ABSTRACT
Non-small cell lung cancer (NSCLC) is a common and lethal malignancy. The carcinogenic roles of lncRNA CALML3 antisense RNA 1 (CALML3-AS1) have been documented. However, the function and potential mechanisms of CALML3-AS1 in the progression of NSCLC need to be further explored. The molecule expression was assessed by qRT-PCR and Western blot. The subcellular localization of CALML3-AS1 was observed by fluorescence in situ hybridization (FISH). The malignant behaviors of NSCLC cells were evaluated by CCK-8, colony formation, EdU, wound healing and transwell assays. In vivo xenograft tumor and liver metastatic models were established. The molecular mechanisms were investigated by RIP, RNA pull-down and ChIP assays. The methylation level was detected by MSP. Herein, we found that CALML3-AS1 was upregulated, while butyrophilin-like 9 (BTNL9) was downregulated in NSCLC. Functionally, CALML3-AS1 depletion repressed NSCLC cell malignant phenotypes, in vivo tumor growth, and liver metastasis. Mechanistically, AlkB homolog 5 (ALKBH5) enhanced CALML3-AS1 stability via N6-methyladenosine (m6A) demethylation, whereas m6A reader YTH domain-containing 2 (YTHDC2) destabilized CALML3-AS1. Moreover, CALML3-AS1 inhibited BTNL9 transcription and expression through the recruitment of Zeste homolog 2 (EZH2). Rescue experiments demonstrated that BTNL9 downregulation counteracted sh-CALML3-AS1-mediated antitumor effects on NSCLC. Taken together, CALML3-AS1 modulated by ALKBH5 and YTHDC2 in an m6A modification dependent manner drives NSCLC progression via epigenetically repressing BTNL9.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , MicroRNAs , RNA Methylation , RNA, Long Noncoding , Humans , Butyrophilins/genetics , Butyrophilins/metabolism , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , In Situ Hybridization, Fluorescence , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Methylation , MicroRNAs/genetics , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA Methylation/geneticsABSTRACT
Gefitinib has been widely applied for the treatment of lung adenocarcinoma (LUAD). However, the long-term application of gefitinib usually leads to acquired drug resistance in tumour patients, resulting in clinical treatment failure. Small nucleolar host gene 17 (SNHG17) has been shown to play a regulatory role in LUAD progression. Nevertheless, the role of SNHG17 in LUAD gefitinib resistance remains elusive. The expression pattern of SNHG17 was examined in tissues and cell lines of gefitinib-sensitive and gefitinib-resistant LUAD, respectively. Gain- and loss-of-function experiments were employed to assess the biological functions of SNHG17 in cell proliferation and apoptosis, as well as aggressive phenotypes of LUAD cells. MeRIP-qPCR and colorimetric quantificational analysis were performed to detect m6A modifications and contents. Fluorescence in situ hybridisation (FISH) and subcellular fractionation analysis were used to reveal the distribution of SNHG17. RIP and ChIP assays were performed to further validate the SNHG17/EZH2/LATS2 regulatory axis. A xenograft tumour growth assay was conducted to evaluate the role of SNHG17 in LUAD gefitinib resistance in vivo. SNHG17 was upregulated in gefitinib-resistant LUAD tissues and cell lines. Functional assays showed that SNHG17 aggravated the malignant phenotypes of gefitinib-resistant LUAD cells. In addition, METTL3-mediated N6-methyladenosine modification could induce the upregulation of SNHG17by stabilising its RNA transcript. Mechanistically, SNHG17 epigenetically repressed the expression of LATS2 by recruiting EZH2 to the promoter region of LATS2. The regulatory role of the SNHG17/EZH2/LATS2 axis in LUAD gefitinib resistance was further supported in vivo. Collectively, our findings suggested that SNHG17 induced by METTL3 could promote LUAD gefitinib resistance by epigenetically repressing LATS2 expression.
Subject(s)
Adenocarcinoma , Lung Neoplasms , RNA, Long Noncoding , Adenocarcinoma/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Gefitinib/pharmacology , Gene Expression Regulation, Neoplastic , Humans , Lung/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Methyltransferases/metabolism , Protein Serine-Threonine Kinases/genetics , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
This study investigated the regulation of GRP78 in tumour-associated macrophage polarization in lung cancer. First, our results showed that GRP78 was upregulated in macrophages during M2 polarization and in a conditioned medium derived from lung cancer cells. Next, we found that knocking down GRP78 in macrophages promoted M1 differentiation and suppressed M2 polarization via the Janus kinase/signal transducer and activator of transcription signalling. Moreover, conditioned medium from GRP78- or insulin-like growth factor 1-knockdown macrophages attenuated the survival, proliferation, and migration of lung cancer cells, while conditioned medium from GRP78-overexpressing macrophages had the opposite effects. Additionally, GRP78 knockdown reduced both the secretion of insulin-like growth factor 1 and the phosphorylation of the insulin-like growth factor 1 receptor. Interestingly, insulin-like growth factor 1 neutralization downregulated GRP78 and suppressed GRP78 overexpression-induced M2 polarization. Mechanistically, insulin-like growth factor 1 treatment induced the translocation of GRP78 to the plasma membrane and promoted its association with the insulin-like growth factor 1 receptor. Finally, IGF-1 blockade and knockdown as well as GRP78 knockdown in macrophages inhibited M2 macrophage-induced survival, proliferation, and migration of lung cancer cells both in vitro and in vivo.
Subject(s)
Biomarkers, Tumor/metabolism , Endoplasmic Reticulum Chaperone BiP/metabolism , Gene Expression Regulation, Neoplastic , Lung Neoplasms/pathology , Macrophage Activation , Macrophages/immunology , Animals , Apoptosis , Biomarkers, Tumor/genetics , Cell Movement , Cell Proliferation , Endoplasmic Reticulum Chaperone BiP/genetics , Humans , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , Janus Kinases/genetics , Janus Kinases/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/immunology , Lung Neoplasms/metabolism , Mice , Mice, Nude , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Chromosome 14 ORF 166 (C14orf166), a protein involved in the regulation of RNA transcription and translation, has been reported to possess the potency to promote tumorigenesis; however, the role of C14orf166 in non-small-cell lung cancer (NSCLC) remains unknown. The purpose of the present study was to assess C14orf166 expression and its clinical significance in NSCLC. Immunohistochemical staining, quantitative real-time PCR (qRT-PCR), and Western blotting were used to detect the C14orf166 protein and mRNA expression levels in NSCLC tissues compared with adjacent normal tissues, as well as in NSCLC cells lines compared with normal human bronchial epithelial cells (HBE). Then, the correlations between the C14orf166 expression levels and the clinicopathological features of NSCLC were analyzed. Additionally, the Cox proportional hazard model was used to evaluate the prognostic significance of C14orf166. We found that C14orf166 expression increased in carcinoma tissues compared with their adjacent normal tissues at the protein (P<0.001) and mRNA levels (P<0.001). High expression of C14orf166 was significantly associated with the T stage (P=0.006), lymph node metastasis (P=0.001), advanced TNM stage (P<0.001), and chemotherapy (P<0.001). Moreover, according to the survival analysis, patients with overexpressed C14orf166 were inclined to experience a shorter overall survival and disease-free survival time (P<0.001). Multivariate COX analysis implied that C14orf166 was an independent prognostic biomarker. Taken together, our findings indicate that the overexpression of C14orf166 may contribute to the disease progression of NSCLC, represent a novel prognostic predictor and help high-risk patients make better decisions for subsequent therapy.
Subject(s)
Biomarkers, Tumor/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Prognosis , Trans-Activators/genetics , Adult , Aged , Carcinoma, Non-Small-Cell Lung/pathology , Disease Progression , Disease-Free Survival , Female , Gene Expression Regulation, Neoplastic , Humans , Kaplan-Meier Estimate , Lymphatic Metastasis , Male , Middle AgedABSTRACT
Platelet-derived growth factor receptor (PDGFR)-ß is an important tyrosine kinase and its downregulation has been reported to alter the radiosensitivity of glioma cells, although the underlying mechanism is unclear. In order to investigate the effect of PDGFR-ß on the radiosensitivity of glioblastoma, the present study transfected C6 glioma cells with a PDGFR-ß-specific small interfering (si)RNA expression plasmid, and downregulation of the expression of PDGFR-ß in C6 glioma cells was confirmed by western blotting and immunohistochemical analysis. Clone formation assays and xenograft growth curves demonstrated that PDGFR-ß-siRNA enhanced the radiosensitivity of C6 glioma cells in vitro and in vivo. Furthermore, MTT and xenograft growth curves demonstrated that PDGFR-ß-siRNA inhibited the proliferation of C6 glioma cells in vitro and in vivo, and terminal deoxynucleotidyl transferase dUTP nick end-labeling and immunohistochemical analyses demonstrated that PDGFR-ß-siRNA induced apoptosis and inhibited the expression of Ki-67, cyclin B1 and vascular endothelial growth factor in C6 glioma cell xenografts. Taken together, these results suggested that PDGFR-ß may be used as a target for the radiosensitization of glioblastoma.
ABSTRACT
C14orf166, a 28 kD protein regulating RNA transcription and translation, may serve a critical role in oncogenesis. The aim of the current study was to explore the association between C14orf166 expression and esophageal squamous cell carcinoma (ESCC) and to draw attention to the association between C14orf166 and the initiation, progression and prognosis of ESCC. C14orf166 expression in ESCC and paired normal tissues was detected by immunohistochemical staining, western blotting and reverse transcriptionquantitative polymerase chain reaction, and the association between C14orf166 expression and clinicopathological characters of ESCC was analyzed. Survival analysis was used to assess the prognostic significance of C14orf166 and it was observed that C14orf166 expression was higher in the ESCC tissues when compared with adjacent noncancerous tissues at protein (P<0.001) and mRNA levels (P<0.001). There was a significant difference in T stage, lymph node metastasis and TNM stage in patients categorized according to different C14orf166 expression levels. The overexpression of C14orf166 was associated with a shorter overall survival and diseasefree survival, and multivariate analysis indicated that C14orf166 was an independent prognostic indicator. The present study indicates that the expression of C14orf166 is elevated in ESCC, and is potentially a valuable prognostic predictor for ESCC.
Subject(s)
Carcinoma, Squamous Cell/pathology , Esophageal Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Trans-Activators/genetics , Trans-Activators/metabolism , Adult , Aged , Biomarkers, Tumor/metabolism , Blotting, Western , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/mortality , Disease-Free Survival , Esophageal Neoplasms/metabolism , Esophageal Neoplasms/mortality , Esophageal Squamous Cell Carcinoma , Female , Follow-Up Studies , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Lymphatic Metastasis , Male , Middle Aged , Neoplasm Staging , Prognosis , Proportional Hazards Models , RNA, Messenger/metabolism , Up-RegulationABSTRACT
Recently H19 has been demonstrated to be up-regulated in esophageal squamous cell carcinoma (ESCC) and shown to be the precursor of miR-675 that encodes miR-675-5p conservatively. miR-675 is overexpressed in many human cancers; however, the function of miR-675-5p is largely unknown in ESCC. In this study, we found that miR-675-5p expression was significantly increased in ESCC tissues and cell lines and related with ESCC progression and poor prognosis. We also showed here that down-regulation of miR-675-5p in ESCC cells dramatically induced cell G1 arrest and reduced cell proliferation, colony formation, migration and invasion in vitro as well as tumorigenesis and tumor metastasis in vivo. We subsequently identified that REPS2 was a target gene of miR-675-5p. We found that inhibition of miR-675-5p up-regulated the expression of REPS2, inhibited RalBP1/RAC1/CDC42 signaling pathway. Inversely, interference of REPS2 abrogated the effect induced by miR-675-5p inhibition, which resembled the function of miR-675-5p up-regulation. Taken together, our findings suggested that miR-675-5p might play an oncogenic role in ESCC through RalBP1/RAC1/CDC42 signaling pathway by inhibiting REPS2 and might serve as a valuable prognostic biomarker and therapeutic target for ESCC patients.
Subject(s)
Carcinogenesis/genetics , Carcinoma, Squamous Cell/genetics , Esophageal Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Intracellular Signaling Peptides and Proteins/genetics , MicroRNAs/metabolism , ATP-Binding Cassette Transporters/metabolism , Adult , Aged , Biomarkers, Tumor/metabolism , Calcium-Binding Proteins , Carcinoma, Squamous Cell/mortality , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/surgery , Cell Line, Tumor , Disease-Free Survival , Down-Regulation , Esophageal Neoplasms/mortality , Esophageal Neoplasms/pathology , Esophageal Neoplasms/surgery , Esophageal Squamous Cell Carcinoma , Female , G1 Phase Cell Cycle Checkpoints/genetics , GTPase-Activating Proteins/metabolism , Humans , Kaplan-Meier Estimate , Lymphatic Metastasis , Male , Middle Aged , Neoplasm Staging , Prognosis , RNA Interference , RNA, Small Interfering , Signal Transduction/genetics , Up-Regulation , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolismABSTRACT
OBJECTIVE: To observe the effects of intermittent one-lung ventilation (OLV) before and after surgery on the inflammatory cytokines and biomarkers of oxidative stress in serum of lung cancer patients undergoing open thoracotomy. METHODS: Between June 2011 to March 2012, 80 patients undergoing lobectomy were classified into four groups nonrandomly: Group A, control group; B, OLV preconditioning group; C, OLV postconditioning group; D, OLV preconditioning-combined-with-postconditioning group. Neutrophil granulocyte (PMN), interleukin 6 (IL-6), superoxide dismutase (SOD), and malondialdehyde (MDA) were assayed in plasma samples taken preoperatively (T1), intraoperatively (T2), and 2 hours postoperatively (T3). RESULTS: Comparison of T1 with T2 and T3 documented significant increase in MDA, PMN, and IL-6 levels and decrease in SOD in the control group (p < 0.01). Compared with the control group, the levels of IL-6 and MDA decreased and SOD increased significantly at T2 in the OLV preconditioning group, at T3 in the OLV preconditioning combined postconditioning group (p < 0.05). CONCLUSION: Preconditioning of intermittent OLV before thoracotomy combined with postconditioning of intermittent returning two-lung ventilation after surgery maybe alleviate systematic inflammatory response and oxidative stress for lung cancer patients.
Subject(s)
Acute Lung Injury/prevention & control , Ischemic Postconditioning , Ischemic Preconditioning , One-Lung Ventilation , Thoracotomy , Biomarkers/blood , Female , Humans , Lung Neoplasms/surgery , Male , Middle Aged , Oxidative Stress/physiologyABSTRACT
BACKGROUND: Heat shock-related 70 kDa protein 2 (HSPA2) has been identified as a potential cancer-promoting protein expressed at abnormal levels in a subset of cancers. However, its important role in esophageal squamous cell carcinoma (ESCC) is hardly known by people. The purpose of this study is to assess HSPA2 expression and to explore its role in ESCC. METHODS: Thirty ESCC samples, paired adjacent non-cancerous tissues and normal esophageal tissues, were collected for HSPA2 detection by quantitative RT-PCR (qRT-PCR) and western blotting. Additionally, the expression of HSPA2 in ESCC and adjacent non-cancerous tissues from 120 patients was analyzed by immunohistochemistry, and correlated with clinicopathological parameters and patients' outcome. RESULTS: HSPA2 mRNA and protein were overexpressed in ESCC tissues. Overexpression of HSPA2 was significantly associated with primary tumor, TNM stage, lymph node metastases and recurrence, respectively (all, P <0.05). Kaplan-Meier curves showed that elevated HSPA2 expression was associated with shorter disease-free survival and overall survival in ESCC patients. Cox multivariate regression analysis revealed that overexpression of HSPA2 was an independent prognostic factor in disease-free survival and overall survival for ESCC patients (hazard ratio was 2.115 and 2.210, respectively, P <0.05). CONCLUSIONS: Our findings demonstrate that overexpression of HSPA2 may contribute to the malignant progression of ESCC and present a novel prognostic indicator for ESCC patients.
Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma, Squamous Cell/mortality , Esophageal Neoplasms/mortality , Esophagus/metabolism , HSP70 Heat-Shock Proteins/metabolism , Neoplasm Recurrence, Local/mortality , Adult , Aged , Biomarkers, Tumor/genetics , Blotting, Western , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Esophageal Neoplasms/metabolism , Esophageal Neoplasms/pathology , Esophagus/pathology , Female , Follow-Up Studies , HSP70 Heat-Shock Proteins/genetics , Humans , Immunoenzyme Techniques , Lymphatic Metastasis , Male , Middle Aged , Neoplasm Grading , Neoplasm Recurrence, Local/metabolism , Neoplasm Recurrence, Local/pathology , Neoplasm Staging , Prognosis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Survival RateABSTRACT
OBJECTIVE: REPS2 plays important roles in inhibiting cell proliferation, migration and in inducing apoptosis of cancer cells, now being identified as a useful biomarker for favorable prognosis in prostate and breast cancers. The purpose of this study was to assess REPS2 expression and to explore its role in esophageal squamous cell carcinoma (ESCC). METHODS: Protein expression of REPS2 in ESCCs and adjacent non-cancerous tissues from 120 patients was analyzed by immunohistochemistry and correlated with clinicopathological parameters and patient outcome. Additionally, thirty paired ESCC tissues and four ESCC cell lines and one normal human esophageal epithelial cell line were evaluated for REPS2 mRNA and protein expression levels by quantitative RT-PCR and Western blotting. RESULTS: REPS2 mRNA and protein expression levels were down-regulated in ESCC tissues and cell lines. Low protein levels were significantly associated with primary tumour, TNM stage, lymph node metastasis and recurrence (all, P < 0.05). Survival analysis demonstrated that decreased REPS2 expression was significantly associated with shorter overall survival and disease-free survival (both, P < 0.001), especially in early stage ESCC patients. When REPS2 expression and lymph node metastasis status were combined, patients with low REPS2 expression/lymph node (+) had both poorer overall and disease-free survival than others (both, P < 0.001). Cox multivariate regression analysis further revealed REPS2 to be an independent prognostic factor for ESCC patients. CONCLUSIONS: Our findings demonstrate that downregulation of REPS2 may contribute to malignant progression of ESCC and represent a novel prognostic marker and a potential therapeutic target for ESCC patients.
Subject(s)
Apoptosis/genetics , Carcinoma, Squamous Cell/genetics , Esophageal Neoplasms/genetics , Intracellular Signaling Peptides and Proteins/biosynthesis , Lymphatic Metastasis/genetics , Neoplasm Recurrence, Local/genetics , Adult , Aged , Biomarkers, Tumor/genetics , Calcium-Binding Proteins , Carcinoma, Squamous Cell/mortality , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Disease-Free Survival , Down-Regulation , Esophageal Neoplasms/mortality , Esophageal Neoplasms/pathology , Esophageal Squamous Cell Carcinoma , Female , Gene Expression Regulation, Neoplastic , Humans , Intracellular Signaling Peptides and Proteins/genetics , Lymph Nodes/pathology , Lymphatic Metastasis/pathology , Male , Middle Aged , Neoplasm Recurrence, Local/pathology , Neoplasm Staging , RNA, Messenger/biosynthesisABSTRACT
UNLABELLED: The SH2B1 adaptor protein is recruited to multiple ligand-activated receptor tyrosine kinases that play important role in the physiologic and pathologic features of many cancers. The purpose of this study was to assess SH2B1 expression and to explore its contribution to the non-small cell lung cancer (NSCLC). METHODS: SH2B1 expression in 114 primary NSCLC tissue specimens was analyzed by immunohistochemistry and correlated with clinicopathological parameters and patients' outcome. Additionally, 15 paired NSCLC background tissues, 5 NSCLC cell lines and a normal HBE cell line were evaluated for SH2B1 expression by RT-PCR and immunoblotting, immunofluorescence being applied for the cell lines. RESULTS: SH2B1 was found to be overexpressed in NSCLC tissues and NSCLC cell lines. More importantly, high SH2B1 expression was significantly associated with tumor grade, tumor size, clinical stage, lymph node metastasis, and recurrence respectively. Survival analysis demonstrated that patients with high SH2B1 expression had both poorer disease- free survival and overall survival than other patients. Multivariate Cox regression analysis revealed that SH2B1 overexpression was an independent prognostic factor for patients with NSCLC. CONCLUSIONS: Our findings suggest that the SH2B1 protein may contribute to the malignant progression of NSCLC and could offer a novel prognostic indicator for patients with NSCLC.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Adenocarcinoma/metabolism , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Squamous Cell/metabolism , Lung Neoplasms/metabolism , Lung/metabolism , Neoplasm Recurrence, Local/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adenocarcinoma/mortality , Adenocarcinoma/secondary , Blotting, Western , Carcinoma, Non-Small-Cell Lung/mortality , Carcinoma, Non-Small-Cell Lung/secondary , Carcinoma, Squamous Cell/mortality , Carcinoma, Squamous Cell/secondary , Cells, Cultured , Female , Fluorescent Antibody Technique , Follow-Up Studies , Humans , Immunoenzyme Techniques , Lung Neoplasms/mortality , Lung Neoplasms/pathology , Lymphatic Metastasis , Male , Middle Aged , Neoplasm Grading , Neoplasm Recurrence, Local/mortality , Neoplasm Recurrence, Local/pathology , Neoplasm Staging , Prognosis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Retrospective Studies , Reverse Transcriptase Polymerase Chain Reaction , Survival RateABSTRACT
The receptor for advanced glycation end products (RAGE) is oncogenic and overexpressed in human cancers, but its role in hepatocellular carcinoma remains unclear. Here we demonstrated that RAGE is overexpressed in primary hepatocellular carcinoma (PHC) compared to adjacent para-neoplastic liver samples. Serum endogenous secretory RAGE levels were also increased in PHC patients (p < 0.01). Moreover, we demonstrated that RAGE regulates cellular proliferation in Hepatocellular carcinoma (HCC). Knockdown of RAGE by specific siRNA inhibited cellular growth in the hepatocellular carcinoma cell line, Huh7, whereas the RAGE ligand, high mobility group box 1 protein (HMGB1) increased cellular proliferation. In addition, knockdown of RAGE by siRNA arrested cells in the G1 phase and inhibited DNA synthesis (p < 0.01), while HMGB1 protein decreased the number of cells in the G1 phase and increased the number in the S phase (p < 0.05). Furthermore, quantitative real time RT-PCR (qRT-PCR) and Western Blot results demonstrated that RAGE and HMGB1 positively regulate NF-κB p65 expression in Huh7 cells. These studies suggest that RAGE and RAGE ligands are important targets for therapeutic intervention in hepatocellular carcinoma.
Subject(s)
Carcinoma, Hepatocellular/pathology , HMGB1 Protein/metabolism , Liver Neoplasms/pathology , Receptor for Advanced Glycation End Products/genetics , Transcription Factor RelA/metabolism , Adult , Carcinoma, Hepatocellular/blood , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Cell Proliferation , Female , Gene Knockdown Techniques , HMGB1 Protein/genetics , Hep G2 Cells , Humans , Liver Neoplasms/blood , Liver Neoplasms/genetics , Male , Middle Aged , Transcription Factor RelA/genetics , Up-RegulationABSTRACT
OBJECTIVE: To investigate the differential protein profile of human lung squamous carcinoma (HLSC) and normal bronchial epithelium (NBE) and provide preliminary results for further study to explore the carcinogenic mechanism of HLSC. METHODS: Laser capture microdissection (LCM) was used to purify the target cells from 10 pairs of HLSC tissues and their matched NHBE, respectively. A stable-isotope labeled strategy using iTRAQ, followed by 2D-LC/Q-STAR mass spectrometry, was performed to separate and identify the differential expression proteins. RESULTS: A total of 96 differential expression proteins in the LCM-purified HLSC and NBE were identified. Compared with NBE, 49 proteins were upregulated and 47 proteins were downregulated in HLSC. Furthermore, the expression levels of the differential proteins including HSPB1, CKB, SCCA1, S100A8, as well as S100A9 were confirmed by western blot and tissue microarray and were consistent with the results of quantitative proteomics. CONCLUSION: The different expression proteins in HLSC will provide scientific foundation for further study to explore the carcinogenic mechanism of HLSC.
Subject(s)
Carcinoma, Squamous Cell/metabolism , Laser Capture Microdissection/methods , Lung Neoplasms/metabolism , Neoplasm Proteins/metabolism , Proteomics/methods , Respiratory Mucosa/metabolism , Analysis of Variance , Antigens, Neoplasm/analysis , Antigens, Neoplasm/metabolism , Blotting, Western , Bronchi/cytology , Carcinoma, Squamous Cell/chemistry , Female , Humans , Immunohistochemistry , Isotope Labeling , Lung Neoplasms/chemistry , Male , Mass Spectrometry , Middle Aged , Neoplasm Proteins/analysis , Reproducibility of Results , Respiratory Mucosa/chemistry , Serpins/analysis , Serpins/metabolismABSTRACT
The stroma surrounding cancer cell population is increasingly recognized as playing an important role in cancer proliferation, invasion, and metastasis. To identify the stromal proteins involved in nasopharyngeal carcinoma (NPC) carcinogenesis, differences in protein expression of the stroma from NPC and normal nasopharyngeal epithelium tissues (NNET) were assessed using a comparative proteomic approach combined with laser capture microdissection (LCM). LCM was performed to purify stromal cells from NPC and NNET, respectively. Proteins between the pooled microdissected tumor and normal stroma were separated by two-dimensional electrophoresis (2-DE) and differential proteins were identified by mass spectrometry (MS). Sixty differential proteins between normal stroma (NS) and tumor stroma (TS) were identified, and the expression of CapG protein was further confirmed by western blotting and immunohistochemical analysis. Our results will be helpful to study the role of stroma in the NPC carcinogenesis and may provide helpful clues for pathogenesis, early diagnosis, and progression of NPC.
Subject(s)
Carcinoma, Squamous Cell/chemistry , Carcinoma, Squamous Cell/pathology , Nasopharyngeal Neoplasms/chemistry , Nasopharyngeal Neoplasms/pathology , Nasopharynx/chemistry , Nasopharynx/cytology , Proteome/analysis , Adult , Aged , Amino Acid Sequence , Carcinoma, Squamous Cell/metabolism , Electrophoresis, Gel, Two-Dimensional , Epithelium/chemistry , Epithelium/metabolism , Female , Humans , Male , Mass Spectrometry , Microdissection , Microfilament Proteins/analysis , Microfilament Proteins/biosynthesis , Middle Aged , Molecular Sequence Data , Nasopharyngeal Neoplasms/metabolism , Nasopharynx/metabolism , Neoplasm Proteins/analysis , Neoplasm Proteins/biosynthesis , Nuclear Proteins/analysis , Nuclear Proteins/biosynthesis , Proteome/biosynthesis , Stromal Cells/chemistry , Stromal Cells/metabolismABSTRACT
The importance of stromal cells and the factors that they expressed during cancer initiation and progression have been highlighted by recent literature. To identify the stromal proteins involved in nasopharyngeal carcinoma (NPC) carcinogenesis, we assessed differences in protein expression of the stroma from NPC and normal nasopharyngeal epithelium tissues (NNET) using a quantitative proteomic approach combined with laser capture microdissection (LCM). LCM was performed to purify stromal cells from the NPC and NNET, respectively. The differential proteins between the pooled microdissected tumor and normal stroma were analyzed by two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry (MS). Twenty differential proteins were identified, and the expression and location of two differential proteins (L-plastin and S100A9) were further confirmed by Western blotting and immunohistochemical analysis. Our results will be helpful to study the role of stroma in the NPC carcinogenesis, as well as discover the interaction between NPC cells and their surrounding microenvironment.
Subject(s)
Calgranulin B/analysis , Connective Tissue/chemistry , Membrane Glycoproteins/analysis , Microfilament Proteins/analysis , Nasopharyngeal Neoplasms/chemistry , Neoplasm Proteins/analysis , Proteomics/methods , Electrophoresis, Gel, Two-Dimensional , Epithelium/chemistry , Female , Humans , Male , Mass Spectrometry , Middle Aged , Nasopharyngeal Neoplasms/pathology , Nasopharynx/chemistry , Nasopharynx/cytologyABSTRACT
N,N'-Dinitrosopiperazine (DNP) induces nasopharyngeal carcinoma (NPC) and shows organ specificity to the nasopharyngeal epithelium. To investigate its mechanism, the rat NPC model was induced using DNP. Rat NPC and normal nasopharyngeal cells were obtained from the NPC model using laser capture. The total proteins from these cell samples were separated with two-dimension polyacrylamide gel electrophoresis techniques, and highly expressed proteins (> five-fold) were analyzed using matrix-assisted laser desorption/ionization time of flight and bioinformatics. The results showed that HSP70 and mucin 5B expression increased not only in rat NPC but also in atypical hyperplasia nasopharyngeal tissues, a precancer stage of NPC. High-expression of heat shock protein 70 (HSP70) and mucin 5B was further supported by western blot analysis. The immunofluorescence and western-blotting studies further showed that DNP induced the expression of HSP70 and mucin 5B in a dosage-dependent manner in normal nasopharyngeal epithelia cells. Our data indicate that DNP triggers over-expression of HSP70 and mucin 5B, and is involved in nasopharyngeal tumorigenesis. HSP70 and mucin 5B may be important targets in nasopharyngeal tumorigenesis induced by DNP.
Subject(s)
Carcinogens/toxicity , HSP70 Heat-Shock Proteins/metabolism , Mucin-5B/metabolism , Nasopharyngeal Neoplasms/metabolism , Precancerous Conditions/metabolism , Animals , Blotting, Western , Cell Proliferation , Electrophoresis, Gel, Two-Dimensional , Female , Fluorescent Antibody Technique , Male , Nasopharyngeal Neoplasms/chemically induced , Nasopharyngeal Neoplasms/pathology , Nitrosamines/toxicity , Precancerous Conditions/chemically induced , Precancerous Conditions/pathology , Proteomics , Rats , Rats, Wistar , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
OBJECTIVE: To screen the effective target sequences of laryngeal carcinoma related gene LCRG1 using RNAi. METHODS: PCR site mutation method was used to reconstruct pSuper vector. Five pairs of siRNA sequences designed by siRNA software were annealed and inserted into the reconstructed pSuper vector. The reconstructed pSuper 362,398,432,789,903,and pSuper vectors were transfected into Hela cell lines and selected with the appropriate drugs to get resistant and pool cells, respectively. The colonies were identified by RT-PCR or real-time RT-PCR analysis. The silence effects were observed by cloning formation analysis. RESULTS: pSuper vector was reconstructed to restore Bgl II restriction enzyme sites using PCR mutation. The RT-PCR or real-time RT-PCR Results of pool clones showed 362, 398, and 432 pool clones all had better effects of LCRG1 gene-silence, especially 362 pool clones. The expression level of LCRG1 mRNA of selected 362 group anti-puromycin clones A2 and A5 was decreased. The Results of clone forming efficiency revealed that the cellular proliferation in A2 of 362 group was significantly higher than that of the vector and control Hela cells (P<0.05). CONCLUSION: The reconstructed pSuper vector is successfully constructed. The 362 group has better gene silence and has 2 effective 362 group anti-clones, suggesting that methodology has important values in studYing the function and molecular mechanism of LCRG1.
Subject(s)
Gene Silencing , Laryngeal Neoplasms/genetics , Mutagenesis, Site-Directed , RNA, Small Interfering/genetics , Tumor Suppressor Proteins/genetics , Adaptor Proteins, Signal Transducing , Base Sequence , Cloning, Molecular , HeLa Cells , Humans , Molecular Sequence Data , RNA Interference , Transfection , Tumor Cells, CulturedABSTRACT
In this study, we applied laser capture microdissection and a proteomic approach to identify novel nasopharyngeal carcinoma (NPC) biomarkers. Proteins from pooled microdissected NPC and normal nasopharyngeal epithelial tissues (NNET) were separated by two-dimensional gel electrophoresis, and differential proteins were identified by mass spectrometry. Expression of the differential protein cathepsin D in the above two tissues as well as four NPC cell lines was determined by Western blotting. Next, siRNA was used to inhibit the expression of cathepsin D in highly metastatic NPC cell line 5-8F to examine whether it associates with NPC metastasis. Immunohistochemistry was also performed to detect the expression of cathepsin D in 72 cases of primary NPC, 28 cases of NNET, and 20 cases of cervical lymph node metastases, and the correlation of its expression level with clinicopathologic features and clinical outcomes were evaluated. Thirty-six differential proteins between the NPC and NNET were identified. The expression level of cathepsin D in the two types of tissues was confirmed by Western blotting and related to differentiation degree and metastatic potential of the NPC cell lines. Down-regulated cathepsin D expression by siRNA significantly decreased in vitro invasive ability of 5-8F cells. Significant cathepsin D down-regulation was observed in NPC versus NNET, whereas significant cathepsin D up-regulation was observed in lymph node metastasis versus primary NPC. In addition, cathepsin D down-regulation was significantly correlated with poor histological differentiation, whereas cathepsin D up-regulation was significantly correlated with advanced clinical stage, recurrence, and lymph node and distant metastasis. Furthermore, survival curves showed that patients with cathepsin D up-regulation had a poor prognosis. Multivariate analysis confirmed that cathepsin D expression was an independent prognostic indicator. The data suggest that cathepsin D is a potential biomarker for the differentiation and prognosis of NPC, and its dysregulation might play an important role in the pathogenesis of NPC.
Subject(s)
Biomarkers/metabolism , Cathepsin D/metabolism , Nasopharyngeal Neoplasms/metabolism , Proteomics/methods , Antigens, Neoplasm/analysis , Antigens, Neoplasm/metabolism , Biomarkers/analysis , Cathepsin D/analysis , Cathepsin D/genetics , Cell Line, Tumor , Diagnosis, Differential , Electrophoresis, Gel, Two-Dimensional , Female , Humans , Kaplan-Meier Estimate , Keratin-8/analysis , Keratin-8/metabolism , Lasers , Male , Mass Spectrometry/methods , Microdissection/methods , Middle Aged , Nasopharyngeal Neoplasms/diagnosis , Nasopharyngeal Neoplasms/enzymology , Nasopharynx/enzymology , Nasopharynx/metabolism , Nasopharynx/pathology , Neoplasm Invasiveness/genetics , Neoplasm Metastasis/pathology , Neoplasm Staging , Prognosis , RNA, Small Interfering/genetics , Respiratory Mucosa/enzymology , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Serpins/analysis , Serpins/metabolism , TransfectionABSTRACT
A proteomics-based approach has been used to identify proteins that commonly elicit a humoral immune response in nasopharyngeal carcinoma (NPC). Sera from 19 newly diagnosed NPC patients and 19 healthy individuals were analyzed for IgG autoantibodies against NPC proteins resolved by 2-DE. Protein spots that exhibited selective reactivity with sera from NPC patients were identified by MS. Among nine identified proteins, cytokeratin 19 (CK19), Erb3 binding protein (EBP1), and Rho GDP dissociation inhibitor-beta (Rho-GDI-2) induced autoantibodies in more than 36.8% of NPC patients but not in healthy individuals. Furthermore, Western blot analysis and immunohistochemical staining were performed to determine the expression and localization of CK19, EBP1, and Rho-GDI-2 in NPC and normal nasopharyngeal mucosal tissues. Up-regulated CK19 and EBP1, but not Rho-GDI-2, were observed in NPC vs. normal tissue. Subcellular localization of the three proteins in NPC tissue was same as that in the normal tissue. Thus, overexpression of CK19 and EBP1 may be one of the mechanisms for their autoantibody development in NPC. To validate the findings of a proteomic analysis, occurrence of autoantibodies against these three proteins was detected by immunoprecipitation and Western blot analysis in additional 30 NPC patients, 23 other types of cancer patients and 20 healthy individuals. Results showed that frequency of autoantibodies against CK19, EBP1 and Rho-GDI-2 in NPC patients was significantly higher than that in other types of cancer patients and healthy individuals. We conclude that CK19, EBP1 and Rho-GDI-2 may have utility in NPC screening and diagnosis.
ABSTRACT
OBJECTIVE: This study was designed to establish the two-dimensional electrophoresis profiles with high resolution and reproducibility from human lung squamous cell carcinoma tissue and paired tumor-adjacent normal bronchial epithelial tissue, and to identify differential expression of tumor-associated proteins by using proteome analysis. METHODS: Comparative proteome analysis of human lung squamous carcinoma and paired normal bronchial mucosa adjacent to tumors from 20 cases were carried out. Total proteins of the carcinoma tissue and normal bronchial mucosa were separated by means of immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were analyzed and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). RESULTS: (1) Seventy-six differentially expressed proteins were screened by analyzing the electrophoretic maps of the 20 carcinoma and control mucosa tissues. (2) Sixty-eight differential proteins were identified by peptide mass fingerprinting (PMF). Some proteins were products of oncogenes and others were involved in the regulation of cell cycle and signal transduction. (3) The expression of three proteins mdm2, c-Jun and EGFR, correlated with lung squamous carcinoma, were detected by immunohistochemical staining and Western blot analysis. The results showed that the expression of mdm2, c-Jun and EGFR were up-regulated in lung squamous carcinomas, whereas down-regulated in control normal mucosa. It was consistent with our proteome analysis results. Those results suggested that those proteins may play roles in the carcinogenesis of lung squamous carcinoma. CONCLUSION: sixty-eight differentially expressed proteins were successfully characterized by comparative proteome analysis. Those results may provide scientific foundation for screening the molecular biomarkers which can be used in diagnosis and treatment of lung squamous carcinoma, as well as to improve patients' prognosis and provide a new clue for carcinogenesis research of lung squamous cell carcinoma.