Human Non-small Cell Lung Cancer Cell Lines A549 Research Articles

“Crosstalk between non-coding RNAs and transcription factor LRF in non-small cell lung cancer”

Epigenetic approaches in direct correlation with assessment of critical genetic mutations in non-small cell lung cancer (NSCLC) are currently very intensive, as the epigenetic components underlying NSCLC development and progression have attained high recognition. In this level of research, established human NSCLC cell lines as well as experimental animals are widely used to detect novel biomarkers and pharmacological targets to treat NSCLC. The epigenetic background holds a great potential for the identification of epi-biomarkers for treatment response however, it is highly complex and requires precise definition as these phenomena are variable between NSCLC subtypes and systems origin.We engaged an in-depth characterization of non-coding (nc)RNAs prevalent in human KRAS-mutant NSCLC cell lines A549 and H460 and mouse KRAS-mutant NSCLC tissue by Next Generation Sequencing (NGS) and quantitative Real Time PCRs (qPCRs). Also, the transcription factor (TF) LRF, a known epigenetic silencer, was examined as a modulator of non-coding RNAs expression. Finally, interacting networks underlying epigenetic variations in NSCLC subtypes were created. Data derived from our study highlights the divergent epigenetic profiles of NSCLC of human and mouse origin, as well as the significant contribution of 12qf1: 109,709,060–109,747,960 mouse chromosomal region to micro-RNA upregulated species. Furthermore, the novel epigenetic miR-148b-3p/lncPVT1/ZBTB7A axis was identified, which differentiates human cell line of lung adenocarcinoma from large cell lung carcinoma, two characteristic NSCLC subtypes.The detailed recording of epigenetic events in NSCLC and combinational studies including networking between ncRNAs and TFs validate the identification of significant epigenetic features, prevailing in NSCLC subtypes and among experimental models. Our results enrich knowledge in the field and empower research on the epigenetic prognostic biomarkers of the disease progression, NSCLC subtypes discrimination and advancement to patient-tailored treatments.

MiR-183-5p promotes the progression of non-small cell lung cancer through targeted regulation of FOXO1

Abstract Objective To investigate miR-183-5p targeting to forkhead box protein O1 (FOXO1) and its corresponding effect on the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of non-small cell lung cancer (NSCLC) cells. Methods NSCLC tissues and adjacent normal tissues from 60 patients with NSCLC adenocarcinoma were obtained via pathological biopsy or intraoperative resection. Several cell lines were cultured in vitro, including the human normal lung epithelial cell line BEAS-2B and human NSCLC cell lines A549, SPCA-1, PC-9, and 95-D. miR-183-5p and FOXO1 mRNA expression in tissues and cells were detected by qRT-PCR; the corresponding correlations in NSCLC tissues were analyzed using the Pearson test, and the relationship between miR-183-5p expression and clinicopathological parameters was analyzed. The miR-183-5p-mediated regulation of FOXO1 was verified by bioinformatics prediction alongside double luciferase, RNA-binding protein immunoprecipitation (RIP) assay, and pull-down experiments. A549 cells were divided into control, anti-miR-NC, anti-miR-183-5p, miR-NC, miR-183-5p, miR-183-5p+pcDNA3.1, and miR-183-5p+pcDNA3.1-FOXO1 groups. Cell proliferation, invasion, migration, apoptosis, and cell cycle distribution were detected using an MTT assay, clone formation assay, Transwell assay, scratch test, and flow cytometry, respectively. The expression of EMT-related proteins in the cells was analyzed by western blotting. The effect of miR-185-3p silencing on the development of transplanted tumors was detected by analyzing tumor formation in nude mice. Results miR-183-5p expression was significantly higher in NSCLC tissues and cells than in adjacent normal tissues, whereas FOXO1 mRNA expression was significantly down-regulated. There was a significant negative correlation between miR-183-5p and FOXO1 mRNA in NSCLC tissues (P < 0.05). Additionally, the expression of miR-183-5p was significantly correlated with tumor size, tumor differentiation, and tumor-node-metastasis stage in patients with NSCLC (P < 0.05). miR-183-5p targeted and inhibited FOXO1 expression. Compared to the anti-miR-NC group, the cell proliferation, scratch healing rate, N-cadherin and vimentin protein expression, and the proportion of S phase cells were significantly lower in the anti-miR-183-5p group, whereas the protein expression of E-cadherin and α-catenin and the proportion of G0/G1 phase cells were significantly higher; additionally, the frequency of colony formation and invasion were significantly lower in the anti-miR-183-5p group (P < 0.05). Compared to the miR-NC group, the cell proliferation, scratch healing rate, N-cadherin and vimentin protein expression, and the proportion of S phase cells in the miR-183-5p group were significantly higher, whereas the E-cadherin and α-catenin protein expression and the proportion of G0/G1 phase cells were significantly lower; furthermore, the frequency of colony formation and invasion were significantly higher in the miR-183-5p group (P < 0.05). Compared with the miR-183-5p+pcDNA3.1 group, the OD value, scratch healing rate, N-cadherin and vimentin protein expression, and the proportion of S phase cells were significantly lower in the miR-183-5p+pcDNA3.1-FOXO1 group, whereas E-cadherin and α-catenin protein expression and the proportion of G0/G1 phase cells were significantly higher; additionally, the frequency of colony formation and invasion was significantly lower in the miR-183-5p+pcDNA3.1-FOXO1 group (P < 0.05). Overall, silencing miR-185-3p inhibited the growth of transplanted tumors and promoted FOXO1 expression. Conclusion Overexpression of miR-183-5p can inhibit apoptosis and promote the proliferation, migration, invasion, and EMT, of NSCLC cells by down-regulating FOXO1 expression.

MiR-181a-5p restrains non-small cell lung cancer cell invasion and migration by regulating the GTSE1/p53/NF-κB axis

microRNAs (miRNAs) are relevant to metastasis and invasion of non-small cell lung cancer (NSCLC). This study investigated the role of miR-181a-5p in lung cancer. Expression patterns of miR-181a-5p and GTSE1 in the human NSCLC cell line A549 and normal lung epithelial cell line BASE-2B were detected. miR-181a-5p mimic was delivered into A549 cells utilizing Lipofectamine 2000 to overexpress miR-181a-5p, followed by analysis of cell viability, proliferation, apoptosis, invasion, and migration. GTSE1 (G2 and S phase-expressed-1) was predicted as the downstream target gene of miR-181a-5p using bioinformatics analysis software. Targeting relationship between miR-181a-5p and GTSE1 was validated via dual-luciferase assay, RIP assay, and RNA pull-down. Activation of the p53/NF-κB pathway was determined. miR-181a-5p was weakly-expressed in NSCLC cells relative to normal lung epithelial cells. miR-181a-5p overexpression prevented NSCLC cell proliferation, migration, and invasion. Mechanically, miR-181a-5p targeted GTSE1. GTSE1 overexpression partly annulled repression of miR-181a-5p overexpression on NSCLC cell malignant behavior. miR-181a-5p activated the p53 pathway and inhibited the NF-κB pathway by targeting GTSE1. Overall, this study for the first time validated that miR-181a-5p impeded NSCLC cell invasion and migration through activation of the p53 pathway and inhibition of the NF-κB pathway by targeting GTSE1, which may provide a potential novel insight into NSCLC treatment.

CRABP2 involvement in a mechanism of Golgi stress and tumor dry matter in non-small cell lung cancer cells via ER dependent Hippo pathway.

The paper aimed to explore the mechanism of cellular retinoic acid binding protein 2 (CRABP2) involvement in Golgi stress and tumor dryness in non-small cell lung cancer (NSCLC) cells through the estrogen receptor (ER) dependent Hippo pathway. Human NSCLC cell line A549 was purchased from ATCC andcultured in RPMI-1640 with 10% FBS. Attractene reagent was used for plasmid transfection. ER (sh) RNA was designed using RNAi Designer. Seventy-six hours after infection, stable cells were obtained after treated with puromycin for 3 weeks. ER silencing cells (with inhibited ER expression) were compared to the control cells (normal cultured NSCLC cell line A549, CRABP2 normal expression). CRABP2 and ER expression levels were detected by RT-PCR. MTT assay was used to detect cell proliferation, and the cell localization of ER and Golgi was observed by confocal microscopy. The invasion and metastasis of cells were analyzed by Boden chamber invasion and migration assays. Western blotting assays was used for detecting the protein expression of E-cadherin, vimentin, ZO-1 protein and epithelial-mesenchymal transition (EMT) related factors. The lower expression level of mRNA was detected in the ER-silencing group compared to the control group (P<0.05). We also found a higher proliferation level of cells, the number of invading and metastatic cells, the expression of vimentin, p-Lats1T1079, Lats1 and p-YAPS127 mRNA in the control group compared to the ER silencing group (P<0.05). And the expression level of protein kinase RNA-like endoplasmic reticulum kinase (PERK), phosphorylate eukaryotic initiation factor 2 (p-eIF2 alpha), activating transcription factor 4 (ATF4) and C/EBP-homologous protein (CHOP) in the control group was higher than that in the ER silencing group (P<0.05). Adversely, a lower expression level of E-cadherin and ZO-1 protein was found in the control group compared to the ER silencing group (P<0.05). The expression of CRABP2 in NSCLC cells was regulated by ER, and cell proliferation and invasion were regulated by the Hippo pathway. At the same time, it was found that decreased expression of CRABP2 enhanced endoplasmic reticulum/Golgi stress response.

FOXD3-AS1 suppresses the progression of non-small cell lung cancer by regulating miR-150/SRCIN1axis.

Long non-coding RNA (lncNRA) forkhead box D3 antisense RNA 1 (FOXD3-AS1) has been proved to promote or suppress the occurrence and development of multiple types of human tumors. However, the function and mechanism of FOXD3-AS1 in non-small cell lung cancer (NSCLC) are scarcely understood. qRT-PCR was used for detecting FOXD3-AS1, miR-150 and SRC kinase signaling inhibitor 1 (SRCIN1) mRNA expression in NSCLC tissues, and the relationship between pathological characteristics of NSCLC patients and FOXD3-AS1 expression level was analyzed. With human NSCLC cell lines H1299 and A549 as cell models, CCK-8 and BrdU assays were employed for detecting cancer cell proliferation, and Transwell assay was employed for detecting cell invasion ability. Dual luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay were used for the verification of the targeting relationshipe between FOXD3-AS1 and miR-150, and Western blot was employed for detecting SRCIN1 protein expression. FOXD3-AS1 expression was significantly reduced in NSCLC tissues and cell lines, and low expression of FOXD3-AS1 was closely related to positive lymph node metastasis and relatively high tumor grade. FOXD3-AS1 over-expression inhibited the proliferation and invasion of H1299 cell lines, while its knockdown promoted the proliferation and invasion of A549 cells. Additionally, it was confirmed that FOXD3-AS1 suppressed the expression of miR-150 by targeting it, and up-regulated the expression of SRCIN1. FOXD3-AS1 indirectly enhances the expression of SRCIN1 by targeting miR-150, thereby inhibiting NSCLC progression.

The Potential Role of Cathepsin K in Non-Small Cell Lung Cancer.

(1) Background: Cathepsin K has been found overexpressed in several malignant tumors. However, there is little information regarding the involvement of Cathepsin K in non-small cell lung cancer (NSCLC). (2) Methods: Cathepsin K expression was tested in human NSCLC cell lines A549 and human embryo lung fibroblast MRC-5 cells using Western blot and immunofluorescence assay. Cathepsin K was transiently overexpressed or knocked down using transfection with a recombinant plasmid and siRNA, respectively, to test the effects on cell proliferation, migration, invasion, and on the mammalian target of rapamycin (mTOR) signaling pathway. (3) Results: Expression of Cathepsin K was increased significantly in A549 cells and diffused within the cytoplasm compared to the MRC-5 cells used as control. Cathepsin K overexpression promoted the proliferation, migration, and invasion of A549 cells, accompanied by mTOR activation. Cathepsin K knockdown reversed the above malignant behavior and inhibited the mTOR signaling activation, suggesting that Cathepsin K may promote the progression of NSCLC by activating the mTOR signaling pathway. (4) Conclusion: Cathepsin K may potentially represent a viable drug target for NSCLC treatment.

CXCR4/MIF axis amplifies tumor growth and epithelial-mesenchymal interaction in non-small cell lung cancer

Overexpression of C-X-C chemokine receptor type 4 (CXCR4) has been shown in several cancers, including non-small cell lung cancer (NSCLC) and is linked to early metastasis and worse prognosis. The crosstalk between cancer cells and tumor stroma promotes the growth and metastasis and CXCR4 signaling is a key element of this crosstalk.To test the effects of CXCR4 overexpression (CXCR4-OE), we transduced the human NSCLC cell line A549 by using a lentiviral vector. A 3D cell culture model showed generations of tumorspheres and the effects derived by the co-culturing of lung fibroblasts. Using a xenograft mouse model, we also studied the effects of CXCR4-OE in pulmonary cell engraftment and tumor burden in vivo.Our data indicate that CXCR4-OE leads to increased tumorsphere formation and epithelial-mesenchymal transition (EMT). CXCR4-OE by A549 cells resulted in a significant increase in the production of the CXCR4-ligand macrophage migration inhibitory factor (MIF) compared to those transduced with an empty vector (EV) or in which the CXCR4 expression was deleted (KO). In our in vitro system, we did not detect any production of the canonical CXCR4 ligand CXCL12. Autocrine MIF production and CXCR4 signaling are part of a self-perpetuating loop that amplifies tumor growth and EMT. Co-culture with lung fibroblasts further increased tumorsphere formation, partially driven by an increase in IL-6 production. When A549 cells were injected into murine lungs, we observed more abundant and significantly larger tumor lesions in recipients of CXCR4-OE A549 cells compared to those receiving EV or KO cells, consistent with our in vitro findings. Treatment of mice with the MIF antagonist ISO-1 resulted in significantly less tumor burden.In conclusion, our data highlight the role of the CXCR4-OE/MIF/IL-6 axis in epithelial mesenchymal crosstalk and NSCLC progression.

Influence of EGFR-activating mutations on sensitivity to tyrosine kinase inhibitors in a KRAS mutant non-small cell lung cancer cell line.

In non-small cell lung cancer (NSCLC), oncogenic driver mutations including those in KRAS and EGFR are typically mutually exclusive. However, recent reports indicate that multiple driver mutations are found in a certain percentage of cancers, and that the therapeutic responses of such cases with co-mutations of driver genes are largely unclear. Here, using CRISPR-Cas9-mediated genome editing, we generated isogenic cell lines harboring one or two copies of an EGFR-activating mutation from the human NSCLC cell line A549, which is known to harbor a homozygous KRAS gene mutation. In comparison with parent cells with KRAS mutation alone, cells with concomitant EGFR mutation exhibited higher sensitivity to EGFR-tyrosine kinase inhibitors (TKIs) but not to conventional anti-cancer drugs. In particular, cells with two copies of EGFR mutation were markedly more sensitive to EGFR-TKIs compared with parent cells. Thus, the presence of concomitant EGFR mutation can affect the TKI response of KRAS-mutated cells, implying that EGFR-TKI may represent an effective treatment option against NSCLC with EGFR/KRAS co-mutation.

Circ_0000376 enhances the proliferation, metastasis, and chemoresistance of NSCLC cells via repressing miR-384.

Non-small cell lung cancer (NSCLC) is the most common cancer worldwide. Circular RNAs (circRNAs) are recently identified as important gene regulators with critical roles in cancer biology. In this study, we focus on the effect of circ_0000376 targeting miR-384 on malignant phenotypes of NSCLC cells. Circ_0000376 and miR-384 expression in NSCLC tissue samples were measured using qRT-PCR. The association between pathological parameters and the circ_0000376 expression was analyzed as well. Human NSCLC cell lines A549 and NCI-H460 were used as cell models. CCK-8 and BrdU assay were used to assess the effect of circ_0000376 on NSCLC cell line proliferation and drug sensitivity. Transwell assay was conducted to detect the effect of circ_0000376 on migration and invasion. Further, luciferase reporter assay was employed to validate the targeting of miR-384 by circ_0000376. Circ_0000376 expression in NSCLC clinical samples was up-regulated and this was linked to unfavorable pathological parameters. Circ_0000376 markedly accelerated the proliferation and metastasis, and enhanced chemoresistance of NSCLC cells. Mechanically, circ_0000376 overexpression could bind with miR-384 and repress its expression. Circ_0000376 is a newly discovered oncogenic circRNA in NSCLC, and can be potentially regarded as a diagnostic biomarker and therapy target.

MicroRNA‑4500 suppresses tumor progression in non‑small cell lung cancer by regulating STAT3.

Research has revealed that microRNA (miR)-4500 is downregulated in non-small cell lung cancer (NSCLC), and miR-4500 suppresses tumor growth by targeting lin-28 homolog B and NRAS proto-oncogene, GTPase. In the present study, it was reported that signal transducer and activator of transcription 3 (STAT3) may function as a novel target gene for miR-4500 in NSCLC. The experiments conducted in the present study confirmed that the miR-4500 expression was decreased in NSCLC tissues and cells compared with adjacent normal tissues and a normal lung cell line. miR-4500 suppressed the cell proliferation, migration, invasion and promoted apoptosis of the human NSCLC cell lines A549 and H1975. Expression of STAT3 was negatively correlated with miR-4500 expression in vivo. A luciferase reporter assay suggested that miR-4500 directly targeted the 3′ untranslated region of STAT3. The tumor inhibition effect of small interfering RNA STAT3 in A549 and H1975 lines may be partially impaired by a miR-4500 inhibitor. The results of the present study suggests that miR-4500 may be a tumor suppressor and a potential therapeutic target in NSCLC.

IL‑21/IL‑21R inhibit tumor growth and invasion in non‑small cell lung cancer cells via suppressing Wnt/β‑catenin signaling and PD‑L1 expression.

Lung cancer is considered to be one of the world's deadliest diseases, with non-small cell lung cancer (NSCLC) accounting for 85% of all lung cancer cases. The present study aimed to investigate the role and underlying mechanisms of interleukin-21 (IL-21), and its receptor IL-21R, in NSCLC. Lung tissues and blood samples of NSCLC were used to measure IL-21, IL-21R and programmed death 1 ligand 1 (PD-L1) expression using ELISA, western blot and immunohistochemistry analyses. Following treatment with different doses of IL-21, the proliferation, invasion and migration of human NSCLC cell line A549 was evaluated using a cell counting kit-8, colony formation, Transwell and scratch wound healing assays, respectively. Additionally, IL-21R and PD-L1 expression in A549 cells was detected using western blot analysis and immunofluorescence. IL-21R silencing was subsequently used to investigate its effects in cell proliferation, invasion and migration. PD-L1, IL-1β and tumor necrosis factor α (TNF-α) expression were measured. Finally, Wnt/β-catenin signaling expression was evaluated using western blot analysis following treatment with IL-21. Cells were then treated with lithium chloride (LiCl), which is an agonist of Wnt/β-catenin signaling, and the levels of PD-L1, IL-1β and TNF-α were detected. The results revealed that IL-21 and IL-21R expression in the lung tissues and blood samples of patients with NSCLC were decreased, while PD-L1 expression was increased, compared with normal tissues or healthy controls. Treatment of A549 cells with IL-21 upregulated IL-21R expression, downregulated PD-L1 and inhibited cell growth and metastasis in a dose-dependent manner. Following IL-21R silencing, the effects of IL-21 treatment were reversed, suggesting that IL-21 acted on A549 cells through binding to IL-21R. In addition, the results demonstrated that IL-21 treatment reduced the expression levels of proteins associated with the Wnt/β-catenin signaling, whereas activation of Wnt/β-catenin signaling with the LiCl agonist upregulated PD-L1, IL-1β and TNF-α expression. In conclusion, the IL-21/IL-21R axis reduced the growth and invasion of NSCLC cells via inhibiting Wnt/β-catenin signaling and PD-L1 expression. The present results may provide a novel molecular target for NSCLC diagnosis and therapy.

Impact and mechanism of PMs-3 to epithelial mesenchymal transformation of non-small cell lung cancer cell line A549

Objective To explore Impact and mechanism of PMs-3 to epithelial mesenchymal transformation (EMT) of non-small cell lung cancer (NSCLC) cell line A549. Methods Nanocomposite structure synthetized by our team was utilized on human NSCLC cell line A549 and human normal pulmonary epithelial cell line BEAS-2B with concentrations of 0, 20, 40, 60, 80, 100, 120 nmol/L for 48 h. Methyl thiazol tetrazolium (MTT) assay was employed to detect the effect of PMs-3 on A549 cells. Wound assay and Transwell chamber assay were used to test the migration and invasion of PMs-3 on A549 cells. EMT related genes were detected with real time PCR (RT-qPCR) and Western blotting. Results Results of MTT assay detected that PMs-3 could inhibit activity of A549 cells significantly, and inhibition rate was (3.203±1.222)% (0 nmol/L), (11.958±4.000)% (20 nmol/L), (25.348±7.710)% (40 nmol/L), (34.775±9.155)% (60 nmol/L), (41.163±10.988)% (80 nmol/L), (50.430±9.743)% (100 nmol/L), (51.895±6.161)% (120 nmol/L), which showed a dose-dependent manner (F=35.451, P 0.05). Conclusion PMs-3 could inhibit NSCLC cells via regulating EMT. Key words: Non-small cell lung cancer; PMs-3; Epithelial mesenchymal transformation; Drug treatment

Abstract 1027: Inhibiting pathways predicted from a steroid hormone gene signature yields synergistic antitumor effects in lung cancer

Abstract Mounting preclinical and clinical evidence suggest an involvement of estrogen signaling in promoting non-small cell lung cancer (NSCLC) progression. In estrogen receptor β-positive (ERβ) cases, we previously found seven genes within the prediction analysis of microarrays 50 gene (PAM50) panel that showed strong association in predicting prognosis of NSCLC patients, with Myc, MIA, FGFR4, CXXC5, Grb-7 and FOXC1 being upregulated while PR was downregulated in highly aggressive lung tumors. These genes described one network containing ERβ and human epidermal growth factor receptor 2/3 (HER2/HER3) signaling, which suggests that these two interacting pathways define lung tumors with more aggressive biology. Here, we sought to evaluate the therapeutic potential of combining the pan-HER tyrosine kinase inhibitor, Dacomitinib, and the ER antagonist, Fulvestrant in NSCLC. Cell viability assay in three different human NSCLC cell lines 201T (EGFR wild-type), A549 (K-ras mutant) and HCC827 (EGFR exon 19 deletion) showed strong synergy between Dacomitinib and Fulvestrant, as determined by a combination index &amp;lt; 1. The combination strongly inhibited p-EGFR, p-HER2, p-HER3, p-HER4, and the downstream signaling p-AKT and p-MAPK compared to single treatments. Mechanistically, the transcription activity of activator protein-1 (AP-1) family members Fos and Jun were significantly reduced, as detected by the ability of AP-1 to bind to DNA. Expression of c-Myc and CyclinD1 were also significantly inhibited by the combination. In NSCLC cell lines, the combination reversed the gene signature associated with poor prognosis with c-Myc, MIA, CXXC5, FGFR4, Grb-7, FOXC1 being downregulated while PR was upregulated. Similarly, the AP-1 inhibitor t-5224 mimicked the effects of the combination in reversing the PAM50 gene signature, suggesting that the combination’s effect is largely mediated by AP-1 downregulation. In vivo, the combination also revealed a synergistic effect, measured by the combination ratio method (&amp;gt; 1), and induced tumor regression in 201T and A549 xenografts, with average tumor volume significantly lower than the single treatment groups. The immunohistochemical analysis of the xenografts revealed significant downregulation in Ki67. Western blot analysis of the tumor lysates from the combination treatment groups showed an increase in cleaved caspase-3 and a decrease in both ER and HER signaling. The PAM50 gene signature was also reversed in both xenograft studies detected by real-time-qPCR. Similar effects were seen for c-Myc and PR at the protein levels detected by immunohistochemistry staining. These observations encouragingly support the use of this combination clinically in NSCLC, considering its ability to suppress tumor growth and produce a gene signature that predicts better clinical outcomes, as assessed by the PAM50 genes. Citation Format: Abdulaziz A. Almotlak, Mariya Farooqui, Jill M. Siegfried. Inhibiting pathways predicted from a steroid hormone gene signature yields synergistic antitumor effects in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1027.

Hsa_circ_0001946 Inhibits Lung Cancer Progression and Mediates Cisplatin Sensitivity in Non-small Cell Lung Cancer via the Nucleotide Excision Repair Signaling Pathway.

Background: Despite great advances in the diagnosis and treatment of non-small cell lung cancer (NSCLC), early diagnosis remains a challenge because patients usually have advanced lung cancer at the time they are diagnosed. The limited efficacy of conventional chemotherapy is another major problem in the treatment of NSCLC. Based on a published set of sequencing data, we find that hsa_circ_0001946 is a circRNA molecule with a significantly different expression level in three cell lines (human normal lung fibroblasts cell line MRC-5, human NSCLC cell line A549, cisplatin-resistant cell line A549/DDP), NSCLC tissues and paired adjacent normal tissues. We believe that hsa_circ_0001946 may have an effect on the progression of NSCLC and its sensitivity to cisplatin.Methods: We focused on investigating the circular RNA, hsa_circ_0001946. RNA interference of hsa_circ_0001946 was carried out in A549 cell lines to determine the effect of reduced hsa_circ_0001946 expression on lung cancer progression and was analyzed by Cell Counting Kit-8 (CCK-8), 5-ethynyl-20-deoxyuridine, clone formation, Hoechst, wound healing, and transwell assays. The nucleotide excision repair (NER) signaling pathway was identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Moreover, cellular responses to cisplatin were assessed through CCK-8 and flow cytometry assays. Western blot analysis and host-cell reactivation assay were used to determine the effect of hsa_circ_0001946 on NER signaling.Results: In this study, we found that the reduced expression of hsa_circ_0001946 promoted the viability, proliferation, migration, and invasion of NSCLC cells, as well as inhibition of cell apoptosis. Our findings suggest that hsa_circ_0001946 can affect the sensitivity of NSCLC cells to the chemotherapeutic drug cisplatin via modulation of the NER signaling pathway.Conclusions: Our study demonstrated the role of hsa_circ_0001946 in NSCLC pathogenesis, development, and chemosensitivity, and suggests that hsa_circ_0001946 may serve as a novel biomarker for the diagnosis and prediction of platinum-based chemosensitivity in patients with NSCLC.

MiR-875 can regulate the proliferation and apoptosis of non-small cell lung cancer cells via targeting SOCS2.

To investigate the effect of miR-875 on the proliferation and apoptosis of non-small cell lung cancer (NSCLC) cancer cell line A549 and the related mechanism. 30 paired tumor tissue and the adjacent tissue were collected. Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) has been performed to detect the expression of miR-875 in NSCLC tissues and adjacent normal tissues. Moreover, suppressor of cytokine signaling 2 (SOCS2) has been predicted as a target of miR-875, and Dual-Luciferase reporter assay has been performed to confirm the targeting relationship; furthermore, the expression of SOCS2 in tumor tissue and the adjacent tissue were compared. Next, human NSCLC cell line A549 cells were cultured and transfected with miR-875 inhibitor with or without SOCS2 siRNA, and the proliferation and apoptosis of the cells were evaluated by Cell Counting Kit (CCK-8) and flow cytometry methods. Finally, the relative protein expression of Wnt and β-catenin were analyzed by Western blot analysis. MiR-875 was significantly up-regulated in NSCLC tissues compared with the adjacent tissues. SOCS2 was confirmed as a target of miR-875, and the expression of SOCS2 was markedly decreased in NSCLC tissues. Moreover, the knockdown of miR-875 inhibited the proliferation and promoted the apoptosis of A549 cells, while transfection of SOCS2 siRNA can block miR-875 inhibitor-induced anti-proliferative effects. Finally, the transfection of miR-875 inhibitor decreased the expression of Wnt and β-catenin, and SOCS2 siRNA can reverse the effect. MiR-875 may regulate the proliferation and apoptosis of NSCLC cells via targeting SOCS2, suggesting that miR-875 has the potential to become a therapeutic target for the treatment in NSCLC.

Hydroxysafflor yellow A inhibited lipopolysaccharide-induced non-small cell lung cancer cell proliferation, migration, and invasion by suppressing the PI3K/AKT/mTOR and ERK/MAPK signaling pathways.

BackgroundChronic inflammation plays a significant role in the occurrence and development of non‐small cell lung cancer (NSCLC). Hydroxysafflor yellow A (HSYA), a chemical compound of the yellow color pigments extracted from the safflower, has been widely used in clinical treatment with positive antioxidation, anti‐inflammation, and antitumor effects. However, the role and underlying mechanisms of HYSA on development and progress in inflammation‐mediated NSCLC are unknown.MethodsCell counting kit‐8, colony formation, EdU, cell apoptosis, wound healing, Transwell migration and invasion, and enzyme‐linked immunosorbent assays; flow cytometry; and Western blotting were conducted using human NSCLC cell lines A549 and H1299.ResultsLipopolysaccharide (LPS) significantly promoted the proliferation and enhanced colony formation of A549 and H1299 cells, while HYSA notably reversed the effects of LPS. HYSA induced apoptosis of LPS‐mediated A549 and H1299 cells in a dose dependent manner; and remarkably suppressed migration, invasion, and epithelial–mesenchymal transition (EMT), significantly regulated production of LPS‐induced inflammation cytokines, and downregulated protein expression of PI3K/Akt/mTOR and ERK/MAPK signaling pathways in LPS‐induced A549 and H1299 cells. Furthermore, PI3K (LY294002) and ERK (SCH772984) inhibitors remarkably inhibited proliferation, migration, invasion, and EMT, and induced apoptosis in LPS‐mediated A549 and H1299 cells. These effects were even more obvious in the presence of HYSA and LY294002 or SCH772984 compared to those of either agent alone.ConclusionHYSA suppressed LPS‐mediated proliferation, migration, invasion, and EMT in A549 and H1299 cells by inhibiting the PI3K/Akt/mTOR and ERK/MAPK signaling pathways, indicating that HYSA may be a potential candidate to treat inflammation‐mediated NSCLC.

Salidroside represses proliferation, migration and invasion of human lung cancer cells through AKT and MEK/ERK signal pathway

Salidroside, a glycoside of tyrosol, is isolated from Rhodiola rosea and shows anti-cancer functions in several cancers. However, the potentials of salidroside in the migration and invasion of lung cancer cells and its underlying mechanisms remain unknown. We aimed to investigate the functions and mechanisms of salidroside in non-small cell lung cancer (NSCLC). Human NSCLC cell line A549 was treated with different doses of salidroside. Cell viability, colony formation, apoptosis, migration and invasion were detected by CCK-8, crystal violet-staining assay, flow cytometry and transwell assay, respectively. qRT-PCR and western blot analysis were performed to assess the regulatory effects of salidroside on miR-195 expression and the activation of AKT and the MEK/ERK signal pathway. We found that, salidroside remarkably reduced cell viability, colony formation and Cyclin D1 expression, but increased p21 expression and apoptosis in A549 cells. Additionally, salidroside inhibited the migration and invasion of A549 cells by regulating expressions of migration- and invasion-related proteins. Finally, salidroside inhibited phosphorylation of AKT, MEK and ERK by upregulating miR-195 expression in A549 cells. In conclusion, salidroside inhibited the survival, migration and invasion of NSCLC cells. Salidroside blocked AKT and the MEK/ERK signal pathway by upregulating miR-195 expression in A549 cells.

&lt;p&gt;Rhein shows potent efficacy against non-small-cell lung cancer through inhibiting the STAT3 pathway&lt;/p&gt;

Non-small-cell lung cancer (NSCLC) comprises about 85% of all lung cancers and is usually diagnosed at an advanced stage with poor prognosis. The IL-6/STAT3 signaling pathway plays a pivotal role in NSCLC biology. Rhein is a lipophilic anthraquinone extensively found in medicinal herbs. Emerging evidence suggests that Rhein has significant antitumor effects, supporting the potential uses of Rhein as an antitumor agent. Cell viability and colony formation were performed to examine Rhein's potent anti-proliferative effect in human NSCLC cell lines PC-9, H460 and A549. Flow cytometry-based assay was employed to study whether Rhein could affect cell apoptosis and cycle. The expression level of P-STAT3, apoptosis and cycle-related proteins Bcl-2, Bax, MDM2, CDC2, P53 and CyclinB1 were detected by Western blotting. The xenograft models were used to evaluate the in vivo effect of Rhein. We found that Rhein could significantly reduce the viability and stimulate apoptosis in human NSCLC cells in a dose-dependent manner. Western blot analysis results suggested that the antitumor effect of Rhein might be mediated via STAT3 inhibition. Rhein upregulated the expression of the proapoptotic protein Bax and downregulated the expression of the antiapoptotic protein Bcl-2. In addition, Rhein induced the arrest of NSCLC cells in the G2/M phase of the cell cycle and dose dependently inhibited the expression of cycle-related proteins. The Rhein also inhibited tumor growth in H460 xenograft models. Rhein shows potent efficacy against NSCLC through inhibiting the STAT3 pathway. Our results also suggest that Rhein has a promising potential to be used as a novel antitumor agent for the treatment of NSCLC.

Physcion 8-O-β-Glucopyranoside Exerts Anti-Tumor Activity Against Non-Small Cell Lung Cancer by Targeting PPARγ.

Lung cancer is one of the most common causes of cancer related mortality. The present study is designed to investigate whether a naturally occurring anthraquinone compound, physcion 8-O-β-glucopyranoside (PG) could exert anti-cancer activity against non-small cell lung cancer (NSCLC). Cell viability was determined by Cell Counting Kit-8 (CCK-8) assay. Cell cycle distribution and cell apoptosis were determined by flow cytometry. Expressions of marker proteins were assessed by western blot analysis. To examine the role of PPARγ (peroxisome proliferator-activated receptor γ) in PG-induced apoptosis and cell cycle arrest, PPARγ was knockdown using siRNA. In addition, a xenograft model was established to investigate the effect of PG in vivo. The results showed that PG markedly induced cell cycle arrest and apoptosis in human NSCLC cell lines A549 and H358. The anti-tumor effect of PG in NSCLC cells was mediated by upregulation of PPARγ. Besides, in NSCLC cell lines, the anti-cancer activity of PG was also examined in the xenograft mice model, which showed that PG could significantly reduce tumor burden and activate apoptotic signaling. Our results demonstrated that PG can be regarded as a candidate chemotherapeutic agent for lung cancer. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc. Anat Rec, 302:785-793, 2019. © 2018 Wiley Periodicals, Inc.

MicroRNA-145-3p inhibits non-small cell lung cancer cell migration and invasion by targeting PDK1 via the mTOR signaling pathway.

The mammalian target of rapamycin (mTOR) pathway is dysregulated in more than 50% of all human malignancies and is a major target in cancer treatment. In this study, we explored the underlying mechanism involving microRNA-145-3p (miR-145-3p) in the development and progression of non-small cell lung cancer (NSCLC) by targeting PDK1 via the mTOR signaling pathway. NSCLC tissues and adjacent normal tissues were obtained from 83 NSCLC patients. miR-145-3p, PDK1, and mTOR levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry. Human NSCLC cell lines A549 and H1299 were transfected with miR-145-3p and siPDK1 to confirm the effect of miR-145-3p and PDK1 on NSCLC cells in vitro. Cell growth was evaluated by a CCK8 assay. Cell motility and chemotaxis analysis were determined by the scratch test and chemotaxis assay, respectively. The protein levels of PDK1 and mTOR were measured using the western blotting. Results showed lower level of miR-145-3p and higher levels of PDK1 and mTOR in NSCLC tissues compared to the adjacent normal tissues. In vitro results showed that cell growth, cell motility, and chemotaxis were all inhibited in cells transfected with miR-145-3p and those transfected with siPDK. Additionally, dual luciferase reporter gene assay helped confirmed that PDK1 is a target of miR-145. Finally, levels of PDK1, mTOR, and phosphorylated-mTOR were lower in cells transfected with miR-145-3p as well as those with siPDK1. These findings indicate that miR-145-3p may inhibit cell growth, motility, and chemotaxis in NSCLC by targeting PDK1 through suppressing the mTOR pathway.