Alisol B as a novel inhibitor of TβRs suppresses the growth and metastasis of non-small cell lung cancer by inhibiting TGF-β-induced epithelial-mesenchymal transition.

Objective To explore the role and molecular mechanism of circ_001042 in lung adenocarcinoma (LUAD). Methods The expression level of circ_001042 and linear RNA MRPS35 in cells and clinical tissues was detected by real-time PCR (qRT-PCR). The expression of circ_001042 and transforming growth factor β1 (TGF-β1) in LUAD cells was elevated by the respective transfection of overexpression vectors OE-circ_001042 and TGF-β1; MTT and transwell assays were applied to test the proliferation, migration, and invasion abilities of cells, respectively. The E-cadherin expression level in the cells was assessed by immunofluorescence staining, and western blot was utilized to determine the expression level of epithelial-mesenchymal transition (EMT) and TGF-β1/P38 MAPK signaling axis-related proteins in the cells. Results Circ_001042 was significantly downregulated in LUAD tissues and cells, and high circ_001042 expression could inhibit the proliferation, invasion, and migration of LUAD cells. In addition, circ_001042 also inhibited the EMT process (the E-cadherin level was upregulated; and the levels of N-cadherin, vimentin, and Snail were downregulated) and TGF-β1/P38 MAPK signaling axis activity in LUAD cells. Moreover, circ_001042 could suppress the promotion of TGF-β1 on the proliferation, invasion, migration, and EMT process of LUAD cells and the activation of TGF-β1/P38 MAPK signaling axis. Conclusion By inhibiting TGF-β1, circ_001042 not only suppresses the proliferation, migration, invasion, and EMT of LUAD but also inhibits the activation of TGF-β1/P38 MAPK signaling axis. Therefore, circ_001042 can act as a potential target for early diagnosis and targeted therapy of LUAD.

P2.14-06 The Study of Rac3 Molecular Mechanism Through ERK Signaling Pathway in Lung Adenocarcinoma

背景与目的肺腺癌（lung adenocarcinoma, LUAD）是一种致病率和死亡率都极高的癌症，尽管现代医学的治疗手段在不断进步，但患者的5年生存率仍不高，因此我们想通过探究LUAD发生发展的分子机制进而鉴定新的治疗靶标。我们前期的研究报道了ZSWIM1（zinc finger SWIM-type containing 1）是一个促进LUAD细胞增殖、迁移、侵袭的新蛋白。本研究将着重探究E3泛素连接酶TRIM21（tripartite-motif protein 21）对ZSWIM1在LUAD细胞中促增殖、迁移功能的影响。方法利用蛋白免疫共沉淀技术（co-immunoprecipitation, Co-IP）和免疫荧光技术（immunofluorescence, IF）验证TRIM21和ZSWIM1之间的相互作用和共定位；利用MTT和Transwell实验检测TRIM21及TRIM21、ZSWIM1协同对LUAD增殖、迁移的影响；利用蛋白印迹实验（Western blot, WB）检测TRIM21和ZSWIM1对LUAD细胞上皮间充质转化（epithelial-mesenchymal transition, EMT）标志物表达的影响；利用Co-IP联合WB检测TRIM21对ZSWIM1泛素化的影响。结果TRIM21与ZSWIM1存在相互作用和共定位。TRIM21的上调可以抑制LUAD细胞的增殖和迁移。过表达TRIM21能够降低ZSWIM1对LUAD细胞增殖、迁移和侵袭的促进作用，并逆转ZSWIM1对E-cadherin和Vimentin表达的影响。敲低TRIM21则可以增强ZSWIM1对LUAD细胞增殖和迁移的促进作用。机制方面，我们发现过表达TRIM21可明显增强ZSWIM1的泛素化水平，下调ZSWIM1的蛋白表达量。结论TRIM21通过结合并促进ZSWIM1的泛素化，进而降低ZSWIM1的蛋白表达，这抑制了ZSWIM1对LUAD细胞增殖、迁移、侵袭表型的促进作用。

Immunotherapy has become the first-line treatment for advanced non-small-cell lung cancer (NSCLC), but most patients still fail to benefit or have disease progression following treatment. M2 phenotype tumor-associated macrophages (M2-TAMs) are important cellular components in the immunosuppressive microenvironment of NSCLC, but how they contribute to immunoresistance remains unclear. This study was conducted to investigate the role and mechanism of M2-TAMs in NSCLC immunoresistance. We collected postoperative tumor samples for detection of M2-TAMs and other immune cells infiltration by immunofluorescence detection and flow cytometry. We then constructed a non-contact cell co-culture system using Transwell chambers. CCK-8, colony formation, wound healing and invasion assays were performed to evaluated the effect of M2-TAMs on the proliferation, migration and invasion abilities of lung adenocarcinoma (LUAD) cells in vitro. Xenograft model were performed to analysis the effect of M2-TAMs on the tumorigenesis and metastasis of LUAD cells in vivo. M2-TAMs were greatly increased in the tumor tissue of patients with immunoresistant LUAD. They could significantly promote the proliferation, invasion, and migration of LUAD cells, and improve their resistance to cytotoxic T lymphocytes (CTL) cytotoxicity. Further research showed M2-TAMs could considerably enhance the expression of METTL3 and total m6A RNA level in LUAD cells and interfering with METTL3 could significantly reverse the impairment of M2-TAMs on the efficacy of CTL in killing tumor cells. In conclusion, M2-TAMs could promote LUAD immunoresistance by enhancing METTL3-mediated m6A methylation. Our results suggest METTL3 could be a potential therapeutic target for reversing immunoresistance and shed new light on the mechanism of M2-TAMs promoting LUAD immunoresistance.

Lung adenocarcinoma (LUAD) is a malignant tumor that occurs in the lungs. Numerous reports have substantiated the participation of long non-coding RNAs (lncRNAs) in the tumorigenesis of LUAD. Previously, lncRNA alpha-2-macroglobulin antisense RNA 1 (A2M-AS1) was confirmed to be an important regulator in the biological processes of LUAD and dysregulation of A2M-AS1 was associated with non-small cell lung cancer (NSCLC) progression. However, the precise mechanism of A2M-AS1 in LUAD has not been elucidated. Therefore, our study was designed to investigate the detailed molecular mechanism of A2M-AS1 in LUAD. Herein, the expression of lncRNA A2M-AS1, microRNA (miRNA) miR-587, and bone morphogenetic protein 3 (BMP3) in LUAD cell lines and tissues were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. The viability, proliferation, migration and invasion of LUAD cells were tested by cell counting kit-8 (CCK-8), colony formation and Transwell assays. In vivo tumor growth was investigated by xenograft animal experiment. Interactions among A2M-AS1, miR-587 and BMP3 were measured by RNA pulldown and luciferase reporter assays. In this study, A2M-AS1 was downregulated in LUAD tissues and cells and related to poor prognosis in LUAD patients. A2M-AS1 overexpression suppressed LUAD cell proliferation, migration and invasion in vitro and inhibited tumor growth in vivo. Mechanistically, A2M-AS1 directly bound with miR-587 to promote BMP3 expression in LUAD cells. Low expression of BMP3 was found in LUAD tissues and cells and was closely correlated with poor prognosis in LUAD patients. BMP3 deficiency reserved the inhibitory influence of A2M-AS1 overexpression on LUAD cell behaviors. Overall, A2M-AS1 inhibits cell growth and aggressiveness via regulating the miR-587/BMP3 axis in LUAD.

NFAT5 promotes proliferation and migration of lung adenocarcinoma cells in part through regulating AQP5 expression

High circ_0020123 expression is associated with poor prognosis in patients with non-small cell lung cancer (NSCLC) as previously reported. Whether circ_0020123 also plays an oncogenic role in lung adenocarcinoma (LUAD) is still unknown. Additionally, circ_0020123 is derived from part of exon (1312–3851) from its host gene PDZ domain-containing protein 8 (PDZD8). We hypothesized that circ_0020123 might affect malignant behaviors of LUAD cells by regulating PDZD8. Reverse transcription quantitative polymerase chain reaction revealed that PDZD8 was highly expressed in LUAD tissues and cells. PDZD8 knockdown suppressed LUAD cell proliferation and migration as shown by colony formation assays, Ethynyl deoxyuridine incorporation assays, Transwell assays, and wound healing assays. circ_0020123 was also found to be upregulated in LUAD tissues and cells. Moreover, circ_0020123 positively regulated PDZD8 expression in LUAD cells but exerted no significant effect on the transcriptional level of PDZD8. Mechanistically, circ_0020123 act as a competing endogenous RNA (ceRNA) to interact with miR-1283, thereby releasing the repression on PDZD8. Moreover, PDZD8 overexpression rescued the suppressive effect of circ_0020123 knockdown on LUAD cell proliferation and migration. In conclusion, circ_0020123 interacts with miR-1283 as a ceRNA to regulate PDZD8 expression, thus promoting the proliferation and migration of LUAD cells. The study might provide new biomarkers for future LUAD investigation.

Lung adenocarcinoma (LUAD) is prone to bone metastasis, resulting in poor prognosis. The present study aimed to detect the expression of deoxyribonuclease 1-like 3 (DNASE1L3) and forkhead-box P2 (FOXP2) in LUAD cells to investigate the role of DNASE1L3 in the regulation of proliferation, migration, invasion and tube formation of LUAD cells and how FOXP2 affects DNASE1L3 expression. The expression of DNASE1L3 and FOXP2 in LUAD cells was analyzed by reverse transcription-quantitative PCR (RT-qPCR) and western blotting. The transfection efficiency of DNASE1L3 overexpression plasmids, FOXP2 overexpression or interference plasmids into A549 cells was also confirmed by RT-qPCR and western blotting. The viability, proliferation, migration and invasion and tube formation of LUAD cells following transfection was in turn detected by MTT, EdU staining, wound healing, Transwell and tube formation assay. The expression of proteins associated with epithelial-mesenchymal transformation and tube formation was detected by western blotting. Binding between DNASE1L3 and FOXP2 was confirmed by dual-luciferase reporter assay and chromatin immunoprecipitation. Gene Expression Profiling Interactive Analysis database predicted that underexpression of DNASE1L3 in LUAD was associated with poor prognosis. DNASE1L3 expression was decreased in LUAD cells and overexpression of DNASE1L3 inhibited the proliferation, migration, invasion and tube formation of LUAD cells. Transcription factor FOXP2 positively regulated DNASE1L3 transcription in LUAD cells. FOXP2 was also underexpressed in LUAD cells and downregulation of FOXP2 promoted proliferation, migration, invasion and tube formation of LUAD cells, which was reversed by overexpression of DNASE1L3. In conclusion, DNASE1L3 was positively regulated by transcription factor FOXP2 and overexpression inhibited proliferation, migration, invasion and tube formation of LUAD cells.

Non-small-cell lung cancer (NSCLC) is one of the most common types of malignant cancer, characterized by high rates of metastasis and mortality. However, the molecular mechanisms underlying NSCLC growth and progression remain largely unclear. Here, EP300-AS1 is identified as a critical tumor-suppressive long non-coding RNA (lncRNA) in NSCLC. EP300-AS1 inhibits NSCLC cell growth and metastasis both in vitro and in vivo, and is associated with better clinical outcomes. The function of EP300-AS1 depends on EP300-AS1-PTBP1 interaction and PTBP1-mediated PRMT5 mRNA stability. EP300-AS1 binds directly to PTBP1, preventing its cytoplasmic translocation and PTBP1-PRMT5 mRNA complex formation in NSCLC. In the absence of PTBP1 binding to the PRMT5 mRNA 3’-UTR, PRMT5 mRNA stability and expression are reduced. PTBP1 knockdown or PRMT5 inhibition abolishes EP300-AS1-regulated NSCLC cell proliferation, migration, and invasion. In patients with lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), EP300-AS1 expression is negatively correlated with PRMT5 expression. Overall, these findings establish the EP300-AS1-PTBP1-PRMT5 axis as a key regulatory pathway in NSCLC progression, providing a novel regulatory mechanism and a promising target for NSCLC prediction and therapy.

ObjectiveTo investigate the effects of miR-490-3p on the proliferation, migration, invasion and apoptosis of lung adenocarcinoma (LUAD) cells through the Wnt/β-catenin signaling pathway.MethodsDifferentially expressed miRNAs in LUAD tissues were analyzed by bioinformatics and the target miRNA went through GSEA enrichment analysis. qRT-PCR was used to detect the expression of miR-490-3p in human LUAD cells and normal bronchial cells. The constructed vectors were transfected into the LUAD cell lines using Lipofectamine 2000. Cell viability was detected by MTT, cell migration and invasion were detected by transwell assay, and cell apoptosis was detected by flow cytometry. Western blot was performed to detect the expression levels of the proteins related to the Wnt/β-catenin pathway and cell apoptosis. Xenograft tumor mouse models were used for in vivo validation.ResultsThe results of qRT-PCR showed that miR-490-3p was relatively lowly expressed in LUAD cells, and the expression level was different in different LUAD cell lines. The results of MTT, transwell and flow cytometry exhibited that miR-490-3p could significantly inhibit the proliferation, migration, invasion and increase cell apoptosis rate of LUAD cells. Western blot results showed that miR-490-3p promoted the expression of Bax, Caspase-3 and E-cadherin as well as the phosphorylation of GSK-3β and inhibited the expression of Bcl-2, β-catenin and C-myc. Additionally, animal experiments were performed to prove that miR-490-3p suppressed LUAD malignant progression in vivo.ConclusionMiR-490-3p inhibited the proliferation, migration, invasion and promoted the apoptosis of LUAD cells by down-regulating the Wnt/β-catenin signaling pathway, suggesting that miR-490-3p may be an indicator for early diagnosis and prognosis of LUAD.

FAM207BP, a pseudogene-derived lncRNA, facilitates proliferation, migration and invasion of lung adenocarcinoma cells and acts as an immune-related prognostic factor

Harringtonine (HT) targets PARP-1 to inhibit TS mRNA m6A modification, blocks STAT3-induced miR-4521/TS axis and attenuates 5-fluorouracil (5-FU) resistance in NSCLC.

The hidden potential of solasonine: Targeting non-small cell lung cancer (NSCLC) metastasis through GATM and Smad2 pathways.

The potential of Humulus scandens in delaying non-small cell lung cancer: Targeting the PI3K/Akt signaling pathway to inhibit tumor growth and metastasis.

Simple SummaryNon-small-cell lung carcinoma (NSCLC) covers for almost 85% of all lung cancers and a major contributor to the overall cancer death rate. Erlotinib is used to treat NSCLC via inhibition of epithelial growth factor receptor (EGFR) kinase activity. Despite its high efficacy, recurrence can occur in patients who become resistant to the drug. We performed DARTS LC-MS/MS with SWATH of DIA analysis and identified a novel binding protein of Erlotinib that may underlie NSCLC resistance. Our study indicated that Erlotinib binds POLA2 in addition to EGFR. This was confirmed by DARTS and CETSA results. Importantly, POLA2 expression levels in four NSCLC cell lines were positively correlated with anti-proliferative Erlotinib efficacy (Pearson correlation coefficient, R = 0.9886). These results suggest that POLA2 is a novel complementary target protein of Erlotinib, and could clinically provide validity as a surrogate marker for drug resistance in patients with NSCLC.Erlotinib inhibits epithelial growth factor receptor (EGFR) kinase activity and is used to treat non-small cell lung cancer (NSCLC). Despite its high efficacy, recurrence can occur in patients who become resistant to the drug. To address the underlying mechanism of Erlotinib resistance, we investigated additional mechanisms related to mode-of-drug-action, by multiple protein-binding interactions, besides EGFR by using drug affinity responsive target stability (DARTS) and liquid chromatography-mass spectrometry (LC-MS/MS) methods with non-labeled Erlotinib. DNA polymerase alpha subunit B (POLA2) was identified as a new Erlotinib binding protein that was validated by the DARTS platform, complemented with cellular thermal shift assays. Genetic knock-down of POLA2 promoted the anti-proliferative effect of the drug in the Erlotinib-resistant cell line H1299 with high POLA2 expression, whereas the overexpression of POLA2 restored anti-proliferative effects in the Erlotinib-sensitive cell line HCC827 with low POLA2 expression. Importantly, POLA2 expression levels in four NSCLC cell lines were positively correlated with anti-proliferative Erlotinib efficacy (Pearson correlation coefficient, R = 0.9886). These results suggest that POLA2 is a novel complementary target protein of Erlotinib, and could clinically provide validity as a surrogate marker for drug resistance in patients with NSCLC.