Migration Of Non-small Cell Lung Cancer Cells Research Articles

Circular RNA hsa_circ_0000317 inhibits non-small cell lung cancer progression through regulating microRNA-494-3p/phosphatase and tensin homolog deleted on chromosome 10 axis

BackgroundCircular RNA (circRNA), a group of non-coding RNA, is pivotal in the progression of various cancers, including Non-Small Cell Lung Cancer (NSCLC). Some circRNAs have been reported to be implicated in the progression of NSCLC, however, the function and molecular mechanism of hsa_circ_0000317 (circ_0000317) in NSCLC have not been fully understood. MethodsThe significantly differentially expressed circRNA in NSCLC tissues, circ_0000317, was screened out by microarray. Circ_0000317, microRNA(miR)-494-3p and Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN) expressions in NSCLC tissues were respectively probed by quantitative real-time polymerase chain reaction and western blot assay. MTT and Transwell assays were adopted to examine the growth, migration, and invasion of NSCLC cells. Bioinformatics, luciferase reporter gene assay, RNA immunoprecipitation, and RNA pull-down assay were conducted to probe the relationships among circ_0000317, miR-494-3p, and PTEN. ResultsCirc_0000317 expression level was reduced in NSCLC tissues and cell lines. Circ_0000317 expression in NSCLC patients was associated with TNM stage and lymphatic metastasis. Circ_0000317 overexpression restrained the proliferation, migration, and invasion of NSCLC cells, but co-transfection of miR-494-3p mimics partially reversed this effect. In addition, circ_0000317, was identified as a competitive endogenous RNA, which could sponge miR-494-3p to increase PTEN expression and activate PI3K/AKT pathway. ConclusionCirc_0000317, inhibits NSCLC progression via modulating miR-494-3p/PTEN/PI3K/AKT pathway.

IncRNA Linc00511 Upregulation Elevates TGFBR1 and Participates in the Postoperative Distant Recurrence of Non-Small-Cell Lung Cancer by Targeting miR-98-5p.

Long non-coding RNA (lncRNA) Linc00511 has been demonstrated as an oncogenic lncRNA in some types of cancer, such as bladder cancer and breast cancer. However, whether Linc00551 plays critical roles in non-small-cell lung cancer (NSCLC) is still unknown. To examine the role of Linc00511 in the pathogenesis of NSCLC, 62 stage I NSCLC patients and the age- and gender-matched healthy controls were enrolled in this investigation. Patients were followed up for three years. Linc00511, microRNA-98-5p (miR-98-5p) and transforming growth factor beta receptor 1 (TGFBR1) mRNA levels were measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR). TGFBR1 protein level was determined by Western blot. The putative interaction between Linc00511 and miR-98-5p was predicted by bioinformatics and confirmed by dual luciferase activity and RNA pull-down. Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) assay. Cell migration and invasion were measured by Transwell assay. Serum Linc00511 level at admission was higher in patients than in healthy controls and decreased on the day of discharge than before surgical resection. During the 3-years of follow-up, plasma Linc00511 level was upregulated only in patients with distant recurrence. Linc00511 was predicted to interact with miR-98-5p, and Linc00511 overexpression inhibited miR-98-5p expression but upregulated TGFBR1, a downstream target for miR-98-5p. Moreover, the elevation of Linc00511 and TGFBR1 both promoted NSCLC cell proliferation, invasion, and migration. However, miR-98-5p overexpression had opposite effects, reversing the effects of TGFBR1. In conclusion, Linc00511 was elevated in NSCLC, which might participate NSCLC postoperative distant recurrence and promoted NSCLC cell proliferation, migration and invasion via targeting and regulating miR-98-5p/TGFBR1 axis.

Silencing tumor-intrinsic CD73 enhances the chemosensitivity of NSCLC and potentiates the anti-tumoral effects of cisplatin: An in vitro study.

Besides suppressing anti-tumoral immune responses, tumor-intrinsic inhibitory immune checkpoints have been implicated in tumor development. Herein, we aimed to investigate the significance of tumor-intrinsic CD73, as an inhibitory immune checkpoint, in non-small cell lung cancer (NSCLC) development and propose a novel therapeutic approach. We investigated the cell viability, chemosensitivity, apoptosis, migration, and the cell cycle of A-549 and NCI-H1299 following treatment with cisplatin and CD73-small interfering RNA (siRNA) transfection. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to study the viability of studied groups and chemosensitivity of tumoral cells. Flow cytometry and 4',6-diamidino-2-phenylindole (DAPI) staining were used to investigate the apoptosis of NSCLC cells. Flow cytometry and the wound-healing assay were used to investigate the cell cycle and migration of NSCLC cells, respectively. The mRNA expression levels of c-Myc, caspase 3, ROCK, and MMP-9 were investigated to study the underlying molecular mechanism. CD73-siRNA transfection has significantly decreased the cell viability and enhanced the chemosensitivity of A-549 and NCI-H1299 cells to cisplatin. CD73-siRNA has considerably stimulated apoptosis, arrested the cell cycle, inhibited tumor migration, downregulated the mRNA expression of c-Myc, MMP-9, and ROCK, and upregulated caspase 3 expression in NSCLC cells. Besides, combined cisplatin therapy with CD73-siRNA transfection has potentiated the aforementioned anti-tumoral effects of cisplatin on NSCLC cells. Besides suppressing anti-tumoral immune responses, tumor-intrinsic CD73 can facilitate NSCLC development, and the combined cisplatin therapy with CD73-siRNA transfection can substantially enhance the chemosensitivity of NSCLC to cisplatin and potentiates cisplatin-induced anti-tumoral effects on NSCLC.

Functions of long non-coding RNA LNC11649 in non-small cell lung cancer cells as a reprocessed form of MALAT1.

Long non-coding RNAs (lncRNAs) have received much attention concerning their expression mechanisms in tumor formation. It is known that lncRNAs are involved in the occurrence, development, and prognosis of various tumors, including lung cancer. In our study, one non-coding RNA, LNC11649 (649 nt in length), was identified in non-small cell lung cancer (NSCLC) by PacBio third-generation sequencing technology. Both northern blot and quantitative PCR analyses confirmed the presence of LNC11649 in NSCLC tissues and cells with high expression. Its sequence was found to be highly homologous to lncRNA MALAT1. Knocking down MALAT1 could lead to a significant downregulation of LNC11649 content, revealing the possibility that LNC11649 could originate from MALAT1 reprocessing. RNA immunoprecipitation and electrophoretic mobility shift assays confirmed an interaction between LNC11649 and the MSI1 protein. Further experiments revealed that LNC11649 promoted the cytoplasmic distribution of MSI1 through its interaction with MSI1 and then activated the Akt signaling pathway to regulate the proliferation and migration of NSCLC cells. Our study reveals the possibility that LNC11649 plays a cancer-promoting role as a reprocessed form of MALAT1 in NSCLC cells and suggests that the MALAT1/LNC11649/MSI1/Akt regulatory axis becomes a potential therapeutic target for lung cancer.

A novel C-terminal heat shock protein 90 inhibitor that overcomes STAT3-Wnt-β-catenin signaling-mediated drug resistance and adverse effects.

Rationale: The heat shock protein (Hsp) system plays important roles in cancer stem cell (CSC) and non-CSC populations. However, limited efficacy due to drug resistance and toxicity are obstacles to clinical use of Hsp90 inhibitors, suggesting the necessity to develop novel Hsp90 inhibitors overcoming these limitations.Methods: The underlying mechanism of resistance to Hsp90 inhibitors was investigated by colony formation assay, sphere formation assay, western blot analysis, and real-time PCR. To develop anticancer Hsp90 inhibitors that overcome the signal transducer and activator of transcription 3 (STAT3)-mediated resistance, we synthesized and screened a series of synthetic deguelin-based compounds in terms of inhibition of colony formation, migration, and viability of non-small cell lung cancer (NSCLC) cells and toxicity to normal cells. Regulation of Hsp90 by the selected compound NCT-80 [5-methoxy-N-(3-methoxy-4-(2-(pyridin-3-yl)ethoxy)phenyl)-2,2-dimethyl-2H-chromene-6-carboxamide] was investigated by immunoprecipitation, drug affinity responsive target stability assay, binding experiments using ATP-agarose beads and biotinylated drug, and docking analysis. The antitumor, antimetastatic, and anti-CSC effects of NCT-80 were examined in vitro and in vivo using various assays such as MTT, colony formation, and migration assays and flow cytometric analysis and tumor xenograft models.Results: We demonstrated a distinct mechanism in which Hsp90 inhibitors that block N-terminal ATP-binding pocket causes transcriptional upregulation of Wnt ligands through Akt- and ERK-mediated activation of STAT3, resulting in NSCLC cell survival in an autocrine or paracrine manner. In addition, NCT-80 effectively reduced viability, colony formation, migration, and CSC-like phenotypes of NSCLC cells and their sublines with acquired resistance to anticancer drugs by inducing apoptosis and inhibiting epithelial-mesenchymal transition and the growth of NSCLC patient-derived xenograft tumors without overt toxicity. With regards to mechanism, NCT-80 directly bound to the C-terminal ATP-binding pocket of Hsp90, disrupting the interaction between Hsp90 and STAT3 and degrading STAT3 protein. Moreover, NCT-80 inhibited chemotherapy- and EGFR TKI-induced programmed cell death ligand 1 expression and potentiated the antitumor effect of chemotherapy in the LLC-Luc allograft model.Conclusions: These data indicate the potential of STAT3/Wnt signaling pathway as a target to overcome resistance to Hsp90 inhibitors and NCT-80 as a novel Hsp90 inhibitor that targets both CSCs and non-CSCs in NSCLC.

Epithelial-mesenchymal reprogramming by KLF4-regulated Rictor expression contributes to metastasis of non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) is one of the deadliest cancers in the world. Metastasis is considered one of the leading causes of treatment failure and death in NSCLC patients. A crucial factor of promoting metastasis in epithelium-derived carcinoma has been considered as epithelial-mesenchymal transition (EMT). Rictor, one of the components of mTORC2, has been reportedly involved in EMT and metastasis of human malignancies. However, the regulatory mechanisms of Rictor, Rictor-mediated EMT and metastasis in cancers remain unknown. Our present study indicates that Rictor is highly expressed in human NSCLC cell lines and tissues and is regulated, at least partially, at the transcriptional level. Knockdown of Rictor expression causes phenotype alterations through EMT, which is accompanied by the impairment of migration and invasion ability in NSCLC cells. Additionally, we have cloned and identified the human Rictor core promoter for the first time and confirmed that transcription factor KLF4 directly binds to the Rictor promoter and transcriptionally upregulated Rictor expression. Knockdown of KLF4 results in Rictor's downregulation accompanied by a series of characteristic changes of mesenchymal-epithelial transition (MET) and significantly decreases migration, invasion as well as metastasis of NSCLC cells. Re-introducing Rictor in KLF4-knockdown NSCLC cells partially reverses the epithelial phenotype to the mesenchymal phenotype and attenuates the inhibition of cell migration and invasion caused by KLF4 knocking down. Knockdown of KLF4 prevents mTOR/Rictor interaction and metastasis of NSCLC in vivo. The understanding of the regulator upstream of Rictor may provide an opportunity for the development of new inhibitors and the rational design of combination regimens based on different metastasis-related molecular targets and finally prevents cancer metastasis.

SIX1 Predicts Poor Prognosis and Facilitates the Progression of Non-small Lung Cancer via Activating the Notch Signaling Pathway.

Background: Many transcription factors involved in embryonic development and reactivated in tumors are considered potential prognostic biomarkers and novel therapeutic targets in various cancers. Sine oculis homeobox homolog 1 (SIX1), a developmentally restricted transcriptional regulator, plays a critical role during tumor initiation and development. However, the prognostic value and biological function of SIX1 in non-small cell lung cancer (NSCLC) remain unclear.Methods: Bioinformatic analyses were conducted to investigate the expression of SIX1 in cancer and adjacent normal tissues of NSCLC and further explore the correlations between SIX1 expression and clinical outcomes. Western blotting and RT-PCR analysis were performed to detect of SIX1 expression level in NSCLC cell lines and normal bronchial epithelial cell. EdU, CCK-8, clonal formation assay, wound healing and transwell assay were performed to explore the effects of gain- or loss-of-function of SIX1 on cellular proliferation, migration and invasion in vitro. Gene set enrichment analysis (GSEA) was used to identify the potential signaling pathways involved in SIX1 mediated biological function and the correlation was confirmed by western blotting and RT-PCR analysis. In vivo experiment was conducted to further validate the tumor-promoting effects of SIX1.Results: Bioinformatic analysis indicated that SIX1 was markedly upregulated in NSCLC tissues of and positively correlated with poor prognosis of patients with NSCLC. Ectopic expression of SIX1 facilitated proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of NSCLC cells. On the contrary, knocking down SIX1 exhibited the opposite effects. Mechanistic studies suggested that SIX1 activated the Notch pathway to promote the malignant biological behaviors of NSCLC, which could be reversed by inhibiting the Notch signaling with γ-secretase inhibitor.Conclusions: SIX1 could facilitate multiple malignant biological behaviors by activating the Notch signaling pathway and function as a promising prognostic biomarker.

RIOK1 is associated with non-small cell lung cancer clinical characters and contributes to cancer progression.

Lung cancer is one of the most common malignant tumors and is currently the leading cause of cancer-related deaths worldwide. Although the treatment strategy has been significantly improved, the prognosis of lung cancer patients is still quite poor. RIOK1 has been reported to be highly expressed in non-small cell lung cancer (NSCLC), however, its clinical significance and biological function are still largely unknown in lung cancer. Using western blot and immunohistochemistry, we showed that RIOK1 was highly expressed in NSCLC tissues and correlated with advanced stage and poor prognosis. Furthermore, knockdown of RIOK1 could inhibit proliferation, migration, and invasion in NSCLC cells and tumorigenesis in vivo through AKT, Cyclin B1, MMP2, and EMT pathway. Furthermore, cell viability and apoptosis assays demonstrated that RIOK1 maintained NSCLC cell survival and reduced apoptosis rate when cells were treated with cisplatin. Western blot analysis demonstrated that RIOK1 depletion caused up-regulated protein expression of cleaved PARP and Caspase-3 in NSCLC cells. These findings revealed a novel function of RIOK1 in non-small cell lung cancer progression and suggest that RIOK1 might become a promising diagnostic and therapeutic target for this disease.

Knockdown of LINC00511 decreased cisplatin resistance in non-small cell lung cancer by elevating miR-625 level to suppress the expression of leucine rich repeat containing eight volume-regulated anion channel subunit E.

LINC00511 has been reported as a biomarker related to the prognosis of non-small cell lung cancer (NSCLC), but the molecular mechanism and exact functions of LINC00511 in chemoresistance of NSCLC remain to be elucidated. RT-qPCR was used to evaluate the mRNA expression of LINC00511, miR-625, and leucine rich repeat containing 8 volume-regulated anion channel subunit E (LRRC8E). Western blotting detected the protein levels of Ki-67, MMP-9, cleaved-caspase-3. The interaction between miR-625 and LINC00511 or LRRC8E was verified by luciferase reporter assays. CCK-8, TUNEL, and Transwell assays were used to evaluate IC50 value, proliferation, migration, and invasion of NSCLC cells. In our study, it was discovered that the levels of LINC00511 and LRRC8E were increased, while miR-625 expression was decreased in NSCLC tissues, DDP-resistant NSCLC cells, and non-resistant NSCLC cells. LINC00511 depletion significantly curbed cell growth, IC50 value, and metastasis in DDP-resistant NSCLC cells. In addition, the influence of LINC00511 deficiency on the DDP resistance in NSCLC was overturned by suppressing miR-625. Furthermore, LRRC8E overexpression abolished the promotive effect of miR-625 abundance on the DDP sensitivity in DDP-resistant NSCLC cells. Our results demonstrated that LINC00511 increased DDP resistance in NSCLC by suppressing miR-625 to upregulate LRRC8E.

A Peptide Encoded by a Long Non-Coding RNA DLX6-AS1 Facilitates Cell Proliferation, Migration, and Invasion by Activating the wnt/β-Catenin Signaling Pathway in Non-Small-Cell Lung Cancer Cell.

Recently, accumulating study shows that some long non-coding RNAs (lncRNAs) have potential protein/peptide-coding capacities. In this study, the coding potential of lncRNA distal-less homeobox 6 antisense 1 (DLX6-AS1) was examined and the roles and downstream pathways of a DLX6-AS1-encoded peptide in non-small-cell lung cancer (NSCLC) cell development were investigated. The peptide-coding potential of lncRNA DLX6-AS1 was extrapolated based on prior ribosome footprint and ribosome sequencing data, IPX0002962000 mass spectrometry dataset, and Getorf bioinformatics analysis. The peptide-coding abilities of several DLX6-AS1 open reading frame (ORF) fragments, as well as protein levels were detected by Western blot assay. Cell proliferative, migratory, and invasive abilities were tested by CCK-8 or Transwell assays, respectively. Potential key biological processes and pathways related to DLX6-AS1 expression were identified by single-gene gene set enrichment analysis (GSEA) based on RNA-seq data of 510 lung adenocarcinoma samples in the TCGA GDC database. The results showed that an ORF of lncRNA DLX6-AS1 could encode a short peptide. The exogenous overexpression of this ORF-encoded peptide promoted NSCLC cell proliferation, migration, and invasion. GSEA analysis suggested that DLX6-AS1 might play crucial roles in cancer progression and wnt signaling pathway. Further analysis revealed that the exogenous overexpression of a DLX6-AS1-encoded peptide could exert its functions by activating the wnt/β-catenin pathway in NSCLC cells. In conclusion, the exogenous overexpression of a DLX6-AS1-encoded peptide could facilitate NSCLC cell growth by activating wnt/β-catenin pathway.

Fuzzy Planar Cell Polarity Gene (FUZ) Promtes Cell Glycolysis, Migration, and Invasion in Non-small Cell Lung Cancer via the Phosphoinositide 3-Kinase/Protein Kinase B Pathway.

Purpose: Fuzzy planar cell polarity gene (FUZ) is regarded as a planar cell polarity effector that controls multiple cellular processes during vertebrate development. The role of FUZ in glucose metabolism, invasion, and metastasis of non-small cell lung cancer (NSCLC) is unclear. The aims of this study were to investigate the relationship between FUZ and glucose metabolism and its mechanism of action.Materials and methods: Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to detect FUZ expression in A549 and H1299 cells. Additionally, qRT-PCR and western blot analysis were used to detect the expression of related glucose metabolism indicators, and lactate and 18 Fluorine fludeoxyglucose (18F-FDG) uptake assays used to detect changes in glucose metabolites. Further, cell invasion and migration behavior were evaluated by transwell and wound healing assays. In vivo tumor growth assay was conducted to assess the effect of FUZ.Results: We found that FUZ was significantly upregulated in the NSCLC cell lines compared to that in the normal HBE cells. FUZ was found to promote energy metabolism through the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway, and overexpression of FUZ increased both lactic acid and 18F-FDG uptake. Moreover, FUZ knockdown significantly inhibited the migration and invasion of NSCLC cells. In vivo, FUZ knockdown can significantly inhibit tumor proliferation in the xenograft model, which was well identified by Micro-PET scan.Conclusion: The present finding in vitro and vivo show that FUZ is involved in NSCLC cell energy metabolism, invasion and migration via the PI3K/AKT signaling pathway, suggesting that FUZ can be a potential therapeutic target for NSCLC.

Long noncoding RNA TFAP2A AS1 exerts promotive effects in non small cell lung cancer progression via controlling the microRNA 548a 3p/CDK4 axis.

In this study, we mainly focus on probing expression profile and detailed functions of long non coding RNA TFAP2A antisense RNA 1 (TFAP2A AS1) in non small cell lung cancer (NSCLC). Moreover, the mechanisms played by TFAP2A AS1 were unraveled comprehensively. Herein, a notable overexpressed TFAP2A AS1 in NSCLC was observed by TCGA and our own cohort. An increased TFAP2A AS1 level displayed a negative correlation with the overall survival of patients with NSCLC. Loss of function approaches illustrated that the absence of TFAP2A AS1 weakened NSCLC cell proliferation, colony formation, migration and invasion in vitro. Also, interference of TFAP2A AS1 caused in vivo tumor growth suppression. Mechanistically, TFAP2A AS1 could negative regulate microRNA 584 3p (miR 584 3p) as a competitive endogenous RNA. Furthermore, cyclin dependent kinase 4 (CDK4), a direct target of miR 584 3p, was positively controlled by TFAP2A AS1 in a miR 5184 3p dependent manner. Rescue function experiments corroborated that the anticancer activities of TFAP2A AS1 deficient on the oncogenicity of NSCLC cells were reversed by downregulating miR 584 3p or overexpressing CDK4. To sum up, TFAP2A AS1 exhibits cancer promoting roles in NSCLC through the adjustment of miR 584 3p/CDK4 axis.

MiR-769-5p, Which Targets HDGF, Inhibits Cell Proliferation and Invasion in Nonsmall Cell Lung Cancer.

MiR-769-5p regulates tumor correlative genes, which plays a critical role in the progression of various types of tumor. However, the precise regulatory mechanism of miR-769-5p on nonsmall cell lung cancer (NSCLC) is unknown. This study was to discover the role and underlying mechanisms of miR-769-5p in NSCLC. MiR-769-5p expression was shown to be reduced, according to our findings. MiR-769-5p overexpression inhibited NSCLC cell proliferation while promoting NSCLC cell apoptosis. Furthermore, NSCLC cell migration and invasion were reduced when miR-769-5p was overexpressed. Furthermore, HDGF was confirmed as a miR-769-5p target gene that was negatively regulated by miR-769-5p. Furthermore, more research revealed that HDGF overexpression reduced the inhibitory effect of miR-769-5p on NSCLC cell biofunction. Finally, miR-769-5p inhibited NSCLC cell proliferation and invasion by targeting HDGF, indicating that NSCLC could benefit from miR-769-5p as a diagnostic and prognostic biomarker.

E3 ubiquitin-protein ligase 2 inhibits cell proliferation, migration, and invasion of non-small cell lung cancer through ubiquitination of Notch1

This study aimed to explore the role of MIB2 in non-small cell lung cancer (NSCLC) and the underlying mechanism. Quantitative real-time PCR (QRT-PCR) and western blot were first performed to detect MIB2 expression in tumor tissues obtained from NSCLC patients (n = 30) and NSCLC cells, respectively. 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and transwell assays were then used to examine the effect of MIB2 on the proliferation, migration and invasion of NSCLC cells. Western blot was further performed to examine the effect of Mind bomb 2 (MIB2), an E3 ligase on Notch1 protein and its ubiquitination. MIB2 was significantly down-regulated in NSCLC tissues and cells, both in mRNA and protein level. MIB2 also note worthily inhibited the proliferation, migration, and invasion of NSCLC cells. Furthermore, MIB2 only down-regulated Notch1 protein level, while facilitated the ubiquitination of Notch1. Additionally, Notch1 significantly relieved the repressed proliferation, migration and invasion of NSCLC cells induced by MIB2. Conclusively, MIB2 inhibited cell proliferation, migration and invasion via inducing Notch1 ubiquitination and degradation in NSCLC.

TdIF1-LSD1 Axis Regulates Epithelial-Mesenchymal Transition and Metastasis via Histone Demethylation of E-Cadherin Promoter in Lung Cancer.

We have previously found that TdT-interacting factor 1 (TdIF1) is a potential oncogene expressed in non-small cell lung cancer (NSCLC) and is associated with poor prognosis. However, its exact mechanism is still unclear. The lysine-specific demethylase 1 (LSD1) is a crucial mediator of the epithelial–mesenchymal transition (EMT), an important process triggered during cancer metastasis. Here, we confirm that TdIF1 is highly expressed in NSCLC and related to lymph node metastasis through The Cancer Genome Atlas (TCGA) analysis of clinical samples. Silencing TdIF1 can regulate the expression of EMT-related factors and impair the migration and invasion ability of cancer cells in vitro. An analysis of tumor xenografts in nude mice confirmed that silencing TdIF1 inhibits tumor growth. Furthermore, we determined the interaction between TdIF1 and LSD1 using immunoprecipitation. Chromatin immunoprecipitation (ChIP) revealed that TdIF1 was enriched in the E-cadherin promoter region. The knockdown of TdIF1 repressed the enrichment of LSD1 at the E-cadherin promoter region, thereby regulating the level of promoter histone methylation and modulating E-cadherin transcription activity, ultimately leading to changes in EMT factors and cancer cell migration and invasion ability. The LSD1 inhibitor and TdIF1 knockdown combination showed a synergistic effect in inhibiting the growth, migration, and invasion of NSCLC cells. Taken together, this is the first demonstration that TdIF1 regulates E-cadherin transcription by recruiting LSD1 to the promoter region, thereby promoting EMT and tumor metastasis and highlighting the potential of TdIF1 as a therapeutic target for NSCLC.

Circular RNA hsa_circ_0002360 promotes non-small cell lung cancer progression through upregulating matrix metalloproteinase 16 and sponging multiple micorRNAs

ABSTRACT Dysregulated circular RNAs (circRNAs) are involved in the progression of non-small cell lung cancer (NSCLC). However, the role of has_circ_0002360 (circ_0002360) in NSCLC has rarely been reported. In this study, circ_0002360 expression in NSCLC tissues and cell lines was measured using microarray data and quantitative real-time PCR (qRT-PCR). After gain-of-function and loss-of-function, cell models were established; 5-bromo-2-deoxyuridine (BrdU) and transwell assays were conducted to detect NSCLC cell growth, migration, and invasion. What is more, bioinformatic analysis and dual-luciferase reporter assay were adopted to show how circ_0002360, microRNAs (miR-127-5p, miR-145-5p, miR-585-3p, and miR-758-3p), and matrix metalloproteinase 16 (MMP16) 3ʹUTR interact with each other. Western blotting was executed to probe the regulatory effects of circ_0002360 and these miRNAs on MMP16 protein expression in NSCLC cells. We found that circ_0002360 expression was raised in NSCLC tissues. High circ_0002360 expression predicted a short overall survival time for NSCLC patients. Circ_0002360 overexpression promoted NSCLC cell proliferative, migrative, and invasive abilities, and circ_0002360 depletion worked oppositely. MiR-127-5p, miR-145-5p, miR-585-3p, and miR-758-3p were the targets of circ_0002360, and circ_0002360 could regulate MMP16 expression by competitively binding with the above miRNAs. In summary, circ_0002360 serves as a competitive endogenous RNA to raise MMP16 expressions by competitively binding to miR-127-5p, miR-145-5p, miR-585-3p, and miR-758-3p, thereby promoting NSCLC progression.

Long non-coding RNA LINC00511 promotes proliferation, invasion, and migration of non-small cell lung cancer cells by targeting miR-625-5p/GSPT1.

BackgroundLung cancer is a malignant tumor with a high rate of mortality and metastasis. Recently, extensive research has shown that long non-coding RNAs (lncRNAs) play a crucial role in the development and progression of non-small cell lung cancer (NSCLC). In this paper, we aimed to explore the impact of long intergenic non-coding RNA 00511 (LINC00511) on the development and metastasis of NSCLC.MethodsA dataset containing 501 lung squamous cell carcinoma (LUSC) samples and 49 normal samples was downloaded from The Cancer Genome Atlas (TCGA). The differential gene expression and prognostic potential of LINC00511 in LUSC were analyzed by “limma” in R software. Samples of tumor tissues and normal tissues from 67 patients with NSCLC were obtained, along with clinical features. NSCLC cell proliferation, cell cycle, migration, and invasion were detected by LINC00511 knockdown with Cell Counting Kit-8 (CCK-8), flow cytometry, wound-healing assay, and Transwell experiment. The regulatory relationship between LINC00511 and microRNA (miR)-625-5p, or between miR-625-5p and G1 to S phase transition 1 (GSPT1), was detected by luciferase reporter gene assay. LINC00511, miR-625-5p, and GSPT1 expression in tumor and normal tissues and cells was determined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. A xenograft experiment in nude mice was performed. Ki67 and GSPT1 expression in the tumor tissues of the nude mice was assessed by immunohistochemistry.ResultsLINC00511 expression was clearly higher in the tumor tissues of the NSCLC patients than in normal tissues (P<0.001). High LINC00511 expression was related to larger tumor size, positive lymph node metastasis, advanced TNM stage, and a lower 5-year survival rate. Compared with those of the shNC group, the NSCLC cells of the shLINC00511 group had a prominently lower optical density (OD) 450 value at 72 h, a lower percentage of cells in S phase, a higher relative wound width, and a lower invasive cell number (P<0.01 or P<0.001). LINC00511 promoted GSPT1 expression via suppressing miR-625-5p. Compared with those of the shNC group, the nude mice of the shLINC00511 group had a much lower subcutaneous tumor volume and weight (P<0.05 or P<0.001).ConclusionslncRNA LINC00511 promotes proliferation, invasion, and migration of NSCLC cells by targeting miR-625-5p/GSPT. LINC00511 may be a potential diagnostic marker and therapeutic target for NSCLC.

A small-molecule compound D6 overcomes EGFR-T790M-mediated resistance in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a deadly and highly prevalent malignancy. Targeting activated-EGFR mutations in NSCLC via EGFR tyrosine kinase inhibitor (EGFR-TKI) initially achieves a profound therapeutic response, but resistance frequently evolves, reducing treatment options. Here, we present a small-molecule compound D6 which selectively inhibits tumor cell growth and migration in NSCLC cells with EGFR-TKI-resistant T790M-EGFR-activated mutations (T790M-EGFR-AM), e.g., L858R/T790M, 19Del/T790M and L858R/T790M/C797S. D6 mimics a natural product isolated from the roots of Codonopsis pilosula and selectively competes with T790M-EGFR-AM to bind to HSP90, thus facilitating the ubiquitination dependent proteasomal degradation of T790M-EGFR-AM. By contrast, D6 has little impact on typical HSP90 chaperone activity, suggesting low systemic toxicity. Promisingly, D6 combined with erlotinib or osimertinib shows efficacy in overcoming the EGFR-TKIs-resistance in NSCLCs. Our study raises an alternative strategy to overcome T790M-mediated EGFR-TKI resistance in NSCLC via targeting the protein–protein interaction of HSP90 and T790M-EGFR by intervention with D6.

Circ_0000376 downregulation inhibits the progression of non-small cell lung cancer by mediating the miR-488-3p/BRD4 axis and the PI3K/PKB signaling pathway.

The involvement of circular RNAs (circRNAs) in the development of cancers has attracted much interest. This study aimed to determine the role of circ_0000376 in non-small cell lung cancer (NSCLC) and provide a new mechanism. The expression of circ_0000376, miR-488-3p and bromodomain containing 4 (BRD4) mRNA was measured by quantitative real-time PCR (qPCR). Cell behaviors, including cell proliferation, invasion, migration, apoptosis and cell cycle progression were investigated using cell counting kit-8 (CCK-8) assay and colony formation assay, transwell assay, wound healing assay and flow cytometry assay, respectively. The putative relationship between miR-488-3p and circ_0000376 or BRD4 was verified by dual-luciferase reporter assay. The protein levels of BRD4 and phosphorylated PI3K/PKB were detected by western blot. Xenograft model was constructed to determine the role of circ_0000376 in vivo. Circ_0000376 was highly expressed in NSCLC tissues and cells. Circ_0000376 downregulation inhibited NSCLC cell proliferation, invasion and migration, promoted cell apoptosis and cell cycle arrest and slowed tumor growth in vivo. Circ_0000376 competitively bound to miR-488-3p to regulate the expression of BRD4. Rescue experiments showed that miR-488-3p deficiency reversed the effects of circ_0000376 downregulation, and miR-488-3p restoration-suppressed cell proliferation, migration and invasion were recovered by BRD4 overexpression. Moreover, circ_0000376 downregulation weakened the levels of phosphorylated PI3K and PKB, thus reducing the activity of the PI3K/PKB pathway. Circ_0000376 downregulation blocked the development of NSCLC by targeting the miR-488-3p/BRD4 network and suppressing the PI3K/PKB pathway, which broadens knowledge into the understanding of the role of circ_0000376 in NSCLC.

Baicalin Inhibits EMT through PDK1/AKT Signaling in Human Nonsmall Cell Lung Cancer.

Background Baicalin is a naturally occurring compound with anticancer, antioxidant, and anti-inflammatory properties. However, the mechanism underlying its anticancer activity on nonsmall cell lung cancer (NSCLC) remains unclear. Methods The effects of baicalin on the progression and metastasis of experimental NSCLC cell lines were studied in vitro and in vivo. Wound-healing and transwell assays were performed to evaluate the potency of baicalin and the motility and migration ability of NCI-H460 cells. Immunofluorescence assay, western blot assay, and immunohistochemistry test were conducted to investigate the inhibiting effect of baicalin on the epithelial-mesenchymal transition (EMT) of NSCLC. Results Baicalin inhibited the proliferation and migration of NCI-H446 human NSCLC cells in a dose-dependent manner, reduced the expression levels of phospho-3-phosphoinositide-dependent protein kinase 1 (p-PDK1) and phosphor-serine/threonine-protein kinase (p-AKT), reversed the levels of EMT markers, and inhibited the migration of NSCLC cells. Conclusions Baicalin impedes EMT by inhibiting the PDK1/AKT pathway in human NSCLC and thus may be an effective alternative treatment for carcinoma and a new candidate antimetastasis drug.