Non-small Cell Lung Cancer Invasion Research Articles

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.

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.

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.

Circular RNA circ_0001287 inhibits the proliferation, metastasis, and radiosensitivity of non-small cell lung cancer cells by sponging microRNA miR-21 and up-regulating phosphatase and tensin homolog expression

ABSTRACT As a type of non-coding RNA, circular RNA (circRNA) figures prominently in human cancer progression. Nonetheless, the expression, function, and regulatory mechanism of circ_0001287 in non-small cell lung cancer (NSCLC) remain obscure. In this work, quantitative real-time polymerase chain reaction (qRT-PCR) was implemented to quantify circ_0001287 and miR-21 expressions in NSCLC tissues and cells. The relationship between circ_0001287 expression and the clinicopathological parameters of NSCLC patients was examined. Cell counting kit-8 (CCK-8), 5-bromo-2©-deoxyuridine (BrdU), and Transwell experiments were conducted to detect the multiplication, migration, and invasion of NSCLC cells after circ_0001287 was overexpressed or knocked down. The survival of NSCLC cells was studied using colony formation experiment under different doses of radiation. RNA immunoprecipitation (RIP) experiment and luciferase reporter gene experiment verified the binding relationship between circ_0001287 and miR-21. Western blot was employed to examine the regulatory effects of circ_0001287 and miR-21 on phosphatase and tensin homolog (PTEN) expression. We reported that circ_0001287 expression was down-modulated in NSCLC tissues and cell lines. Besides, circ_0001287 low expression was associated with low differentiation and positive lymph node invasion of NSCLC. Circ_0001287 overexpression suppressed the multiplication, migration, invasion, and radioresistance of NSCLC cells, whereas circ_0001287 knockdown promoted the above phenotypes. Circ_0001287 could adsorb miR-21 and repress its expression, and indirectly up-modulate PTEN expression in NSCLC cells. Taken together, circ_0001287/miR-21/PTEN axis is probably involved in regulating NSCLC cell multiplication, metastasis, and radioresistance.

Oncoprotein HBXIP promotes tumorigenesis through MAPK/ERK pathway activation in non-small cell lung cancer.

Objective:The oncoprotein, hepatitis B X-interacting protein (HBXIP), has been reported to play an important role in human malignancies. However, its functions in non-small cell lung cancer (NSCLC) are poorly understood. The goal of the present study was to identify the role of HBXIP in the regulation of NSCLC development.Methods:The level of HBXIP expression in NSCLC tissue was assessed by immunohistochemical and Western blot analyses, and its relationships with clinicopathological features and outcomes were statistically evaluated. The effects of HBXIP on NSCLC cell progression were assessed through cell viability, colony formation, and flow cytometry analyses in vitro. The mechanism by which HBXIP regulated the MAPK pathway was studied by Western blot, immunofluorescence, and immunoprecipitation assays. In addition, in vivo experiments were performed to evaluate the progression of NSCLC and ERK signaling pathway activation after HBXIP knockdown.Results:HBXIP was overexpressed in human NSCLC and was correlated with the invasiveness of NSCLC. The high expression of HBXIP in NSCLC was significantly correlated with gender (P = 0.033), N stage (P = 0.002), and tumor-node-metastasis stage (P = 0.008). In vitro experiments using an NSCLC cell line revealed that HBXIP knockdown resulted in the suppression of cell proliferation and colony formation, which was consistent with the enhanced cell cycle arrest in G1 phase. The results of a mechanistic investigation suggested that binding of HBXIP to MEK1 protein promoted MAPK/ERK signaling pathway activation in NSCLC by preventing the proteasome-mediated degradation of MEK1. In addition, the results obtained using in vivo subcutaneous tumor xenografts confirmed that HBXIP deficiency decreased MEK1 protein levels and NSCLC tumor growth.Conclusions:Taken together, our results showed that the HBXIP-MEK interaction promoted oncogenesis via the MAPK/ERK pathway, which may serve as a novel therapeutic target for cancers in which MAPK/ERK signaling is a dominant feature.

LINC01296/miR-141-3p/ZEB1-ZEB2 axis promotes tumor metastasis via enhancing epithelial-mesenchymal transition process.

Purpose: Tumor metastasis seriously affects the survival of patients. In recent years, some studies confirmed that long non-coding RNA (lncRNA) played an essential role in tumor progression. A few studies reported that LINC01296 acted as an oncogenic regulator of cancer. However, its in-depth specific biological mechanism in tumor metastasis is still unknown.Methods: Real-time quantitative PCR (qPCR) was performed to detect the expression of LINC01296 and miR-141-3p in NSCLC, CRC tissues and cell lines, and the dual luciferase report was used to evaluate the relationship between LINC01296, miR-141-3p and ZEB1/ZEB2 relationship. Western blot experiments are used to detect changes in protein levels. Transwell and wound healing measures migration and invasion capabilities.Results: In this study, we used non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) as the research objects, LINC01296 was found to be highly expressed in NSCLC and CRC tissues and positively related to poor prognosis. We also demonstrated LINC01296 regulated NSCLC and CRC invasion and metastasis by modulating epithelial-mesenchymal transition (EMT) by up-regulating ZEB1 and ZEB2. Consequently, LINC01296 acted as a sponge of miR-141-3p, which negatively regulates EMT process.Conclusions: The report revealed a new mechanism by which LINC01296 regulates the EMT process through miR-141-3p/ZEB1-ZEB2 axis and affects cancer metastasis.

Insufficient ablation promotes the metastasis of residual non-small cell lung cancer (NSCLC) cells via upregulating carboxypeptidase A4

Background Thermal ablation is a potentially curative therapy for early-stage non-small cell lung cancer (NSCLC). Early recurrence after thermal ablation necessitates our attention. Methods The invasion and migration abilities of NSCLC after sublethal heat stimulus were observed in vitro and in vivo. Sublethal thermal stimulus molecular changes were identified by RNA sequencing. A xenograft model of NSCLC with insufficient ablation was established to explore the epithelial-to-mesenchymal transition (EMT) and metastasis-related phenotypes alteration of residual tumors. Results In vitro, the invasion and migration abilities of NSCLC cells were enhanced 72 h after 44 °C and 46 °C thermal stimulus. Epithelial–mesenchymal transition (EMT) phenotypes were also upregulated under these conditions. RNA sequencing revealed that the expression of carboxypeptidase A4 (CPA4) was significantly upregulated after thermal stimulus. Significant upregulation of CPA4 and EMT phenotypes was also found in the xenograft model of insufficient NSCLC ablation. The EMT process and invasion and migration abilities can be reversed by silencing CPA4. Conclusions This study demonstrates that sublethal heat stimulus caused by insufficient ablation can promote EMT and enhance the metastatic capacity of NSCLC. CPA4 plays an important role in these biological processes. Inhibition of CPA4 might be of great significance for improving early-stage NSCLC survival after ablation.

Circular RNA circ_0010235 sponges miR-338-3p to play oncogenic role in proliferation, migration and invasion of non-small-cell lung cancer cells through modulating KIF2A

Background Circular RNA microarray analysis showed hsa_circ_0010235 (circ_0010235) was highly upregulated in non-small-cell lung cancer (NSCLC) patients; however, its role in carcinogenesis and development of NSCLC cells was unrevealed. Here, we intended to investigate role and mechanism of circ_0010235 in NSCLC proliferation, migration and invasion. Methods and results Expression of circ_0010235, microRNA (miR)-338-3p and kinesin family member 2A (KIF2A) was detected by quantitative real-time PCR, western blotting and immunohistochemistry (IHC). Cell progression was measured by cell-counting kit-8 assay, 5-ethynyl-2-deoxyuridine (EdU) assay, flow cytometry, transwell assay, western blotting, IHC and xenograft experiment. The relationship among circ_0010235, miR-338-3p and KIF2A was determined by dual-luciferase reporter assay, RNA immunoprecipitation and Pearson’s correlation analysis. Expression of circ_0010235 was increased in human NSCLC tissues and cells, accompanied with miR-338-3p downregulation and KIF2A upregulation. Essentially, circ_0010235 could sponge miR-338-3p via target binding, and miR-338-3p downstream targeted KIF2A. Functionally, exhaustion of circ_0010235 induced apoptosis rate of NSCLC cells and curbed cell viability, EdU incorporation, migration rate and invasion rate, accompanied with higher E-cadherin and lower N-cadherin expression. Additionally, re-expression of miR-338-3p prompted above similar effects in NSCLC cells in vitro. Contrarily, miR-338-3p blockage partially counteract the effects of circ_0010235 exhaustion; plus, restoration of KIF2A could attenuate miR-338-3p role, as well. Notably, interfering circ_0010235 delayed tumour growth of NSCLC cells by promoting miR-338-3p and E-cadherin expression, and depressing KIF2A, ki-67 and N-cadherin expression. Conclusions circ_0010235 could be a novel identified oncogenic circRNA in NSCLC, and targeting miR-338-3p/KIF2A axis was one regulatory mechanism underlying circ_0010235. KEY MESSAGE Circ_0010235 was an upregulated circRNA in NSCLC patients and cells. Interfering circ_0010235 restrained NSCLC cell proliferation and metastasis in vitro and in vivo. miR-338-3p per se suppressed NSCLC in vitro and its downregulation diminished the tumour-suppressive role of circ_0010235 blockage in NSCLC cells. miR-338-3p could downstream target KIF2A and be sponged by circ_0010235.

MicroRNA-32-5p inhibits epithelial-mesenchymal transition and metastasis in lung adenocarcinoma by targeting SMAD family 3.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-associated death worldwide. MicroRNA (miRNA)-32-5p is as an important cancer-associated miRNA in different types cancer. To date, the role of miR-32-5p in the migration and invasion of NSCLC remains unknown. In the present study, a Transwell assay was performed to investigate the role of miR-32-5p in lung adenocarcinoma. miR-32-5p expression level was determined via reverse transcription-quantitative PCR in 24 pairs of NSCLC and adjacent normal tissues. SMAD family member 3 (SMAD3) was considered as a novel target gene by luciferase reporter assay and western blot in NSCLC. The present study demonstrated that miR-32-5p is frequently downregulated in NSCLC tissues. The overexpression of miR-32-5p resulted in the inhibition of migratory and invasive abilities in NSCLC cells. Thus, SMAD3 was identified as a target of miR-32-5p, and its expression was negatively correlated with miR-32-5p expression in clinical NSCLC tissues. Overall, these findings indicate that miR-32-5p serves as a tumor suppressor by targeting SMAD3. Thus, miR-32-5p may be a potential therapeutic target for the treatment of lung adenocarcinoma.

ZCCHC14 regulates proliferation and invasion of non-small cell lung cancer through the MAPK-P38 signalling pathway.

ZCCHC14 is a CCHC‐type zinc finger protein which is expressed in tissues in human and mouse. The function of ZCCHC14 in tumours remains unclear. In this research, we explored the expression, function and related molecular mechanisms of ZCCHC14 in human non–small cell lung cancer (NSCLC). Immunochemistry staining showed that ZCCHC14 was low‐expressed or absent in NSCLC tissues. In NSCLC patients, the low expression of ZCCHC14 in tumour tissues was significantly correlated with TNM stage, differentiation degree and adverse clinical outcome (P < .05). The proliferation and invasion ability of cancer cells transfected with ZCCHC14 CRISPR/Ca9 KO plasmids was significantly enhanced (P < .05). Immunoblotting analysis indicated that the expression of p‐P38, cyclinD1 and MMP7 were significantly up‐regulated after disabling ZCCHC14 (P < .05). We used MAPK‐P38 pathway inhibitor doramapimod (BIRB 796) to inhibit P38 signalling pathway activity and determined that the agent significantly disrupted the function of ZCCHC14 and hindered the proliferation and invasion of the tumour. The finding revealed that ZCCHC14 can regulate proliferation and invasion of NSCLC through the P38 pathway. ZCCHC14 plays a crucial regulatory role in the development of NSCLC and may become a zinc finger target for clinical treatment.

Long Noncoding RNA DANCR Activates Wnt/β-Catenin Signaling through MiR-216a Inhibition in Non-Small Cell Lung Cancer.

Long noncoding RNA differentiation antagonizing nonprotein coding RNA (lncRNA-DANCR) is associated with poor prognosis in multiple cancers, and promotes cancer stemness and invasion. However, the exact mechanisms by which DANCR promotes non-small cell lung cancer (NSCLC) remain elusive. In this study, we determined that DANCR knockdown (KD) impeded cell migration and reduced stem-like characteristics in two NSCLC cell lines, A549 and H1755. Wnt signaling was shown to promote NSCLC proliferation, stemness, and invasion; therefore, we hypothesized that DANCR may regulate these activities through induction of the Wnt/β-catenin pathway. DANCR KD reduced β-catenin signaling and protein expression, and decreased the expression of β-catenin gene targets c-Myc and Axin2. One of the well-defined functions of lncRNAs is their ability to bind and inhibit microRNAs. Through in silico analysis, we identified tumor suppressor miR-216a as a potential binding partner to DANCR, and confirmed this binding through coimmunoprecipitation and luciferase-reporter assays. Furthermore, we show that DANCR-induced β-catenin protein expression may be blocked with miR-216a overexpression. Our findings illustrate a role of DANCR in NSCLC migration and stemness, and suggest a novel DANCR/miR-216a signaling axis in the Wnt/β-catenin pathway.

FKBP4 Accelerates Malignant Progression of Non-Small-Cell Lung Cancer by Activating the Akt/mTOR Signaling Pathway.

Objective To study the expression, biological function, and mechanism of FKBP4 in non-small-cell lung cancer (NSCLC). Methods First of all, the expression of FKBP4 in NSCLC tissues and cell lines was detected by qRT-PCR; then, the effects of FKBP4 on proliferation, apoptosis, migration, and invasion of NSCLC were studied by CCK-8 assays, flow cytometry assays, wound-healing assays, and Transwell assays. After that, tumor xenografts were used to explore the effect of FKBP4 on NSCLC tumor growth in vivo, and the phosphorylation of Akt and mTOR was measured by western blot. Results FKBP4 was highly expressed in NSCLC tissues and cells, and its expression was closely related to NSCLC tumor size, lymph node metastasis, and patient prognosis. In vitro, FKBP4 can promote NSCLC cell proliferation, migration, and invasion and inhibit NSCLC cell apoptosis. In vivo, FKBP4 can promote NSCLC tumor growth. Furthermore, FKBP4 can promote Akt and mTOR phosphorylation and activate the Akt/mTOR signaling pathway and an mTOR inhibitor can neutralize the functions of FKBP4 in NSCLC cells. Conclusion FKBP4 serves as an oncogene to promote malignant processes in NSCLC, and it has the potential to be used as a biological marker and therapeutic target for NSCLC.

LncRNA NEAT1/miR-204/NUAK1 Axis is a Potential Therapeutic Target for Non-Small Cell Lung Cancer.

BackgroundLong non-coding RNA (lncRNA) is a key part of non-coding RNA, and more and more evidence has revealed that it plays a vital role in tumors. NEAT1 is a lncRNA discovered in the early stage. However, it is still unclear whether NEAT1 and miR-204 play a regulatory role in lung cancer (LC). This research aimed to determine the biological function of NEAT1/miR-204 in non-small cell lung cancer (NSCLC).Materials and MethodsIn order to research the function of NEAT1 in NSCLC, RT-PCR, Western blot, luciferase assay and RNA immunoprecipitation assay were used to determine the relationship between NEAT1, miR-204 and NUAK1. CCK8 test, cell migration and invasion test were used to explore the influence of NEAT1 on proliferation and metastasis of LC cells. Tumor allotransplantation was used to detect the influence of NEAT1 on the growth of LC.ResultsThe results revealed that NEAT1 was obviously enhanced in LC cell lines. Further functional analysis showed that low expression of NEAT1 obviously suppressed the growth, migration and invasion of NSCLC and facilitated cell apoptosis. Determination of luciferase reporter gene revealed that miR-204 was the direct target of NEAT1 in LC. In addition, NUAK1 was called the direct target of miR-204, and miR-204/NUAK1 had saved the role of NEAT1 in NSCLC cells. Tumor allotransplantation experiments showed that knocking down NEAT1 could inhibit the growth of LC.ConclusionIn summary, our results showed that the down-regulation of NEAT1 in NSCLC inhibited its growth, migration and invasion through the miR-204/NUAK1 axis.

Fentanyl Inhibits Lung Cancer Viability and Invasion via Upregulation of miR-331-3p and Repression of HDAC5

PurposeNon-small cell lung cancer (NSCLC) accounts for more than 80% of lung cancer cases and remains the primary cause of cancer-related deaths worldwide. Fentanyl is a commonly utilized anesthetic during the process of tumor resection, and exhibits inhibitory effects on the progression of numerous cancer types, including pancreatic cancer, colorectal cancer and gastric cancer. However, the effects of fentanyl on the cell viability and invasion of NSCLC has not been investigated. Current study aimed to investigate the effects and the mechanisms underlying the effects of fentanyl on NSCLC.MethodsThe expression of μ-opioid receptor (MOR) was proved by flow cytometry. The expression of microRNA-331-3p (miR-331-3p) and histone deacetylase 5 (HDAC5) in NSCLC tissues and cell lines are evaluated by reverse transcription-quantitative PCR (RT-qPCR) and Western blot, respectively. Cell viability and invasion are measured by cell counting kit-8 (CCK-8) assay and transwell assay, respectively. The interaction between miR-331-3p and 3ʹ-untranslated region (UTR) of HDAC5 is predicted by TargetScan 7.1 (http://www.targetscan.org/vert_71/), validated by dual luciferase assay, RT-qPCR and Western blot.ResultsThere was lower miR-331-3p expression and higher HDAC5 expression in NSCLC cell lines A549 and CALU-1 compared with BEAS-2B, which was reversed by fentanyl administration. miR-331-3p targeted 3ʹ-UTR of HDAC5 in NSCLC cell lines A549 and CALU-1. miR-331-3p inhibitor partially abrogated the inhibitory effects of fentanyl on NSCLC cell viability and invasion by targeting HDAC5. In addition, there was higher HDAC5 expression and lower miR-331-3p expression in tumor tissues which were isolated from patients with NSCLC compared to the adjacent normal tissues, and miR-331-3p was negatively correlated with HDAC5 in NSCLC tumor tissues.ConclusionFentanyl inhibits the viability and invasion of NSCLC cells by induction of miR-331-3p and reduction of HDAC5.

EPB41 suppresses the Wnt/β-catenin signaling in non-small cell lung cancer by sponging ALDOC.

Despite advancements in therapeutic options, the overall prognosis for non-small-cell lung cancer (NSCLC) remains poor. Further exploration of the etiology and targets for novel treatments is crucial for managing NSCLC. In this study, we revealed the significant potential of EPB41 for inhibiting NSCLC proliferation, invasion and metastasis in vitro and in vivo. Consistent with its tumor suppressor role in NSCLC, the expression of EPB41 in NSCLC specimens evidently decreased compared to that in normal tissues, and low EPB41 expression was associated with poor prognoses for NSCLC patients. We further demonstrated the importance of EPB41 protein as a novel inhibitor of the Wnt signaling, which regulates β-Catenin stability, and elucidated the crucial role of the EPB41/ALDOC/GSK3β/β-Catenin axis in NSCLC. Suppression of EPB41 expression in cancer cells elevated the levels of free ALDOC protein released from the EPB41-ALDOC complex, leading to disassembly of the β-catenin destruction complex, reduced proteolytic degradation of β-catenin, elevated cytoplasmic accumulation and nuclear translocation of β-catenin, thereby activating the expression of multiple oncogenes and, thus, NSCLC pathogenesis. Our study highlights the potential of EPB41 as a future therapeutic target for lung cancer.

MiR-371b-5p promotes cell proliferation, migration and invasion in non-small cell lung cancer via SCAI.

Objective: Multiple gene targets have been reported for treatment of non-small cell lung cancer (NSCLC), however, the accompanying genetic tolerance was reported increasingly. Therefore, it is important to find new biomarkers or therapeutic targets in treatment of NSCLC.Methods: The expression levels of miR-371b-5p were detected by qRT-PCR in NSCLC tissues and cell lines. To evaluate the effect of miR-371b-5p on NSCLC progression, we first transfected the miR-371b-5p inhibitor for construction of the miR-371b-5p down-regulated cell model. Then the cell proliferation, migration, invasion and cell apoptosis were detected. In addition, the expression levels of adhesion factors were detected. The target gene of miR-371b-5p was identified by bioinformatics analysis, and rescue experiment was conducted to validate the effect of miR-371b-5p on proliferation, migration and invasion of NSCLC.Results: Our findings revealed that the miR-371b-5p was overexpressed in NSCLC and could markedly promote the cell proliferation, migration and invasion. Expression levels of both intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were significantly down-regulated when treated by miR-371b-5p inhibitor. Moreover, dual-luciferase reporter assay showed that the miR-371b-5p targeted SCAI in regulation of cell proliferation, migration and invasion, and the expression of miR-371b-5p was negatively associated with SCAI in NSCLC tissues and cell lines. Rescue experiment revealed that the miR-371b-5p could rescue the effect of SCAI on cell proliferation, migration and invasion.Conclusion: Our results suggest that the miR-371b-5p and SCAI may serve as novel prognostic biomarkers and therapeutic targets for NSCLC.

Serum microRNA-365 suppresses non-small-cell lung cancer metastasis and invasion in patients with bone metastasis of lung cancer.

ObjectiveIn the present investigation, we evaluated the effects of microRNA-365 (miR-365) on non-small-cell lung cancer (NSCLC) cell metastasis and invasion in patients with bone metastasis of lung cancer.MethodsBlood samples from patients with NSCLC and healthy controls and the A549 adenocarcinoma cell line were included in this study. Quantitative real-time PCR and microarray were performed on blood samples. The MTT assay, luciferase reporter assay, Transwell assay, ELISA, and western blot were performed to evaluate expression of associated factors.ResultsExpression of miR-365 was reduced in patients with bone metastasis of NSCLC. Downregulation of miR-365 promoted cell growth, metastasis, and invasion of NSCLC. Upregulation of miR-365 reduced cell growth, metastasis, and invasion of NSCLC. Downregulation of miR-365 induced expression of NKX homeobox-1 (NKX2-1), epidermal growth factor receptor (EGFR), phosphoinositide-3-kinase (PI3K), and p-Akt proteins in an in vitro model of NSCLC. Inhibition of NKX2-1 reduced the effects of miR-365 on cell growth, metastasis, and invasion of NSCLC. Activation of EGFR reduced the effects of miR-365 on cell growth, metastasis, and invasion of NSCLC.ConclusionsThe study established that the serum miR-365 suppresses NSCLC cell metastasis and invasion in patients with bone metastasis of lung cancer via EGFR/PI3K through NKX2-1.

PWP1 Promotes the Malignant Phenotypes of Lung Cancer Cells by Interacting with DVL2 and Merlin.

PurposeThe significance of periodic tryptophan protein 1 (PWP1) expression in human cancer and its molecular mechanism of action have not been reported so far.Materials and MethodsImmunohistochemistry was performed to analyze the expression of PWP1 in non-small cell lung cancer (NSCLC) tissues and statistical analysis was applied to analyze the relationship between PWP1 expression and the clinicopathological factors. The effects of PWP1 on NSCLC proliferation and invasion were determined by colony formation, transwell and MTT assays. Western blot analysis (WB), dual-luciferase reporter gene assays and immunofluorescence staining were performed to demonstrate whether PWP1 stimulates Wnt pathway and inhibits Hippo pathway. Co-immunoprecipitation (Co-ip) assays were used to confirm the potential role of PWP1 in Wnt and Hippo signaling pathways.ResultsPWP1 expression in NSCLC was higher than that in normal bronchial epithelium and normal submucosal glands. In addition, PWP1 expression had a positive correlation with poor differentiation, high pathological tumor-node-metastasis (TNM) stage, and lymph node metastasis. Kaplan–Meier database demonstrated that the overall survival time of patients with high PWP1 expression was significantly shorter than that of patients with low PWP1 expression. Mechanistically, we found that PWP1 could interact with DVL2 to upregulate β-catenin (thereby activating the Wnt pathway), whereas PWP1 could interact with Merlin (NF2) to downregulate p-MST1 (thereby inhibiting the Hippo signaling pathway). The effects of PWP1 on promoting the Wnt pathway or inhibiting the Hippo pathway were offset in DVL2- or Merlin-knockdown cells transiently overexpressing PWP1.ConclusionPWP1 expression in NSCLC was correlated with poor prognosis. PWP1 enhanced the activity of the Wnt pathway by interacting with DVL2, whereas PWP1 inhibited the activity of the Hippo pathway by interacting with Merlin. Together, these two effects promoted the detrimental biological behaviors of NSCLC cells.

High-mobility group box 3 (HMGB3) silencing inhibits non-small cell lung cancer development through regulating Wnt/β-catenin pathway.

It has been reported that high-mobility group box 3 is overexpressed in various cancers. This study aimed to explore its function in non-small cell lung cancer (NSCLC). A546 and H460cell lines were used for in vivo experiments, scratch healing tests, transwell migration and invasion experiments. It was first found that HMGB3 was highly expressed in tumor tissues in the patients and associated with NSCLC stage. Silencing of HMGB3 significantly slowed the growth, proliferation and invasion of NSCLC in vitro, and repressed cell growth in vivo. Mechanistic studies suggest that the observed effects were mediated by inhibiting the expression of β-catenin/MMP7/c-Myc in Wnt pathway. Our study highlights the role of HMGB3 in NSCLC, which may provide a therapeutic target for the treatment of NSCLC.

Down-Regulation of SREBP via PI3K/AKT/mTOR Pathway Inhibits the Proliferation and Invasion of Non-Small-Cell Lung Cancer Cells.

BackgroundLung cancer is one of the most common causes of cancer-related deaths worldwide, metabolic disorders are also a problem that puzzles mankind. SREBP is overexpressed in non-small-cell lung cancer (NSCLC) and is also a key regulator of lipid synthesis. However, the mechanisms by which SREBP regulates the proliferation, migration and invasion in NSCLC remain unclear.Materials and MethodsCCK-8, colony formation assay, soft agar assay, scratch wound healing assay and transwell assays were performed to detect proliferation, and invasion in NSCLC cells, respectively. In addition, Western blotting assay, qPCR and immunofluorescence were applied to detect the expressions of SREBP1, SREBP2, ki-67, PCNA, Bax, bcl-2, E-cadherin, N-cadherin, Vimentin, PI3K, p-PI3k, AKT, p-AKT, mTOR, p-mTOR in NSCLC cells.ResultsIn this study, downregulation of SREBP significantly inhibited the proliferation, migration and invasion of A549 and H1299 cells. Moreover, the method of piecewise inhibition was adopted to prove that SREBP is a downstream molecule of the PI3K/Akt/mTOR signaling pathway.ConclusionOur study indicated that downregulation of SREBP inhibited the growth in NSCLC cells via PI3K/AKT/mTOR signaling pathway. Thus, we suggested SREBP may serve as a potential target for the treatment of patients with NSCLC.