Adjacent Normal Lung Tissues Research Articles

5-HT7 Receptor Contributes to Proliferation, Migration and Invasion in NSCLC Cells.

IntroductionBecause only a small portion of NSCLC (non-small-cell lung cancer) patients benefit from molecular targeted therapy or immunotherapy and do not develop therapeutic resistance, continued research on new targets is warranted. Serotonin has recently emerged as a growth factor for tumor cells, and its receptors may be potential therapeutic targets. The mechanism related to the behavior of the 5-HT7 receptor in NSCLC remains unknown.MethodsBoth gene expression analysis and immunohistochemical analysis were conducted to evaluate 5-HT7 receptor expression in NSCLC tissues. The correlation between 5-HT7 receptor expression and clinicopathological features was also examined. Cell proliferation was measured using a CCK8 (Cell Counting Kit-8) assay and colony formation, migration and invasion were evaluated by the Transwell assay. siRNA transfection and stimulation with the selective agonist LP211 were used to identify the involvement of molecules in proliferation, migration and invasion. Quantitative real-time chain reaction (qRT-PCR) and Western blotting were used to quantifiy mRNA and protein levels, respectively. Pathway inhibitors facilitated the exploration of possible signaling pathways regulated by the 5-HT7 receptor in migration and invasion.ResultsThe 5-HT7 receptor was overexpressed in NSCLC tumor tissues compared with adjacent normal lung tissues. High 5-HT7 receptor expression levels were correlated with lymph node metastasis (P=0.007) and advanced TNM stage (P=0.000) in NSCLC patients. The 5-HT7 receptor positively regulated cell proliferation, migration and invasion in NSCLC cells. The stimulatory effect of the 5-HT7 receptor on A549 cell migration and invasion may occur through the P38 pathway. In H1299 cells, the 5-HT7 receptor might positively regulate Src to promote cell migration and invasion.ConclusionOur findings suggest that the 5-HT7 receptor, which mediates NSCLC progression, may be a potential therapeutic target.

Analysis of Wnt7B and BMP4 expression patterns in congenital pulmonary airway malformation.

Congenital pulmonary airway malformation (CPAM) is a rare disorder characterized by aberrant overgrowth of terminal bronchioles. The objective of this study was to describe wingless-type MMTV integration site family 7B (Wnt7B) and bone morphogenetic protein 4 (BMP4) expression patterns in human CPAM lesions and to explore the possible roles of Wnt7B and BMP4 in the pathogenesis of CPAM. Fifteen tissue samples from patients with CPAM were obtained from the Pathology Department of Shengjing Hospital of China Medical University. Samples representing CPAM lesions and adjacent normal lung tissues were collected and Wnt7B and BMP4 expression was detected through immunohistochemical (IHC) staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. IHC revealed that Wnt7B immunopositive cells were only detected in epithelial cells, whereas BMP4 immunopositive cells were detected in epithelial and mesenchymal cells. Expression of Wnt7B and BMP4 immunopositive cells was higher in CPAM lesions than that in adjacent normal lung tissue. qRT-PCR and Western blotting showed that Wnt7B and BMP4 mRNA and protein expression were significantly higher in CPAM lesions than in adjacent normal lung tissue (P < .05). Overall, the level of BMP4 was higher than that of Wnt7B. Increased expression of Wnt7B and BMP4 appear to be related to the pathogenesis of CPAM and abnormal pulmonary development. Upregulation of Wnt7B and BMP4 could play an important role in the development of the bronchial-alveolar structures that characterize CPAM.

Combined evaluation of the expression status of CD155 and TIGIT plays an important role in the prognosis of LUAD (lung adenocarcinoma)

The interaction between CD155 and its high-affinity ligand TIGIT is being increasingly investigated in various solid tumors. However, the prognostic significance of CD155 and TIGIT in lung adenocarcinoma (LUAD) remains unclear. In this study, immunohistochemistry was applied in 334 LUAD cases to evaluate the expression of CD155 and TIGIT. Western blotting was conducted in 5 paired primary LUAD and adjacent normal lung tissues. Our results reveal that CD155 and TIGIT are overexpressed in LUAD tissues and that aberrant overexpression is closely correlated with poor clinical outcomes (P < 0.01). The multivariate model also shows that CD155 expression is an independent risk factor for LUAD (RR, 1.34; P = 0.036). Moreover, patients expressing high CD155 and TIGIT simultaneously presented shorter overall survival (OS) (P < 0.01) and progression-free survival (PFS) (P < 0.01). These findings suggest that CD155 and TIGIT can make up a prognosticating tool to predict clinical outcomes, thereby contributing to personalized medical care in LUAD.

Cyclin K interacts with β-catenin to induce Cyclin D1 expression and facilitates tumorigenesis and radioresistance in lung cancer.

Rationale: Radioresistance remains the major cause of local relapse and distant metastasis in lung cancer. However, the underlying molecular mechanisms remain poorly defined. This study aimed to investigate the role and regulatory mechanism of Cyclin K in lung cancer radioresistance.Methods: Expression levels of Cyclin K were measured by immunohistochemistry in human lung cancer tissues and adjacent normal lung tissues. Cell growth and proliferation, neutral comet and foci formation assays, G2/M checkpoint and a xenograft mouse model were used for functional analyses. Gene expression was examined by RNA sequencing and quantitative real-time PCR. Protein-protein interaction was assessed by immunoprecipitation and GST pull-down assays.Results: We report that Cyclin K is frequently overexpressed and correlates with poor prognosis in lung cancer patients. Functionally, we demonstrate that Cyclin K depletion results in reduced proliferation, defective G2/M checkpoint and enhanced radiosensitivity in lung cancer. Mechanistically, we reveal that Cyclin K interacts with and promotes the stabilization of β-catenin protein, thereby upregulating the expression of Cyclin D1. More importantly, we show that Cyclin D1 is the major effector that mediates the biological functions of Cyclin K in lung cancer.Conclusions: These findings suggest that Cyclin K positively modulates the β-catenin/Cyclin D1 axis to promote tumorigenesis and radioresistance in lung cancer, indicating that Cyclin K may represent a novel attractive biomarker for lung cancer radiotherapy.

FANCI Cooperates with IMPDH2 to Promote Lung Adenocarcinoma Tumor Growth via a MEK/ERK/MMPs Pathway.

PurposeFanconi anemia complementation group I (FANCI) is a key protein in ribosome biogenesis and DNA repair. Here, we aimed to determine the clinical significance, prognostic value and biology functions of FANCI in lung adenocarcinoma (LUAD).MethodsThe expression of FANCI in LUAD tissue and its relationship with patient outcomes were assessed using bioinformatics analysis, as well as quantitative reverse-transcription PCR (qRT-PCR) and Western blot analysis of LUAD tissue and adjacent normal lung tissue. The chi-squared test and Cox regression analysis were used to analyze the clinical significance of FANCI expression. The biological effects of FANCI knockdown in human LUAD cell lines were investigated by analysis of proliferation, colony formation, cell cycle distribution, migration, and invasion in vitro, and monitoring of tumor xenograft growth in vivo. FANCI interactions with IMPDH2 and involvement in MEK/ERK/MMPs signaling were analyzed using co-immunoprecipitation assays, immunofluorescence microscopy, and Western blotting.ResultsFANCI was identified as a hub gene for LUAD. FANCI expression was upregulated in LUAD tissues compared with normal lung tissues and was positively associated with lymphatic metastasis, distant metastasis, and poor outcome. FANCI was also an independent prognostic factor in LUAD patients. Knockdown of FANCI in LUAD cell lines decreased their proliferation, migration, invasion, and cell cycle progression in vitro, and decreased the growth of xenografts in mice. Direct binding of FANCI to IMPDH2 decreased IMPDH2 degradation, regulated activation of MEK/ERK/MMPs signaling. Overexpression of IMPDH2 reversed the inhibitory effects of FANCI knockdown.ConclusionFANCI may act as an oncogene in LUAD by cooperating with IMPDH2 to promote cell proliferation via the MEK/ERK/MMPs pathway. These results identify FANCI as a potential prognostic biomarker and therapeutic target for LUAD.

Increases in mRNA and Protein Levels of the Genes for the Actin-Binding Proteins Profilin, Fascin, and Ezrin Promote Metastasis in Non-Small Cell Lung Cancer

The actin-binding proteins profilin, fascin, and ezrin were tested for involvement in metastasis of non-small cell lung cancer (NSCLC). The levels of the PFN1, FSCN1, and EZR mRNAs and respective proteins were determined by real-time PCR and Western blotting; tumor and adjacent normal lung tissue samples were obtained from 46 NSCLC patients. Patients with lymphatic metastasis had higher expression levels of the profilin, fascin, and ezrin mRNAs and the profilin and fascin proteins. Both mRNA and protein expression levels increased in patients with distant metastasis. The molecules may serve as predictors to evaluate the prognosis in NSCLC.

Prognostic model based on circular RNA circPDK1 for resected lung squamous cell carcinoma.

Circular RNA has been revealed as a potential biomarker in multiple malignancies. However, few studies have focused on its potential to be prognostic markers in lung squamous cell carcinoma (LSCC). In this work, we aimed to build a prognostic model of resected LSCC based on circular RNA pyruvate dehydrogenase kinase 1 (circPDK1) and other clinicopathological factors. circPDK1 was identified via next-generation sequencing. Three hundred two cases of LSCC tissue and their adjacent normal lung tissues were obtained from multiple medical centers and divided into study cohort (n=232) and validation cohort (n=70). The expression of circPDK1 was detected for analyzing its potential prognostic value for recurrence-free survival (RFS) and overall survival (OS) in LSCC. Finally, combined with circPDK1, T staging, lymph nodes (LN) metastasis status, age, and serum squamous cell Carcinoma Antigen (SCCAg), we built a prognostic model by nomograms method and confirmed it in the validation cohort. CircPDK1 was identified to be overexpressed (P<0.01) in LSCC. Through analysis in study cohort, circPDK1low patients (less than the mean expression, n=124) showed more lymph nodes metastasis (P=0.025), more vascular invasion (VI) (P=0.047), more visceral pleural invasion (VPI) (P=0.015) and poorer prognosis (P=0.003) than circPDK1high ones (n=108). Univariate and multivariate analysis showed that circPDK1, T staging, LN status, age, and SCCAg were significant prognostic factors for RFS and OS. The prognostic model based on these factors showed the concordance index (C-index) of 0.8214 and 0.8359 for predicting 5-year RFS and OS, respectively. Finally, the calibration curves were performed in the study cohort and a validation cohort to evaluate the model's efficiency. circPDK1 was identified as a potential biomarker of resected LSCC. The prognostic model including circPDK1, T staging, LN status, age, and SCCAg could effectively predict prognosis of resected LSCC.

T-cell immunoglobulin and mucin domain 1 expression in human non-small-cell lung cancer tissues and its clinical significance

Objective To investigate the expression of T-cell immunoglobulin and mucin domain 1 (TIM-1) in non-small-cell lung cancer (NSCLC) tissues and to analyze its clinical significance. To further explore the regulatory effect of TIM-1 intervention on the biological function of NSCLC cell lines. Methods The immunohistochemistry assay and the tissue micro-array were used to examine the TIM-1 expression in lung cancer tissues as well as adjacent normal lung tissues. The correlation between the expression level of TIM-1 and patients’ clinic pathological parameters was evaluated by using chi-square test. The lung cancer cell lines A549 and SK-MES-1 were used to construct the stable knockdown expression of TIM-1 using RNA interference (RNAi) method. The cell counting kit-8 (CCK-8), wound healing, and Transwell were used to examine the cellular function after TIM-1 knockdown of these cells. Results Higher TIM-1 expression in lung squamous cell carcinoma tissues is significantly correlated with advanced TNM stage (Ⅲ+ Ⅳ: 33.3%, Ⅰ+ Ⅱ: 13.2%) (χ2=3.969, P<0.05), but not any other parameters. CCK-8 assay showed that, knockdown expression of TIM-1 make the cell proliferation rate decreased significantly. The wound healing assay showed that the cell migration abilities of LV-TIM-1-shRNA group was significantly lower than LV-NC group (A549, 24 h: t=17.540, P<0.01; SK-MES-1, 24 h: t=11.450, P<0.01). The Transwell assay showed that the cell invasion abilities was significantly decreased after knockdown expression of TIM-1. Conclusion Our results showed that the TIM-1 was highly expressed in non-small-cell lung cancer tissues, and its expression level was significantly correlated with TNM stage in squamous cell carcinoma patients. TIM-1 expression in lung adenocarcinoma tissues could be used as independent prognostic predictor for the patients, while higher TIM-1 expression in lung squamous cell carcinoma tissues trends significant for the prognostic prediction for the patients. Knockdown expression of TIM-1 could inhibit the cell viability, and the abilities of migration and invasion, and thus TIM-1 could be used as potential therapeutic target for lung cancer. Key words: T-cell immunoglobulin and mucin domain 1; Lung cancer; Immunohistochemistry; RNA interference; Prognosis

LncRNA LINC00857 regulates lung adenocarcinoma progression, apoptosis and glycolysis by targeting miR-1179/SPAG5 axis.

Long non-coding RNA (lncRNA), a member of non-coding RNA family with over 200 nucleotides in length, typically serves as an oncogene or tumor suppressor in tumor progression, such as cancer cell proliferation, apoptosis and glycolysis. Recent studies manifested lncRNA LINC00857 was involved in cell cycle regulation of lung cancer. Due to complicated networks in tumorigenesis, the potential roles of LINC00857 underlying lung cancer progression still need further investigation. In this study, we explored the expression of LINC00857 in lung adenocarcinoma (LUAD) tissues and LUAD cell lines and found a dramatical upregulation of LINC00857 compared with the adjacent normal lung tissues and BEAS-2B cell line, respectively. Then, LINC00857 knockdown led to the cell proliferation and glycolysis was repressed, while the apoptosis was elevated in LUAD cell lines. Furthermore, we identified a direct interaction between LINC00857 and miR-1179 in LUAD cells using bioinformatic method and report assay. Finally, we evidenced LINC00857 promoted cell growth and glycolysis and repressed apoptosis via sponging miR-1179 and further regulating sperm-associated antigen 5 (SPAG5) expression in LUAD cell lines. Hence, our results authenticated that lncRNA LINC00857 regulated the cell proliferation, glycolysis and apoptosis of LUAD cells mainly through targeting the miR-1179/SPAG5 axis, which might be a novel insight into lung cancer progression and provided a potential target for clinical treatment of LUAD patients.

Overexpression of Raf-1 kinase inhibitor protein inhibits cell invasion and migration in lung cancer cells through suppressing epithelial-mesenchymal transition

Background: Raf-1 kinase inhibitor protein (RKIP) is a small evolutionary conserved protein that was associated with the Raf-MEK-ERK pathway. However, whether RKIP would alter the invasion and metastasis of non-small cell lung cancer (NSCLC) and play the role through suppressing epithelial-mesenchymal transition (EMT) remains to be explored. Methods: A549 cells were transfected with RKIP-GV141 plasmid for overexpression of RKIP. Colony formation assay and MTT assay were performed to measure the effects of RKIP on the proliferation and cell viability assay of A549 cells. Transwell, Migration Assay and wound healing assay were performed to analyze the effects of RKIP on the invasion and metastasis of A549 cells. E-cadherin and vimentin were measured by Western blot to conform RKIP affects invasion and metastasis of NSCLC via inhibiting EMT. Results: RKIP is downregulated in NSCLC tissues compared to adjacent normal lung tissues by IHC. Decreased expression of RKIP contributes to poor prognosis in lung adenocarcinoma patients. Age and pTNM stage were independent prognostic factors for adenocarcinoma patients. Overexpression of RKIP reduces the cell viability and limits the proliferation, invasion, and migration of A549 cells in vitro . Wound healing assay showed the degressive ability of metastasis. High expression of E-cadherin and low expression of vimentin indicated that RKIP affects invasion and metastasis of NSCLC via inhibiting EMT. Conclusions: RKIP is decreased in NSCLC tissues. Overexpression of RKIP in A549 cells would decrease the cellular proliferation, viability, invasion ability, and metastasis ability probably via inhibiting EMT through upregulating E-cadherin expression and downregulating vimentin expression.

Systematic analysis of concentrations of 52 elements in tumor and counterpart normal tissues of patients with non-small cell lung cancer.

BackgroundMany studies have documented the abnormal concentrations of major/trace elements in serum or malignant tissues of patients, but very few works systematically tested the concentrations of elements in tumor tissues in comparison with paired adjacent normal tissues from the same patients.MethodsTumor and adjacent normal lung tissues were obtained from 93 patients with previously untreated NSCLC, and 43 patients whose tumor and paired normal lung tissues reached 200 mg or more were selected for measurement of the elements' concentrations using an inductively coupled plasma‐atomic emission spectrometer.ResultsWe found that the concentrations of the 52 elements varied from 0.4 ng/g tissue (Lu, Pd, and Tm) to 1 658 000 ng/g (Na), 1 951 000 ng/g (P), and 2 495 000 ng/g (K). Thirty eight of the 52 (73.1%) elements showed approximately equal concentrations in tumor and adjacent normal lung tissues of the patients. The concentrations of nine elements (K, P, Mg, Zn, Rb, Cu, Se, Cs, and Tl) in tumor samples were significantly higher than their paired normal lung tissues, and five elements (Na, Fe, Cr, Cd, and Ge) exhibited decreased concentrations in cancer samples compared to counterpart normal lung tissues. Low Fe in tumor samples was associated with smoking history, whereas low Cr was associated with histology (squamous cell carcinoma) of the patients.ConclusionsOur results demonstrate that measurement of elements’ concentrations in both cancer and paired normal tissues is important to get insights into the roles of these elements in carcinogenesis, and therapeutic approaches to normalize the elements are warranted to treat NSCLCs.

Long Non-coding RNA JHDM1D-AS1 Interacts with DHX15 Protein to Enhance Non-Small-Cell Lung Cancer Growth and Metastasis

JHDM1D antisense 1 (JHDM1D-AS1), a long non-coding RNA (lncRNA), has been shown to promote pancreatic cancer growth by inducing an angiogenic response. However, its biological and clinical significance in non-small-cell lung cancer (NSCLC) is still unclear. In this study, we examined the expression and prognostic significance of JHDM1D-AS1 in NSCLC. The effects of JHDM1D-AS1 knockdown or overexpression on NSCLC growth and metastasis were investigated. We show that JHDM1D-AS1 is upregulated in NSCLC relative to adjacent normal lung tissues. High JHDM1D-AS1 expression is significantly correlated with advanced tumor, node, and metastasis (TNM) stage and lymph node metastasis. JHDM1D-AS1 expression serves as an independent prognostic factor for overall survival of patients with NSCLC. Functionally, JHDM1D-AS1 knockdown inhibits NSCLC cell aggressiveness both in vitro and in vivo, which is rescued by ectopic expression of JHDM1D-AS1. JHDM1D-AS1 binding stabilizes DHX15 protein in NSCLC cells. DHX15 overexpression enhances NSCLC cell proliferation and invasion, whereas knockdown of DHX15 exerts opposite effects. JHDM1D-AS1-mediated aggressive phenotype is impaired when DHX15 is silenced. Ectopic expression of DHX15 restores the defects in proliferation and invasion of JHDM1D-AS1-depleted NSCLC cells. Collectively, the interaction between JHDM1D-AS1 and DHX15 accounts for NSCLC growth and metastasis. This work provides potential additional therapeutic targets for treatment of NSCLC.

The Immune Contexture Associates with the Genomic Landscape in Lung Adenomatous Premalignancy.

Epithelial cells in the field of lung injury can give rise to distinct premalignant lesions that may bear unique genetic aberrations. A subset of these lesions may escape immune surveillance and progress to invasive cancer; however, the mutational landscape that may predict progression has not been determined. Knowledge of premalignant lesion composition and the associated microenvironment is critical for understanding tumorigenesis and the development of effective preventive and interception strategies. To identify somatic mutations and the extent of immune cell infiltration in adenomatous premalignancy and associated lung adenocarcinomas, we sequenced exomes from 41 lung cancer resection specimens, including 89 premalignant atypical adenomatous hyperplasia lesions, 15 adenocarcinomas in situ, and 55 invasive adenocarcinomas and their adjacent normal lung tissues. We defined nonsynonymous somatic mutations occurring in both premalignancy and the associated tumor as progression-associated mutations whose predicted neoantigens were highly correlated with infiltration of CD8+ and CD4+ T cells as well as upregulation of PD-L1 in premalignant lesions, suggesting the presence of an adaptive immune response to these neoantigens. Each patient had a unique repertoire of somatic mutations and associated neoantigens. Collectively, these results provide evidence for mutational heterogeneity, pathway dysregulation, and immune recognition in pulmonary premalignancy.Significance: These findings identify progression-associated somatic mutations, oncogenic pathways, and association between the mutational landscape and adaptive immune responses in adenomatous premalignancy.See related commentary by Merrick, p. 4811.

P2.03-49 Roles of CENPU in Lung Adenocarcinoma Progression and Invasion

Centromere protein U (CENPU), a centromere protein mediating kinetochore-microtubule interaction, is critical for proper cell cycle and mitosis. It has been implicated that CENPU promotes tumorigenesis in variant malignancies. However, roles of CENPU in lung adenocarcinoma progression and underlying mechanisms remain to be elucidated. CENPU expression in 90 pair lung adenocarcinoma/adjacent normal lung samples was examined with immunohistochemistry (IHC). Then CENPU expression was inhibited with lentiviral-mediated shRNA strategy in human lung adenocarcinoma cell line H1299 to examine the impact of CENPU knockdown for lung adenocarcinoma progression and metastasis. Cell proliferation, colony formation, cell cycle and cell survival were analyzed by Cellomics cell counting method, colonogenesis assay, PI and Annexin V-APC staining respectively while cellular migration and invasion were determined by cell scratch and transwell test. Furthermore, expression of critical factors involved in epithelial-to-mesenchymal transition (EMT) were determined with western blot. CENPU expression was significantly increased in lung adenocarcinomas as compared to adjacent normal lung tissues (fold change=8.54, P<0.0001) (Fig. 1A). Functionl analysis revealed that in human lung adenocarcinoma cell line H1299, CENPU knockdown impaired cell proliferation (Fig. 1B), inhibited colony formation ability (Fig. 1C) and induced cell cycle arrest (Fig. 1D). Additionally, cellular migration and invasion was also inhibited by CENPU knockdown (Fig. 1E-F). It is further shown that E-Cadherin was induced while N-Cadherin and vimentin were inhibited by CENPU knockdown (Fig. 1G), indicating that CENPU was important for EMT process and cancer metastasis. It showed that CENPU expression is significantly upregulated lung adenocarcinoma tissue. Functional analysis indicated that CENPU is critical for cell proliferation, survival, migration and metastasis in lung adenocarcinoma cell line H1299. CENPU represents a promising target for lung adenocarcinoma therapy.

Overexpression of Raf-1 kinase inhibitor protein inhibits cell invasion and migration in lung cancer cells through suppressing epithelial-mesenchymal transition.

BackgroundRaf-1 kinase inhibitor protein (RKIP) is a small evolutionary conserved protein that was associated with the Raf-MEK-ERK pathway. However, whether RKIP would alter the invasion and metastasis of non-small cell lung cancer (NSCLC) and play the role through suppressing epithelial-mesenchymal transition (EMT) remains to be explored.MethodsA549 cells were transfected with RKIP-GV141 plasmid for overexpression of RKIP. Colony formation assay and MTT assay were performed to measure the effects of RKIP on the proliferation and cell viability assay of A549 cells. Transwell, Migration Assay and wound healing assay were performed to analyze the effects of RKIP on the invasion and metastasis of A549 cells. E-cadherin and vimentin were measured by Western blot to conform RKIP affects invasion and metastasis of NSCLC via inhibiting EMT.ResultsRKIP is downregulated in NSCLC tissues compared to adjacent normal lung tissues by IHC. Decreased expression of RKIP contributes to poor prognosis in lung adenocarcinoma patients. Age and pTNM stage were independent prognostic factors for adenocarcinoma patients. Overexpression of RKIP reduces the cell viability and limits the proliferation, invasion, and migration of A549 cells in vitro. Wound healing assay showed the degressive ability of metastasis. High expression of E-cadherin and low expression of vimentin indicated that RKIP affects invasion and metastasis of NSCLC via inhibiting EMT.ConclusionsRKIP is decreased in NSCLC tissues. Overexpression of RKIP in A549 cells would decrease the cellular proliferation, viability, invasion ability, and metastasis ability probably via inhibiting EMT through upregulating E-cadherin expression and downregulating vimentin expression.

LETM1 is a potential biomarker of prognosis in lung non-small cell carcinoma

BackgroundAlthough the leucine zipper-EF-hand-containing transmembrane protein 1 (LETM1) is one of the mitochondrial inner membrane proteins that is involved in cancer prognosis in various tumors, LETM1 as a biomarker for prognostic evaluation of non-small cell lung carcinoma (NSCLC) has not been well studied.MethodsTo address this issue, we used 75 cases NSCLC, 20 cases adjacent normal lung tissues and NSCLC cell lines. We performed immunohistochemistry staining and western blot analysis as well as immunofluorescence imaging.ResultsOur studies show that expression of LETM1 is significantly correlated with the lymph node metastasis (p = 0.003) and the clinical stage (p = 0.005) of NSCLC. The Kaplan-Meier survival analysis revealed that NSCLC patients with positive expression of LETM1 exhibits a shorter overall survival (OS) rate (p = 0.005). The univariate and multivariate Cox regression analysis indicated that LETM1 is a independent poor prognostic marker of NSCLC. In addition, the LETM1 expression is correlated with cancer stemness-related gene LGR5 (p < 0.001) and HIF1α expression (p < 0.001), but not with others. Moreover, LETM1 expression was associated with the expression of cyclin D1 (p = 0.003), p27 (p = 0.001), pPI3K(p85) (p = 0.025), and pAkt-Thr308 (p = 0.004). Further, our studies show in LETM1-positive NSCLC tissues the microvessel density was significantly higher than in the negative ones (p = 0.024).ConclusionThese results indicate that LETM1 is a potential prognostic biomarker of NSCLC.

Expression of SUMO1P3 Compared with SUMO1 is an Independent Predictor of Patient Outcome in Lung Adenocarcinoma.

BackgroundThe small ubiquitin-like modifier 1 (SUMO1) and small ubiquitin-like modifier 1 pseudogene 3 (SUMO1P3) are long noncoding RNAs (lncRNAs). The prognostic significance of SUMO1 and SUMO1P3 expression in non-small cell lung cancer (NSCLC) remains unclear. This study aimed to use clinical, genetic, and survival data from the Cancer Genome Atlas (TCGA), to analyze the prognostic significance of SUMO1 and SUMO1P3 expression in the two main subtypes of NSCLC, lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC).Material/MethodsData were acquired from TCGA and in silico survival analysis was performed. SUMO1 and SUMO1P3 expression were compared between patients with LUAD and LUSC. Patient outcome was assessed as complete remission (CR), partial remission (PR), stable disease (SD), and progressive disease (PD). Recurrence-free survival (RFS) was defined as the survival time from primary surgery to the time of locoregional or distant recurrence of lung cancer.ResultsSUMO1P3 was significantly increased in LUSC and LUAD tissues compared with adjacent normal lung tissue and was significantly co-expressed with SUMO1. SUMO1P3 expression was significantly increased in patients with LUAD but not LUSC with reduced RFS after primary or follow-up treatment. Although patients with LUAD who had high SUMO1 or SUMO1P3 expression had reduced RFS compared with low expression groups, univariate and multivariate analysis showed that only SUMO1P3 expression was independently associated reduced RFS (HR, 1.418; 95% CI, 1.041–1.930; p=0.027).ConclusionsSUMO1P3 expression was an independent indicator of reduced RFS in patients with LUAD.

LINC01123, a c-Myc-activated long non-coding RNA, promotes proliferation and aerobic glycolysis of non-small cell lung cancer through miR-199a-5p/c-Myc axis

BackgroundLong non-coding RNAs (lncRNAs) have been associated with non-small cell lung cancer (NSCLC), but the underlying molecular mechanisms of their specific roles in mediating aerobic glycolysis have been poorly explored.MethodsNext-generation RNA sequencing assay was performed to identify the differentially expressed RNAs between NSCLC tissues with high 18F-fluorodeoxyglucose (FDG) uptake and their adjacent normal lung tissues. LINC01123 expression in NSCLC tissues was measured by real-time PCR and in situ hybridization (ISH) assay. The biological role of LINC01123 in cell growth and aerobic glycolysis capability was determined by performing functional experiments in vitro and in vivo. Further, the transcription of LINC01123 was explored by bioinformatics analysis, dual-luciferase reporter assay, and chromatin immunoprecipitation (ChIP) assay. RNA immunoprecipitation (RIP) and luciferase analyses were used to confirm the predicted competitive endogenous RNA (ceRNA) mechanisms between LINC01123 and c-Myc.ResultsThree hundred sixty-four differentially expressed genes were identified in RNA-seq assay, and LINC01123 was one of the most overexpressed lncRNAs. Further validation in expanded NSCLC cohorts confirmed that LINC01123 was upregulated in 92 paired NSCLC tissues and associated with poor survival. Functional assays showed that LINC01123 promoted NSCLC cell proliferation and aerobic glycolysis. Mechanistic investigations revealed that LINC01123 was a direct transcriptional target of c-Myc. Meanwhile, LINC01123 increased c-Myc mRNA expression by sponging miR-199a-5p. In addition, rescue experiments showed that LINC01123 functioned as an oncogene depending on miR-199a-5p and c-Myc.ConclusionSince LINC01123 is upregulated in NSCLC, correlates with prognosis, and controls proliferation and aerobic glycolysis by a positive feedback loop with c-Myc, it is expected to be a potential biomarker and therapeutic target for NSCLC.

Genome-wide profiling reveals alternative polyadenylation of mRNA in human non-small cell lung cancer

BackgroundLung cancer is the second most common cancer with an extremely poor overall survival rate. Post-transcriptional regulation of gene expression play many important roles in human cancer, and one of the potential mechanisms underlying this is alternative mRNA maturation at its 3′ untranslated regions (3′-UTRs).MethodsCancer tissues and paired adjacent normal lung tissues from 26 patients diagnosed with non-small cell lung cancer (NSCLC) were analyzed by in vitro transcription-sequencing alternative polyadenylation sites (IVT-SAPAS). 41,773,101 reads in average were obtained from each paired sample. A potential regulation of Cleavage Stimulation Factor Subunit 2 (CSTF2) on 3′UTR length of genes was tested in H460 cells.Results1439 (10.26%) genes showed up-regulated expression and 1364 (9.72%) genes showed down-regulated expression in lung cancer tissue versus normal lung tissue, and shorten 3′UTR in cancer tissue was detected in cancer tissues collected from 96.2% (25/26) patients, indicating lung cancer tend to have shortened 3′UTRs of these identified genes. KEGG analysis showed 1855 genes with shorten 3′UTR were enriched in mTOR signaling, ubiquitin mediated proteolysis and RNA degradation. Knocking down CSTF2 expression in H460 cells results in 3′UTR elongation of genes that was identified to be with shortened length in cancer tissues.ConclusionAlternative polyadenylation (APA) site-switching of 3′UTRs is prevalent in NSCLC, and CSTF2 may serve as an oncogene regulates the 3′UTR length of cancer related genes in NSCLC.

Synaptotagmin 7 in twist-related protein 1-mediated epithelial – Mesenchymal transition of non-small cell lung cancer

BackgroundTwist-related protein 1 (TWIST1) plays an essential role in the carcinogenesis and metastasis of NSCLC. Our aims were to identify the molecule at the downstream of TWIST1 and to evaluate its potential as a diagnostic and a prognostic marker in NSCLC. MethodsThe functional genes at the downstream of TWIST1 were obtained via microarray gene expression analyses in the NSCLC cell line. The expression levels of synaptotagmin 7 (SYT7) in a cohort of patients with NSCLC (n = 154) were examined using immunohistochemistry staining and assessed by Kaplan-Meier survival analysis and Cox regression analysis. The effects of SYT7 on the tumorigenesis and metastasis of NSCLC were measured in NSCLC cells. In vivo xenograft lung cancer models were used to study the tumorigenesis role of SYT7. FindingsWe discovered that SYT7 is significantly altered by TWIST1 expression. We further confirmed that SYT7 protein was significantly higher in NSCLC than that in the adjacent normal lung tissue, and higher SYT7 expression was associated with poor survival of NSCLC patients. The protein level of SYT7 was positively correlated with TWIST1 in NSCLC tissue. Functional experiments indicated that SYT7 promoted proliferation, invasion, and metastasis and inhibited cell apoptosis of NSCLC cells in vitro. In vivo experiments showed that shSYT7 inhibited the xenograft tumor growth of NSCLC cells. Knocking down of SYT7 increased the expression of E-cadherin and decreased the level of N-cadherin and Vimentin in cultured cells. InterpretationOur data indicate that SYT7 is an important promoter for EMT and tumor progression in NSCLC. FundThis project was supported by grants from the Major Scientific and Technological Innovation Project of Shandong Province (2018CXGC1212), Science and Technology Foundation of Shandong Province (2014GSF118084, 2016GSF121043), Medical and Health Technology Innovation Plan of Jinan City (201805002) and the National Natural Science Foundation of China (81372333).