NSCLC Tumorigenesis Research Articles

MiR-6884-5p inhibits proliferation and epithelial-mesenchymal transition in non-small cell lung cancer cells

BackgroundNon-small cell lung cancer (NSCLC) is associated with a high mortality and morbidity rate. MicroRNAs participate in tumorigenesis, progression and metastasis of NSCLC. However, miR-6884-5p has not been previously studied. This study aimed to investigate the role of miR-6884-5p in NSCLC and explore its underlying mechanisms. MethodsWe used miR-6884-5p mimics and inhibitors to assess its effects in NSCLC. miR-6884-5p expression levels in NSCLC cell lines were quantified using qRT-PCR. Cell viability was determined using a cell-counting kit 8 assay. Western blot analysis was employed to measure apoptotic proteins. The impact of miR-6884-5p on cell proliferation was assessed via colony formation assay. Furthermore, Transwell assays were utilized to visualize and quantify the effects of miR-6884-5p on NSCLC migration and invasion. ResultsmiR-6884-5p mimic significantly inhibited NSCLC cell proliferation to 71.21 % and 72.26 % of control at 5 days of culture time in H460 and HC9 cells (both p < 0.01), respectively, while miR-6884-5p inhibitor significantly promoted cell proliferation to 119.66 % and 126.44 % of control at 5 days of culture time in H460 and HC9 cells (both p < 0.05), respectively. In addition, miR-6884-5p promoted apoptosis by reducing the anti-apoptotic protein B-cell lymphoma 2 (BCL2) protein and increasing apoptotic protein BCL2 associated X protein (all p < 0.01 at least). Moreover, miR-6884-5p effectively suppressed transforming growth factor β1-induced epithelial-mesenchymal transition, as evidenced by the restored expression of E-cadherin (p < 0.01), N-cadherin (p < 0.01) and Vimentin (p < 0.05), leading to the inhibition of migration and invasion in NSCLC cell lines. ConclusionsOur findings demonstrate that miR-6884-5p can inhibit NSCLC cell proliferation, migration, and invasion, suggesting its potential as a therapeutic target for NSCLC treatment.

RAGE potentiates EGFR signaling and interferes with the anticancer effect of gefitinib on NSCLC cells.

The receptor for advanced glycation end-products (RAGE) has been implicated in tumorigenesis, while EGFR signaling plays a vital role in lung cancer progression. Both RAGE and EGFR are transmembrane receptors that transmit intracellular signals through ligand binding, and their downstream signaling cascades show substantial overlap. However, the interplay between these two molecules remains poorly understood. In the present study, we evaluated the correlation between RAGE and EGFR in the tumorigenesis of NSCLC and evaluated the impact of RAGE on the response of NSCLC cells to gefitinib, an EGFR-tyrosine kinase inhibitor (TKI). The expression and activation of EGFR and the phosphorylation of its downstream molecules, STAT3 and Erk, were increased in RAGE-overexpressed A549 (A549-RAGE) cells. Notably, ligand-triggered activation of EGFR signaling was significantly greater in A549-RAGE compared to A549-parental cells. In addition, gefitinib had less effect on the inhibition of EGFR signaling in A549-RAGE cells. These findings were validated in other NSCLC cell lines, H1299 and H1975. Furthermore, upon gefitinib administration, the anti-apoptotic marker Bcl-2 expression was up-regulated in A549-RAGE cells, while the apoptotic markers Bcl-2 associated X protein (Bax) and Bcl-2 interacting mediator (Bim) remained at lower levels compared to A549-parental cells. Importantly, our findings provide evidence that RAGE interferes with the anticancer effect of gefitinib by modulating the activation of EGFR-STAT3 and EGFR-Erk pathways. Overall, these significant findings deepen our understanding of the intricate relationship between RAGE and EGFR signaling in NSCLC tumorigenesis and provide new considerations for the clinical treatment of NSCLC.

Fibronectin promotes tumor progression through integrin αvβ3/PI3K/AKT/SOX2 signaling in non-small cell lung cancer

The tumor microenvironment, especially the extracellular matrix (ECM), is strongly associated with tumor cell proliferation and metastasis. Numerous studies have provided evidence suggesting that fibronectin (FN) in ECM supports cancer cell escape and contributes to cell migration, resulting in distant cancer metastasis and poor outcomes in patients. In our study, it was demonstrated that FN expression was elevated in tumor tissues from highly malignant NSCLC patients, compared to those with low malignancy (p = 0.0076). Importantly, FN promoted proliferative phenotypes and strengthened tumorigenesis capabilities in NSCLC cells, including A549 and Lewis cells, leading to sustained tumor growth in vivo. Mechanistically, it was identified that FN facilitated the activation of the integrin αvβ3/PI3K/AKT signaling pathway, which subsequently upregulated tumor stemness through the downstream transcription factor SOX2. Blockade of integrin αvβ3 signal efficiently suppressed NSCLC proliferation and tumorigenesis both in vitro and in vivo. In conclusion, our study demonstrated that extracellular FN could facilitate NSCLC development through the integrin αvβ3/PI3K/AKT/SOX2 signaling pathway. Blockade of integrin αvβ3 could efficiently enhance the anticancer effects of chemotherapy, offering an innovative approach for clinical NSCLC therapy.

MIB2 promotes the progression of non-small cell lung cancer by regulating cell cycle control pathways.

Although numerous measures have been used to improve the outcome of lung cancer patients, lung cancer, as the second most common diagnosed cancer, is still the main cause of cancer death. It becomes increasingly urgent for us to deeply deplore the molecular mechanism of lung cancer and to discover the potential therapeutic targets. In our study, we are dedicated to discovering the role of MIB2 in lung cancer development. The public databases were used to compare the expression level of MIB2 in cancer and non-cancer tissue. We analyzed the expression of MIB2 in lung cancer samples by performing Rt-PCR and western blot. We carried out CCK8 and clone assays to study the influence of MIB2 in lung cancer proliferation. The transwell assays and wound healing assays were implemented to study the function of MIB2 in metastasis and invasion. Proteins of cell cycle control pathways are detected to verify the potential mechanism of MIB2 in lung cancer progression. MIB2 is up regulated in lung cancer tissue compared to adjacent normal lung tissue according to both public databases and our clinical lung cancer samples. Knockdown of MIB2 inhibits proliferation, metastasis, and invasion of lung cancer cell lines. Cyclins and cyclin dependent kinases (CDK) including CDK2, CDK4, and cyclinB1 were down regulated in MIB2 knockdown cells. Our results prove that MIB2 acts as a driver in NSCLC tumorigenesis by regulating cell cycle control pathways.

Dysregulation of SWI/SNF Chromatin Remodelers in NSCLC: Its Influence on Cancer Therapies including Immunotherapy.

Lung cancer is the leading cause of cancer death worldwide. Molecularly targeted therapeutics and immunotherapy revolutionized the clinical care of NSCLC patients. However, not all NSCLC patients harbor molecular targets (e.g., mutated EGFR), and only a subset benefits from immunotherapy. Moreover, we are lacking reliable biomarkers for immunotherapy, although PD-L1 expression has been mainly used for guiding front-line therapeutic options. Alterations of the SWI/SNF chromatin remodeler occur commonly in patients with NSCLC. This subset of NSCLC tumors tends to be undifferentiated and presents high heterogeneity in histology, and it shows a dismal prognosis because of poor response to the current standard therapies. Catalytic subunits SMARCA4/A2 and DNA binding subunits ARID1A/ARID1B/ARID2 as well as PBRM1 were identified to be the most commonly mutated subunits of SWI/SNF complexes in NSCLC. Mechanistically, alteration of these SWI/SNF subunits contributes to the tumorigenesis of NSCLC through compromising the function of critical tumor suppressor genes, enhancing oncogenic activity as well as impaired DNA repair capacity related to genomic instability. Several vulnerabilities of NSCLCS with altered SWI/SNF subunits were detected and evaluated clinically using EZH2 inhibitors, PROTACs of mutual synthetic lethal paralogs of the SWI/SNF subunits as well as PARP inhibitors. The response of NSCLC tumors with an alteration of SWI/SNF to ICIs might be confounded by the coexistence of mutations in genes capable of influencing patients' response to ICIs. High heterogenicity in the tumor with SWI/SNF deficiency might also be responsible for the seemingly conflicting results of ICI treatment of NSCLC patients with alterations of SWI/SNF. In addition, an alteration of each different SWI/SNF subunit might have a unique impact on the response of NSCLC with deficient SWI/SNF subunits. Prospective studies are required to evaluate how the alterations of the SWI/SNF in the subset of NSCLC patients impact the response to ICI treatment. Finally, it is worthwhile to point out that combining inhibitors of other chromatin modulators with ICIs has been proven to be effective for the treatment of NSCLC with deficient SWI/SNF chromatin remodelers.

KLF4 suppresses the proliferation and metastasis of NSCLC cells via inhibition of MSI2 and regulation of the JAK/STAT3 signaling pathway

BackgroundNon-small cell lung cancer (NSCLC) remains an aggresive tumor with poor survival rates. Krüppel-like factor 4 (KLF4) is known to be involved in progression of NSCLC; however, the detailed mechanism by which KLF4 regulates the progression of NSCLC remains unclear. MethodsIn order to investigate the function of KLF4 in NSCLC, cell proliferation was measured by MTT and colony formation assays. The migration and invasion of NSCLC cells were detected via wound healing and Transwell assays, respectively. Then, the interaction between KLF4 and MSI2 was confirmed using a dual-luciferase reporter assay, and the mechanism by which KLF4 regulates the tumorigenesis of NSCLC was assessed by RT-qPCR and Western blotting. ResultsThe results showed that KLF4 was downregulated, while MSI2 was upregulated in NSCLC. Additionally, KLF4 could inhibit transcription of MSI2, and overexpression of KLF4 or knockdown of MSI2 could inhibit the proliferation, migration and invasion of NSCLC cells. Moreover, KLF4 could inhibit JAK2/STAT3 signalling pathway. ConclusionsIn conclusion, KLF4 significantly inhibited the proliferation, invasion and migration of NSCLC cells via inactivation of MSI2/JAK2/STAT3 signalling pathway. Thereby, our finding might shed new lights on exploring the new strategies against NSCLC.

MiR-489-3p promotes malignant progression of non-small cell lung cancer through the inactivation of Wnt/\u03b2-catenin signaling pathway via regulating USP48

BackgroundNon-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer globally, with average age of cancer patients becoming younger gradually. It is of significance to gain a comprehensive understanding of molecular mechanism underlying NSCLC.MethodsQuantitative polymerase chain reaction (qPCR) and western blot were applied to measure RNA and protein levels separately. Functional assays and western blot were performed to determine the effects of miR-489-3p and USP48 on cell growth, migration and epithelial-mesenchymal transition (EMT) in NSCLC. TOP/FOP flash luciferase reporter assay was carried out to detect the activity of Wnt pathway. Besides, qPCR, RNA pulldown and luciferase reporter assays were conducted to probe into the target gene of miR-489-3p. Immunoprecipitation-western blot (IP-western blot) analysis was implemented to assess the effect of USP48 on the ubiquitination of β-catenin.ResultsmiR-489-3p hampers NSCLC cell proliferation, migration and EMT in vitro and NSCLC tumorigenesis and metastasis in vivo. Additionally, miR-489-3p inactivates Wnt/β-catenin signaling pathway and regulates USP48 to inhibit the ubiquitination of β-catenin. Moreover, USP48 propels the development of NSCLC cells.ConclusionsThe current study demonstrated that miR-489-3p promotes the malignant progression of NSCLC cells via targeting USP48, which might offer a new perspective into NSCLC treatment.Graphical abstract

Glycogen synthase kinase GSK3\u03b1 promotes tumorigenesis by activating HIF1/VEGFA signaling pathway in NSCLC tumor

BackgroundLung cancer is one of the most common cancers and the leading cause of cancer-related death. Glycogen synthase kinase-3 (GSK-3) α, a member of the glycogen synthase kinase-3 family, reportedly plays a role in tumorigenesis. However, its biological function in tumorigenesis requires deeper exploration. Hypoxia is a major feature of solid tumor, along with decreasing availability of oxygen, inducing treatment resistance, and tumor progress.MethodsLevels of GSK3α expression in clinical samples were detected using western blot and IHC assays, while its biological function and underlying mechanism of action in tumor progression were investigated using western blot, CCK8, cell cycle, colony formation, Transwell, ELISA and tube formation assays. Furthermore, we investigated the relationship between GSK3α expression and the HIF1α/VEGFA signaling pathway in vivo using a mouse xenograft model.ResultsGSK3α was significantly upregulated in NSCLC patients with cases that exhibited high GSK3α levels recording shorter survival times. Moreover, GSK3α overexpression promoted proliferation, migration, invasion and clone formation ability of NSCLC cells, while its silencing resulted in an opposite phenomenon. Moreover, GSK3α not only activated the HIF1α/VEGFA signaling pathway, but also regulated HIF1α stabilization independently via the PHDs-pVHL signaling pathway. Moreover, GSK3α-mediated tumor angiogenesis depended on HIF1α expression both in vitro and in vivo.ConclusionGSK3α functioned as an oncogene in NSCLC tumorigenesis by regulating the HIF1/VEGFA signaling pathway in an independent manner through the PHDs-pVHL signaling pathway. These findings were expected to provide novel sights to guide future development of therapies for effective treatment of NSCLC.BdenY92FCzrTCxoDyoPgeDVideo abstract

LINC00152 knockdown suppresses tumorigenesis in non-small cell lung cancer via sponging miR-16-5p.

BackgroundNon-small cell lung cancer (NSCLC) is one of the most aggressive types of cancer worldwide. It has been reported that long non-coding RNAs (lncRNAs) are involved in the pathogenesis of NSCLC. In addition, LINC00152 is known to be upregulated in NSCLC. However, the mechanism underlying the effect of LINC00152 on NSCLC tumorigenesis remains to be elucidated.MethodsIn the present study, cell viability, apoptosis and invasion were investigated by CCK-8, flow cytometry and Transwell assays, respectively. Reverse transcription‑quantitative polymerase chain reaction and Western blotting were performed to determine the mRNA and protein expression levels. In addition, the association between LINC00152, microRNA (miR)-16-5p and BCL2-like 2 (BCL2L2) was evaluated using a dual-luciferase assay.ResultsThe results demonstrated that LINC00152-knockdown significantly attenuated the viability of NSCLC cells via promoting cell apoptosis. In addition, the migration and invasion ability of NSCLC cells was also decreased following transfection of cells with LINC00152 siRNA. Furthermore, miR-16-5p inhibitor or BCLCL2-overexpression reversed LINC00152 siRNA-induced NSCLC cell growth inhibition.ConclusionsThe findings of the present study demonstrated that LINC00152-silencing suppressed NSCLC tumorigenesis via regulating the miR-16-5p/BCL2L2 axis. Therefore, linc00152 has the potential as a molecular marker and may be a potential target for the treatment of non-small cell lung cancer.

Circ_0020123 promotes NSCLC tumorigenesis via up-regulating KIAA1522 expression through miR-940

ABSTRACT Circ_0020123 was highly expressed in NSCLC tissues and cell lines, and knockdown of circ_0020123 abolished cell growth, migration and invasion in vitro and hindered tumor growth in nude mice. Mechanically, circ_0020123 directly targeted miR-940, and KIAA1522 was a target of miR-940. Thereafter, a series of rescue experiments showed that circ_0020123 served its biological functions by miR-940/KIAA1522 axis. In all, circ_0020123 acted as an oncogene to promote the tumorigenesis of NSCLC via miR-940/KIAA1522 axis, suggesting a potential therapeutic target for NSCLC treatment.

Circular RNA hsa_circ_0000190 Facilitates the Tumorigenesis and Immune Evasion by Upregulating the Expression of Soluble PD-L1 in Non-Small-Cell Lung Cancer.

Lung cancer is the leading cause of death from cancer in Taiwan and throughout the world. Immunotherapy has revealed promising and significant efficacy in NSCLC, through immune checkpoint inhibition by blocking programmed cell death protein (PD)-1/PD-1 ligand (PD-L1) signaling pathway to restore patients’ T-cell immunity. One novel type of long, non-coding RNAs, circular RNAs (circRNAs), are endogenous, stable, and widely expressed in tissues, saliva, blood, urine, and exosomes. Our previous results revealed that the plasma level of hsa_circ_0000190 can be monitored by liquid-biopsy-based droplet digital PCR and may serve as a valuable blood-based biomarker to monitor the disease progression and the efficacy of immunotherapy. In this study, hsa_circ_0000190 was shown to increase the PD-L1 mRNA-mediated soluble PD-L1 (sPD-L1) expression, consequently interfering with the efficacy of anti-PD-L1 antibody and T-cell activation, which may result in immunotherapy resistance and poor outcome. Our results unraveled that hsa_circ_0000190 facilitated the tumorigenesis and immune evasion of NSCLC by upregulating sPD-L1 expression, potentially developing a different aspect in elucidating the molecular immunopathogenesis of NSCLC. Hsa_circ_0000190 upregulation can be an effective indicator for the progression of NSCLC, and hsa_circ_0000190 downregulation may possess a potential therapeutic value for the treatment of NSCLC in combination with immunotherapy.

Knockdown of hsa_circ_0000729 Inhibits the Tumorigenesis of Non-Small Cell Lung Cancer Through Mediation of miR-1281/FOXO3 Axis.

BackgroundNon-small cell lung cancer (NSCLC) is a subtype of lung cancer which seriously threatens the health of people. Circular RNAs (CircRNAs) are endogenous RNAs which have stable closed structure; they are known to be involved in tumorigenesis of NSCLC. Meanwhile, hsa_circ_0000729 was reported to be upregulated in NSCLC. Nevertheless, the function of hsa_circ_0000729 in NSCLC remains unclear.MethodsWestern blot and RT-qPCR were performed to investigate protein and mRNA levels, respectively. CCK-8 assay was performed to test the cell viability and cell death was investigated by flow cytometry. NSCLC cell pyroptosis was observed by electron microscope. In addition, the migration and invasion of NSCLC cells were detected by wound healing and transwell assay. The relation among hsa_circ_0000729, miR-1281 and FOXO3 was explored by dual luciferase reporter assay and RNA pull-down.ResultsHsa_circ_0000729 was found to be upregulated in NSCLC cells, and hsa_circ_0000729 knockdown obviously suppressed the proliferation of NSCLC cells through inducing pyroptosis. In addition, silencing of hsa_circ_0000729 notably inhibited the invasion and migration of NSCLC cells. Meanwhile, hsa_circ_0000729 could bind with miR-1281, and FOXO3 was directly targeted by miR-1281. Moreover, the anti-tumor effect of hsa_circ_0000729 siRNAs on NSCLC was markedly reversed by miR-1281 antagomir. Furthermore, silencing of hsa_circ_0000729 inhibited the tumor growth of NSCLC in vivo.ConclusionKnockdown of hsa_circ_0000729 inhibits the tumorigenesis of NSCLC through mediation of miR-1281/FOXO3 axis. Thus, hsa_circ_0000729 might be served as a crucial mediator in NSCLC.

Long non-coding RNA DLX6-AS1 knockdown suppresses the tumorigenesis and progression of non-small cell lung cancer through microRNA-16-5p/BMI1 axis.

BackgroundNon-small cell lung cancer (NSCLC) is a huge threat to sufferers’ life and overall health. Long non-coding RNA (lncRNA) distal-less homeobox 6 antisense RNA 1 (DLX6-AS1) has been revealed to function as a carcinogenesis factor in some cancers. This research aimed to scrutinize the role and mechanism underlying DLX6-AS1 in NSCLC tumorigenesis and progression.MethodsThe levels of DLX6-AS1, microRNA-16-5p (miR-16-5p), and BMI1 mRNA were estimated via reverse transcription-quantitative PCR (RT-qPCR) assay. The protein levels were disclosed by western blot assay. Cell proliferative potential was estimated by colony formation and Cell Counting Kit-8 (CCK-8) assays. Cell migration was estimated by Transwell and wound healing assay. A Transwell assay was executed to estimate cell invasion. The relationships of DLX6-AS1, miR-16-5p, and BMI1 were forecasted by bioinformatics analysis, and confirmed by luciferase reporter assay and RNA immunoprecipitation (RIP) assay. A xenograft mice model was employed to to inspect the function of DLX6-AS1 knockdown on NSCLC tumorigenesis in vivo.ResultsDLX6-AS1 was overexpressed in NSCLC tissues and cells, and was inextricably linked with the poor prognosis of NSCLC patients. Depletion of DLX6-AS1 oppressed cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT) but promoted apoptosis in NSCLC. MiR-16-5p is a target of DLX6-AS1 and directly targets BMI1. Moreover, the anti-tumor impacts of miR-16-5p were overturned by overexpression of DLX6-AS1 or BMI1 in NSCLC cells. Additionally, DLX6-AS1 silencing inhibited tumor growth of NSCLC in vivo.ConclusionsIn conclusion, lncRNA DLX6-AS1 downregulation suppressed the tumorigenesis and progression of NSCLC via miR-16-5p/BMI1 axis in vitro and in vivo, elucidating the vital roles and downstream targets of DLX6-AS1 in NSCLC.

The prognostic impact of KRAS, TP53, STK11 and KEAP1 mutations and the influence of the NLR in NSCLC patients treated with immunotherapy.

e21010 Background: ICIs changed the way NSCLC is treated, but not all patients benefit from it. PD-L1 level is used to predict response to therapy, but its performance is sub-optimal. KRAS is important in NSCLC tumorigenesis, but the impact of its mutations in patients treated with ICIs is unclear. Similarly, studies evaluating co-mutations in TP53, STK11 and KEAP1 as well as the NLR showed that they may predict the benefit of ICIs. Methods: We conducted a retrospective study including all consenting patients with NSCLC treated with ICIs at the CHUM between July 2015 and June 2020. OS and PFS were compared in co-mutation subgroups using Kaplan-Meier and logrank methods. Co-mutations in TP53, STK11 and KEAP1 as well as the NLR were accounted for. Overall response rate (ORR) and safety data was also compared in subgroups and will be detailed at the meeting. Results: We included 100 patients with known KRAS status. From these, 50 were wild-type ( KRASWT) and 50 were mutated ( KRASMut). The most frequent mutation was G12C (54%). Co-mutation status for TP53, STK11 and KEAP1 were known for, respectively, 40, 39 and 38 patients. Co-mutations for these genes were present in respectively 19 (47.5%), 8 (20.5%) and 4 (10.5%). Data comparing KRASMut and KRASWT showed non-significant differences in survival (median OS of respectively 21.1 vs. 17.7 months, p = 0.27). The presence of STK11 and/or KEAP1 mutations was associated with a negative impact on survival when compared with wild-type (median OS 7.4 vs 20.4 months, p = 0.001). When the presence of a KRAS mutation was compounded with STK11 and KEAP1, KRASMut (vs KRASWT) trended to a better prognosis in STK11+KEAP1WT tumors (median OS of 21.1 for KRASMut vs 15.8 for KRASWT, p = 0.15), but not in STK11+/-KEAP1Mut tumors (7.4 for KRASMut vs 7.0 for KRASWT). No influence on survival was seen in relationship to the TP53 co-mutation. Interestingly, the NLR was significantly higher with STK11 mutations (6.66Mut vs 3.59WT, p = 00012), slightly lower with TP53 mutations (3.23Mut vs 4.82WT, p = 0.047) but not impacted by KEAP1 (3.72Mut vs 4.20WT, p = 0.72) or KRAS mutations (4.32Mut vs 5.21WT, p = 0.34). Conclusions: The STK11 and KEAP1 mutations are significant adverse predictors of ICI therapy benefit. The NLR is strongly impacted by STK11 mutations but not by KEAP1 mutations suggesting marked differences in the resistance mechanism for both mutations. In STK11-KEAP1WT tumors, KRAS mutations seems to be associated with improved survival in NSCLC patient treated with ICIs.

Knockdown of SNORA47 Inhibits the Tumorigenesis of NSCLC via Mediation of PI3K/Akt Signaling Pathway.

BackgroundNon-small cell lung cancer (NSCLC) is a frequently diagnosed aggressive cancer all over the world. Small nucleolar RNAs (snoRNAs) are a group of non-coding mediatory RNAs. A previous report indicated that small nucleolar RNA 47 (SNORA47) is upregulated in NSCLC. However, the role of SNORA47 in NSCLC is unclear.Material and MethodsCell proliferation was measured by immunofluorescence staining. Cell apoptosis and cycle of NSCLC were tested by flow cytometry and the protein expressions were investigated by Western-blot. Meanwhile, cell migration and invasion were detected by transwell assay. Xenograft mice model was established to detect the effect of SNORA47 knockdown on tumor growth of NSLC in vivo.ResultsKnockdown of SNORA47 significantly inhibited the proliferation of NSCLC cells via inducing cell apoptosis. Moreover, migration and invasion of NSCLC cells were notably decreased by SNORA47 shRNA. SNORA47 knockdown significantly induced G1 arrest in NSCLC cells via regulation of p27 Kip1, CDK2, and cyclin D1. Meanwhile, SNORA47 shRNA inhibited EMT process and PI3K/Akt signaling in NSCLC cells. Finally, silencing of SNORA47 significantly inhibited the tumor growth of NSCLC in vivo.ConclusionKnockdown of SNORA47 significantly inhibited the tumorigenesis of NSCLC via inhibition of PI3K/Akt signaling and EMT process. Thereby, our finding might shed a new light on exploring the strategies for the treatment of NSCLC.

LncRNA WT1-AS/miR-494-3p Regulates Cell Proliferation, Apoptosis, Migration and Invasion via PTEN/PI3K/AKT Signaling Pathway in Non-Small Cell Lung Cancer.

BackgroundNon-small cell lung cancer (NSCLC) is one of the most common malignancies with the highest morbidity and mortality worldwide. Long non-coding RNAs (lncRNAs) are recently recognized as noteworthy regulators of different tumors, counting NSCLC. However, the biological functions and regulatory mechanism of lncRNA WT1-AS in NSCLC progression still stay uninvestigated.MethodsWT1-AS and miR-494-3p levels in NSCLC cell lines were detected by real-time quantitative polymerase chain reaction (RT-qPCR). In the current study, the regulatory effects of WT1-AS/miR-494-3p axis on cellular behaviors of NSCLC cell lines (A549 and NCI-H1975) were evaluated by a variety of methods. Cell counting kit-8 (CCK-8) and EDU assays were adopted to assess NSCLC cell proliferation. Tunnel staining and flow cytometry assay were applied to determine cell apoptosis and cell cycle distribution. Besides, cell migration and invasion abilities were analyzed by performing wound healing and transwell assays. Meanwhile, the levels of key proteins related to NSCLC cell apoptosis and PTEN/PI3K/AKT pathway were examined using Western blot assay. In addition, luciferase reporter assays were used to determine the interaction between WT1-AS and miR-494-3p or miR-494-3p and PTEN.ResultsVisibly downregulated WT1-AS in NSCLC cell lines was obtained from Broad Institute Cancer Cell Line Encyclopedia (CCLE) database and further verified by performing RT-qPCR. Besides, miR-494-3p was the downstream target gene of WT1-AS and obviously upregulated miR-494-3p in NSCLC cell lines was confirmed. WT1-AS overexpression suppressed cell proliferation, migration and invasion abilities while enhanced cell apoptosis of A549 and NCI-H1975 cells. Furthermore, upregulation of miR-494-3p distinctly reversed these inhibitory effects of WT1-AS overexpression on the tumorigenesis and progression of NSCLC. In addition, WT1-AS promoted PTEN expression and thereby inhibited activation of PI3K/AKT pathway by sponging miR-494-3p.ConclusionTo conclude, lncRNA WT1-AS impeded cell proliferation, migration, invasion but accelerated cell apoptosis via negatively regulating miR-494-3p to mediate PTEN/PI3K/AKT pathway in NSCLC.

Down Regulation of SIRT2 Reduced ASS Induced NSCLC Apoptosis Through the Release of Autophagy Components via Exosomes

Metastasis of cancer is the main cause of death in many types of cancer. Acute shear stress (ASS) is an important part of tumor micro-environment, it plays a crucial role in tumor invasion and spread. However, less is known about the role of ASS in tumorigenesis and metastasis of NSCLC. In this study, NSCLC cells were exposed to ASS (10 dyn/cm2) to explore the effect of ASS in regulation of autophagy and exosome mediated cell survival. Finally, the influence of SIRT2 on NSCLC cell metastasis was verified in vivo. Our data demonstrates that ASS promotes exosome and autophagy components releasing in a time dependent manner, inhibition of exosome release exacerbates ASS induced NSCLC cell apoptosis. Furthermore, we identified that this function was regulated by sirtuin 2 (SIRT2). And, RNA immunoprecipitation (RIP) assay suggested SIRT2 directly bound to the 3′UTR of transcription factor EB (TFEB) and facilitated its mRNA stability. TFEB is a key transcription factor involved in the regulation of many lysosome related genes and plays a critical role in the fusion of autophagosome and lysosome. Altogether, this data revealed that SIRT2 is a mechanical sensitive protein, and it regulates ASS induced cell apoptosis by modulating the release of exosomes and autophagy components, which provides a promising strategy for the treatment of NSCLCs.

ERK3/MAPK6 is required for KRAS-mediated NSCLC tumorigenesis

KRAS is one of the most frequently mutated oncogenes, especially in lung cancers. Targeting of KRAS directly or the downstream effector signaling machinery is of prime interest in treating lung cancers. Here, we uncover that ERK3, a ubiquitously expressed atypical MAPK, is required for KRAS-mediated NSCLC tumors. ERK3 is highly expressed in lung cancers, and oncogenic KRAS led to the activation and stabilization of the ERK3 protein. In particular, phosphorylation of serine 189 in the activation motif of ERK3 is significantly increased in lung adenocarcinomas in comparison to adjacent normal controls in patients. Loss of ERK3 prevents the anchorage-independent growth of KRAS G12C-transformed human bronchial epithelial cells. We further find that loss of ERK3 reduces the oncogenic growth of KRAS G12C-driven NSCLC tumors in vivo and that the kinase activity of ERK3 is required for KRAS-driven oncogenesis in vitro. Our results demonstrate an obligatory role for ERK3 in NSCLC tumor progression and suggest that ERK3 kinase inhibitors can be pursued for treating KRAS G12C-driven tumors.

Downregulation of lncRNA FGF12-AS2 suppresses the tumorigenesis of NSCLC via sponging miR-188-3p.

BackgroundNon-small-cell lung carcinoma (NSCLC) seriously threatens the health of human beings. Aberrant expression of lncRNAs has been confirmed to be related with the progression of multiple malignant tumors, including NSCLC. LncRNA FGF12-AS2 has been considered to be upregulated in NSCLC. However, the mechanism by which FGF12-AS2 promotes the tumorigenesis of NSCLC remains elusive.MethodsGene and protein expressions in NSCLC cells were measured by q-PCR and western blot, respectively. CCK-8 and immunofluorescence staining were performed to detect the cell proliferation. Cell apoptosis was tested by flow cytometry. Transwell assay was used to detect the cell migration and invasion. Finally, the dual luciferase report assay was used to verify the relation among FGF12-AS2, miR-188-3p, and NCAPG2.ResultsDownregulation of FGF12-AS2 significantly inhibited the proliferation of NSCLC cells via inducing apoptosis. In addition, FGF12-AS2 silencing notably suppressed the migration and invasion of A549 cells. Meanwhile, FGF12-AS2 modulated the progression of NSCLC via regulation of miR-188-3p/NCAPG2 axis. Finally, knockdown of FGF12-AS2 inhibited the tumorigenesis of NSCLC via suppressing the EMT process of NSCLC.ConclusionDownregulation of lncRNA FGF12-AS2 suppressed the tumorigenesis of NSCLC via sponging miR-188-3p. Thus, FGF12-AS2 may serve as a potential target for the treatment of NSCLC.

Hsa_circ_0018818 knockdown suppresses tumorigenesis in non-small cell lung cancer by sponging miR-767-3p

To identify potential therapeutic targets in non-small cell lung cancer NSCLC, we conducted a bioinformatics analysis of circRNAs differentially expressed between NSCLC tissues and adjacent normal tissues. Cell proliferation and apoptosis was assessed using CCK-8 and flow cytometry, respectively. A connection between hsa_circ_0018818 and miR-767-3p was confirmed in dual luciferase reporter assays. Gene and protein expression in NSCLC cells were measured using quantitative PCR and Western-blotting, respectively. And a xenograft tumor model was established to assess the function of hsa_circ_0018818 in NSCLC in vivo. Hsa_circ_0018818 was greatly upregulated in NSCLC tumor tissues. Knocking down hsa_circ_0018818 using a targeted shRNA inhibited the proliferation and invasiveness of NSCLC cells and induced their apoptosis via the miR-767-3p/Nidogen 1 (NID1) signaling axis. Hsa_circ_0018818 knockdown also inactivated Epithelial-mesenchymal transition (EMT) process and PI3K/Akt signaling. In summary, hsa_circ_0018818 knockdown inhibited NSCLC tumorigenesis in vitro and in vivo, which suggests it could potentially serve as a target for the treatment of NSCLC.