Non-small Cell Lung Cancer Tissue Samples Research Articles

Evaluation of the relationship between the IL-17A gene expression level and regulatory miRNA-9 in relation to tumor progression in patients with non-small cell lung cancer: a pilot study

A pro-inflammatory cytokine, IL-17A, is associated with increased risk of developing numerous cancers, including non-small cell lung cancer (NSCLC). IL-17A is a target gene for miR-9. This encouraged us to analyze these two genes in terms of their usefulness as prognostic markers in NSCLC. The expression levels of IL-17A gene and miR-9 was assessed in 26 NSCLC tissue samples and 26 unchanged lung tissue adjacent to lung tumors (control tissue), using qPCR. In both tissue groups, a decreased expression of IL-17A was observed in 100% of samples. Increased expression of miRNA-9 was observed in 92% of tumor samples, and in 100% of control samples. Neither statistical differences in the level of expression IL-17A depending on the patient’s age, gender, smoking status, nor histopathology of the cancer was found. Regarding the presence of nodule metastasis (‘N’ value in TNM classification), significantly lower expression level of IL-17A was observed in cN2 as compared with cN1 group. Additionally, statistically lower IL-17A expression was found in III versus II tumor stage (cAJCC classification). Significant negative correlation between both studied genes was revealed in SCC subgroup. This leads to the conclusion that miRNA-9 can regulate the expression of IL-17A as an IL-17A mRNA antagonistic mediator. Inhibition of proinflammatory action of IL-17A in correlation with tumor progression can be related to various activity of Th17 cells on cancer development according to its immunogenicity, and also may suggest suppressive role of IL-17A in tumor progression. However, because of low number of analyzed samples, further studies on the functional role of IL-17A in development and/or progression NSCLC seem warranted.

MicroRNA‑718 serves a tumor‑suppressive role innon‑smallcell lung cancer by directly targeting CCNB1.

MicroRNA-718 (miR-718) serves crucial roles in tumorigenesis and in the progression of a number of cancers. However, the expression profile, specific functions and mechanisms of action of miR-718 in non-small cell lung cancer (NSCLC) are still elusive. The aims of the present study were to quantify the expression of miR-718, determine its biological roles and elucidate the molecular mechanisms responsible for its activities in NSCLC cells. Reverse transcription-quantitative PCR was carried out to assess miR-718 expression in NSCLC tissue samples and cell lines. The Cell Counting Kit-8 assay, flow cytometry, cell migration and invasion assays, and a tumor xenograft experiment were performed to evaluate the effects of miR-718 overexpression on the malignant biological behaviors of NSCLC cells. miR-718 expression was demonstrated to be significantly decreased in NSCLC tissue samples and cell lines. This reduced expression was significantly associated with tumor, node, metastasis stage, tumor size, lymph node metastasis and poor overall survival among patients with NSCLC. Exogenous miR-718 expression suppressed NSCLC cell proliferation, migration and invasion, and promoted apoptosis in vitro; whereas it hindered tumor growth in vivo. Experiments to elucidate the mechanisms involved revealed that miR-718 functions by directly targeting cyclin B1 (CCNB1) mRNA. CCNB1 expression was found to be upregulated in NSCLC and inversely correlated with miR-718 levels. CCNB1 depletion had effects similar to those of miR-718 overexpression in NSCLC cells. Furthermore, restoration of CCNB1 expression attenuated the tumor-suppressive effects of miR-718 overexpression in NSCLC cells. These results indicated that miR-718 suppressed NSCLC progression in vitro and in vivo by directly targeting CCNB1 mRNA, which may indicate a potential target for the diagnosis and treatment of this fatal disease.

SPINK1 is a prognosis predicting factor of non-small cell lung cancer and regulates redox homeostasis.

Serine protease inhibitor Kazal-type 1 (SPINK1) is a trypsin kinase inhibitor, which is involved in the development of inflammation, cell proliferation and cancer development and progression. However, the prognostic value of SPINK1 in non-small cell lung cancer (NSCLC) and its ability to regulate intrinsic redox homeostasis have, to the best of our knowledge, not been previously investigated. In the present study, it was revealed that SPINK1 is highly expressed in NSCLC tissue samples compared with normal tissue samples, and may be a potential prognostic marker of NSCLC. Functional analyses demonstrated that SPINK1 promoted tumor cell growth and inhibited apoptosis through maintaining redox homeostasis by regulating the nuclear factor erythroid 2-related factor 2 pathways. It has been proposed that SPINK1 could be a prognostic marker of NSCLC and a novel antioxidant promoter under oxidative stress conditions in NSCLC.

Upregulated miRNA-182-5p expression in tumor tissue and peripheral blood samples from patients with non-small cell lung cancer is associated with downregulated Caspase 2 expression.

Lung cancer has the highest morbidity and mortality rates among all malignant tumors worldwide. Previous studies demonstrated that microRNA (miR)-182-5p may serve different roles in different types of cancer, including renal cell carcinoma and liver cancer. However, the functional role of miR-182-5p in non-small cell lung cancer (NSCLC) remains unknown. In the current study, the expression level of miR-182-5p in tumor tissue and peripheral blood samples obtained from patients with NSCLC was examined. The biological function of miR-182-5p on NSCLC cell proliferation was also investigated. Tissue and adjacent normal tissue samples were collected from 33 patients with NSCLC. In addition, peripheral blood samples were obtained from patients with NSCLC and 26 healthy control patients. The NSCLC cell line H1299 was used for all functional assays. Reverse transcription-quantitative polymerase chain reaction was used to determine the miR-182-5p or Caspase 2 (CASP2) mRNA expression levels in NSCLC tissue and peripheral blood samples, as well as in the NSCLC cell line. Western blotting was used to examine the protein expression level of CASP2 in tissue samples and cells, and ELISA was performed to measure the protein level of CASP2 in peripheral blood samples. MTT assay was performed to examine NSCLC cell proliferation. Flow cytometry was used to detect apoptosis. Dual-luciferase reporter assay was used to examine whether miRN182-5p directly interacts with CASP2. The current study demonstrated that miR-182-5p expression was upregulated in NSCLC tissue and peripheral blood samples from patients with NSCLC, which suggests that miR-182-5p, may serve a functional role in NSCLC. In addition, inhibition of miR-182-5p expression suppressed cell proliferation and enhanced cell apoptosis in NSCLC cells. CASP2 expression was downregulated in NSCLC tissue and peripheral blood samples from patients with NSCLC. The current study demonstrated that miR-182-5p may regulate NSCLC cell proliferation and apoptosis by regulating CASP2 expression as miR-182-5p directly binds with the 3′-untranslated region of CASP2, thereby regulating CASP2 expression.

PHLPP2 as a novel metastatic and prognostic biomarker in non‐small cell lung cancer patients

BackgroundPH domain and leucine‐rich repeat protein phosphatase 2 (PHLPP2) has been reported to be a potent tumor suppressor in many human cancers. However, PHLPP2 has not been fully researched as a putative clinical prognostic biomarker of lung cancer.MethodsThe Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases including data on 1383 non‐small cell lung cancer (NSCLC) patients were used to determine PHLPP2 expression. PHLPP2 expression was then examined by immunohistochemistry, and its clinical significance analyzed in 134 NSCLC patients, including 73 patients with adenocarcinoma and 81 with squamous cell carcinoma.ResultsWe found PHLPP2 expression to be less pronounced in NSCLC tissue samples than that in nontumoral lung tissues according to data taken from TCGA and GEO datasets; this outcome was further validated by immunohistochemistry assay. The low PHLPP2 expression level was found to be associated with the presence of lymph node metastasis (P = 0.003). Importantly, PHLPP2 was found to be an independent indicator of prognosis for overall (hazard ratio [HR] = 0.520, 95% confidence interval [Cl] = 0.327–0.827; P = 0.006) and disease‐free survival (HR = 0.489, 95% Cl = 0.308–0.775; P = 0.002) in patients with surgically‐resected NSCLC by multivariate analysis.ConclusionTaken together, our findings show that PHLPP2 is a robust clinical marker for NSCLC survival and could serve as a potential therapeutic target.

Prognostic value of insulin-like growth factor 2 mRNA-binding protein 3 and vascular endothelial growth factor-A in patients with primary non-small-cell lung cancer.

Insulin-like growth factor 2 mRNA-binding protein 3 (IMP3) and vascular endothelial growth factor-A (VEGF-A) may play important roles in the process of tumor progression and tumor angiogenesis. The aim of the present study was to examine the co-expression of IMP3 and VEGF-A in primary human non-small cell lung cancer (NSCLC), to investigate the association between these two expression levels and determine the clinicopathological implications, including changes to microvessel density (MVD), and to assess the prognostic value of co-expression. Using immunohistochemical staining, the expression of IMP3, VEGF-A and CD34 expression was detected in 128 primary NSCLC tissue samples. According to the expression of IMP3 and VEGF-A, the cases were divided into four groups. Next, the clinicopathological features, MVD and survival time were investigated across the different groups. The immunohistochemical analyses demonstrated that there was a significant correlation between IMP3 and VEGF-A expression in NSCLC (r=0.181; P=0.041). Co-expression of IMP3 and VEGF-A was significantly associated with larger primary tumor size (P=0.016), poorer differentiation (P=0.014), more advanced Tumor-Node-Metastasis stage (P=0.012), increased MVD (P=0.004) and positive lymph node metastasis (P=0.002). Survival analysis demonstrated that cases with IMP3 and VEGF-A double-positive staining were significantly associated with lower survival rates compared with cases with double-negative staining (P=0.039). In the early NSCLC (I–IIa) subgroup, the mean survival time of the double-positive staining group was significantly shorter compared with that of the double-negative staining group (P=0.015). Co-expression of IMP3 and VEGF-A was associated with angiogenesis and a poorer prognosis in NSCLC, and may therefore play a critical role in NSCLC progression.

PARK2 Suppresses Proliferation and Tumorigenicity in Non-small Cell Lung Cancer

Aims: PARK2 mutation is originally associated with the progression of Parkinson's disease. In recent years, PARK2 has been reported as a tumor suppressor gene in various cancers, including lung cancer. However, the biological functions and potential molecular mechanisms of PARK2 in non-small cell lung cancer (NSCLC) are still unclear.Methods: The level of PARK2 expression in 32 tissue samples of NSCLC and matched non-tumor lung tissues was detected by Western blot, and 64 specimens of NSCLC tissues were detected by immunohistochemistry. H1299 and H460 cell lines were used to PARK2 overexpression models, and H460 cell line was also used to PARK2 knockdown model. Using cell viability, colony formation, cell cycle, apoptosis, migration, and invasion assay, the biological functions of PARK2 were evaluated and the potential molecular mechanism of PARK2 was investigated in vitro. Meanwhile, 22 nude mice were employed for in vivo studies.Results: Western blot analysis revealed a decrease of PARK2 protein expression in human NSCLC samples. Immunohistochemistry also identified a vastly reduced expression of PARK2 in NSCLC (72%) and low PARK2 expression was significantly associated with tumor histological grade, lymph node metastasis and advanced TNM stage. Overexpression of PARK2 suppressed cell proliferation, colony formation, migration, and invasion, arrested cell cycle progression in the G1 phase, and induced apoptosis in human non-small cell lines H1299 and H460 in vitro. Meanwhile, knockdown of PARK2 had the opposite biological functions. In addition, PARK2 significantly decreased the tumor volumes in subcutaneous xenograft model and reduced the incidence of metastatic tumors in the transfer model. Exploration of the molecular mechanism of PARK2 in NSCLC showed that PARK2 negatively regulated the EGFR/AKT/mTOR signaling pathway.Conclusions: PARK2 was an important tumor suppressor in NSCLC, which might inhibit cancer growth and metastases through the down regulation of the EGFR/AKT/mTOR signaling pathway.

NF-κB-Induced Upregulation of miR-548as-3p Increases Invasion of NSCLC by Targeting PTEN.

Non-Small Cell Lung Cancer (NSCLC) is an aggressive cancer type due to high metastatic capacity. Nuclear Factor Kappa B (NF-κB) is a consistently active transcription factor in malignant lung cancer cells and has crucial significance in NSCLC progression. It is also implicated in the transcriptional regulation of many genes including microRNAs (miRNAs) that function as tumor suppressor or oncogene. It has been increasingly reported that several miRNAs defined as gene members are induced by NF-κB. The present study aimed to find novel miRNAs that are regulated by NF-κB. Chromatin İmmunoprecipitation Sequencing (ChIP-Seq) experiment and bioinformatic analysis were used to determine NF-κB-dependent miRNAs. Western blot analysis, quantitative real-time polymerase chain reaction (qRT-PCR), luciferase reporter gene assays were carried out to investigate the target genes of miRNAs. To determine biologic activity, transwell invasion and MTT assay were carried out on H1299 NSCLC cell line. miRNA expression level was evaluated in metastatic and non-metastatic tissue samples of NSCLC patients. ChIP-Seq and qRT-PCR experiments showed that miR-548as-3p is transcriptionally regulated by NF- κB in response to Tumor Necrosis Factor-α (TNF-α) treatment. Then, we found that tumor suppressor Phosphatase and Tension homolog (PTEN) is a direct target of miR-548as-3p. Furthermore, miR-548as-3p mediates phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway and NF-κB-implicated genes including Matrix Metalloproteinases 9 (MMP9), Slug and Zeb1. We further showed that miR-548as-3p increased invasiveness of NSCLC cells and was upregulated in metastatic tumor tissues compared to non-metastatic ones. All these findings provide a miRNAs-mediated novel mechanism for NF-κB signaling and that miR-548as-3p could be a biomarker for NSCLC metastasis.

LncRNA H19 Interaction with Mutant p53 Promotes Non-Small Cell Lung Cancer Progression

Background: Long non-coding RNAs (lncRNA) regulate gene expression, while p53 is a tumor suppressor gene. Alterations in p53 expression could be important in lung carcinogenesis. This study explored the association and interaction of lncRNA and p53 in non-small cell lung cancer (NSCLC) development and progression. Methods: A eukaryotic expression vector carrying lncRNA H19 was constructed and transfected into NSCLC cells for the assessment of cell phenotype and gene expression through qRT-PCR, Western Blot, RNA-pull down, and immunoprecipitation-Western blot or qRT-PCR. Mutated p53 (p53mt) was assessed by using RT-PCR in H19-transfected tumor cells and 200 cases of NSCLC tissue samples for association with H19 expression. Results: p53 was over-expressed in NSCLC tissues, while H19 overexpression resulted in an increase in tumor cell growth and colony formation. H19 was able to promote p53-mediated transcriptional activity and H19 overexpression led to increased levels of p53 mRNA and protein. The RNA pull down-Western blot and immunoprecipitation-Western blot assays revealed the H19 was associated an increase in p53 expression. Ex vivo data showed that the p53 level was associated with advanced tumor-node-metastasis (TNM) stages and tobacco smoking or poor overall survival of patients. Conclusion: Both H19 and p53 are overexpressed in NSCLC tissues and their interaction promotes the p53 half-life and transcriptional activity, leading to NSCLC progression. Furthermore, high p53 and H19 expression is associated with poor overall survival of patients. Funding Statement: This study was supported in part by a grant from The Foundation of Medical Engineering Cross-work (#YG2016QN07). Declaration of Interests: The authors have no conflicts of interest to declare. Ethics Approval Statement: The study was approved by the Research Ethics Committee of Shanghai Jiaotong University (Shanghai, China) and written informed consent was obtained from all of the included patients.

Long non-coding RNA MIAT promotes non-small cell lung cancer progression by sponging miR-1246.

Recently, long non-coding ribonucleic acids (lncRNAs) have attracted more attention for their roles in tumor progression. The aim of this study was to investigate the exact role of lncRNA MIAT in the progression of non-small cell lung cancer (NSCLC) and to explore the possible underlying mechanism. MIAT expression in NSCLC tissue samples was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The association between the expression of MIAT and the prognosis of NSCLC patients were explored. Furthermore, the wound healing assay and the transwell assay were conducted in vitro. In addition, the luciferase assay and the RNA immunoprecipitation assay (RIP) were used to elucidate the underlying mechanism. The MIAT expression in NSCLC tissues was significantly higher than that of the corresponding normal tissues. Meanwhile, the MIAT expression was associated with the overall survival time of NSCLC patients. The migration and invasion of cells were significantly promoted after MIAT was over-expressed in vitro. Meanwhile, the cell migration and cell invasion were obviously remarkedly inhibited after MIAT knock-down in vitro. Bioinformatics analysis predicted that microRNA-1246 (miR-1246) was as a novel target for MIAT. The expression of miR-1246 was significantly down-regulated or up-regulated after the overexpression or down-expression of MIAT, respectively. Further mechanism assays showed that miR-1246 was a direct target of MIAT in NSCLC. MIAT enhanced the NSCLC cell migration and invasion via targeting miR-1246, which might be a potential biomarker in NSCLC.

Hypermethylation of the PTTG1IP promoter leads to low expression in early-stage non-small cell lung cancer.

Despite the clinical requirement for early diagnosis, the early events in lung cancer and their mechanisms are not fully understood. Pituitary tumor transforming gene 1 binding factor (PTTG1IP) is a tumor-associated gene; however, to the best of our knowledge, its association with lung cancer has not been reported. The present study analyzed PTTG1IP expression in early-stage non-small cell lung cancer (NSCLC) samples and investigated its epigenetic regulatory mechanisms. The results revealed that the mRNA level of PTTG1IP in NSCLC tissues was significantly downregulated by 43% compared with that in adjacent tissues. In addition, overexpression of this gene significantly inhibited cell proliferation. According to data from The Cancer Genome Atlas, a significant negative correlation was identified between the PTTG1IP gene methylation level and expression level in lung adenocarcinoma and lung squamous cell carcinoma cases. Reduced representation bisulfite sequencing (RRBS) analysis of six paired early-stage NSCLC tissue samples indicated that the CpG island shore of the PTTG1IP promoter is hypermethylated in lung cancer tissues, which was further validated in 12 paired early-stage NSCLC samples via bisulfite amplicon sequencing. Following treatment with 5-aza-2′-deoxycytidine to reduce DNA methylation in the promoter region, the PTTG1IP mRNA level increased, indicating that the PTTG1IP promoter DNA methylation level negatively regulates PTTG1IP transcription. In conclusion, in early-stage NSCLC, the PTTG1IP gene is regulated by DNA methylation in its promoter region, which may participate in the development and progression of lung cancer.

High developmental pluripotency‑associated 4 expression promotes cell proliferation and glycolysis, and predicts poor prognosis in non‑small‑cell lung cancer.

The developmental pluripotency-associated 4 (Dppa4) gene serves critical roles in cell self-renewal, as well as in cancer development and progression. However, the regulatory role of Dppa4 in non-small-cell lung cancer (NSCLC) and its underlying mechanisms remain elusive. The aim of the present study was to investigate the biological function of Dppa4 in NSCLC and its underlying mechanism of action. Dppa4 expression was measured in NSCLC tissue samples and cell lines, and its effect on cell proliferation and the expression of glycolytic enzymes was determined. In addition, the underlying mechanisms of Dppa4-induced alterations in glycolysis were analyzed. Univariate and multivariate analyses were also performed to analyze the prognostic significance of clinicopathological characteristics. Dppa4 was found to be highly expressed in NSCLC tissues and cell lines. Furthermore, it was observed that Dppa4 was correlated with the degree of tumor differentiation and TNM stage. Univariate and multivariate analyses identified Dppa4 expression and clinical stage as prognostic factors for NSCLC patients. Kaplan-Meier analysis further revealed that patients with lower Dppa4 expression exhibited a better prognosis. In NSCLC cells, Dppa4 knockdown inhibited cell proliferation, while Dppa4 overexpression enhanced cell proliferation, which was likely mediated by glycolysis promotion. Dppa4 knockdown had no evident effect on the majority of enzymes examined; however, glucose transporter type 4 (GLUT-4) and pyruvate kinase isozyme M2 were significantly upregulated, and hexokinase II (HK-II) and lactate dehydrogenase B (LDHB) were downregulated following Dppa4 knockdown. By contrast, Dppa4 overexpression resulted in downregulation of GLUT-4, and upregulation of HK-II, enolase and LDHB, whereas it had no effect on other enzymes. Since the most evident effect was observed on LDHB, further functional experiments demonstrated that this enzyme reversed the promoting effects of Dppa4 in NSCLC. In conclusion, Dppa4 promotes NSCLC progression, partly through glycolysis by LDHB. Thus, the Dppa4-LDHB axis critically contributes to glycolysis in NSCLC cells, thereby promoting NSCLC development and progression.

Immunohistochemical expression of GRP78 in relation to angiogenesis markers VEGF-a and CD31 and other histopathological parameters in NSCLC.

e20500 Background: Neovascularization is essential for the growth and progression of tumor tissues. GRP78 is frequently overexpressed in various types of cancers and has been suggested as a proangiogenic factor. Methods: In order to evaluate GRP78 expression and its role in angiogenesis of non-small cell lung cancer (NSCLC), paraffin-embedded NSCLC tissue samples (66 adenocarcinoma and 24 squamous cell carcinoma) were retrospectively collected from 90 patients who underwent surgical resection between 2008 and 2015; and did not receive chemotherapy or radiotherapy prior to surgery. Then we performed immunohistochemistry to examine GRP78 expression and we analyzed the relationships between GRP78 and the angiogenesis marker VEGF-A and the microvessel density marker (MVD) CD31. Additionally, we analyzed the association of GRP78 with clinicopathological characteristics of the patients. Results: Strong GRP78 expression was evident in about 86% of the tumor tissues (77/90). There was significant difference in GRP78 expression between poorly-differentiated and well-differentiated tumors (OR = 0.023, 95% CI = 0.001-0.419, p = 0.011). Moreover, there was a statistically significant association between VEGF-A and CD31 (χ² = 12.00, p = 0.001). Indeed, CD31 and VEGF-A expression significantly associated with histological type. Among the tissues expressing high CD31, approximately 86% of them were adenocarcinoma (χ² = 5.009, p = 0.025); and among the tissues expressing positive VEGF-A, about 79% were adenocarcinoma tissues (χ² = 6.545, p = 0.021). Conclusions: Strong GRP78 may be related to good prognosis of NSCLC. Nevertheless, further studies investigating large number of samples of all histological subtypes are required.

ΜicroRNA‑889 plays a suppressive role in cell proliferation and invasion by directly targeting TAB1 in non‑small cell lung cancer.

MicroRNAs (miRNAs) are frequently reported to be aberrantly expressed in non‑small cell lung cancer (NSCLC) and are closely associated with aggressive tumor phenotypes. Hence, identification of cancer‑related miRNAs in NSCLC may be helpful for improving the cure rate of NSCLC treatments. miR‑889 has been demonstrated to be a novel cancer‑associated miRNA that is aberrantly expressed and plays an important role in esophageal squamous cell carcinoma and hepatocellular carcinoma. However, the exact functions and precise molecular mechanisms through which miR‑889 affects NSCLC progression are still unknown. In the present study, we report for the first time that miR‑889 expression is low in NSCLC tissues and cell lines. Clinically, low miR‑889 expression was found to be correlated with the TNM stage and distant metastasis in NSCLC patients. Functionally, miR‑889 overexpression suppressed the proliferation and invasiveness of NSCLC cells invitro and decreased NSCLC xenograft tumor growth in mice. Furthermore, TGF‑β‑activated kinase 1‑binding protein 1 (TAB1) was confirmed as a direct target gene of miR‑889 in NSCLC cells. TAB1 was revealed to be overexpressed in NSCLC tissue samples and was inversely correlated with miR‑889 levels. Moreover, a TAB1 knockdown had effects similar to that of miR‑889 overexpression, whereas restoration of TAB1 expression counteracted the actions of miR‑889 in NSCLC cells. Overall, the present results indicated that miR‑889 inhibits the aggressive behaviors of NSCLC by directly targeting TAB1 mRNA, thus highlighting the importance of the miR‑889/TAB1 pathway in the malignant progression of NSCLC.

MED12 exerts an emerging role in actin-mediated cytokinesis via LIMK2/cofilin pathway in NSCLC

BackgroundMediator complex subunit 12 (MED12) is an essential hub for transcriptional regulation, in which mutations and overexpression were reported to be associated with several kinds of malignancies. Nevertheless, the role of MED12 in non-small cell lung cancer (NSCLC) remains to be elucidated.MethodsMED12 mutation was detected by Next-generation sequencing. The expression of MED12 in 179 human NSCLC tissue samples and 73 corresponding adjacent normal lung tissue samples was measured by immunohistochemistry (IHC). CRISPR-Cas9 was used to knock out MED12 in PC9 and SPC-A1 cells. MED12 rescued stable cell lines were generated by lentivirus infection. We traced cell division process by live cell imaging. The molecular mechanism of aborted cytokinesis resulted by MED12 knockout was investigated by RNA-seq. Effects of MED12 deletion on the proliferation of NSCLC cells were determined by MTT assay and Colony-formation assay in vitro and xenograft tumor model in nude mouse. Cell senescence was measured by SA-β-gal staining.ResultsIn our study, no MED12 exon mutation was detected in NSCLC samples, whereas we found that MED12 was overexpressed in human NSCLC tissues, which positively correlated with the tumor volume and adversely affected patient survival. Furthermore, knockout MED12 in NSCLC cell lines resulted in cytokinesis failure, displayed a multinuclear phenotype, and disposed to senescence, and become non-viable. Lack of MED12 decreased the proliferative potential of NSCLC cells and limited the tumor growth in vivo. Mechanism investigations revealed that MED12 knockout activated LIMK2, caused aberrant actin cytoskeleton remodeling, and disrupted the abscission of intercellular bridge, which led to the cytokinesis failure. Reconstitution of exogenous MED12 restored actin dynamics, normal cytokinesis and cell proliferation capacity in MED12 knockout cells.ConclusionsThese results revealed a novel role of MED12 as an important regulator for maintaining accurate cytokinesis and survival in NSCLC cells, which may offer a therapeutic strategy to control tumor growth for NSCLC patients especially those highly expressed MED12.

PELI3 mediates pro-tumor actions of down-regulated miR-365a-5p in non-small cell lung cancer

BackgroundTo analyze the relative expression of PELI3 and its mechanistic involvement in the non-small cell lung cancer (NSCLC).MethodsPELI3 expression in NSCLC tissue samples was determined by the immunohistochemistry. The transcripts abundance of PELI3 was measured with real-time PCR. The protein intensity was analyzed by western blot. The overall survival in respect to PELI3 or miR-365a-5p expression was plotted by the Kaplan–Meier’s analysis. Cell growth was determined by colony formation assay. Cell viability was measured by MTT assay. The migration and invasion were evaluated by wound healing and transwell assay respectively. The regulatory effect of miR-365a-5p on PELI3 was interrogated with luciferase reporter assay. The direct binding between miR-365a-5p and PELI3 was analyzed by pulldown assay.ResultsPELI3 was aberrantly up-regulated in NSCLC both in vivo and in vitro. High level of PELI3 associated with poor prognosis. PELI3-deficiency significantly inhibited cell viability, colony formation, migration and invasion. We further identified that miR-365a-5p negatively regulated PELI3 in this disease. Ectopic expression of miR-365a-5p in both A549 and H1299 phenocopied PELI3-deficiency. Meanwhile, PELI3-silencing significantly abolished the pro-tumoral effect elicited by miR-365a-5p inhibition.ConclusionOur results highlighted the importance of dysregulated miR-365a-5p-PELI3 signaling axis in NSCLC.

Evidence for an oncogenic role of HOXC6 in human non-small cell lung cancer.

BackgroundIdentification of specific biomarkers is important for the diagnosis and treatment of non-small cell lung cancer (NSCLC). HOXC6 is a homeodomain-containing transcription factor that is highly expressed in several human cancers; however, its role in NSCLC remains unknown.MethodsThe expression and protein levels of HOXC6 were assessed in NSCLC tissue samples by Quantitative real-time PCR (qRT-PCR) and immunohistochemistry, respectively. HOXC6 was transfected into the NSCLC cell lines A549 and PC9, and used to investigate its effect on proliferation, migration, and invasion using CFSE, wound healing, and Matrigel invasion assays. Next-generation sequencing was also used to identify downstream targets of HOXC6 and to gain insights into the molecular mechanisms underlying its biological function.ResultsHOXC6 expression was significantly increased in 66.6% (20/30) of NSCLC tumor samples in comparison to normal controls. HOXC6 promoted proliferation, migration, and invasion of NSCLC cells in vitro. RNA-seq analysis demonstrated the upregulation of 310 and 112 genes in A549-HOXC6 and PC9-HOXC6 cells, respectively, and the downregulation of 665 and 385 genes in A549-HOXC6 and PC9-HOXC6 cells, respectively. HOXC6 was also found to regulate the expression of genes such as CEACAM6, SPARC, WNT6, CST1, MMP2, and KRT13, which have documented pro-tumorigenic functions.DiscussionHOXC6 is highly expressed in NSCLC, and it may enhance lung cancer progression by regulating the expression of pro-tumorigenic genes involved in proliferation, migration, and invasion. Our study highlighted the oncogenic potential of HOXC6, and suggests that it may be a novel biomarker for the diagnosis and treatment of NSCLC.

HMOF reduction enhances radiosensitivity through the homologous recombination pathway in non-small-cell lung cancer.

PurposeHuman males absent on the first (hMOF) is a histone acetyltransferase (HAT) and is responsible for acetylating histone H4 at lysine 16 (H4K16). Recent studies have indicated that hMOF is overexpressed in non-small-cell lung cancer (NSCLC) as an oncogene. The aim of this study is to profile the prognostic roles of hMOF in patients with unresectable stage III NSCLC undergoing definitive radiotherapy (RT) and in the radiosensitivity of human NSCLC cells.Materials and methodsThe expression of hMOF was detected in 24 normal and tumor-paired fresh-frozen NSCLC tissue samples. The immunohistochemistry was conducted, and the correlation of hMOF with clinicopathological parameters was studied in tissues from 90 patients with unresectable stage III NSCLC who underwent definitive RT. Radiation sensitivity was monitored using clonogenic assays in NCI-H1299 and A549 NSCLC cell lines with hMOF knockdown.ResultshMOF was overexpressed in NSCLC tissues compared with non-cancerous tissues. Compared to patients with downregulated hMOF, upregulated hMOF was observed in 51.1% (46/90) of the patients, who showed a significantly worse 5-year survival rate (5.4% vs 22.9%, P=0.025). hMOF expression was an independent prognostic factor of unresectable stage III NSCLC patients who underwent definitive RT. Silencing hMOF increased in vitro the sensitive enhancing ratio (SER) of NSCLC cell lines and downregulated the expression of phospho-ataxia telangiectasia mutated (p-ATM) and RAD51 after irradiation (IR).ConclusionOverexpression of hMOF predicts poor prognosis in patients with unresectable stage III NSCLC undergoing definitive RT. Downregulating hMOF might be a promising intervention to improve the outcome after RT.

MicroRNA-345 suppresses cell invasion and migration in non-small cell lung cancer by directly targeting YAP1.

To investigate the roles and the underlying mechanism of miR-345 in regulating non-small cell lung cancer (NSCLC) cell migration and invasion. Fifty-two pairs of NSCLC tissues and matched normal lung tissue samples were collected from NSCLC patients who had undergone surgical resection between June 2015 and August 2017 in our hospital. Human NSCLC cell lines (NCI-H460, NCI-H1299, A549 and GLC-82) and normal human bronchial epithelium cell BEAS-2B were cultured. NSCLC cell migration and invasion capacities were determined by transwell assays. The relative protein and mRNA expression level of yes-associated protein 1 (YAP1) were detected by Western blot and Quantitative Real-time polymerase chain reaction (qRT-PCR) analysis, respectively. MiR-345 was prominently downregulated in NSCLC tissue specimens. Results of transwell assays showed that miR-345 overexpression could dramatically inhibit NSCLC cell migration and invasion. Additionally, data in current study also identified YAP1 as a direct functional target of miR-345 in NSCLC cells. YAP1 expressions were negatively correlated with the miR-345 expressions in NSCLC tissue samples. Moreover, YAP1 was found to be involved in the functions of miR-345 in inhibiting NSCLC cell invasion and migration. All the above results indicated that miR-345 inhibited NSCLC cell migration and invasion by targeting YAP1, suggesting that miR-345/YAP1 axis might be a promising biomarker for NSCLC treatments.

MiR-34b inhibits the migration/invasion and promotes apoptosis of non-small-cell lung cancer cells by YAF2.

Lung cancer is the leading cause of cancer death in the world and microRNAs (miRNA) have been found to be involved in the initiation and development of cancer by acting as potential oncogenes or tumor suppressor genes. In this study, we investigated the expression of miR-34b in non-small cell lung cancer (NSCLC) patients and discussed the molecular mechanism of miR-34b in the invasion and migration of A549 cells in vitro. Our results showed that miR-34b was significantly down-regulated in primary cancer tissues when compared with the normal lung tissues. The over-expression of miR-34b inhibited migration and invasion, and promoted apoptosis of A549 lung cancer cells. Furthermore, Luciferase reporter assay validated YY1-associated factor 2 (YAF2) as a direct target of miR-34b and YAF2 expression was significantly increased in clinical NSCLC tissue samples. In addition, the over-expression of miR-34b inhibited YAF2, p-Jak2, p-STAT3 and MMP2 protein expression and promoted caspase 3 protein expression in cancer cells. Our results suggest that miR-34b may inhibit migration and invasion of NSCLC cells by targeting YAF2. Thus, our findings provide new insight into the molecular mechanisms of lung cancer metastasis and miR-34b may serve as a potential target in the treatment of human lung cancer.