Migration Of Non-small Cell Lung Cancer Cells Research Articles

Insulin-Like Growth Factor Binding Protein-3 Exerts Its Anti-Metastatic Effect in Aerodigestive Tract Cancers by Disrupting the Protein Stability of Vimentin.

Simple SummaryLocal invasion and distal metastasis are the main causes of cancer-related death and the poor prognosis of patients with aerodigestive tract cancers. Therefore, understanding the biology of invasion and metastasis is important for the development of effective therapeutic strategies. The present study shows that insulin-like growth factor binding protein-3 (IGFBP-3) inhibits the migration and invasion of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) cells in vitro and the development of metastasized tumors in vivo. Mechanistic studies suggest vimentin as a cellular target for the antimetastatic effect of IGFBP-3. These results contribute to a better understanding on the regulation of metastasis of cancer cells, providing the rationale to utilize IGFBP-3 as an effective therapeutic strategy targeting migration and metastasis of aerodigestive tract cancers.The proapoptotic, antiangiogenic, and antimetastatic activities of insulin-like growth factor binding protein-3 (IGFBP-3) through IGF-dependent or -independent mechanisms have been suggested in various types of human cancers. However, a mechanistic explanation of and downstream targets involved in the antimetastatic effect of IGFBP-3 is still lacking. In this study, by applying various in vitro and in vivo models, we show that IGFBP-3 suppresses migration and invasion of human head and neck squamous carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) cells. Silencing IGFBP-3 expression elevated the migration and invasion of NSCLC and HNSCC cells in vitro and their local invasion and metastasis in vivo, whereas overexpression of IGFBP-3 decreased such prometastatic changes. Local invasion of 4-nitroquinoline-1-oxide (4-NQO)-induced HNSCC tumors was consistently significantly potentiated in Igfbp3 knockout mice compared with that in wild-type mice. Mechanistically, IGFBP-3 disrupted the protein stability of vimentin via direct binding and promoting its association with the E3 ligase FBXL14, causing proteasomal degradation. The C-terminal domain of IGFBP-3 and the head domain of vimentin are essential for their interaction. These results provide a molecular framework for IGFBP-3′s IGF-independent antimetastatic and antitumor activities.

Isochorismatase domain-containing protein 1 (ISOC1) participates in DNA damage repair and inflammation-related pathways to promote lung cancer development.

BackgroundThe advent of novel molecular targets has dramatically changed the treatment landscape of lung cancer in recent years. Isochorismatase domain-containing protein 1 (ISOC1) has been reported as a potential biomarker in gastrointestinal cancer, while its function in lung cancer has not been determined.MethodsThe expression levels and prognostic significance of ISOC1 were assessed using bioinformatic analysis. Overexpression of ISOC1 and miR-4633, and knockdown of ISOC1 in non-small cell lung cancer (NSCLC) cell lines were generated by lentiviral infection with overexpressed or shRNA plasmids. CRISPR/Cas9 system was applied to knockout ISOC1 in A549 cells. The functions of ISOC1 and miR-4633 in lung cancer development were investigated using cell proliferation, migration, and invasion assays. Xenograft tumor growth assays in nude mice were further assessed the effect of ISOC1 in the tumorigenesis of NSCLC in vivo. Cell cycle distribution analysis was performed to uncover the underlying mechanism of ISOC1 and miR-4633 in promoting NSCLC cell proliferation. Co-immunoprecipitation combined with mass spectrometry and RNA sequencing were performed to uncover the potential mechanism of ISOC1 in lung cancer development.ResultsOur results found that ISOC1 expression was upregulated in NSCLC tissues and that increased expression of ISOC1 was significantly associated with worse disease-free survival in NSCLC patients. Overexpression of ISOC1 could increase the proliferation, viability, migration, and invasion of NSCLC cells. Furthermore, miR-4633, located in the first intron of ISOC1, could also promote tumor cell progression and metastasis. Mice xenograft tumor assay showed that knockout of ISOC1 could significantly inhibit tumor growth in vivo. Besides, co-immunoprecipitation combined with mass spectrometry assay revealed that ISOC1 interacted with the proteins of DNA damage repair pathways and that upregulated ISOC1 expression could significantly increase the number of DNA damage lesions. RNA sequencing analysis showed that the downstream signaling pathways mediated by ISOC1 were mainly inflammation-related.ConclusionsWe demonstrated that ISOC1 and its intronic miR-4633, both of them could promote NSCLC cell proliferation, migration, invasion, and cell cycle progression. ISOC1 participates in DNA damage repair and inflammation to promote lung cancer development.

Tumor-associated macrophages secret exosomal miR-155 and miR-196a-5p to promote metastasis of non-small-cell lung cancer.

BackgroundUnderstanding the molecular basis underlying metastasis of non-small cell lung cancer (NSCLC) may provide a new therapeutic modality for the treatment of NSCLC. However, the mechanisms by which tumor-associated macrophages (TAMs) affect NSCLC metastasis remain undefined. In this study, we aimed to discover a novel regulatory pathway involved in NSCLC metastasis.MethodsCell Counting Kit-8 (CCK-8), Transwell, western blot assays were used to assess cell viability, migration, invasion and epithelial-mesenchymal transition (EMT). Exosomes from macrophages medium were characterized, and in vitro cell coculture was further conducted to investigate M2 derived exosomes mediated crosstalk between TAMs and tumor cells. Besides, miRNA microarray was used to analyze miRNA expression profiles of M0 and M2 derived exosomes. Luciferase reporter assay was used to verify the potential binding between miRNA and mRNA. Moreover, 6-week-old male BALB/c nude mice were performed to establish transplantation tumor model using tail vein injection. Hematoxylin & eosin staining was used to detect the metastasis of tumor tissues.ResultsWe found that M2 TAMs were the main TAMs in metastatic tissues of NSCLC patients and exosomes derived from M2 TAMs were able to promote cell viability, cell migration, cell invasion and EMT in NSCLC. We demonstrated that miR-155 and miR-196a-5p were abundant in M2 TAMs and exosomes secreted by M2 TAMs. Functional experiments demonstrated that the deletion of miR-155 and miR-196a-5p in M2 TAMs significantly prevented NSCLC metastasis in vitro and in vivo. To clarify the mechanism governing miR-155 and miR-196a-5p from M2 TAMs, we carried out bioinformatics analysis to predict potential target genes. Mechanistically, miR-155 and miR-196a-5p directly bound to the 3'-UTR of Ras association domain family member 4 (RASSF4), and negatively regulating RASSF4 expression. At last, rescue assays demonstrated that miR-155 and miR-196a-5p exerted its performance by RASSF4.ConclusionsOverall, we revealed a new regulatory pathway that was M2 TAMs secreted exosomal miR-155 and miR-196a-5p to promote NSCLC metastasis. This dynamic and reciprocal cross-talk between NSCLC and macrophages innovatively provided a potential opportunity for diagnosis and treatment of NSCLC.

MiR-199a-5p-HIF-1α-STAT3 Positive Feedback Loop Contributes to the Progression of Non-Small Cell Lung Cancer.

Background: Non-small cell lung cancer (NSCLC) is the most common malignancy worldwide. MiR-199a-5p has been reported to play important roles in multiple tumors, inclusive of NSCLC. However, little is definitively known pertaining to its explicit mechanism of action in NSCLC.Methods: The expressions of miR-199a-5p and hypoxia-inducible factor-1α (HIF-1α) mRNA were quantified employing qRT-PCR. H1299 and A549 cells were transiently transfected with miR-199a-5p mimics or inhibitors. Then, CCK-8 assays, flow cytometry analysis, and Transwell assay were performed for detecting cell proliferation, cell cycle, apoptosis, migration, and invasion of NSCLC cells, respectively. HIF-1α, signal transducer and activator of transcription 3 (STAT3), and p-STAT3 expressions were detected via Western blotting. Bioinformatic analysis and dual-luciferase assay were performed to investigate the interactions among miR-199a-5p, HIF-1α, and STAT3. Xenograft models were established with nude mice for further analyzing the bevacizumab resistance of NSCLC cells.Results: MiR-199a-5p expression was markedly attenuated in NSCLC tissues and cell lines. Overexpression of miR-199a-5p repressed the proliferation, migration, and invasion but induced the apoptosis of NSCLC cells. HIF-1α was identified as a direct target of miR-199a-5p. There was a positive feedback loop among miR-199a-5p, HIF-1α, and STAT3. Co-transfection of HIF-1α or STAT3 overexpression plasmids counteracted the effects of miR-199a-5p. In vivo experiments indicated that the feedback loop was in association with the bevacizumab resistance of NSCLC cells.Conclusion: MiR-199a-5p blocked the progression of NSCLC and sensitized NSCLC cells to bevacizumab by suppressing HIF-1α and STAT3, while the HIF-1α/STAT3 axis suppressed the expression of miR-199a-5p, which forms a positive feedback loop to promote the sustaining progression of NSCLC.

Circ_0000735 enhances the proliferation, metastasis and glycolysis of non-small cell lung cancer by regulating the miR-635/FAM83F axis

Background Circular RNA (circRNA) is considered to be an important regulator of cancer malignant progression, including non-small cell lung cancer (NSCLC). Circ_0000735 has been found to be associated with NSCLC progression. Therefore, its role and molecular mechanism in NSCLC deserve further exploration. Methods Quantitative real-time PCR (qRT-PCR) was used to measure the expression of circ_0000735, microRNA (miR)-635 and family with sequence similarity 83 member F (FAM83F). Cell proliferation, migration, invasion and apoptosis were determined using cell counting kit 8 assay, colony formation assay, transwell assay and flow cytometry. Cell glycolysis were measured by detecting the glucose consumption and lactate production of cells. Western blot analysis was utilized to test the protein levels of glycolysis markers and FAM83F. The relationship between circ_0000735 and miR-635 or miR-635 and FAM83F was verified by dual-luciferase reporter assay. The effect of circ_0000735 on NSCLC tumor growth was evaluated by constructing xenograft models. Results Circ_0000735 was a highly expressed circRNA in NSCLC. Silenced circ_0000735 could inhibit NSCLC cell proliferation, migration, invasion, glycolysis, and increase apoptosis. MiR-635 could be sponged by circ_0000735, and its inhibitor could reverse the regulation of circ_0000735 silencing on NSCLC progression. Moreover, FAM83F was a target of miR-635, and circ_0000735 positively regulated FAM83F by sponging miR-635. The inhibitory effect of miR-635 on NSCLC progression could also be reversed by FAM83F overexpression. Additionally, circ_0000735 knockdown reduced NSCLC tumor growth through regulating miR-635/FAM83F axis. Conclusion Circ_0000735 promoted NSCLC progression by the miR-635/FAM83F axis, showing that circ_0000735 might be a promising biomarker for NSCLC. Highlights: Circ_0000735 knockdown represses NSCLC cell progression and tumor growth. Circ_0000735 functions as a miR-635 sponge. FAM83F is targeted by miR-635.

E3 ubiquitin ligase HECW1 promotes the metastasis of non-small cell lung cancer cells through mediating the ubiquitination of Smad4.

Lung cancer is the leading cause of cancer-related death globally. Ubiquitin modification plays a crucial role in the regulation of gene expression, and is closely associated with cancer pathogenesis. The aim of our study was to clarify the role and mechanisms of action for HECT, C2 and WW domain containing E3 ubiquitin protein ligase 1 (HECW1) in non-small cell lung cancer (NSCLC). Herein, we demonstrate that the expression of HECW1 was significantly increased in NSCLC cell lines and tissues. Upregulation of HECW1 markedly enhanced the proliferation of NSCLC cells, whereas downregulation of HECW1 significantly inhibited proliferation. Moreover, the expression levels of HECW1 positively correlated with the migration and invasiveness of NSCLC cells. Upregulation or downregulation of HECW1 only affected the protein expression levels of SMAD family member 4 (Smad4), but had no effect on the mRNA expression levels. Furthermore, after treatment with MG-132, the relative protein level of Smad4 significantly increased in NSCLC cells. HECW1 promoted the proliferation, migration, and invasiveness of NSCLC cells by inducing the ubiquitination and degradation of Smad4, thus our data provide a novel target for NSCLC treatment.

Salvianolic acid A blocks vasculogenic mimicry formation in human non-small cell lung cancer via PI3K/Akt/mTOR signalling.

Vasculogenic mimicry (VM) is associated with aggressive cancer cells. Salvianolic acid A (Sal-A), an antioxidant and anti-inflammatory agent, has bioactive properties from Salvia miltiorrhiza Bunge. Current investigation aspired to explore the activity of Sal-A in the VM formation of non-small cell lung cancer (NSCLC) and the mechanism underling this function. The CCK8, the scratch and boyden chemotaxis assay were presented to describe NSCLC cells viability, migration and invasion capabilities, respectively. The protein expression was verified by western blotting. In this report, Sal-A caused a reduction in viability, metastasis and capillaries structure formation of NSCLC cells. Additionally, Sal-A markedly prevented the key VM related proteins, containing EphA2, VE-cadherin and MMP2. Besides, Sal-A significantly diminished p-PI3K, p-Akt and p-mTOR level in NSCLC cells. More importantly, SC79 pretreatment reversed Sal-A inhibits NSCLC cells viability, metastasis and VM formation. These data exhibit that Sal-A could block VM network formation in NSCLC cells through modulating the PI3K/Akt/mTOR signalling pathway.

MicroRNA-99b inhibits NSCLC cell invasion and migration by directly targeting NIPBL.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. microRNAs (miRNAs) have been confirmed as vital regulators of multiple tumors, including NSCLC. The aim of the current study was to explore the biological mechanisms of miR-99b in NSCLC progression. NSCLC tissues and adjacent matched human non-neoplastic lung tissues used in this study were collected from 50 cases of NSCLC patients. The expression of miR-99b and NIPBL in NSCLC tissues and cell lines (A549, NCI-H460, NCI-H1299 and SPC-A1) were determined by real-time-polymerase chain reaction (qRT-PCR). The NIPBL protein level was measured by Western blot. Dual-Luciferase reporter, Western blotting and qRT-PCR were carried out to verify the potential target of miR-99b. Transwell assay was used for investigating miR-99b effect on cell migration and invasion in NSCLC cells. The results of qRT-PCR indicated that the expression of miR-99b was downregulated in the NSCLC tissues and cell lines. Overexpression of miR-99b could significantly inhibit the invasion and migration capacities in NSCLC cells. Furthermore, we also determined that NIPBL was a direct target of miR-99b. Additionally, we found NIPBL was implicated in the suppressive effects on NSCLC cell invasion and migration mediated by miR-99b. In summary, miR-99b exerted anti-tumor functions in NSCLC via regulation of NIPBL, suggesting that miR-99b/NIPBL axis may be novel biomarkers for NSCLC treatments.

Long Non-Coding RNA CAR10 Facilitates Non-Small Cell Lung Cancer Cell Migration and Invasion by Modulating the miR-892a/GJB2 Pathway.

IntroductionNon-coding RNAs, including long non-coding (lnc)RNAs and microRNAs (miRs), play crucial roles in numerous malignant tumors, including non-small cell lung cancer (NSCLC).MethodsThe expression levels of chromatin-associated RNA Intergenic 10 (CAR10), gap junction protein beta 2 (GJB2) and miR-892a in NSCLC were evaluated by reanalyzing three Gene Expression Omnibus (GEO) datasets, and performing reverse transcription-quantitative PCR, immunohistochemistry staining and Western blot analysis, accordingly. Functionally, Transwell and Matrigel assays were performed to measure changes in the migration and invasion abilities of the A549 and H1299 cell lines. The targeted binding effects between CAR10 and miR-892a, as well as between miR-892a and GJB2 were confirmed by conducting dual-luciferase reporter and RNA pull-down assays, respectively.ResultsThe present study demonstrated that CAR10 was upregulated in patients with NSCLC, which was also associated with a poor prognosis. Functionally, CAR10 was confirmed to be oncogenic and promoted NSCLC cell migration and invasion, using overexpression and knockdown Transwell assays. Furthermore, GJB2 expression was revealed to be upregulated and was positively correlated with CAR10 expression in NSCLC. A further mechanistic study revealed that GJB2 was a downstream target of CAR10, which induced the migration and invasive potential of the A549 and H1299 cell lines. More specifically, miR-892a was found to serve as a bridge between CAR10 and GJB2, via similar miRNA response elements. The RNA pull-down and luciferase assays indicated that miR-892a directly binds both CAR10 and GJB2.ConclusionCAR10 promoted NSCLC cell migration and invasion by upregulating GJB2 and sponging miR-892a. These findings illustrated that the CAR10/miR-892a/GJB2 axis may be a novel molecular target for the treatment of NSCLC.

LncRNA WT1-AS downregulates lncRNA UCA1 to suppress non-small cell lung cancer and predicts poor survival

BackgroundLncRNA WT1-AS inhibits gastric cancer, while its role in other cancers is unknown. We investigated the role of WT1-AS in non-small cell lung cancer (NSCLC).MethodsSixty-six NSCLC patients (40 males and 26 females; 36 to 68 years old; mean age 52.7 ± 6.4 years old) were selected from the 178 NSCLC patients operated on for lung cancer between 2010 and 2013. RT-qPCR was used to analyze the expression of lncRNA. Overexpression experiments were performed to assess interactions between lncRNAs. CCK-8 assay was carried to evaluate the roles of WT1-AS and UCA1 in regulating cell proliferation. Cell invasion and migration assays were performed to assess the roles of WT1-AS and UCA1 in regulating cell invasion and migration. Western-blot was performed to illustrate the effect of WT1-AS and UCA1 in EMT.ResultsWT1-AS was downregulated in NSCLC and was correlated with poor survival. The expression of WT1-AS in NSCLC was not correlated with clinical stages. LncRNA UCA1 was upregulated in cancer tissues and inversely correlated with WT1-AS. Overexpression of UCA1 did not affect WT1-AS, while overexpression of WT1-AS led to inhibited expression of UCA1. Overexpression of UCA1 resulted in increased proliferation, EMT, migration and invasion of NSCLC cells, while overexpression of WT1-AS showed opposite effects. In addition, overexpression of UCA1 inhibited the role of overexpression of WT1-AS.ConclusionsTherefore, overexpression of WT1-AS may inhibit the cell proliferation and EMT to decrease cell migration and invasion of NSCLC cells by downregulating UCA1.

MiR-758-3p inhibits the proliferation and invasion of non-small cell lung cancer cells by targeting NUSAP1

Objective: To investigate the biological function of miR-758-3p and the underlying mechanism in non-small cell lung cancer (NSCLC). Methods: miR-758-3p mimics was transfected to A549 NSCLC cells, miRNA control was used as a negative control, cells transfected with nucleolar and spindle associated protein 1 (NUSAP1)-overexpression vector indicated as NUSAP1 group, cells co-transfected with miR-758-3p mimics and NUSAP1-overexpression vector indicated as miR-758-3p mimics+ NUSAP1 group. The effects of miR-758-3p on the proliferation, migration and invasion abilities of A549 cells were detected by cell counting kit-8 (CCK-8) and Transwell assays, respectively. Bioinformatics and dual luciferase reporter assay were used to predict and verify the target gene of miR-758-3p. Results: The expressions of miR-758-3p and NUSAP1 in A549 cells were significantly up-regulated at 24 hours after transfection with miR-758-3p mimics (P<0.05). Compared with the miRNA control group (1.15±0.06), the OD value of miR-758-3p mimic group (0.78±0.06) was significantly decreased at 72 hours after transfection (P<0.05). The number of migrated cells of miR-758-3p mimic group (119.04±11.49) was significantly lower than that of the control group (271.38±19.05) (P<0.05). The number of invaded cells of miR-758-3p mimic group (71.33±5.36) was significantly lower than the control group (164.30±8.11) (P<0.05). The result of dual-luciferase reporter assay showed that NUSAP1 was a direct target of miR-758-3p. Moreover, up-regulation of NUSAP1 abolished the inhibitory effects of miR-758-3p on the proliferation, migration and invasion abilities of A549 cells. Conclusion: miR-758-3p inhibits the proliferation, migration and invasion of NSCLC cells by targeting NUSAP1.

Long non-coding RNA linc00665 inhibits CDKN1C expression by binding to EZH2 and affects cisplatin sensitivity of NSCLC cells

Long non-coding RNAs (lncRNAs) can play significant regulatory roles in cells that affect the development and acquired drug resistance of lung cancer. Herein, we report that lncRNA linc00665 is significantly upregulated in non-small cell lung cancer (NSCLC) tissues compared with adjacent normal tissues. linc00665 affects the sensitivity of NSCLC cells to the chemotherapy drug cisplatin (DDP), making it a potential target for the treatment of NSCLC. Functional experiments showed that linc00665 enhanced the proliferation and migration of NSCLC cells in vivo and in vitro, and knocking down linc00665 could enhance the drug sensitivity of NSCLC cells to DDP. Further work revealed that linc00665 could recruit enhancer of zeste homolog 2 (EZH2) to the promoter region of cyclin-dependent kinase inhibitor 1C (CDKN1C) to inhibit its transcription and thus carry out its tumorigenic role. In conclusion, our study elucidated the carcinogenic role of the linc00665-EZH2-CDKN1C axis in NSCLC tumors and its ability to influence the sensitivity of these tumors to DDP. These results suggest that linc00665 may be a potential diagnostic marker and therapeutic target in NSCLC, and they also provide a new direction for the development of clinical reversal methods for acquired drug resistance in patients with NSCLC.

Long non‑coding RNA PITPNA‑AS1 silencing suppresses proliferation, metastasis and epithelial‑mesenchymal transition in non‑small cell lung cancer cells by targeting microRNA‑32‑5p.

Lung cancer is one of the most common types of cancer and has a high mortality rate, worldwide. The major histopathological subtype is non-small cell lung cancer (NSCLC). The aim of the present study was to investigate the role of long non-coding (lnc) RNA PITPNA antisense RNA 1 (PITPNA-AS1) in NSCLC and elucidate its potential mechanisms. The expression of PITPNA-AS1 was determined in several NSCLC cell lines. Following PITPNA-AS1-silencing, cell proliferation, invasion and migration were evaluated using Cell Counting Kit-8, colony formation, Transwell assay and wound healing assays, respectively. The expression levels of proliferation-, migration- and epithelial-mesenchymal transition (EMT)-associated proteins were examined using immunofluorescence assay or western blot analysis. A luciferase reporter assay was conducted to verify the potential interaction between PITPNA-AS1 and microRNA(miR)-32-5p. Subsequently, rescue assays were performed to investigate the effects of PITPNA-AS1 and miR-32-5p on NSCLC progression. The results demonstrated that PITPNA-AS1 was highly expressed in NSCLC tissues and cell lines. It was found that PITPNA-AS1 silencing inhibited the proliferation, invasion and migration of NSCLC cells. Furthermore, the protein expression of E-cadherin was upregulated, while the expression levels N-cadherin and vimentin were downregulated. The luciferase reporter assay confirmed that miR-32-5p was a direct target of PITPNA-AS1. The rescue experiments suggested that a miR-32-5p inhibitor significantly reversed the inhibitory effects of PITPNA-AS1 silencing on proliferation, invasion, migration and EMT in NSCLC cells. Collectively, the present results demonstrated that PITPNA-AS1 silencing could suppress the progression of NSCLC by targeting miR-32-5p, suggesting a promising biomarker in NSCLC diagnosis and treatment.

CDCA4 suppresses epithelial\u2013mesenchymal transtion (EMT) and metastasis in Non-small cell lung cancer through modulating autophagy

BackgroundCell division cycle associated 4 (CDCA4) has been reported to be engaged into the progression of several cancers. The function of CDCA4 in Non-small cell lung cancer (NSCLC) was unknown. We aimed to explore the critical role of CDCA4 in NSCLC.MethodsCDCA4 stably knocking down and overexpression cell lines were established and Western blotting assay was applied to measure relevant protein expression of Epithelial-Mesenchymal Transtion (EMT) and cell autophagy. Staining of acidic vacuoles, transmission electron microscopy and immunofluorescence staining were employed to detect autophagy. The ability of cells to migrate and invade were detected by Transwell migration and invasion assays. The interaction of CDCA4 with CARM1 was identified by immunoprecipitation and Western blotting analysis.ResultsIn the present study, it was found that inhibition of CDCA4 induced EMT, migration and invasion of NSCLC cells while inhibiting autophagy of NSCLC cells. Meanwhile, overexpression of CDCA4 in NSCLC cells showed the opposite function. More importantly, the inhibition of autophagy could promote the EMT, migration and invasion of NSCLC cells, which should be impaired via the activation of autophagy. In addition, CDCA4-inhibited EMT, migration and invasion could be partially aggravated by autophagy activator, rapamycin, and reversed by autophagy inhibitor, 3-MA. Correspondingly, the application of rapamycin or 3-MA to CDCA4 knockdown cells showed the opposite effects. Further investigation suggested that CDCA4 could interact with coactivator associated arginine methyltransferase 1 (CARM1). Autophagy was induced while cell migration and invasion were inhibited in CARM1 knockdown cells. CDCA4 could suppress the protein expression CARM1 and knocking down of CARM1 could alter cell autophagy, migratory and invasive abilities regulated by CDCA4.ConclusionAll data indicated that CDCA4 inhibited the EMT, migration and invasion of NSCLC via interacting with CARM1 to modulate autophagy.

HOXC-AS2 mediates the proliferation, apoptosis, and migration of non-small cell lung cancer by combining with HOXC13 gene

ABSTRACT Non-small cell lung cancer (NSCLC) is the highest incidence and mortality of malignant tumors worldwide and has become a global public health problem. Long non-coding RNAs (LncRNAs) are expected to participate in the progression of NSCLC. This study aims to explore the effects and underlying mechanisms of LncRNA HOXC-AS2 on NSCLC cell proliferation, apoptosis, and migration. The Cell Counting Kit-8 (CCK-8) and clone formation assay were used to measure the A549 and HCC827 cell proliferation. The cell apoptosis and migration was respectively analyzed by flow cytometry and transwell assay. RNA immunoprecipitation (RIP) was used to detect the interaction between HOXC-AS2 and HOXC13. The expression of β-catenin, α-SMA, MMP-1, MMP-2 expression, E-cadherin, and Ki-67 expression were determined by Western blot or immunohistochemistry (IHC) assay. We found that HOXC-AS2 was significantly up-regulated in NSCLC tissues. Knockdown of HOXC-AS2 expression resulted in significant decreases in NSCLC cell proliferation, migration, and epithelial-mesenchymal transition (EMT) process marker proteins, simultaneously activated A549 and HCC827 cell apoptosis. RIP assay suggested that HOXC13 was a functional target for HOXC-AS2. And HOXC-AS2 and HOXC13 could positively regulate each other. Compared with the normal tissues, the mRNA level of HOXC13 was increased in NSCLC tissues. HOXC13 silencing counteracted increases of A549 and HCC827 cell proliferation and migration, as well as a decrease of cell apoptosis induced by HOXC-AS2 overexpression. Moreover, HOXC-AS2 silencing reduced tumor growth rate and Ki-67 expression in vivo. Taken together, HOXC-AS2 knockdown inhibited NSCLC cell proliferation and migration, as well as stimulated NSCLC cell apoptosis through regulation of HOXC13 expression.

Radiation exposure triggers the malignancy of non‑small cell lung cancer cells through the activation of visfatin/Snail signaling.

It is estimated that one-half of patients with non-small cell lung cancer (NSCLC) undergo radiotherapy worldwide. However, the outcome of radiotherapy alone is not always satisfactory. The aim of the present study was to evaluate the effects of radiotherapy on the malignancy of NSCLC cells. It was demonstrated that radiation therapy could increase the migration and invasion of NSCLC cells in vitro. Moreover, the upregulation of visfatin, a 52-kDa adipokine, mediated radiation-induced cell motility. A neutralizing antibody specific for visfatin blocked radiation-induced cell migration. Radiation and visfatin induced the expression of Snail, a key molecule that regulates epithelial to mesenchymal transition in NSCLC cells. Furthermore, visfatin positively regulated the mRNA stability of Snail in NSCLC cells, but had no effect on its protein degradation. This may be explained by visfatin-mediated downregulation of microRNA (miR)-34a, which was shown to bind the 3′ untranslated region of Snail mRNA to promote its decay. Collectively, these findings suggested that radiation could induce cell motility in NSCLC cells through visfatin/Snail signaling.

2,6-DMBQ suppresses cell proliferation and migration via inhibiting mTOR/AKT and p38 MAPK signaling pathways in NSCLC cells

2,6-Dimethoxy-1,4-benzoquinone (2,6-DMBQ) is the major bioactive compound found in fermented wheat germ extract. Although fermented wheat germ extract has been reported to show anti-proliferative and anti-metabolic effects in various cancers, the anticancer potential and molecular mechanisms exerted by 2,6-DMBQ have not been investigated in non-small cell lung cancer (NSCLC) cells. Here, we report that 2,6-DMBQ suppresses NSCLC cell growth and migration through inhibiting activation of AKT and p38 MAPK. 2,6-DMBQ significantly suppressed anchorage-dependent and independent cell growth. Additionally, 2,6-DMBQ induced G2 phase cell cycle arrest through inhibiting the expression and phosphorylation of cyclin B1 and CDC2, respectively. Furthermore, 2,6-DMBQ strongly suppressed NSCLC cell migration through induction of E-cadherin expression. To determine the molecular mechanism(s) exerted by 2,6-DMBQ upon NSCLC cell lines, various signaling kinases were screened; the results indicate that 2,6-DMBQ strongly inhibits the phosphorylation of AKT and p38 MAPK. Additionally, the growth kinetics of cells treated with an AKT or p38 MAPK inhibitor in combination with 2,6-DMBQ indicate that 2,6-DMBQ suppresses NSCLC cell growth and migration through inhibition of AKT and p38 MAPK. Taken together, our results suggest that 2,6-DMBQ is a potential anticancer reagent against NSCLC cells and could be useful for treating lung cancer patients.

Hypoxia-sensitive long noncoding RNA CASC15 promotes lung tumorigenesis by regulating the SOX4/\u03b2-catenin axis

BackgroundAccumulating evidence has demonstrated that long non-coding RNAs (lncRNAs) are involved in the hypoxia-related cancer process and play pivotal roles in enabling malignant cells to survive under hypoxic stress. However, the molecular crosstalk between lncRNAs and hypoxia signaling cascades in non-small cell lung cancer (NSCLC) remains largely elusive.MethodsFirstly, we identified differentially expressed lncRNA cancer susceptibility candidate 15 (CASC15) as associated with NSCLC based on bioinformatic data. The clinical significance of CASC15 in lung cancer was investigated by Kaplan-Meier survival analysis. Then, we modulated CASC15 expression in NSCLC cell lines by RNAi. CCK-8 and transwell assays were carried out to examine the effects of CASC15 on proliferation and migration of NSCLC cells. Upstream activator and downstream targets of CASC15 were validated by luciferase reporter assay, qRT-PCR, Western blotting, and chromatin immunoprecipitation (ChIP). Lastly, RNA in situ hybridization (RNA-ISH) and immunohistochemistry (IHC) were performed to confirm the genetic relationships between CASC15 and related genes in clinical samples.ResultsCASC15 was highly expressed in NSCLC tissues and closely associated with poor prognosis. Loss-of-function analysis demonstrated that CASC15 was essential for NSCLC cell migration and growth. Mechanistic study revealed that CASC15 was transcriptionally activated by hypoxia signaling in NSCLC cells. Further analysis showed that hypoxia-induced CASC15 transactivation was mainly dependent on hypoxia-inducible factor 1α (HIF-1α) and hypoxia response elements (HREs) located in CASC15 promoter. CASC15 promotes the expression of its chromosomally nearby gene, SOX4. Then SOX4 functions to stabilize β-catenin protein, thereby enhancing the proliferation and migration of NSCLC cells. HIF-1α/CASC15/SOX4/β-catenin pathway was activated in a substantial subset of NSCLC patients.ConclusionsHIF-1α/CASC15/SOX4/β-catenin axis plays an essential role in the development and progression of NSCLC. The present work provides new evidence that lncRNA CASC15 holds great promise to be used as novel biomarkers for NSCLC. Blocking the HIF-1α/CASC15/SOX4/β-catenin axis can serve as a potential therapeutic strategy for treating NSCLC.

Long non‑coding RNA MEG3 inhibits cell migration and invasion of non‑small cell lung cancer cells by regulating the miR‑21‑5p/PTEN axis.

Long non-coding RNAs (lncRNAs) are involved in the occurrence and progression of numerous types of cancer. The aim of the present study was to evaluate the effect of the lncRNA maternally expressed gene 3 (MEG3) on the migration and invasion of non-small cell lung cancer (NSCLC) H1299 and PC9 cells. Reverse transcription-quantitative (RT-q)PCR analysis showed that MEG3 was downregulated in NSCLC PC9 and H1299 cells. Additionally, bioinformatics analysis indicated that MEG3 sponges microRNA (miR)-21-5p; miR-21-5p was predicted to target the phosphatase and tensin homolog (PTEN) 3′-untranslated region sequence. MEG3 overexpression led to miR-21-5p suppression and PTEN upregulation in PC9 and H1299 cells, as detected by RT-qPCR. Subsequently, western blot analysis confirmed that MEG3 overexpression enhanced PTEN expression levels and inhibited the PI3K/AKT signaling pathway in NSCLC cells. These effects were attenuated by miR-21-5p. Dual luciferase assay supported the sponging effect of MEG3 on miR-21-5p and validated the direct interaction between miR-21-5p and PTEN. Furthermore, Transwell assay demonstrated that MEG3 overexpression had an inhibitory effect on cell migration and invasion. MEG3 overexpression also mediated epithelial-to-mesenchymal transition by significantly enhancing E-cadherin and decreasing N-cadherin, Vimentin and matrix metalloprotein 9 expression levels in NSCLC cells, as indicated by western blot analysis. These changes were partially reversed by an miR-21-5p mimic. These results indicated that MEG3 acted as a tumor suppressor that inhibited NSCLC cell migration and invasion via sponging miR-21-5p, which, in turn, enhanced the expression levels of PTEN, in part via the PI3K/AKT signaling pathway. The results of the present study have suggested the potential of MEG3 as a novel therapeutic target for NSCLC treatment.

GABPB1-AS1 acts as a tumor suppressor and inhibits non-small cell lung cancer progression by targeting miRNA-566/F-box protein 47.

It has been certified that GABPB1-AS1 is aberrantly expressed and plays as a vital role in some kinds of cancers. However, its expression pattern and functions in non-small cell lung cancer (NSCLC) are still largely unknown. This study aims to assess GABPB1-AS1 expression and biological roles in NSCLC. The expression of GABPB1-AS1 was detected in NSCLC specimens and adjacent normal specimens. CCK8 and Transwell assays were performed to evaluate the effects of GABPB1-AS1 on NSCLC cell proliferation, migration and invasion. Bioinformatics tools and luciferase reporter assays were applied to predict and verify GABPB1-AS1's direct targets. The results revealed that GABPB1-AS1 is sharply reduced in NSCLC specimens and cell lines. CCK8 assays indicated that overexpression of GABPB1-AS1 dramatically reduced NSCLC cell growth, and Transwell assays proved that NSCLC cell migration and invasion were distinctly inhibited by GABPB1-AS1. Exploration of the mechanism uncovered that miRNA-566 (miR-566)/F-box protein 47 (FBXO47) is directly targeted by GABPB1-AS1 in NSCLC. The study demonstrated that GABPB1-AS1 inhibited NSCLC cell proliferation, migration and invasion by targeting miR-566/FBXO47.