSCLC tissues compared to adjacent healthy tissues. Increasing RBM22 expression in NSCLC cell lines led to a marked decrease in cellular proliferation, invasiveness, and stemness, while silencing RBM22 produced opposing effects. Further investigations confirmed that RBM22 directly interacts with LATS1 mRNA, thereby stabilizing and enhancing its expression. In vivo studies validated that elevated RBM22 expression substantially reduced tumor formation and pulmonary metastases, as evidenced by decreased tumor size, mass, and Ki-67 proliferation marker expression, along with a significant reduction in the number of metastatic nodules in the lungs. Our study demonstrates that RBM22 suppresses NSCLC by stabilizing LATS1 mRNA, which in turn reduces tumor growth and metastasis. Consequently, RBM22 emerges as a valuable therapeutic target for NSCLC, offering new strategies for addressing this challenging condition.

Hou M ，Huang Q ，Chen S ，Lei J ，Zhang Y ... -  《-》

Has_circ_0002360 facilitates immune evasion by enhancing heterogeneous nuclear ribonucleoprotein A1 stability, thereby promoting malignant progression in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is marked by complex molecular aberrations including differential expression of circular RNAs (circRNAs). hsa_circ_0002360, a circRNA, has been identified as overexpressed in NSCLC. This study aimed to evaluate the expression patterns of hsa_circ_0002360 and its potential role as an oncogenic factor in NSCLC. We analyzed two GEO datasets (GSE112214 and GSE158695) using R software to identify differentially expressed circRNAs. Quantitative reverse transcription PCR (qRT-PCR) assessed the expression of hsa_circ_0002360 in NSCLC tissues and cell lines compared to controls. We used siRNA and overexpression vectors to modulate hsa_circ_0002360 levels in A549 cells, followed by assays to assess proliferation, migration, invasion, apoptosis, and epithelial-mesenchymal transition (EMT). Interactions with RNA-binding proteins, specifically HNRNPA1, were investigated using RNA-pull down and RIP assays. In GEO datasets GSE112214 and GSE158695, hsa_circ_0002360 was identified as significantly overexpressed in NSCLC, a finding supported by qRT-PCR analyses showing higher levels in NSCLC tissues and cell lines compared to controls. Functional assays demonstrated that knockdown of hsa_circ_0002360 in A549 cells decreased proliferation, migration, invasion, and altered epithelial-mesenchymal transition marker expression, while inducing apoptosis, suggesting its oncogenic role. Conversely, overexpression promoted tumor characteristics, corroborated by in vivo xenograft models showing increased tumor growth. Hsa_circ_0002360's interaction with HNRNPA1, evidenced through RNA-pull down and RIP assays, implicates it in regulatory pathways that enhance NSCLC progression. This expression was also correlated with advanced TNM stages and metastasis, highlighting its potential as a therapeutic target. hsa_circ_0002360 acts as an oncogene in NSCLC, promoting tumor progression and metastasis through regulation of cell growth, apoptosis, and EMT processes. The interaction between hsa_circ_0002360 and HNRNPA1 suggests a novel mechanism of circRNA-mediated modulation of NSCLC pathology, providing potential targets for therapeutic intervention.

Fan J ，Xue L ，Lu R ，Liu J ，Luo J ... -  《-》

Circular RNA circ_0004630 promotes malignancy and glycolysis of nonsmall cell lung cancer by sponging microRNA-1208 and regulating leucine-rich repeat kinase 2 expression.

Emerging evidence has discovered that circular RNAs play important regulators of nonsmall cell lung cancer (NSCLC), but the role and potential molecular mechanism of hsa_circ_100549 (circ_0004630) involved in NSCLC is poorly defined. In this study, circ_0004630, microRNA-1208 (miR-1208), and leucine-rich repeat kinase 2 (LRRK2) expression were detected using real-time quantitative polymerase chain reaction. Cell proliferation, colony formation, apoptosis, and invasion were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine, colony formation, flow cytometry, and transwell assays. Protein levels of glucose transporter 1, Hexokinase 2, and LRRK2 were detected using western blot assay. Glucose consumption, lactate production, and adenosine triphosphate content were assessed using the corresponding kits. After predicting via bioinformatics software Circinteractome and Targetscan, the binding between miR-1208 and circ_0004630 or LRRK2 was verified by a dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assay. The xenograft tumor model analyzed the biological role circ_000460 on tumor growth in vivo. It was found that circ_0004630 and LRRK2 were increased, and miR-1208 was low expression in NSCLC tissues and cells. Functionally, the downregulation of circ_0004630 inhibited NSCLC cell proliferation, invasion, glycolysis, and accelerated apoptosis in vitro. In mechanism, circ_0004630 might work as a sponge of miR-1208 to modulate LRRK2 expression. In addition, DUXAP8 deficiency cured neuroblastoma tumor growth in vivo. In conclusion, circ_0004630 knockdown might suppress NSCLC cell proliferation, metastasis, and glycolysis partly by the miR-1208/LRRK2 axis. Our findings hinted at an important theoretical basis for further elucidating the pathogenesis of NSCLC and targeted therapy.

Zhang X ，Wu J ，Miao Y ，Wang J ，Wang E ... -  《-》

Exosomal miR-20b-5p Induces EMT and Enhances Progression in Non-Small Cell Lung Cancer Via TGFBR2 Downregulation.

The mechanism by which specific miRNAs in NSCLC exosomes regulate NSCLC progression remains unclear. First, exosomes were isolated and identified. Exosomes were labeled with PKH26 for cell tracking studies. Subsequently, BEAS-2B cells and BEAS-2B cell exosomes were transfected with miR-20b-5p mimics or miR-20b-5p inhibitors, and cell proliferation was measured by EdU and CCK-8. cell migration and invasion were detected by wound healing tests and Transwell. The potential target of miR-20b-5p was predicted and verified by luciferase assay. Relative expression levels of miR-20b-5p and TGFBR2 were detected by qRT-PCR. Protein expression level was detected by Western blot. In addition, A549 cell exosomes were injected into mice through the tail vein and the pathological changes of lung tissue were detected by HE staining. Expression levels of E-cadherin and Vimentin in lung tissues were detected by immunohistochemistry. Our results also showed that high levels of miR-20b-5p in exosomes generated from NSCLC cells conceivably enter the cytoplasm of their own cells and by downregulating TGFBR2, accelerate NSCLC invasion, migration and EMT while promoting NSCLC cell proliferation. Exosome analysis using clinical plasma specimens revealed that miR-20b-5p expression was considerably improved in exosomes from NSCLC patients compared with those from healthy controls. In vitro and in vivo, exosomes with high levels of miR-20b-5p were linked to the progression of NSCLC. Our data suggest that exosomes with high levels of miR-20b-5p can increase development and metastasis of NSCLC cells by downregulating TGFBR2, which would serve as a predictive and diagnostic marker for NSCLC.

Ma H ，Jiang B ，Ren Q ，Sun Y ，Wang M ，Xia S ，Wang D ，Zhang W ... -  《-》