Clinical significance of KLF15 expression in human lung adenocarcinoma

AIM: To explore the clinical significance of Krüpple-like factor 15 (KLF15) protein expression in the patients with lung adenocarcinoma for exploring the therapeutic and prognositic biomarkers of lung cancer. METHODS: Four cases of lung adenocarcinoma tissues and matched adjacent tissues were collected from our hospital, and the expression of KLF15 protein in these tissues was analyzed by Western blot. At the same time, 72 cases of archived paraffin-embedded samples and clinical data of the patients with lung adenocarcinoma were also collected. The KLF15 protein expression in the archived paraffin-embedded lung adenocarcinoma samples was detected by immunohistochemical staining. The correlations between KLF15 protein expression and clinical characteristics of the patients including prognosis were also analyzed. In addition, the KLF15 protein was up-regulated in A549 cells, and then the effects of KLF15 protein on the viability of the cells were measured by CCK-8 assay. RESULTS: The protein expression of KLF15 in the 4 cases of lung adenocarcinoma tissues was significantly lower than that in matched paracancerous tissues. Fifty-three cases of lung adenocarcinoma specimens showed low expression or no expression of KLF15 protein in total 72 cases (73.6%). The 5-year survival rate of the patients with high expression of KLF15 protein in their specimens was higher than that of the patients with the low expression of KLF15 protein (P<0.01), and the expression of KLF15 protein was significantly correlated with the pathological staging (P<0.01) and T stage (P<0.01) of the patients with lung adenocarcinoma. Furthermore, the low expression of KLF15 protein was an important poor prognostic indicator of the patients. Up-regulation of KLF15 protein in the A549 cells significantly inhibited the growth of the cells. CONCLUSION: KLF15 inhibits the growth of lung adenocarcinoma cells. It could be used as a therapeutic target and a prognostic biomarker for the patients with lung adenocarcinoma.     

Am80 inhibits neointima hyperplasia by promoting interaction of KLF4 with RARα

AIM: To investigate the inhibitory effect of Am80 on neointima hyperplasia in carotid arteries after balloon injury and to observe the interaction between Krüppel-like factor 4 (KLF4) and retinoic acid receptor α (RARα).METHODS: Neointima hyperplasia in carotid arteries was observed by hemotoxylin and eosin staining. The expression of KLF4 and cyclin D1 was examined by immunostaining and Western blotting analysis. To detect the interaction between KLF4 and RARα in the vascular tissue, the injured arteries were harvested, and the protein extracts were prepared and subjected to co-immunoprecipitation assay.RESULTS: Compared with injured group, Am80 significantly reduced neointimal hyperplasia and the thickness ratio of intima to media. Am80 not only up-regulated KLF4 or RARα expression in caro-tid arteries, but also increased the interaction between KLF4 and RARα at tissue levels.CONCLUSION: Am80 inhibits neointima hyperplasia in carotid arteries after balloon injury by promoting the interaction between KLF4 and RARα.     

Effect of KLF4 on viability,apoptosis of colorectal cancer cells and chemosensitivity to cisplatin

AIM:To investigate the effect of Krüppel-like factor 4 (KLF4) on the viability, apoptosis and cisplatin chemosensitivity of colorectal cancer cells. METHODS:KLF4 expression in colorectal cancer cell lines Caco2, SW480 and HCT116 was detected by Western blot. The SW480 cells were divided into pcDNA3.1 group (transfected with pcDNA3.1 empty plasmid), pcDNA3.1-KLF4 group (transfected with pcDNA3.1-KLF4 expression plasmid) and pcDNA3.1-KLF4+cisplatin group (treated with 1 mg/L cisplatin for 48 h after pcDNA3.1-KLF4 was transfected into SW480 cells). The protein levels of KLF4, p-IκBα, cyclin D1 and survivin were determined by Western blot. The cell viability was measured by CCK-8 assay. The apoptotic rate was analyzed by flow cytometry. The content of reactive oxygen species(ROS) was measured by DCFH-DA probe. RESULTS:The expression of KLF4 in the colorectal cancer cells were significantly lower than that in the human colon mucosal epithelial NCM460 cells (P<0.05). Compared with pcDNA3.1 group, the protein expression of KLF4 in pcDNA3.1-KLF4 group was significantly increased (P<0.05). Compared with pcDNA3.1 group, the cell viability and the protein expression of cyclin D1 and survivin were significantly decreased, and the apoptotic rate, the content of ROS and the protein level of p-IκBα were significantly increased in pcDNA3.1 group (P<0.05). Compared with pcDNA3.1-KLF4 group, the cell viability and the expression of cyclin D1 and survivin proteins were significantly decreased, and the apoptotic rate, the content of ROS and the protein level of p-IκBα were significantly increased in pcDNA3.1-KLF4+cisplatin group (P<0.05). CONCLUSION:Upregulation of KLF4 gene expression in colorectal cancer cells reduces the cell viability, induces apoptosis and increases the chemosensitivity of the cells to cisplatin. The mechanism may be related to the enhancement of intracellular ROS content and down-regulaton of the phosphorylation level of IκBα, the key molecule of NF-κB signaling pathway.     

Effect of Yiqi Huayu Huatan decoction on expression of KLF15, HMGB1, NF-κB and its downstream inflammatory factors in rats with UUO-induced renal interstitial fibrosis

AIM: To observe the effect of Yiqi Huayu Huatan decoction (YHHD) on unilaterral ureteral obstruction (UUO)-induced renal interstitial fibrosis in rats, and to investigate the possible mechanism. METHODS: Female SD rats (n=48) were randomly divided into sham group, model group, telmisartan group, and low-, middle-and high-dose YHHD groups, with 8 rats in each group. The UUO model rats was established by ligating left ureter. The rats in sham group and model group were treated with equal volume of normal saline, others were treated with the corresponding drugs daily. After 12 weeks, the rats were sacrificed. The serum samples were collected for determining the concentrations of cystatin C (Cys-C) and uric acid (UA). The morphological changes of the renal tissue were observed by PAS staining. The collagen fiber was observed by Masson staining. The mRNA expression of Krüppel-like factor 15 (KLF15), high-mo-bility group box protein 1 (HMGB1), nuclear factor-κB (NF-κB), IκB, monocyte chemotactic protein-1 (MCP-1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), fibronectin (FN), collagen type I (Col I) and Col-Ⅳ was detected by real-time PCR. The protein expression of KLF15, HMGB1 and NF-κB was detected by Western blot. The protein expression of MCP-1 was determined by the method of immunohistochemistry. RESULTS: Compared with sham group, the deposition rate of collagen fibers and the concentration of Cys-C in model group were significantly increased (P<0.05), the mRNA and protein expression of KLF15 was significantly down-regulated (P<0.05), while the mRNA expression of HMGB1, NF-κB, IκB, MCP-1, IL-1β, TNF-α, FN, Col I and Col Ⅳ and the protein expression of HMGB1, NF-κB and MCP-1 were significantly up-regulated (P<0.05). Compared with model group, the deposition rates of collagen fibers in middle-and high-dose YHHD groups and telmisartan group were significantly decreased (P<0.05), with down-regulated protein expression of HMGB1 and NF-κB and mRNA expression of IL-1β and TNF-α (P<0.05). The protein expression of KLF15 was significantly up-regulated in high-dose YHHD group and telmisartan group (P<0.05), while the protein expression of MCP-1 and the mRNA expression of FN were significantly down-regulated (P<0.05). The mRNA expression of KLF15 was significantly up-regulated in high-dose YHHD group (P<0.05), while the mRNA expression of MCP-1, Col I and Col IV was significantly down-regulated (P<0.05). The mRNA expression of NF-κB and IκB was significantly down-regulated and the concentration of Cys-C was significantly decreased in each dose of YHHD groups and telmisartan group (P<0.05). No significant difference of UA level among the groups was observed. CONCLUSION: YHHD alleviates renal interstitial fibrosis in a dose-dependent manner, and YHHD at high dose shows the most obvious effect. The mechanism may be associated with the up-regulation of KLF15 and the down-regulation of HMGB1, NF-κB and its downstream inflammation-related factors in the renal tissue.     

The study on the relationship between transforming growth factor-β1 and lung carcinoma

AIM: To explore the relationship between the expression of transforming growth factor-β1 (TGF-β1) and the pathogenesis of lung carcinoma, and the influence of radiotherapy on plasma TGF-β1 level of patients with lung carcinoma. METHODS: By immunohistochemical method, the expression of TGF-β1 was examined. An enzyme-linked immunoadsorbent assay (ELISA) was used to quantify the plasma TGF-β1 levels in different time as before radiotherapy, at the end of radiotherapy, and at the time of follow-up 6 months after radiotherapy, respectively. The changes of quantity of TGF-β1 in different time above were analysed statistically. RESULTS: There was a significant increase in TGF-β1 expression in the carcinoma compared with normal lung tissue. The mean TGF-β1 level in the 39 lung carcinoma patients before radiotherapy was (11.0±1.5) μg/L, which was significantly higher than control group (3.8±0.2 μg/L) (P＜0.05); but the plasma TGF-β1 level after radiotherapy was significantly lower (5.6±0.5 μg/L) compared with the level before radiotherapy (P＜0.05), and there was not significant differences compared with control group (P＞0.05). At the time of follow-up 6 months, the patients of lung carcinoma had a significantly higher plasma TGF-β1 level (11.3±1.2 μg/L) compared with the level at the end of radiotherapy (P＜0.05), but there was not significant differences compared with before radiotherapy (P＞0.05). Not significant difference was found in TGF-β1 levels among different histologic types. CONCLUSION: These data demonstrated that TGF-β1 was associated with the pathogenesis of lung carcinoma, and it may be a useful tumor marker in patients with lung carcinoma.     

Hypoxia-induced caveolin-1 up-regulation is involved in migration and invasion of lung adenocarcinoma A549 cells

AIM:To investigate the regulatory role of hypoxia mimic reagent cobalt chloride (CoCl2) on caveolin-1 (Cav-1) generation and the influence of Cav-1 on the abilities of migration and invasion of human lung adenocarcinoma A549 cells. METHODS:The concentrations of Cav-1 and hypoxia-inducible factor (HIF)-1α in pleural effusion of the patients with lung cancer (MPE) or tuberculous pleurisy (TBPE) were detected, and the correlation was also compared. A549 cells were treated with CoCl2 at different concentrations and time in the presence or absence of HIF-1α inhibitor YC-1. The concentrations of Cav-1 and HIF-1α in the cell supernatants were measured by ELISA. The effects of Cav-1 induced by CoCl2 on the migration and invasion of A549 cells were determined by scratch test and Transwell invasion trial, respectively. RESULTS:The levels of Cav-1 and HIF-1α in MPE were significantly higher than those in TBPE. There was a highly positive correlation between Cav-1 and HIF-1α levels in the pleural effusion. CoCl2 induced the generation of Cav-1 and HIF-1α in A549 cells in a concentration- and time-dependent manner, the peak occurred at 200 μmol/L or 24 h, while the concentration over 200 μmol/L or after treated over 24 h, a concentration- or time-dependent inhibition was observed. HIF-1α inhibitor YC-1 concentration-dependently inhibited the generation of HIF-1α and Cav-1 induced by CoCl2 in A549 cells. CoCl2 enhanced A549 cells migration and invasion, with 200 μmol/L played the strongest role, which were down-regulated significantly in the presence of YC-1. CONCLUSION: The alteration of hypoxia-induced Cav-1 generation might be involved in the migration and invasion of A549 cells. A possible role for HIF-1α is indicated in Cav-1 generation.     

Identification of specific serum biomarkers in gastric adenocarcinoma patients

AIM: To screen the possible serum biomarkers for the diagnosis of gastric adenocarcinoma. METHODS: The surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was used to screen the serum samples from 109 cases of gastric adenocarcinoma and 106 control subjects (60 healthy subjects, 30 patients with chronic superficial gastritis and 16 cases of chronic atrophic gastritis). The differentially-expressed protein peaks were selected and isolated with high-performance liquid chromatography (HPLC), and processed with enzyme before liquid chromatography-mass spectrometry tandem mass spectrometry (LC-MS/MS) analysis. The data mining was performed with software Xcalibur program component Bioworks 3.2. RESULTS: Three differentially-expressed protein peaks were selected as potential serum biomarkers of gastric adenocarcinoma patients.The m/z peak at 5 906.5 showed the increase (8.53±4.33 in cancer group, and 0.88±0.31 in control group). The m/z peaks at 6 635.7 and 8 716.3 showed the decrease (6.54±2.44 and 0.93 ± 0.29, respectively, in cancer group and 17.56±4.43 and 2.16±0.98, respectively, in control group, P<0.01). The 3 m/z peaks were identified as fibrinogen α-chain, apolipoprotein A-II and apolipoprotein CI,respectively. The combined use of the 3 biomarkers distinguished the samples in the cancer patients out of the controls at a sensitivity of 93.85% (61/65) and a specificity of 94.34% (50/53). CONCLUSION: The fibrinogen α-chain, apolipoprotein A-II and apolipoprotein CI identified as potential markers for gastric adenocarcinoma show diagnostic values in clinical application.     

Effect of nicotine in vitro on expressions of apoptosis-related genes in human lung adenocarcinoma cells by cDNA microarray

AIM: To investigate effect of nicotine on growth of human lung adenocarnoma cells and expressions of apoptosis-related gene. METHODS: Lung adenocarcinoma cell line, SPC-A-1, was cultured in the presence of various concentrations (1-1 000 μg/L) of nicotine for 48 hours. MTT was applied to evaluate effect of nicotine in vitro on growth of SPC-A-1 cell line. After SPC-A-1 cells were treated with 100 μg/L for 48 hours, cDNA expression profile microarray was used to detect the expressions of 451 apoptosis-related genes in SPC-A-1 cell line. RESULTS: Significant proliferation in SPC-A-1 cells treated with nicotine (1-10 μg/L) was observed, but this effect decreased with increase in concentration of nicotine in culture. Growth inhibition rate of 1, 10, 100, 1 000 μg/L of nicotine was 27%, -40%, -40% and -93%. Microarray detection showed that significantly different expressions appeared in 80 of 451 apoptosis-related genes. 29 apoptosis-promoted genes and 26 apoptosis-inhibited genes were up-regulated significantly (CY3/CY5>2.0), and 25 genes were significantly down-regulated (CY3/CY5<0.5). CONCLUSION: Nicotine may promote growth of human lung adenocarcinoma cell through regulating many apoptosis-related gene expressions.     

Significance of TRPM8 protein expression in lung adenocarcinoma

AIM To investigate the significance of transient receptor potential cation channel subfamily M member 8 （TRPM8） protein expression in lung adenocarcinoma. METHODS The tumor samples from 112 cases of patients with lung adenocarcinoma were collected in our hospital, and 4~5 years of follow-up was conducted. The protein expression of TRPM8 was analyzed by immunohistochemical staining, and the correlations between the TRPM8 protein expression and the clinical characteristics including prognosis of the patients with lung adenocarcinoma were investigated. After TRPM8 protein expression was up-regulated in A549 lung adenocarcinoma cells by lentiviral infection, the proliferation of A549 cells was analyzed by CCK-8 assay and colony formation assay, the cell cycle and apoptosis were analyzed by flow cytometry, and the migration and invasion abilities of the cells were measured by scratch experiment and Transwell assay. The TRPM8 protein expression was stably up-regulated in H1299 cells by lentiviral infection, and then the left and right buttocks of the immunodeficient mice were subcutaneously injected with empty vector control cells and TRPM8-overexpressing cells, respectively. The effects of TRPM8 on the growth of H1299 cell-derived xenograft tumor in immunodeficient mice were evaluated. RESULTS The 4~5-year survival rate in the patients with high TRPM8 protein expression was significantly higher than that in the patients with low expression of TRPM8 protein （P=0.017）. The tumor maximum diameter in the patients with high TRPM8 protein expression was significantly smaller than that in the patients with low TRPM8 protein expression （P=0.028）. The viability, the number of colonies and the migration and invasion abilities of TRPM8-overexpressing A549 cells were significantly decreased as compared with empty vector and parental cells （P<0.01）. The results of flow cytometry analysis showed that the proportion of A549 cells at S stage was significantly increased in TRPM8 overexpression group as compared with empty vector group （P<0.01）. The growth rate and the weight of TRPM8-overexpressing H1299 cell-derived xenograft tumor in immunodeficient mice were significantly lower than those in empty vector group （P<0.01）. CONCLUSION TPRM8 is a tumor suppressor in lung adenocarcinoma, and low expression of TRPM8 protein was a poor prognositic indicator of patients with lung adenocarcinoma.     

Expression of CD44s in lung cancer and its significance

AIM: To investigate expression of CD44s in lung cancer and it's clinical significance. METHODS: A total of 117 primary lung cancer from patients were examined for CD44s expression by immunohistochemical staining. RESULTS: CD44s mostly expressed in non-small cell lung cancer (NSCLC) but not in small ecll lung cancer (SCLC), and squamous cell carcinoma(SCC) showed much stronger expression of CD44s than adenocarcinoma(ADC)(P＜0.05). In comparison of the lung cancer with/ without lymph node metastasis, the latter showed stronger expression of CD44s(P＜0.01). According to TNM, there was a distinct statistic difference between early stage and advanced stage(P＜0.05). CONCLUSION: CD44s might be a better indicant in histological classification of lung cancer, lymph node metastasis, clinical stage and prognosis.     

Polarized distribution of M2 macrophages in marginal region around lung adenocarcinoma and its effect on prognosis

AIM: To explore the polarized distribution of M2 macrophages in the marginal region around lung adenocarcinoma, the marginal/central ratio and their effect on the prognosis. METHODS: Double immunohistochemistry staining was used to determine the distribution and the difference of CD163⁺/CD68⁺ (M2) macrophages in the marginal and central regions in 49 cases of lung adenocarcinoma in situ (AIS), 11 cases of minimally invasive adenocarcinoma (MIA) and 57 cases of invasive adenocarcinoma (IA) in order to explore the effect and mechanism of the polarized distribution and the marginal/central ratio on the progression of lung adenocarcinoma. Single-factor Kaplan-Meier survival curve analysis and multivariate Cox survival analysis were employed to explore the relationship between the polarized distribution of M2 macrophages and the prognosis. RESULTS: Polarized aggregation of M2 macrophages was observed in the marginal region of lung adenocarcinoma compared with that in the central region, and the difference was significant (P<0.01). Based on the median level, they were divided into high polarized group and low polarized group. In low polarized group, M2 macrophage count in AIS was not significantly different from that in MIA or IA. However, in high polarized group, M2 macrophage count in AIS was lower than that in MIA and IA in turn and there were statistically significant differences (P<0.01). Single-factor Kaplan-Meier survival curve analysis and log-rank test result showed that the number of M2 macrophages in the marginal region and marginal/central ratio were negatively correlated to the survival time (χ²=44.71, P<0.01; χ²=21.75, P<0.01). Multivariate Cox survival analysis showed that the high polarized distribution of M₂ macrophages in the marginal region and the marginal/central ratio were independent risk factors for the prognosis (P<0.01). CONCLUSION: There is a polarization effect of M2 macrophages on the marginal region of lung adenocarcinoma. The marginal polarization and the marginal/central ratio are independent risk factors of the prognosis. Therefore, it may be an effective method for the evaluation of the prognosis to judge the marginal polarization by preoperative puncture and to determine the marginal/central ratio of M2 macrophages by postoperative biopsy.     

Relationship of expression of extracellular matrix metalloproteinase inducer and hepatocyte growth factor with lymphoid metastasis and prognosis in non-small-cell lung carcinoma

AIM: To investigate the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and hepatocyte growth factor (HGF) in non-small-cell lung carcinoma (NSCLC) and their relationship with lymphoid metastasis and prognosis.METHODS: Expression of EMMPRIN and HGF in 77 cases of patients with NSCLC was detected immunohistochemically.The relationship of expression of EMMPRIN and HGF with tumor size,smoking,histological type,differentiation,lymphoid metastasis,clinical stage,and prognosis was analyzed.RESULTS: The expressive rates of EMMPRIN and HGF were 68% and 44%,respectively.The expressions of EMMPRIN and HGF were associated positively with lymphoid metastasis (r=0.371 and 0.339，P＜0.01),and inversely with survival time (P＜0.01).No relationship was found between the expression of EMMPRIN,HGF and smoking,tumor size,histological type and differentiation (P＞0.05).The expression of EMMPRIN was associated with the expression of HGF in NSCLC.CONCLUSION: The expression of EMMPRIN and HGF is associated with lymphoid metastasis and prognosis in NSCLC.Overexpression of EMMPRIN and HGF implies infavourable prognosis in NSCLC.     

Expression of GATA3 in human breast cancer tissues and its clinical significance

AIM: To investigate the expression of GATA3 in human breast carcinoma and its clinical significance. METHODS: The expression level of GATA3 in breast cancer tissues from 124 patients was detected by the method of immunohistochemistry and the relationships between GATA3 expression and other clinicopathological factors were analyzed. RESULTS: Low expression of GATA3 in breast cancer tissues was associated with estrogen receptor (ER)/progesterone receptor (PR) negative, high histological tumor grade, p53 mutation and vascular invasion (P<005), but not with age, tumor size,human epidermal growth factor receptor 2 (HER-2) expression and lymph node metastasis (P>005). In all breast cancer tissues, the positive expression rate of GATA3 was 56.4%. The positive expression rate of GATA3 in luminal breast cancer is 684%, higher than that in non-luminal breast cancer (326%, P<005). In all breast cancer tissues, the expression of GATA3 in middle recurrence risk group was higher than that in high recurrence risk group (P<005). CONCLUSION: GATA3 expression in breast cancer is related to differentiation and biological characteristics of the tumor, which can be a factor for evaluation of the treatment and prognosis.