Epithelial-mesenchymal Transition Of Non-small Cell Lung Cancer Cells Research Articles

MiR-224-5p Suppresses Non-Small Cell Lung Cancer via IL6ST-Mediated Regulation of the JAK2/STAT3 Pathway.

Our study aimed to explore the specific functions and potential mechanisms of miR-224-5p in non-small cell lung cancer (NSCLC). We first analyzed the expression of miR-224-5p in NSCLC patients and cell lines through the GEO database and qRT-PCR analysis. Then, we used MTT assays, wound healing assays, Transwell assays, and western blotting to evaluate the effects of miR-224-5p on NSCLC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). Furthermore, we used a xenograft tumor model to evaluate the effect of miR-224-5p on NSCLC tumor growth. Potential binding targets of miR-224-5p were further identified through the target prediction databases, and the relationships between miR-224-5p, its targets, and downstream signaling pathways were further verified using luciferase reporter gene assays and western blotting. The GEO database and qRT-PCR analysis indicated that miR-224-5p was significantly downregulated in NSCLC patients and cell lines. Functional assays indicated that inhibiting miR-224-5p could enhance the proliferation, migration, invasion, and EMT of NSCLC cells, as well as accelerate tumor growth. In contrast, overexpression of miR-224-5p inhibited these processes. We identified IL6ST (interleukin 6 signal transducer) as a binding target of miR-224-5p. We observed that miR-224-5p could bind to and inhibit IL6ST expression and JAK2/STAT3 signaling pathway, and the inhibition of NSCLC tumor growth and JAK2/STAT3 pathway by miR-224-5p could be reversed by IL6ST overexpression. Our study demonstrated that miR-224-5p inhibited NSCLC by targeting IL6ST, thereby downregulating the JAK2/STAT3 signaling pathway.

MYL9 binding with MYO19 suppresses epithelial-mesenchymal transition in non-small cell lung cancer.

The elusive function of myosin light chain 9 (MYL9) in cancer is an area ripe for further investigation. Bioinformatics was utilized to compare the expression levels of MYL9 in non-small cell lung cancer (NSCLC) and normal tissues. Gene set enrichment analysis (GSEA) was employed to investigate the pathways associated with MYL9. BioGRID database was utilized to screen for potential targets of MYL9. The expression of MYL9 and myosin 19 (MYO19) mRNA was quantified using quantitative reverse transcriptase PCR. Cell migration was assessed using a scratch wound healing assay. Protein levels of MYL9, MYO19, and epithelial-mesenchymal transition (EMT) biomarkers were examined using western blot (WB). EpCAM expression in different cell groups was profiled using flow cytometry analysis. Co-immunoprecipitation assays were performed to determine the binding affinity between MYL9 and MYO19. Additionally, direct protein interaction between MYL9 and MYO19 was explored using a GST-pull-down assay. In NSCLC patients, MYL9 was significantly downregulated in vivo and in cell cultures, showing high enrichment in the EMT pathway. Scratch assays indicated its inhibitory effect on cell migration. Western blotting revealed that MYL9 suppresses EMT marker protein expression in NSCLC cells. Flow cytometry showed that MYL9 reduced EpCAM levels on the cell surface. MYO19 was identified as a potential target of MYL9 through CoIP and GST-pull-down assays. Rescue experiments demonstrated that MYO19 enhances cell migration, EMT marker expression, and EpCAM levels, but these effects were countered by MYL9 overexpression. MYL9 impedes the migration and EMT in NSCLC cells by binding to MYO19.

Circ-10720 as a ceRNA adsorbs microRNA-1238 and modulates ZEB2 to boost NSCLC development by activating EMT

BackgroundCircular RNAs (circRNAs) are critical regulators in the progression of tumors. This experimental design aimed to explore the mechanism of circ-10720 in non-small cell lung cancer (NSCLC).MethodsWe used RT-qPCR to measure circ-10720 expression in clinical samples and analyzed its relationship with the clinicopathological characteristics of NSCLC patients. The expression levels of microRNA-1238 (miR-1238) and Zinc Finger E-box-binding Homeobox 2 (ZEB2) in clinical samples were detected by RT-qPCR. NSCLC cells were transfected with relevant plasmids or sequences. Circ-10720, miR-1238, and ZEB2 expressions in cells were analyzed via RT-qPCR or western blot. Cell proliferation, apoptosis, migration, and invasion were assessed with CCK-8, flow cytometry, and transwell assay, respectively. The protein expression of ZEB2 and epithelial–mesenchymal transition (EMT)-related markers (E-cadherin, Vimentin, N-cadherin) were detected via western blot. Xenograft assay was used to determine the effect of circ-10720 on NSCLC in vivo. Circ-10720 and ZEB2 expressions in tumors were detected using RT-qPCR or Western blot. Immunohistochemistry was used to evaluate E-cadherin and N-cadherin expression in tumors. Finally, the binding relationship between miR-1238 with circ-10720 or ZEB2 was verified by the bioinformatics website, dual luciferase reporter assay, RNA pull-down assay, and RIP assay.ResultsCirc-10720 was upregulated in NSCLC and correlated with TNM stage of NSCLC patients. MiR-1238 was lowly expressed but ZEB2 was highly expressed in NSCLC. Circ-10720 silencing suppressed the proliferation, metastasis, and EMT of NSCLC cells. Mechanically, circ-10720 was a competitive endogenous RNA (ceRNA) for miR-1238, and ZEB2 was a target of miR-1238. circ-10720-modulated ZEB2 via competitively binding with miR-1238 to control NSCLC progression. In addition, circ-10720 knockdown suppressed tumor growth in vivo.ConclusionsCirc-10720 acts as a ceRNA to adsorb miR-1238 and modulate ZEB2 to facilitate the proliferation, migration, invasion, and EMT of NSCLC cells.Graphical

Musashi-2 in cancer-associated fibroblasts promotes non-small cell lung cancer metastasis through paracrine IL-6-driven epithelial-mesenchymal transition

BackgroundLung cancer, the most common cause of cancer-related mortality worldwide, is predominantly associated with advanced/metastatic disease. The interaction between tumor cells and cancer-associated fibroblasts (CAFs) in tumor microenvironment is known to be essential for regulating tumor progression and metastasis, but the underlying mechanisms, particularly the role of RNA-binding protein Musashi-2 (MSI2) in CAFs in promoting non-small cell lung cancer (NSCLC) invasiveness and metastatic spread, remain obscure.MethodsGenomic and proteomic database analyses were performed to evaluate the potential clinical significance of MSI2 in NSCLC tumor and stromal clinical specimens. Molecular approaches were used to modify MSI2 in CAFs and determine its functional role in NSCLC cell motility in vitro using 2D and 3D models, and in metastasis in a xenograft mouse model using live-cell imaging.ResultsMSI2, both gene and protein, is upregulated in NSCLC tissues and is associated with poor prognosis and high metastatic risk in patients. Interestingly, MSI2 is also upregulated in NSCLC stroma and activated fibroblasts, including CAFs. Depletion of MSI2 in CAFs by CRISPR-Cas9 strongly inhibits NSCLC cell migration and invasion in vitro, and attenuates local and distant metastatic spread of NSCLC cells in vivo. The crosstalk between CAFs and NSCLC cells occurs via paracrine signaling, which is regulated by MSI2 in CAFs via IL-6. The secreted IL-6 promotes epithelial-mesenchymal transition in NSCLC cells, which drives metastasis.ConclusionOur findings reveal for the first time that MSI2 in CAFs is important in CAF-mediated NSCLC cell invasiveness and metastasis via IL-6 paracrine signaling. Therefore, targeting the MSI2/IL-6 axis in CAFs could be effective in combating NSCLC metastasis.

Periostin secreted by cancer-associated fibroblasts promotes cancer progression and drug resistance in non-small cell lung cancer.

Cancer-associated fibroblasts (CAFs) are important components in the tumor microenvironment, and we sought to identify effective therapeutic targets in CAFs for non-small cell lung cancer (NSCLC). In this study, we established fibroblast cell lines from the cancerous and non-cancerous parts of surgical lung specimens from patients with NSCLC and evaluated the differences in behaviors towards NSCLC cells. RNA sequencing analysis was performed to investigate the differentially expressed genes between normal fibroblasts (NFs) and CAFs, and we identified that the expression of periostin (POSTN), which is known to be overexpressed in various solid tumors and promote cancer progression, was significantly higher in CAFs than in NFs. POSTN increased cell proliferation via NSCLC cells' ERK pathway activation and induced epithelial-mesenchymal transition (EMT), which improved migration in vitro. In addition, POSTN knockdown in CAFs suppressed these effects, and in vivo experiments demonstrated that the POSTN knockdown improved the sensitivity of EGFR-mutant NSCLC cells for osimertinib treatment. Collectively, our results showed that CAF-derived POSTN is involved in tumor growth, migration, EMT induction, and drug resistance in NSCLC. Targeting CAF-secreted POSTN could be a potential therapeutic strategy for NSCLC. KEY MESSAGES: • POSTN is significantly upregulated in CAFs compared to normal fibroblasts in NCSLC. • POSTN increases cell proliferation via activation of the NSCLC cells' ERK pathway. • POSTN induces EMT in NSCLC cells and improves the migration ability. • POSTN knockdown improves the sensitivity for osimertinib in EGFR-mutant NSCLC cells.

MiR-22-3p suppresses NSCLC cell migration and EMT via targeting RAC1 expression

Previous studies have demonstrated the tumor-suppressive function of microRNA-22-3p (miR-22-3p) in several cancers, whereas the significance of miR-22-3p in non-small cell lung cancer (NSCLC) remains unclear. In this study, we explored the biological function and molecular mechanism of miR-22-3p in NSCLC cells. First, we assessed the expression of miR-22-3p in NSCLC tissues and cells based on RT-qPCR and TCGA database. Compared with normal lung tissues and cells, miR-22-3p expression was dramatically decreased in lung cancer tissues and cells. miR-22-3p expression was also correlated with lymph node metastasis and tumor size, but not TNM stages. We further explored the in vitro function of miR-22-3p on the migration and epithelial–mesenchymal transition (EMT) of NSCLC cells. The results showed that overexpression of miR-22-3p suppressed the migration and EMT of NSCLC cells, whereas silencing miR-22-3p showed the opposite effect. Luciferase assay demonstrated that RAS-related C3 botulinum toxin substrate 1 (RAC1) was the target gene for miR-22-3p. Mechanistically, we demonstrated that miR-22-3p suppressed the cell migration and EMT via downregulation of RAC1 because the inhibitory effect of miR-22-3p on cell migration and EMT of NSCLC cells was reversed by RAC1 overexpression. Based on these novel data, the miR-22-3p/RAC1 axis may be an alternative target in the therapeutic intervention of NSCLC.

EPN3 plays oncogenic role in non-small cell lung cancer by activating the JAK1/2-STAT3 pathway.

The effect of Epsin 3 (EPN3) on non-small cell lung cancer (NSCLC) has not yet been clearly elucidated. This study identified the exact function of EPN3 on NSCLC progression. EPN3 expression in NSCLC patients were analyzed based on the Cancer Genome Atlas database. Kaplan-Meier analysis was implemented to research the effect of EPN3 on patients' survival. EPN3 expression in clinical tissues of 62 NSCLC cases was monitored by real-time quantitative reverse transcription polymerase chain reaction, immunohistochemistry and Western blot. A549 and H1299 cells were transfected with EPN3 shRNA and treated by RO8191 (20 μM). Proliferation was researched by cell counting kit-8 and 5-ethnyl-2 deoxyuridine assays. Apoptosis was monitored by flow cytometry. Migration and invasion was assessed by Transwell experiment. EPN3 effect on A549 cell in vivo growth was researched using nude mice. RO8191 (200 μg) was intratumoral injected into mice. Immunohistochemistry and Western blot was implemented to monitor protein expression in cells and xenograft tumor tissues. EPN3 was abnormally up-regulated in NSCLC patients and cells, indicating a lower overall survival. Loss of EPN3 weakened proliferation, migration and invasion, induced apoptosis, and repressed epithelial-mesenchymal transition in NSCLC cells. Loss of EPN3 inactivated the JAK1/2-STAT3 pathway in NSCLC cells. RO8191 treatment reversed the inhibition of EPN3 knockdown on the malignant phenotype of NSCLC cells. RO8191 intratumoral injection reversed the suppression of EPN3 silencing on NSCLC cell in vivo growth. EPN3 acted as an oncogene in NSCLC via activating the JAK1/2-STAT3 pathway. EPN3 may be a promising target for NSCLC treatment.

HnRNPAB is an independent prognostic factor in non‑small cell lung cancer and is involved in cell proliferation and metastasis.

Heterogeneous nuclear ribonucleoprotein A/B (hnRNPAB) is an RNA binding protein that is closely associated with the biological function and metabolism of RNA, which is involved in the malignant transformation of various tumor cells. However, the role and mechanisms of hnRNPAB in non-small cell lung cancer (NSCLC) are still unclear. In the present study, the expression levels of hnRNPAB in NSCLC and normal tissues were analyzed using the human protein atlas database and UALCAN database. The clinical significance of hnRNPAB was assayed using the data of NSCLC cases from The Cancer Genome Atlas database. Subsequently, two stable NSCLC cell lines with hnRNPAB knockdown were constructed and the effects of hnRNPAB silencing on cell viability, migration, invasion and epithelial-mesenchymal transition (EMT) were identified. Genes associated with hnRNPAB expression in NSCLC were screened using the Linked Omics database and verified by quantitative real-time PCR (RT-qPCR). The database analysis indicated that hnRNPAB was mainly expressed in the nucleus of NSCLC cells. Compared with the normal tissues, hnRNPAB expression was overexpressed in NSCLC tissues and was closely associated with the overall survival, sex, tumor-node-metastases classification, and poor prognosis of patients with lung adenocarcinoma. Functionally, knockdown of hnRNPAB inhibited the proliferation, migration, invasion and EMT of NSCLC cells and arrested the cell cycle at G1 phase. Mechanistically, the bioinformatics analysis and RT-qPCR verification demonstrated that hnRNPAB knockdown led to a significant expression change of genes associated with tumorigenesis. In conclusion, the present study indicated that hnRNPAB played an important role in the malignant transformation of NSCLC, supporting the significance of hnRNPAB as a novel potential therapeutic target for the early diagnosis and prognosis of NSCLC.

Frizzled-7-targeting antibody (SHH002-hu1) potently suppresses non-small-cell lung cancer via Wnt/β-catenin signaling.

Non-small-cell lung cancer (NSCLC) is one of the deadliest cancers worldwide, and metastasis is considered one of the leading causes of treatment failure in NSCLC. Wnt/β-catenin signaling is crucially involved in epithelial-mesenchymal transition (EMT), a crucial factor in promoting metastasis, and also contributes to resistance developed by NSCLC to targeted agents. Frizzled-7 (Fzd7), a critical receptor of Wnt/β-catenin signaling, is aberrantly expressed in NSCLC and has been confirmed to be positively correlated with poor clinical outcomes. SHH002-hu1, a humanized antibody targeting Fzd7, was previously successfully generated by our group. Here, we studied the anti-tumor effects of SHH002-hu1 against NSCLC and revealed the underlying mechanism. First, immunofluorescence (IF) and near-infrared (NIR) imaging assays showed that SHH002-hu1 specifically binds Fzd7+ NSCLC cells and targets NSCLC tissues. Wound healing and transwell invasion assays indicated that SHH002-hu1 significantly inhibits the migration and invasion of NSCLC cells. Subsequently, TOP-FLASH/FOP-FLASH luciferase reporter, IF, and western blot assays validated that SHH002-hu1 effectively suppresses the activation of Wnt/β-catenin signaling, and further attenuates the EMT of NSCLC cells. Finally, the subcutaneous xenotransplanted tumor model of A549/H1975, as well as the popliteal lymph node (LN) metastasis model, was established, and SHH002-hu1 was demonstrated to inhibit the growth of NSCLC xenografts and suppress LN metastasis of NSCLC. Above all, SHH002-hu1 with selectivity toward Fzd7+ NSCLC and the potential of inhibiting invasion and metastasis of NSCLC via disrupting Wnt/β-catenin signaling, is indicated as a good candidate for the targeted therapy of NSCLC.

Circular RNA hsa_circ_0004689 (circSWT1) promotes NSCLC progression via the miR-370-3p/SNAIL axis by inducing cell epithelial-mesenchymal transition (EMT).

Previous studies have reported the role of circular RNAs (circRNAs) in the progression of non-small-cell lung cancer (NSCLC). SWT1-derived circRNAs were confirmed to affect the apoptosis of cardiomyocytes; however, the biological functions of SWT1-derived circRNAs in cancers are still unknown. Here, we investigated the potential role of SWT1-derived circRNAs in NSCLC. We used quantitative real-time polymerase chain reaction (qRT-PCR) to measure the expression of circSWT1 in NSCLC tissues and paired normal tissues. The potential functions of circSWT1 in tumor progression were assessed by CCK-8, colony formation, wound healing, and matrigel transwell assays in vitro and by xenograft tumor models in vivo. Next, epithelial-mesenchymal transition (EMT) was evaluated by western blotting, immunofluorescence, and immunohistochemistry (IHC). Moreover, circRIP, RNA pulldown assays, luciferase reporter gene assays, and FISH were conducted to illuminate the molecular mechanisms of circSWT1 via the miR-370-3p/SNAIL signal pathway. Then, we knocked out SNAIL in A549 and H1299 cells to identify the roles of circSWT1 in the progression and EMT of NSCLC through SNAIL. Finally, circSWT1 functions were confirmed in vivo using xenograft tumor models. CircSWT1 expression was significantly upregulated in NSCLC tissues, and high expression of circSWT1 predicted poor prognosis in NSCLC via survival analysis. In addition, overexpression of circSWT1 promoted the invasion and migration of NSCLC cells. Subsequently, we found that overexpression of circSWT1 induced EMT and that knockdown of circSWT1 inhibited EMT in NSCLC cells. Mechanistically, circSWT1 relieved the inhibition of downstream SNAIL by sponging miR-370-3p. Moreover, we found that these effects could be reversed by knocking out SNAIL. Finally, we verified that circSWT1 promoted NSCLC progression and EMT in xenograft tumor models. CircSWT1 promoted the invasion, migration, and EMT of NSCLC. CircSWT1 could serve as a potential biomarker and a potential therapeutic target for NSCLC.

Exosomal epidermal growth factor receptor is involved in HPV-16 E7-induced epithelial-mesenchymal transition of non-small cell lung cancer cells: A driver of signaling in vivo?

ABSTRACT Our previous studies have demonstrated that human papillomavirus (HPV)-16 E7 oncoprotein promoted epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC) cells. Moreover, recent studies have found that exosomes can mediate EMT of NSCLC cells and epidermal growth factor receptor (EGFR) is related to the progression of NSCLC. Here, we further investigated the role of exosomal EGFR in HPV-16 E7-induced EMT of NSCLC cells. Our results showed that the exosomes derived from the stable HPV-16 E7-overexpressing A549 and NCI-H460 NSCLC cells (E7 Exo) significantly increased migration, invasion, and proliferation abilities of NSCLC cells as compared with the exosomes derived from empty vector-infected NSCLC cells (ev Exo). Moreover, both in vitro and in vivo results demonstrated that E7 Exo dramatically enhanced EMT of NSCLC cells and promoted the growth of subcutaneous NSCLC xenografts. Additionally, HPV-16 E7 enhanced the expression of EGFR and p-EGFR in both NSCLC cells and exosomes. Furthermore, the inhibition of EGFR activation or exosome secretion suppressed E7 Exo-induced migration, invasion, and EMT of NSCLC. Moreover, 12 kinds of differentially expressed miRNAs between E7 Exo and ev Exo (fold change≥2, P ≤ .05) were screened out, of which 7 miRNAs were up-regulated while 5 miRNAs were down-regulated in A549 E7 Exo. Taken together, our findings suggest that exosomal EGFR is involved in HPV-16 E7-induced EMT of NSCLC cells, which may play a key role in the progression of HPV-related NSCLC.

MiR-657 Targets SRCIN1 via the Slug Pathway to Promote NSCLC Tumor Growth and EMT Induction.

Background MicroRNA- (miR-) 657 has been shown to regulate immunological and inflammatory activity, and it has also been defined to be dysregulated in both non-small-cell lung cancer (NSCLC) and hepatocellular carcinoma. The mechanistic role whereby miR-657 influences NSCLC progression, however, has yet to be clarified. Methods miR-657 and SRCIN1 expression levels were assessed via qPCR in the cell lines and tissues of NSCLC. Besides, correlations between the levels of miR-657 and NSCLC patient pathological characteristics were examined, and the Kaplan-Meier approach was employed for the evaluation of the prognostic utility of miR-657 in these patients. Moreover, the Pearson correlation analyses and dual-luciferase reporter assessments were used for detecting interactive relationships between miR-657 and SRCIN1. In addition, CCK-8, EdU, and Transwell assessments were employed for the appraisal of the ability of miR-657/SRCIN1 to regulate NSCLC cell proliferation and invasion. Western blotting was employed for the assessment of the levels of NSCLC cell proteins associated with the epithelial-mesenchymal transition (EMT) that were influenced by miR-657. The nude mice xenograft tumor model is established to observe the effect of miR-657 on NSCLC growth in vivo. Results NSCLC patient tissues and cell lines exhibited upregulated miR-657 expression that was closely related to tumor differentiation, lymphoid metastasis, and TNM stage. High levels of miR-657 were predictive of a poorer NSCLC patient prognosis, and overexpressing miR-657 resulted in the more rapid growth of NCI-H1650 and A549 cells, with a concomitant increase in their invasion. In addition, miR-657 overexpression raised the levels of Slug, N-cadherin, and Vimentin in these two cell lines while promoting E-cadherin downregulation. Dual-luciferase reporter assays confirmed that miR-657 was capable of binding to the SRCIN1 gene, and SRCIN1 expression levels were negatively associated with those of miR-657, indicating that it acts as a negative regulator of this gene. Knocking down SRCIN1 was capable to reverse the influences of miR-657 inhibitor treatment on NSCLC cell behavior. Finally, in vivo studies showed that miR-657 promoted NSCLC cell growth. Conclusion The obtained findings illuminate that miR-657 can promote the growth of tumors and the induction of the EMT in NSCLC cells by targeting SRCIN1 expression and modulating Slug pathway activation, highlighting this pathway as a promising therapeutic target in cases suffering from NSCLC.

Hsa_circ_0006427 Suppresses Multiplication, Migration and Invasion of Non-Small Cell Lung Cancer Cells through miR-346/VGLL4 Pathway.

Objective Circular RNAs (circRNAs) are identified as key modulators in cancer biology. Nonetheless, the role of circ_0006427 in non-small cell lung cancer (NSCLC) and its modulatory mechanism are undefined. This study aimed to investigate the potential function and mechanism of circ_0006427 in NSCLC. Materials and Methods In this experimental study, circ_0006427, miR-346 and vestigial like family member 4 (VGLL4) mRNA expressions were analyzed in NSCLC tissues and cells, using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Multiplication, migration and invasion of NSCLC cells were examined using the CCK-8 method and Transwell experiment, respectively. Dual-luciferase reporter gene experiments were conducted to identify the paring relationship between circ_0006427 and miR-346. Western blot was employed to determine expressions of VGLL4 and epithelial-mesenchymal transition (EMT) markers on protein levels. Immuno-histochemistry (IHC) method was adopted to assess VGLL4 protein expression in NSCLC tissues. Results Circ_0006427 expression was down-regulated in NSCLC tissues and cells, and circ_0006427 suppressed multiplication, migration, invasion and EMT of NSCLC cells. miR-346 expression was upregulated in NSCLC tissues and cells, and miR-346 worked as a target of circ_0006427. VGLL4 was down-regulated in NSCLC tissues and cells, and knockdown of VGLL4 accelerated multiplication, migration, invasion and EMT of NSCLC cells. Circ_0006427 enhanced VGLL4 expression by competitively binding with miR-346. ConclusionCirc_0006427/miR-346/VGLL4 axis regulated NSCLC progression.

High level of FHL2 exacerbates the outcome of non-small cell lung cancer (NSCLC) patients and the malignant phenotype in NSCLC cells.

Non-small cell lung cancer (NSCLC) is a malignant tumour with high mortality. FHL2 has been identified as a biomarker of lung cancer. This research explored the effects of FHL2 expression on NSCLC. NSCLC-associated data sets were collected from the assistant for clinical bioinformatics and TCGA databases respectively. The association between FHL2 and clinical characteristics, the prognostic significance of FHL2 and the influences of various variables on NSCLC were determined by Pearson's chi-squared test, the Kaplan-Meier curve and the Cox regression model respectively. FHL2 level was altered by cell transfection and was measured by qRT-PCR. Tumour xenograft formation was completed by inoculating sh-FHL2/pcDNA-FHL2 transfected cells into BALB/c nude mice. Protein expression was assessed by western blot. Cell apoptosis, proliferation and epithelial - mesenchymal transition (EMT) characteristics were evaluated employing TUNEL, BrdU+ and microscopic observation respectively. The expression of Ki67 and N-cadherin was assessed by immunohistochemistry. The results showed that FHL2 was highly expressed in NSCLC tissues. Patients with high FHL2 expression experienced lower overall survival probability. FHL2 knockdown promoted apoptosis, but inhibited EMT of A549 and NCI-H460 cells, which was verified by the increased ratios of cleaved caspase 9/caspase 9 and cleaved caspase 3/caspase 3, as well as augmented E-cadherin and reduced N-cadherin. In an in vivo assay FHL2 knockdown decreased tumour volume and weight, repressed EMT, but enhanced apoptosis. FHL2 upregulation showed the opposite effects of FHL2 knockdown. Furthermore, FHL2 upregulation facilitated cell proliferation both in in vitro and in vivo assays. These outcomes indicated that high level of FHL2 facilitated tumorigenesis, as well as the proliferation and EMT of NSCLC cells.

Circular RNA circZCCHC6 contributes to tumorigenesis by regulating LPCAT1 via miR-433-3p in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-associated mortality worldwide. Circular RNA (circRNA) circZCCHC6 has been reported to be upregulated in the plasma from NSCLC patients. This study is designed to explore the role and mechanism of circZCCHC6 in NSCLC. CircZCCHC6, microRNA-433-3p (miR-433-3p), and lysophosphatidylcholine acyltransferase 1 (LPCAT1) level were determined by real-time quantitative polymerase chain reaction. Cell viability, cell cycle progression, migration, and invasion were assessed by 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), flow cytometry, wound healing, and transwell assays, severally. The binding relationship between miR-433-3p and circZCCHC6 or LPCAT1 was predicted by Circinteractome or Starbase, and then verified by a dual-luciferase reporter, RNA pull-down, or RNA Immunoprecipitation (RIP) assays. Protein levels of LPCAT1, Cyclin D1, E-cadherin, and Vimentin were examined by western blot assay. The biological role of circZCCHC6 on NSCLC tumor growth and epithelial-mesenchymal transition (EMT) was examined by the xenograft tumor model in vivo. CircZCCHC6 was highly expressed in NSCLC serum, tissues, and cells. Moreover, circZCCHC6 knockdown could repress cell viability, cell cycle progression, migration, invasion, and EMT in NSCLC cells in vitro. The mechanical analysis suggested that circZCCHC6 acted as a sponge of miR-433-3p to regulate LPCAT1 expression. CircZCCHC6 silencing hindered cell growth and EMT of NSCLC in vivo. CircZCCHC6 inhibited the progression of NSCLC cells partly by regulating the miR-433-3p/LPCAT1 axis, implying a promising therapeutic target for the NSCLC treatment.

Identification of Prognostic Gene Biomarkers in Non-Small Cell Lung Cancer Progression by Integrated Bioinformatics Analysis.

Simple SummaryNon-small cell lung cancer (NSCLC) is a major contributor to cancer related deaths worldwide. The progression of NSCLC is linked to epithelial-mesenchymal transition (EMT), a biologic process that enables tumor cells to acquire an invasive phenotype and resistance to therapies. Discovery of novel biomarkers in NSCLC progression is essential for improved prognosis and pharmacological interventions. We performed an integrated bioinformatics analysis on available gene expression datasets of transforming growth factor β (TGF-β) induced EMT in NSCLC cell lines aiming to establish new prognostic biomarkers in the disease. The retrieved candidate genes were involved in protein modifications, regulation of cell death and cell adhesions, oxidation-reduction reactions of aerobic respiration and mitochondrial translation. Out of these genes, we identified ten prognostic gene biomarkers, mostly involved in protein modifications, whose expressions correlated with patient survival in NSCLC. This ten-gene prognostic signature will be useful to improve risk prediction and guide treatment strategies in NSCLC. Deciphering the exact functions of the biomarker genes previously not linked with NSCLC will also lead to a better understanding of the pathomechanism of NSCLC progression, revealing novel therapeutic targets in the disease.The progression of non-small cell lung cancer (NSCLC) is linked to epithelial-mesenchymal transition (EMT), a biologic process that enables tumor cells to acquire a migratory phenotype and resistance to chemo- and immunotherapies. Discovery of novel biomarkers in NSCLC progression is essential for improved prognosis and pharmacological interventions. In the current study, we performed an integrated bioinformatics analysis on gene expression datasets of TGF-β-induced EMT in NSCLC cells to identify novel gene biomarkers and elucidate their regulation in NSCLC progression. The gene expression datasets were extracted from the NCBI Gene Expression Omnibus repository, and differentially expressed genes (DEGs) between TGF-β-treated and untreated NSCLC cells were retrieved. A protein-protein interaction network was constructed and hub genes were identified. Functional and pathway enrichment analyses were conducted on module DEGs, and a correlation between the expression levels of module genes and survival of NSCLC patients was evaluated. Prediction of interactions of the biomarker genes with transcription factors and miRNAs was also carried out. We described four protein clusters in which DEGs were associated with ubiquitination (Module 1), regulation of cell death and cell adhesions (Module 2), oxidation-reduction reactions of aerobic respiration (Module 3) and mitochondrial translation (Module 4). From the module genes, we identified ten prognostic gene biomarkers in NSCLC. Low expression levels of KCTD6, KBTBD7, LMO7, SPSB2, RNF19A, FOXA2, DHTKD1, CDH1 and PDHB and high expression level of KLHL25 were associated with reduced overall survival of NSCLC patients. Most of these biomarker genes were involved in protein ubiquitination. The regulatory network of the gene biomarkers revealed their interaction with tumor suppressor miRNAs and transcription factors involved in the mechanisms of cancer progression. This ten-gene prognostic signature can be useful to improve risk prediction and therapeutic strategies in NSCLC. Our analysis also highlights the importance of deregulation of ubiquitination in EMT-associated NSCLC progression.

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.

Silencing of Long Non-Coding RNA FGD5-AS1 Inhibits the Progression of Non-Small Cell Lung Cancer by Regulating the miR-493-5p/DDX5 Axis.

Background:Long non-coding RNA FGD5 antisense RNA 1 (FGD5-AS1), identified to be a carcinogenic lncRNA, exhibits a regulatory role in some malignancies including non-small cell lung cancer (NSCLC). The aim of the present research is to decipher the function and underlying mechanism of FGD5-AS1 in progression of NSCLC.Methods:Expression of FGD5-AS1, miR-493-5p and DEAD-box protein 5 (DDX5) in NSCLC tissues and cells was quantified utilizing qRT-PCR. Cell proliferation was assessed by CCK-8 method. Scratch healing test and Transwell assay were used for assaying cell migration and invasion. Expressions of DDX5 and epithelial-mesenchymal transition (EMT)-related proteins were examined by Western blot. Additionally, targeting relationships between FGD5-AS1 and miR-493-5p, miR-493-5p and DDX5 were verified by dual-luciferase reporter gene assay.Results:Expression of FGD5-AS1 in NSCLC tissues and cell lines was up-regulated. Expression of FGD5-AS1 was in association with enlarged tumor size and lymph node metastasis of the patients. Knockdown of FGD5-AS1 led to the inhibition of proliferation, migration, invasion and EMT of NSCLC cells. FGD5-AS1 directly targeted miR-493-5p, while DDX5 was the target of miR-493-5p in NSCLC cells. Additionally, FGD5-AS1 could positively regulate the expression of DDX5 via suppressing miR-493-5p.Conclusion:FGD5-AS1 facilitates the proliferation, migration, invasion and EMT of NSCLC cells by sponging miR-493-5p and up-regulating DDX5.

NORAD regulates epithelial‑mesenchymal transition of non‑small cell lung cancer cells via miR‑422a.

The poor prognosis of non-small cell lung cancer (NSCLC) is related to epithelial-mesenchymal transition (EMT). Recent studies demonstrated that non-coding RNA activated by DNA damage (NORAD) displays a carcinogenic effect and targets microRNA (miR)-422a, which may be involved in tumor cell migration and invasion. The aim of the present study was to investigate the effect of NORAD on NSCLC cell EMT and the underlying mechanism. Reverse transcription-quantitative PCR and western blotting were performed to detect the expression levels of long non-coding RNAs, miRNAs and mRNAs. Cell viability, migration and invasion were detected by conducting Cell Counting Kit-8, wound healing and Transwell assays, respectively. The target of NORAD was predicted using starBase and further confirmed by conducting a dual-luciferase reporter assay. The results indicated that NORAD expression was significantly increased in lung cancer tissues and cells compared with adjacent healthy tissues and cells. Compared with the control groups, NORAD overexpression promoted SK-MES-1 cell viability, migration and invasion, whereas NORAD knockdown resulted in the opposite effects in A549 cells. Moreover, miR-422a, which was predicted to be a target of NORAD, displayed lower expression levels in lung cancer tissues compared with adjacent healthy tissues. In addition, miR-422a overexpression partially reversed NORAD overexpression-induced increases in SK-MES-1 cell viability, migration, invasion and EMT. In addition, miR-422a knockdown partially reversed the effects of NORAD knockdown. The present study suggested that NORAD regulated lung cancer cell EMT by regulating the expression of miR-422a, providing a potential therapeutic target for the intervention of the development of NSCLC.

MicroRNA-877 contributes to decreased non-small cell lung cancer cell growth via the PI3K/AKT pathway by targeting tartrate resistant acid phosphatase 5 activity

ABSTRACT Non-small cell lung cancer (NSCLC) is a leading cause of cancer death in both men and women. microRNAs (miRs) can exert important functions in cancer development. However, the role of miR-877 in NSCLC as it relates to tartrate resistant acid phosphatase 5 (ACP5) is unknown. For this study, the gain-and-loss-of-function experiments were performed to explore the effects of miR-877 and ACP5 on NSCLC. miR-877 expression in LC and paracancerous tissues, lung epithelial cell line and NSCLC cell lines was detected, and the association between miR-877 expression and clinical features of LC patients was analyzed. The levels of ACP5, epithelial-mesenchymal transition (EMT) markers and apoptosis-related proteins were measured. In vivo experiments were conducted for further validation. Consequently, we found that miR-877 expression was lowered in LC tissues and cell lines, and correlated with clinical stage, differentiation, lymph node metastasis and prognosis of NSCLC patients. Additionally, miR-877 was determined to inhibit ACP5 activity, and miR-877 downregulated the PI3K/AKT pathway by silencing ACP5. Furthermore, overexpression of miR-877 inhibited the viability, migration, invasion and EMT of NSCLC cells, but promoted cell apoptosis. In conclusion, miR-877 overexpression inhibited malignant biological behaviors of NSCLC cells by downregulating ACP5 and inactivating the PI3K/AKT pathway.