Wentao Yue

Downregulation of Cyclophilin A by siRNA diminishes non-small cell lung cancer cell growth and metastasis via the regulation of matrix metallopeptidase 9

BackgroundCyclophilin A (CypA) is a cytosolic protein possessing peptidyl-prolyl isomerase activity that was recently reported to be overexpressed in several cancers. Here, we explored the biology and molecular mechanism of CypA in non-small cell lung cancer (NSCLC).MethodsThe expression of CypA in human NSCLC cell lines was detected by real-time reverse transcription PCR. The RNA interference-mediated knockdown of CypA was established in two NSCLC cell lines (95C and A549). 239836 CypA inhibitor was also used to suppress CypA activity. Tumorigenesis was assessed based on cellular proliferation, colony formation assays, and anchorage-independent growth assays; metastasis was assessed based on wound healing and transwell assays.ResultsSuppression of CypA expression inhibited the cell growth and colony formation of A549 and 95C cells. CypA knockdown resulted in the inhibition of cell motility and invasion. Significantly, we show for the first time that CypA increased NSCLC cell invasion by regulating the activity of secreted matrix metallopeptidase 9 (MMP9). Likewise, suppression of CypA with 239836 CypA inhibitor decreased cell proliferation and MMP9 activity.ConclusionsThe suppression of CypA expression was correlated with decreased NSCLC cell tumorigenesis and metastasis.

Downregulation of PAX6 by shRNA Inhibits Proliferation and Cell Cycle Progression of Human Non-Small Cell Lung Cancer Cell Lines

Background The transcription factor PAX6 is primarily expressed in embryos. PAX6 is also expressed in several tumors and plays an oncogenic role. However, little is known about the role of PAX6 in lung cancer. Methods The function of PAX6 in lung cancer cells was evaluated by small interfering RNA-mediated depletion of the protein followed by analyses of cell proliferation, anchorage-independent growth, and cell cycle arrest. The changes of cyclin D1, pRB, ERK1/2, p38 expression caused by PAX6 inhibition were detected using western-blotting. The PAX6 mRNA level in 52 pairs of tumors and corresponding matched adjacent normal tissues from non-small cell lung cancer patients and lung cancer cell lines was detected by real-time PCR. Results Suppression of PAX6 expression inhibited cell growth and colony formation in A549 and H1299 cells. The percentage of cells in G1-phase increased when PAX6 expression was inhibited. The cyclin D1 protein level, as well as the pRB phosphorylation level, decreased as a result of PAX6 down-regulation. The activity of ERK1/2 and p38 was also suppressed in PAX6 knock-down cells. The PAX6 mRNA was highly expressed in lung cancer tissue and lung cancer cell lines. In most patients (about 65%), the relative ratio of PAX6 mRNA in primary NSCLC versus adjacent tissues exceeded 100. Conclusions Our data implicated that PAX6 accelerates cell cycle progression by activating MAPK signal pathway. PAX6 mRNA levels were significantly elevated in primary lung cancer tissues compared to their matched adjacent tissues.

Cell Cycle Protein Cyclin Y Is Associated With Human Non-Small-Cell Lung Cancer Proliferation and Tumorigenesis

BACKGROUND The role of cell cycle protein cyclin Y (CCNY) in non-small-cell lung cancer (NSCLC) is not clear. Hence, the aim of this study was to explore the potential role of CCNY in lung cancer. PATIENTS AND METHODS Real-time quantitative polymerase chain reaction was used for detecting the expression of CCNY mRNA in 60 samples from patients with NSCLC. The functional role of CCNY in NSCLC cells was evaluated by small interfering RNA-mediated depletion of the protein followed by analysis of cell proliferation, anchorage-independent growth, and xenograft growth. RESULTS CCNY mRNA is overexpressed (N = 60) in samples from patients with NSCLC. Furthermore, CCNY mRNA expression positively correlated with histologic types (squamous cell carcinoma vs. adenocarcinomas; P = .048) and with the tumor size (size > 3 cm vs. size ≤ 3 cm; P = .010) in NSCLC. Functionally, CCNY depletion was shown to inhibit cell proliferation and anchorage-independent growth in lung cancer cells. Moreover, the proliferation effects were increased when CCNY was overexpressed in lung cancer cells. Finally, CCNY was shown to support H1299 and 95D xenograft growth in nude mice. CONCLUSION We reported for the first time (to the best of our knowledge) that CCNY was overexpressed in samples of NSCLC. CCNY mRNA expression associated with histologic types of NSCLC and promoted the malignant growth of lung cancer cell line in vivo and in vitro. Thus, these results validated the role of CCNY as a clinically relevant human oncoprotein and warrant further investigation of CCNY as a biomarker and a therapeutic target in NSCLC.

ABCC4 is required for cell proliferation and tumorigenesis in non-small cell lung cancer.

Background Multidrug resistance protein 4 (MRP4), also known as ATP-cassette binding protein 4 (ABCC4), is a member of the MRP/ABCC subfamily of ATP-binding cassette transporters, which are capable of pumping a wide variety of drugs out of the cell. However, little is known about the function of ABCC4 in the proliferation of lung cancer cells. Methods ABCC4 mRNA and protein levels in lung cancer cell lines were measured by real-time polymerase chain reaction and Western blot, respectively. A lentivirus-mediated RNA interference technique was used to inhibit ABCC4 mRNA expression in A549 and 801D cells. The function of ABCC4 in cell growth was investigated by MTS and colony formation assays. The role of ABCC4 in cell cycle progression was evaluated by flow cytometry and Western blot analysis. ABCC4 mRNA levels in 30 pairs of tumors and corresponding matched adjacent normal tissues from non-small cell lung cancer patients were detected by real-time polymerase chain reaction. Results ABCC4 was highly expressed in lung cancer cell lines. ABCC4 expression was markedly downregulated in A549 and 801D cells using the RNA interference technique. Suppression of ABCC4 expression inhibited cell growth. The percentage of cells in G1 phase was increased when ABCC4 expression was suppressed. Phosphorylation of retinoblastoma protein was weakened, originating in the downregulation of ABCC4. ABCC4 mRNA was highly expressed in lung cancer tissue and lung cancer cell lines. Conclusion ABCC4 may play an important role in the control of A549 and 801D cell growth. ABCC4 is a potential target for lung cancer therapy.

PIK3CA gene mutation associated with poor prognosis of lung adenocarcinoma

Purpose PIK3CA gene mutations have been detected in many malignancies, but the frequency of different mutations and their role in the carcinogenesis of lung adenocarcinoma are still unclear. The purpose of this study was to explore the clinical pathological impact and prognostic implications of PIK3CA mutations in lung adenocarcinoma. Methods Five common PIK3CA mutations (E542K, E545K, and E545D mutation in exon 9, H1047R and H1047L mutation in exon 20) were detected by amplification refractory mutation system (ARMS) allele-specific polymerase chain reaction (PCR), in 122 patients with lung adenocarcinoma. The relationships were studied between these mutations and various clinicopathologic variables (age, lymph node status, distant metastasis, clinicopathologic stage, smoking status, and progression-free survival). Results In total, 25 mutations were identified, of which 24 mutations were clustered in exon 20, and one mutation in exon 9. The most common mutations were H1047R (18 out of the 122 patients, 14.8%) in exon 20. PIK3CA-mutated tumors were more frequently found in patients with lymph node positive metastasis status (P < 0.05). There was no significant association between PIK3CA mutations and age, distant metastasis, smoking status, or clinicopathologic stage. However, mutations were found less frequently in the early clinicopathologic stage patients (six in 50 cases, 12%) than in advanced stage (19 in 72 cases, 26.4%). Higher frequency of H1047R mutations was associated with poor prognosis, and this association reached statistical significance (P < 0.05). Conclusion Our data indicate that the PIK3CA mutations H1047R and H1047L are significant genetic alterations in lung adenocarcinoma. Among lung adenocarcinoma patients who underwent curative resection, PIK3CA mutations were associated with shorter progression-free survival. Our findings demonstrated a significant role of PIK3CA in lung adenocarcinoma.

Serological investigation of the clinical significance of fascin in non-small-cell lung cancer

OBJECTIVES Fascin, a conserved actin bundling protein family member, is frequently up-regulated in various cancer types, including non-small-cell lung cancer (NSCLC), and it plays increasingly important roles in the progression of tumor invasion and metastasis. However, variations in the serum fascin level in tumor patients are usually neglected. MATERIALS AND METHODS In the present study, serum samples from 501 stage I-IV NSCLC patients and 109 healthy volunteers were investigated by an enzyme-linked immunosorbent assay. RESULTS The serum fascin level was markedly increased in the NSCLC patients (P < 0.05), particularly in advanced cases (P < 0.01), compared with that in the healthy controls. We also found that the serum fascin level was significantly correlated with female sex (P = 0.02), non-smoking status (P = 0.044), and adenocarcinoma histology (P < 0.001), with a higher positive rate relative to each counterpart. Furthermore, our results suggested that the serum fascin level reflects the aggressive progress of both lymphatic (P < 0.001) and distant (P < 0.001) metastases in NSCLC. A survival analysis of 283 eligible patients who underwent a follow-up examination after 3 years revealed that the patients in the serum fascin-positive group had a significantly lower overall survival rate compared with those in the negative group for 134 non-distant metastatic (stage M0) cases (P = 0.044). A subsequent Cox regression analysis revealed that the serum fascin level was an independent prognostic factor for M0-stage NSCLC (univariate, P = 0.001; multivariate, P = 0.038). CONCLUSION Our study suggests that the serum fascin level is an effective indicator of tumor aggressiveness, and that it plays an important role in the prognosis of NSCLC, particularly for non-distant metastatic patients.

The role of ZFX in non-small cell lung cancer development.

Zinc finger protein, X-linked (ZFX) is a transcription factor encoded by its gene on the mammalian X chromosome, and functions to control survival and activity of stem cells and lymphocytes. However, little is known about the role of ZFX in tumorigenesis. The function of ZFX in cell proliferation was investigated by the lentivirus-mediated short hairpin RNA interference (shRNA) approach in non-small cell lung cancer (NSCLC) cell culture lines. The expression profiles of ZFX in 49 pairs of tumors and corresponding matched adjacent normal tissues from NSCLC patients were examined by real-time PCR. The specific knockdown of ZFX by shRNA significantly inhibited cell viability and reduced colony formation of 95D cells. And ZFX silencing resulted in cell cycle arrest in G0/G1 phase. In addition, ZFX was overexpressed and correlated with lymph node metastasis in samples from 49 NSCLC patients. We reported for the first time that ZFX may play an important role in cell growth control and cell cycle progression of 95D cells. Furthermore, ZFX was overexpressed in samples of NSCLC and ZFX mRNA expression associated with lymph node metastasis. Therefore, our findings suggest that ZFX would be a potential target to development of therapies for NSCLC.

Clinical Significance and Diagnostic Value of Survivin Autoantibody in Non-small Cell Lung Cancer Patients

BACKGROUND AND OBJECTIVE Lung cancer is one of the most common cancers worldwide and causes more deaths per year than other cancers. Autoantibody was one hotspot in tumor marker research. But the clinical value of Survivin autoantibody is not clear in lung cancer patients at present. The aim of this study is to determine whether Survivin autoantibody could be used as a diagnostic factor of lung cancer and what the clinical value of Survivin autoantibody was in nonsmall cell lung cancer (NSCLC). METHODS Survivin cDNA sequence was obtained by RT-PCR and inserted into prokaryotic expression vector pET30a(+). Fusion protein of Survivin was expressed in E. coil BL21(DE3). SDS-PAGE and Western blot were used to confirm the fusion protein. The Survivin protein was purified by Ni-NTA agarose affinity chromatography. At last, indirect ELISA was used to detect Survivin autoantibody level in the serums of 215 samples from NSCLC patients and serums from 20 samples of nonmalignant lung disease patients as well as 89 samples of healthy persons were also detected as control. RESULTS The recombinant vector pET30a(+)/Survivin was contructed and the Survivin protein is successfully expressed in E.coil BL21(DE3) as inclusion body. Indirect ELISA was done to detect Survivin autoantibody in these serums using purified Survivin protein. It was shown that the positive rate of Survivin autoantibody was 19.5%, with a specificity of 88.9% in NSCLC patients. The expression of Survivin autoantibody has correlation with the volume of tumor and the metastasis of tumor in NSCLC patients (P < 0.05). In our research, positive detection rate of NSCLC was much improved by detecting Survivin autoantibody combined with CEA compared to other tumor markers combined with CEA. CONCLUSIONS In this study, purified fusion protein of Survivin was successfully obtained. Indirect ELISA for detecting Survivin autoantibody in serum of NSCLC patients using purified Survivin protein was established. Our results indicated that Survivin autoantibody as a latent value of tumor marker could be used for clinical diagnosis in NSCLC patients.

Association between VEGFR-3 expression and lymph node metastasis in non-small-cell lung cancer.

Vascular endothelial growth factor receptor (VEGFR)-3 is considered to be associated with lymphangiogenesis. The aim of the present study was to identify the clinical significance of VEGFR-3 expression and lymph node metastasis in patients with non-small-cell lung cancer (NSCLC). Lung tumor tissue samples and 196 lymph nodes from 52 patients with NSCLC were analyzed. In addition, lung tissue samples and 8 lymph nodes from 10 patients with lung diseases other than cancer were included as controls. Semiquantitative multiplex reverse transcription technology was applied to measure the mRNA expression levels of VEGFR-3, while VEGFR-3 protein expression levels were assessed immunohistochemically. The total number of lymphatic vessels was counted and the microlymphatic vessel density (MLVD) was calculated. The results indicated that the VEGFR-3 mRNA expression level in lymph node tissue from the group with lymph node metastasis was significantly lower compared with the group without lymph node metastasis (0.281±0.166 vs. 0.158±0.158; t=4.849; P<0.001). The VEGFR-3 mRNA expression levels in the lung tumor tissue of the NSCLC patients exhibited no statistically significant difference between the lymph node metastasis and lymph node non-metastasis groups (0.139±0.137 vs. 0.142±0.123; t=0.08; P>0.05). In addition, in the lymph node metastasis group, there was no statistically significant difference between the metastasis-positive and -negative lymph nodes (0.158±0.158 vs. 0.123±0.115; t=0.993; P>0.05) with regard to VEGFR-3 mRNA expression. Morphologically, VEGFR-3 immunoreactivity was primarily localized in the cytoplasm of the lymphatic endothelial cells, as well as a number of the cancer cells. MLVD was much higher in the lung tissue surrounding the tumor than in the tumor tissue, and was significantly higher in the lymph node metastasis group than in the lymph node non-metastasis group. VEGFR-3 expression levels were shown to correlate with lymph node metastasis in NSCLC patients, thus, may be a useful biomarker for lymph node metastasis prediction in NSCLC. MLVD is a key indictor of lymphatic vessel metastasis in NSCLC. An enhanced MLVD indicates lymphangiogenesis and lymphatic node metastasis, and may be an important predictor for tumor monitoring and prognosis.

Serum anti-CCNY autoantibody is an independent prognosis indicator for postoperative patients with early-stage nonsmall-cell lung carcinoma.

Cyclin Y (CCNY) is a novel cyclin and almost nothing is known about its role in human cancers. To investigate the clinical significance of serum anti-CCNY autoantibodies in nonsmall-cell lung carcinoma (NSCLC), the serum levels of CCNY protein in 264 patients with NSCLC, 103 patients with tuberculosis, and 89 healthy controls were analyzed by immunohistochemistry. The result shows that, compared with normal lung tissues, the NSCLC tissues contained higher levels of CCNY protein. The levels of anti-CCNY autoantibodies were higher in the sera of the patients with NSCLC than in the sera of the healthy controls (P < 0.001) or the patients with tuberculosis (P = 0.027). Moreover, in a Cox regression analysis, anti-CCNY autoantibody was an independent factor that predicted poor prognosis for postoperative patients with early-stage NSCLC (P = 0.026) as well as for those with distant metastasis (P = 0.012). Our data indicated that Anti-CCNY autoantibody may be useful as a latent tumor marker to facilitate diagnosis and may represent a novel prognostic indicator for patients with early stage NSCLC.