Promotes Non-small Cell Lung Cancer Progression Research Articles

Circ_0004015 silencing represses cisplatin chemoresistance and tumor progression by reducing KLF8 in a miR-198-dependent manner in non-small cell lung cancer.

Circular RNA (circRNA) plays vital roles in diverse cancer progression, including non-small cell lung cancer (NSCLC). Herein, the role of circ_0004015 in regulating the sensitivity of NSCLC to cisplatin (DDP) is revealed. The RNA expression of circ_0004015, microRNA-198 (miR-198) and kruppel like factor 8 (KLF8) was detected by quantitative real-time polymerase chain reaction. Protein expression was checked by western blot. The half maximal inhibitory concentration of DDP and cell proliferation were determined by cell counting kit-8 assay. Cell colony formation ability, migration, invasion and apoptosis were investigated by colony-forming assay, transwell assay and flow cytometry analysis, respectively. The effect of circ_0004015 knockdown on DDP sensitivity in vivo was demonstrated by mouse model assay. The interactions among circ_0004015, miR-198 and KLF8 were predicted by bioinformatics methods, and identified by mechanism assays. The expression of circ_0004015 and KLF8 was apparently upregulated, while miR-198 expression was downregulated in DDP-resistant NSCLC tissues and cells compared with control groups. Additionally, circ_0004015 silencing repressed DDP resistance, cell proliferation, migration and invasion, but induced cell apoptosis in DDP-resistant NSCLC cells. Circ_0004015 knockdown promoted the effect of DDP on tumor formation in vivo. Also, miR-198 inhibitors attenuated circ_0004015 depletion-mediated action though associating with circ_0004015. MiR-198 regulated DDP sensitivity and NSCLC progression by targeting KLF8. Furthermore, circ_0004015 modulated KLF8 expression through interaction with miR-198. Circ_0004015 conferred DDP resistance and promoted NSCLC progression by miR-198/KLF8 pathway, proving a potential target for studying DDP-mediated treatment of NSCLC.

Overexpression of miR-328-5p influences cell growth and migration to promote NSCLC progression by targeting LOXL4.

BackgroundLung cancer is the leading cause of cancer-associated mortality worldwide, and most lung cancers are classified as non-small cell lung cancer (NSCLC). MiR-328 influence the progression of multiple tumors, but the role of miR-328-5p in NSCLC has not been elucidated. The aim of this study was to illuminate the oncogenic role and potential molecular mechanisms of the miR-328-5p and lysyl oxidase like 4 (LOXL4) in NSCLC.MethodsExpression of miR-328-5p was detected by real-time quantitative polymerase chain reaction (qRT-PCR) in tumor and non-tumor adjacent tissues. After Lentivirus-miR-328-5p was employed to intervene this miRNA in NSCLC cell lines, RT-qPCR was used to detect the expression levels of miR-328-5p. Cell Counting Kit-8 (CCK-8), cell colony formation, flow cytometry, wound healing, Transwell assays were used to determine the malignant phenotypes of NSCLC cells. Nude mice models of subcutaneous tumors were established to observe the effect of miR-328-5p on tumorigenesis. Targeting the 3'UTR of LOXL4 by miR-328-5p was verified by integrated analysis including transcriptome sequencing, dual-luciferase and western-blot assays.ResultsHigh miR-328-5p level was observed in NSCLC cells from The Cancer Genome Atlas (TCGA) database and tumor tissues collected from NSCLC patients. Overexpressed miR-328-5p promoted NSCLC cell proliferation, survival, and migration, and promoted tumor growth in vivo. Knockdown of miR-328-5p suppressed tumorigenic activities. Transcriptome sequencing analysis revealed that LOXL4 was downregulated by miR-328-5p, which was confirmed by dual-luciferase reporter and western-blot assays.ConclusionsmiR-328-5p showed targeted regulation of LOXL4 to promote cell proliferation and migration in NSCLC.

COL8A1 Promotes NSCLC Progression Through IFIT1/IFIT3-Mediated EGFR Activation.

Activation of EGFR is a major risk factor for non-small cell lung cancer (NSCLC). Understanding the molecular events promoting EGFR activation can help us gain more insights into the progression of NSCLC. In this study, we demonstrate that collagen type VIII alpha 1 chain (COL8A1), an extracellular matrix component, was overexpressed in NSCLC. In NSCLC cells, knockdown of COL8A1 suppressed cell growth, cycle progression, and migration, and induced cell apoptosis. While COL8A1 overexpression promoted cell proliferation and inhibited cell apoptosis. In addition, we found that COL8A1 depletion reduced interferon response signaling and downregulated (IFIT1) and interferon-induced proteins with tetratricopeptide repeats 3 (IFIT3). Moreover, we indicated that COL8A1 could upregulate IFIT1 and IFIT3 mediated EGFR activation in vitro and in vivo. Lastly, there was a positive correlation among COL8A1, IFIT1, and IFIT3 expression, and EGFR activity in patients with NSCLC. Overall, our data demonstrate that COL8A1 contributes to NSCLC proliferation and invasion through EGFR activation, dependent on IFIT1 and IFIT3 expression.

CircCAMSAP1 promotes non-small cell lung cancer proliferation and inhibits cell apoptosis by sponging miR-1182 and regulating BIRC5

ABSTRACT Recently, various studies have suggested that circular RNAs (circRNAs) are ubiquitous in various malignant events, including non-small cell lung cancer (NSCLC) and are closely related to cell proliferation and apoptosis. Unfortunately, the molecular functions involved in this action still have little overlap. Therefore, this study aimed to identify a novel circCAMSAP1 role in NSCLC. Overexpression of circCAMSAP1 has been demonstrated in NSCLC lung tissues and cell lines. Sequencing and RNase R experiments were planned to determine whether circCAMSAP1 is looped and exists in NSCLC. We also found that downregulated circCAMSAP1 repressed cell proliferation and increased apoptosis of NSCLC cells in vitro and suppressed xenograft tumor growth in vivo. Furthermore, a luciferase assay revealed that circCAMSAP1 could regulate baculoviral inhibitor of apoptosis protein (IAP) repeat containing 5 (BIRC5, also known as survivin) expression by directly binding to miR-1182. However, BIRC5 without 3ʹ untranslated regions (3ʹUTR) could reverse the influence of downregulated circCAMSAP1 on proliferation and apoptosis in NSCLC. Together, our findings reveal a novel mechanism by which the circCAMSAP1/miR-1182/BIRC5 axis promotes NSCLC progression.

Circular RNA circTADA2A promotes the proliferation, invasion, and migration of non-small cell lung cancer cells via the miR-450b-3p/HMGN5 signaling pathway.

BackgroundCircular RNAs (circRNAs) have been confirmed to exert important roles in promoting tumor initiation and progression. However, the expression, effect, and underlying mechanism of circTADA2A in non-small cell lung cancer (NSCLC) remain unclear.MethodsA total of 60 paired clinical samples of NSCLC tissues and corresponding normal adjacent tissues were obtained. Quantitative real-time PCR was used to verify circTADA2A, miR-450b-3p, and HMGN5 mRNA expression. The NSCLC cell Lines A549 and H1299 were individually transfected with circTADA2A and HMGN5. The regulatory interaction between circTADA2A and miR-450b-3p was investigated by dual-luciferase reporter assay. HMGN5 protein expression was detected by Western blotting.ResultsCircTADA2A expression was significantly upregulated and correlated with poor overall survival of NSCLC patients. Functionally, circTADA2A inhibition successfully suppressed the proliferation, invasion, and migration of A549 and H1299 cells. circTADA2A functioned as a competing endogenous RNA to sponge miR-450b-3p to promote the expression of HMGN5 mRNA and protein. Furthermore, a positive relationship between circTADA2A and HMGN5 existed in NSCLC tissues. There were negative relationships between circTADA2A and miR-450b-3p as well as miR-450b-3p and HMGN5 in NSCLC tissues.ConclusionsThese findings suggest that circTADA2A might act as an oncogenic circRNA that promotes NSCLC progression by sponging miR-450b-3p and promoting HMGN5 expression, indicating that the suppression of circTADA2A could become a potential therapeutic target for restraining 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.

VIRMA contributes to non-small cell lung cancer progression via N6-methyladenosine-dependent DAPK3 post-transcriptional modification.

N6-methyladenosine (m6A) has been reported to be abnormally expressed in non-small cell lung cancer (NSCLC), and plays a vital role in regulation of cell proliferation, invasion and metastasis. Vir-Like m6A methyltransferase associated (VIRMA, also called KIAA1429) has not been well studied in NSCLC. Thus, in this study, we investigated the biological impact and underlying mechanism of VIRMA in NSCLC. High expression of VIRMA was testified in patients with NSCLC and predicted worse prognosis in patients. VIRMA facilitated cell proliferation and tumor growth both in vitro and in vivo. Furthermore, VIRMA-regulated m6A modifications led to post-transcriptional suppression of death-associated protein kinase 3 (DAPK3, also called ZIP or ZIPK) through the YT521-B homology domain-containing family proteins 2/3(YTHDF2/3). Inhibition of DAPK3 rescued the tumor-suppressive phenotypes induced by VIRMA deficiency. In conclusion, VIRMA-guided m6A modifications promoted NSCLC progression via m6A-dependent degradation of DAPK3 mRNA. Therefore, VIRMA may be a novel therapeutic target in NSCLC.

Microarray Identifies a Key Carcinogenic Circular RNA 0008594 That Is Related to Non-Small-Cell Lung Cancer Development and Lymph Node Metastasis and Promotes NSCLC Progression by Regulating the miR-760-Mediated PI3K/AKT and MEK/ERK Pathways.

PurposeThis study aimed to explore the circular RNA (circRNA/circ) profile engaged in non-small cell lung cancer (NSCLC) development and metastasis and to investigate potentially key carcinogenic circRNAs related to NSCLC.MethodsCircRNA profiles between 10 NSCLC tissues and 10 adjacent tissues and between five NSCLC tissues with lymph node metastasis (LNM) and five NSCLC tissues without LNM were detected by Arraystar Human circRNA Array followed by bioinformatics. Circ_0008594 knockdown, circ_0004293 overexpression, and circ_0003832 overexpression plasmids were transfected into H23 and H460 cells to sort potential oncogenic circRNA. Then circ_0008594 overexpression and knockdown plasmids were transfected, followed by that circ_0008594 knockdown plus miR-760 knockdown plasmids were transfected into these cells. Cell proliferation, apoptosis, invasion, stemness, and pathways were detected. In addition, xenograft mice models were constructed via injecting H23 cells with circ_0008594 overexpression or knockdown to validate the findings.ResultsA total of 455 dysregulated circRNAs in NSCLC tissues versus adjacent tissues and 353 dysregulated circRNAs in NSCLC tissues with LNM versus those without LNM were discovered. Via cross-analysis, 19 accordant circRNAs were uncovered, among which three candidate circRNAs (circ_0008594, circ_0004293, circ_0003832) were chosen for functional experiments, during which it was observed that circ_0008549 affected H23 and H460 cell proliferation and apoptosis more obviously than circ_0004293 and circ_0003832. Subsequent experiments showed that circ_0008594 promoted H23 and H460 cell proliferation and invasion but affected stemness less and negatively regulated miR-760 via direct binding. Furthermore, miR-760 attenuated the effect of circ_0008549 on regulating H23 and H460 cell functions and the PI3K/AKT and MEK/ERK pathways. In vivo experiments further confirmed that circ_0008549 increased tumor volume, epithelial-mesenchymal transition, and the PI3K/AKT and MEK/ERK pathways while reducing tumor apoptosis and miR-760 NSCLC xenograft models.ConclusionOur study identifies several valuable circRNAs related to NSCLC development and LNM. Furthermore, as a key functional circRNA, circ_0008594 was observed to promote NSCLC progression by regulating the miR-760-mediated PI3K/AKT and MEK/ERK pathways.

ZIP4 promotes non-small cell lung cancer metastasis by activating snail-N-cadherin signaling axis.

Non-small cell lung cancer (NSCLC) is one of the most critical health problems worldwide, with high incidence and poor survival rate. A zinc importer ZIP4 has been implicated in the process of tumor growth and metastasis of many cancers. However, its exact role and the underlying mechanism in NSCLC remains to be elucidated. In the present study, we found that human ZIP4 was substantially overexpressed in NSCLC tissues and was correlated with poor overall survival (OS) and progression-free survival (PFS). Overexpression of ZIP4 promoted cell migration, invasion and metastasis both in vitro and in a mouse lung metastasis model. Silencing of ZIP4 attenuated migration, invasion and metastasis. Mechanistically, overexpression of ZIP4 increased the expression of Snail, Slug and N-cadherin while genetic inactivation of ZIP4 downregulated the expression of above-mentioned genes. Further analysis showed that transcriptional factor Snail which modulates N-cadherin was involved in the process of ZIP4-mediated NSCLC migration and invasion. We also demonstrated that ZIP4 positively correlates with the levels of Snail, Slug and N-cadherin in mice lung metastasis tumors. Together, these results suggest that ZIP4 acts as an important regulator of Snail-N-cadherin signaling axis in promoting NSCLC progression and may serve as a novel predictive marker and therapeutic target in NSCLC.

Cigarette smoking induces aberrant N6-methyladenosine of DAPK2 to promote non-small cell lung cancer progression by activating NF-κB pathway.

N6-methyladenosine (m6A) has recently emerged as an important regulatory mechanism for gene expression and aberrant m6A modification plays an important role in tumor progression. Emerging evidence has shown that aberrant m6A modification induced by cigarette smoking is involved in carcinogenesis, but whether cigarette smoking affects m6A modification and thus deteriorates to non-small cell lung cancer (NSCLC) is still unclear. Here, we identified a tumor suppressor gene-DAPK2 which was significantly associated with poor prognosis of NSCLC patients, especially in patients with a smoking history. Low levels of DAPK2 were detected in smokers and in NSCLC tissues. Cigarette smoking induced aberrant N6-methyladenosine modification of DAPK2, which resulted in decreased DAPK2 mRNA stability and expression of its mRNA and protein. This modification was mediated by the m6A "writer" METTL3 and the m6A "reader" YTHDF2. Mechanistically, we further demonstrated that DAPK2 functions as a tumor suppressor and downregulation of DAPK2 substantially enhances the proliferation and migration abilities in vitro and in vivo by activating NF-κB signaling pathway. Notably, the BAY 11-7085, a NF-κB signaling selective inhibitor, was shown to efficiently suppressed downregulation of DAPK2-induced oncogenic phenotypes of NSCLC cells. Our study reveals that cigarette smoking induces aberrant N6-methyladenosine of DAPK2 to promote NSCLC progression, which provides new insight into the mechanisms of NSCLC progression and a specific therapeutic target for NSCLC patients with a smoking history.

TBC1 domain family member 23 interacts with Ras-related protein Rab-11A to promote poor prognosis of non-small-cell lung cancer via β1-integrin.

Non‐small‐cell lung cancer (NSCLC) accounts for approximately 80% of lung cancer cases. TBC1D23, a member of the TBC/RABGAP family, is widely expressed in human tissues; however, its role in NSCLC is currently unknown. Immunohistochemical analysis was conducted on 173 paraffin‐embedded lung tissue sections from patients with NSCLC from 2014 to 2018 at the First Affiliated Hospital of China Medical University. MTT, colony formation assay, cell cycle assay, scratch assay, transwell assay, Western blotting and real‐time PCR were employed on multiple NSCLC cell lines modified to knock down or overexpress TBC1D23/RAB11A. Immunoprecipitation, immunoprecipitation‐mass spectrometry, immunofluorescence and flow cytometry were performed to explore the interaction between TBC1D23 and RAB11A and TBC1D23 involvement in the interaction between RAB11A and β1 integrin in the para‐nucleus. TBC1D23 was correlated with tumour size, differentiation degree, metastasis, TNM stage and poor prognosis. TBC1D23 was involved in the interaction between RAB11A and β1 integrin in the para‐nucleus, thus activating the β1 integrin/FAK/ERK signalling pathway to promote NSCLC. Furthermore, TBC1D23 promoted NSCLC progression by inducing cell proliferation, migration and invasion. This study indicated the relationship between TBC1D23 expression and the adverse clinicopathological characteristics of patients with NSCLC, suggesting that TBC1D23 may be an important target for NSCLC treatment.

MicroRNA-4491 enhances cell proliferation and inhibits cell apoptosis in non-small cell lung cancer via targeting TRIM7.

MicroRNAs (miRNAs) are involved in the development of non-small cell lung cancer (NSCLC). However, the biological roles of several aberrantly expressed miRNAs have not been explored yet. In the present study, miR-4491 was identified as a novel upregulated miRNA in NSCLC tissues and cell lines. Downregulation of miR-4491 by a miR-4491 inhibitor inhibited the proliferation and triggered the apoptosis of NSCLC cells. Tripartite motif containing 7 (TRIM7), a tumor suppressor gene expressed in NSCLC, was demonstrated in the present study to be directly targeted by miR-4491. This finding was verified by bioinformatics analysis, reverse transcription-quantitative PCR, western blotting and dual luciferase reporter assays. Furthermore, downregulation of miR-4491 inactivated nuclear factor-κB signaling via induction of TRIM7. In addition, TRIM7 silencing attenuated the effect of miR-4491 inhibitor in NSCLC cells. The decreased TRIM7 level in NSCLC tissues was negatively correlated with miR-4491 expression in NSCLC tissues. In conclusion, the findings from this study demonstrated that miR-4491 expression was upregulated in NSCLC tissues and cells and that miR-4491 may promote NSCLC progression via targeting TRIM7.

YBX1 regulated by Runx3-miR-148a-3p axis facilitates non-small-cell lung cancer progression

BackgroundY-box binding protein 1 (YBX1) is a common oncogene in non-small-cell lung cancer (NSCLC), which is regulated by microRNAs (miRNAs) and transcription factors. This research aims to explore the function of YBX1, miR-148a-3p and Runt-related transcription factor 3 (Runx3) in NSCLC development, and analyze their interactions. MethodsYBX1, miR-148a-3p and Runx3 levels were detected using quantitative reverse transcription polymerase chain reaction(RT-PCR), Western blotting or immunohistochemical staining. The functions of YBX1, miR-148a-3p and Runx3 were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, wound healing, transwell, flow cytometry, xenograft model and Western blotting analyses. The binding correlation was validated through dual-luciferase reporter analysis and chromatin immunoprecipitation (ChIP). ResultsYBX1 expression was upregulated, and miR-148a-3p and Runx3 levels were reduced in NSCLC samples and cell lines. YBX1 silence restrained NSCLC cell proliferation, migration, invasion and tumor growth, and enhanced apoptosis. YBX1 was targeted via miR-148a-3p. MiR-148a-3p knockdown promoted cell proliferation, migration, invasion and tumor growth, and repressed apoptosis, and these effects were abolished by YBX1 silence. Runx3 upregulation restrained cell proliferation, migration, invasion and tumor growth, and facilitated apoptosis. Runx3 bound with miR-148a-3p promotor to regulate miR-148a-3p expression. Runx3 silence modulated YBX1 expression though miR-148a-3p to promote NSCLC progression by increasing Cyclin D1, Cyclin B1, Slug-1, MMP-2 and MMP-9 levels. ConclusionRunx3-miR-148a-3p axis targeted YBX1 to modulate NSCLC progression.

KLHL38 involvement in non-small cell lung cancer progression via activation of the Akt signaling pathway

Lung cancer is the leading cause of cancer-related death worldwide. KLHL38 has been reported to be upregulated during diapause but downregulated after androgen treatment during the reversal of androgen-dependent skeletal muscle atrophy. This study aimed to clarify the role of KLHL38 in non-small cell lung cancer (NSCLC). KLHL38 expression was evaluated in tumor and adjacent normal tissues from 241 patients with NSCLC using immunohistochemistry and real-time PCR, and its association with clinicopathological parameters was analyzed. KLHL38 levels positively correlated with tumor size, lymph node metastasis, and pathological tumor-node-metastasis stage (all P < 0.001). In NSCLC cell lines, KLHL38 overexpression promoted PTEN ubiquitination, thereby activating Akt signaling. It also promoted cell proliferation, migration, and invasion by upregulating the expression of genes encoding cyclin D1, cyclin B, c-myc, RhoA, and MMP9, while downregulating the expression of p21 and E-cadherin. In vivo experiments in nude mice further confirmed that KLHL38 promotes NSCLC progression through Akt signaling pathway activation. Together, these results indicate that KLHL38 is a valuable candidate prognostic biomarker and potential therapeutic target for NSCLC.

CircAGFG1 sponges miR-28-5p to promote non-small-cell lung cancer progression through modulating HIF-1α level.

Circular RNAs (circRNAs) have gained much attention for their crucial regulatory roles in human diseases and cancers. However, the role and the mechanism of circRNA ArfGAP with FG repeats 1 (circAGFG1) in non-small-cell lung cancer (NSCLC) are still largely unknown. circAGFG1 was highly expressed in NSCLC, and high expression of circAGFG1 was closely related to the low survival rate of NSCLC patients. circAGFG1 knockdown inhibited the proliferation, migration, and invasion and promoted the apoptosis of NSCLC cells. circAGFG1 bound to miR-28-5p in NSCLC cells, and circAGFG1 promoted NSCLC progression partly through sponging miR-28-5p in vitro. HIF-1α was a target of miR-28-5p, and miR-28-5p overexpression-mediated influences in NSCLC cells were partly overturned by the addition of HIF-1α overexpression plasmid. circAGFG1/miR-28-5p/HIF-1α axis regulated cellular glycolytic metabolism in NSCLC cells. circAGFG1 silencing restrained the xenograft tumor growth in vivo. circAGFG1 promoted the proliferation, migration, and invasion and suppressed the apoptosis of NSCLC cells through accelerating the glycolysis via miR-28-5p/HIF-1α axis.

IRS4 promotes the progression of non-small cell lung cancer and confers resistance to EGFR-TKI through the activation of PI3K/Akt and Ras-MAPK pathways

IRS4 is a member of the insulin receptor substrate (IRS) protein family. It acts as a cytoplasmic adaptor protein, integrating and transmitting signals from receptor protein tyrosine kinases to the intracellular environment. IRS4 can induce mammary tumorigenesis and is usually overexpressed in non-small cell lung cancer (NSCLC). However, little is known about the role of IRS4 in the development and progression of lung cancer. In this study, we show that IRS4 knockout suppresses the proliferation, colony formation, migration, and invasion of A549 lung cancer cells, as well as tumor growth in a nude mouse xenograft model. In contrast, stable expression of IRS4 showed the opposite effects. As expected, IRS4 was found to activate the PI3K/Akt and Ras-MAPK pathways, and we also showed that IRS4 depletion significantly enhanced the sensitivity of EGFR tyrosine kinase inhibitor (EGFR-TKI)-resistant cells to gefitinib. Taken together, these results show that IRS4 promotes NSCLC progression and may represent a potential therapeutic target for EGFR-TKI-resistant NSCLC.

SMAD4 mutation correlates with poor prognosis in non-small cell lung cancer

SMAD4 is an intracellular signaling mediator of the TGF-β pathway. Its mutation was commonly observed in gastrointestinal cancers, such as pancreatic cancer. The loss of SMAD4 on immunohistochemical staining is often used to suggest a pancreaticobiliary differentiation in evaluating a metastatic adenocarcinoma with unknown origin. However, the function and molecular mechanism of SMAD4 in non-small cell lung cancer (NSCLC) development are largely unknown. Thus, we studied the correlation between SMAD4 mutations and clinico-molecular features in the patients with NSCLC. We reported the frequencies and prognostic values of SMAD4 mutations in a Chinese NSCLC cohort using next-generation sequencing. The NSCLC cases from several public databases, including The Cancer Genome Atlas and others, were also used in this study to elucidate SMAD4-related molecular partners and mechanisms. Integrated bioinformatics analyses were conducted, such as analysis of Gene Ontology enrichment analysis, gene set enrichment analysis (GSEA), and survival analysis. Immunohistochemistry showed that the tissues harboring SMAD4 mutations tended to show SMAD4 deficiency or loss, while SMAD4 expression was significantly reduced at all stages of NSCLC cases. We found that reduced SMAD4 expression was more frequent in the patients with poor disease-free survival and resistance to platinum-based chemotherapy. SMAD4 mutation was an independent risk factor for the survival of NSCLC patients. The expression of SMAD4 was associated with that of SMAD2. The GSEA showed that SMAD4 might promote NSCLC progression by regulating proliferation, adhesion, and immune response. In conclusion, these data suggest that SMAD4 mutation or loss as well as reduced expression can be used to identify the NSCLC patients with poor survival and resistance to platinum-based chemotherapy. SMAD4 may be a predictive marker or therapeutic target in NSCLC. The source code and user's guide are freely available at Github: https://github.com/wangyue77-ab/smad4.

LncRNA FAM83A-AS1 promotes lung adenocarcinoma progression by enhancing the pre-mRNA stability of FAM83A.

BackgroundLung cancer is the leading cause of cancer deaths worldwide. Long non‐coding RNAs (lncRNAs) affect a series of cellular biological processes, including oncogene function promotion. In this study, we explored the functions and mechanisms of FAM83A antisense RNA 1 (FAM83A‐AS1) in non‐small cell lung cancer (NSCLC) progression.MethodsThe expression of FAM83A‐AS1and FAM83A mRNA was analyzed using the Cancer Genome Atlas (TCGA) data. Proliferation, migration, invasion and Western blotting were measured after treatment with overexpressed or knockdown FAM83A‐AS1. To determine the relationship between FAM83A‐AS1 and FAM83A, RNase protection assay (RPA), amanitin treatment, RNA pulldown assay and RNA immunoprecipitation (RIP) assay were performed.ResultsHigh expression of FAM83A‐AS1 in lung adenocarcinoma (LUAD) was closely associated with low overall survival (OS) and progression‐free survival (PFS). Functionally, high FAM83A‐AS1 expression increased LUAD cell proliferation and metastasis, indicating that FAM83A‐AS1 exerted its oncogenic functions. Furthermore, FAM83A‐AS1 promoted NSCLC progression via ERK signaling pathways. Mechanistically, FAM83A‐AS1 post‐transcriptionally increased FAM83A expression by enhancing pre‐mRNA stability. FAM83A‐AS1 enhanced FAM83A mRNA stability not only by forming an RNA duplex but also by binding to FBL.ConclusionsWe determined that FAM83A‐AS1 increased FAM83A expression by enhancing FAM83A pre‐mRNA stability and promoted the tumorigenesis of LUAD.

Circ-PITX1 Promotes the Progression of Non-Small Cell Lung Cancer Through Regulating the miR-1248/CCND2 Axis.

BackgroundCircular RNA (circRNA) is a key regulator of cancer, and it has been proved to be involved in the regulation of cancer progression including non-small cell lung cancer (NSCLC). Circ-PITX1 was found to be a significantly upregulated circRNA in NSCLC, and its role and potential mechanism in NSCLC progression deserve further investigation.MethodsThe expression levels of circ-PITX1, microRNA (miR)-1248 and cyclin D2 (CCND2) were examined by quantitative real-time PCR (qRT-PCR). Cell proliferation, apoptosis, cell cycle process, migration and invasion were determined using cell counting kit 8 (CCK8) assay, colony formation assay, flow cytometry, wound healing assay and transwell assay. Xenograft models were built to explore the role of circ-PITX1 in NSCLC tumor growth in vivo. The glycolysis and glutamine metabolism of cells were assessed by detecting the consumptions of glucose and glutamine, cell extracellular acidification rate (ECAR), and the productions of lactate, α-ketoglutaric acid (α-KG) and ATP. The protein levels of hexokinase 2 (HK-2), glutaminase 1 (GLS1) and CCND2 were tested by Western blot (WB) analysis. Dual-luciferase reporter assay and RIP assay were employed to verify the interaction between miR-1248 and circ-PITX1 or CCND2.ResultsCirc-PITX1 was upregulated in NSCLC and its silencing could inhibit the proliferation, migration, invasion, cell cycle process, glycolysis, glutamine metabolism, and promote the apoptosis of NSCLC cells in vitro, as well as reduced tumor growth in vivo. In the terms of mechanism, we found that circ-PITX1 could act as a sponge of miR-1248, and miR-1248 could target CCND2. In addition, miR-1248 inhibitor reversed the inhibitory effect of circ-PITX1 knockdown on NSCLC progression. Similarly, CCND2 overexpression also reversed the suppressive effect of miR-1248 on NSCLC progression. Moreover, circ-PITX1 positively regulated CCND2 expression by sponging miR-1248.ConclusionCirc-PITX1 served as a sponge of miR-1248 to promote NSCLC progression by upregulating CCND2.

RETRACTED: MTTL3 upregulates microRNA-1246 to promote occurrence and progression of NSCLC via targeting paternally expressed gene 3

Non-small cell lung cancer (NSCLC) is one of the major causes of morbidity and mortality worldwide. We aimed to investigate the role of N6-methyladenosine (m6A) methyltransferase-like 3 (METTL3) regulating microRNA-1246 (miR-1246) in the progression of NSCLC by targeting paternally expressed gene 3 (PEG3). METTL3, miR-1246, and PEG3 expression in tissues was assessed, and the predictive role of METTL3 in prognosis of patients with NSCLC was detected. NSCLC cells were relatively treated with altered expression of METTL3, miR-1246, or PEG3 to measure their roles in the proliferation, migration, invasion, apoptosis, and in vivo growth of the NSCLC cells. The RNA m6A level was determined, and the targeting relationship between miR-1246 and PEG3 was confirmed. Our results revealed that METTL3 and miR-1246 were upregulated, whereas PEG3 was downregulated in NSCLC tissues. METTL3 knockdown or PEG3 overexpression in NSCLC cells suppressed malignant behaviors of NSCLC cells. METTL3 affected the m6A modification of miR-1246, thus upregulating miR-1246 and miR-1246-targeted PEG3. The elevation of PEG3 reversed the effects of miR-1246 upregulation on NSCLC cells. This study revealed that m6A methyltransferase METTL3 affects the m6A modification of miR-1246, thus upregulating miR-1246 to promote NSCLC progression by inhibiting PEG3.