| BACKGROUND:Lung cancer has become one of the most common malignant tumor in the world according to recent researches, which contains80%-85%of non-small cell lung cancer (NSCLC). As always, surgical resection, chemotherapy and radiotherapy are the main treatment for the patients with non-small cell lung cancer because of lacking effective means in early diagnosis and treatment. However, postoperative survival is not ideal with less than20%of five-year survival rate, and is always determined by tumor metastasis and postoperative recurrence. Therefore, researches about relevant molecular markers that may be related with lung cancer occurrence, development and metastasis, and their interaction mechanism will be extremely important for evaluating the early diagnosis, treatment and prognosis of lung cancer.PBK/TOPK (PDZ-binding kinase/T-LAK cell-originated protein kinase) is a novel protein kinase, early found in lymphokine activated killer T cells (T-LAK) with322amino acids and involved in regulating the proliferation of malignant tumors. PBK/TOPK expression is higher in malignant tumor, but it is difficult to detect in other normal tissues except normal testicular tissue and some of the embryonic tissue. Ki-67, a common cellular proliferation marker, generally applied to the clinicopathologic diagnosis. The p53gene is known as a inhibitory gene, and it contains two types. The one is wild type p53, which is associated with DNA repair and cell apoptosis. The other is mutant p53, which is closely related with the formation of tumor. As present, there is still not a study to report that the molecular mechanism of PBK/TOPK that may be associated with Ki-67and p53in non-small cell lung cancer, so we will carry on this research.OBJECTIVES:1. To explore PBK/TOPK expression and its prognostic significance in NSCLC;2. To explore the correlation of the expression of PBK/TOPK with Ki-67and p53and its prognostic significance in NSCLC;3. To explore the effect of PBK/TOPK down-regulation on the migration, proliferation and survival of non-small cell lung cancer cells;4. To explore the correlation of PBK/TOPK with Ki-67and p53in non-small cell lung cancer cells;MATERIALS AND METHODS:1. The expression of PBK/TOPK in normal lung tissue, lung benign lesion, non-small cell primary lung cancer and lymph node metastases, and its prognostic significanceImmunohistochemical SP method was used to detect the expression of PBK/TOPK in30cases of normal lung tissue,30cases of lung benign lesions,279cases of non-small cell primary lung cancer and32cases of lymph node metastases. Through imageproPlus6.0of computer image analysis software (IPP), the positive unit (PU) of protein expression was quantificationally tested, statistical analysis and postoperative survival analysis were analyzed together with clinical pathological characters and follow-up data.2. The expression relationship and prognostic significance of PBK/TOPK with Ki-67and p53in non-small cell lung cancerImmunohistochemical SP method was used to detect the expression of PBK/TOPK, Ki-67and p53in279cases of non-small cell lung cancer tissues, and their corelation and survival prognosis were analyzed by semi-quantitative method combining with clinical pathological characters and follow-up data.The reference standard of semi-quantitative analysis:Negative:colorless or pale yellow. Positive:tan or brown. The positive percentage of cells with the expression of PBK/TOPK, Ki-67and p53was tested in non-small cell lung cancer, taking the average value as a threshold of semi-quantitative analysis.3. Effect of PBK/TOPK down-regulation on the migration, proliferation, and survival of non-small cell lung cancer cellsOur study was conducted with interference and control groups. Two kinds of human lung adenocarcinoma cell lines A549and GLC-82with high expression of PBK/TOPK were conformed in our study. The interference group was interfered by using small interfering RNA to down-regulate the expression of PBK/TOPK, and the control group did not make any processing. Scratch test and transwell migration assay were used to detect the effect of PBK/TOPK down-regulation on cell migration, meanwhile, the proliferation and survival of cell were determined by MTT assay and trypan blue staining method, respectively.4. The relationship of PBK/TOPK with Ki-67and p53in non-small cell lung cancer cells Using Real-time quantitative PCR (QRT-PCR), Western blot and cell drop sections, the expression of PBK/TOPK and p53was detected in four kinds of lung cancer cell lines:including three kinds of lung adenocarcinoma cells (A549, H358and GLC-82) and a large cell lung cancer cell (H460). At the same time, the expression of Ki-67was detected by using cell drop immunohistochemical method, and its positive unit (PU) value was quantificationally tested by IPP image analysis software. In order to explore the correlation of PBK/TOPK with Ki-67and p53, two cells with high expression of PBK/TOPK will be interfered by siRNA, then the efficiency of interference will be verified by QRT-PCR and Western blot.5. Statistical analysisAll statistical analysis were carried out with using SPSS13.0statistical software. Two independent-sample t-tests were used between the two sample means. One-way ANOVA test was used in the comparison of multiple sample averages. When the variances are heterogeneous, Dunnett’s T3test were used for multiple comparisons between groups. Otherwise, LSD test were used. The correlation analysis was assessed using Spearman rank correlation. Survival analysis was estimated by Kaplan-Meier method with log-rank test, and Cox’s proportional hazards model was for multivariate analysis. Statistical significance was defined as the2-sided p-value lower than0.05.RESULTS:1. The expression of PBK/TOPK in normal lung tissue, lung benign lesion, non-small cell primary lung cancer and lymph node metastases, and its prognostic significanceThe results of quantitative analysis showed that there was an obvious difference (P=0.000) about PBK/TOPK expression in normal lung tissue, lung benign lesion, non-small cell primary lung cancer and lymph node metastases, and the PU value of PBK/TOPK expressed in the lymph node metastases was higher than those in non-small cell primary lung cancer, lung benign lesion and normal lung tissue. PBK/TOPK expression was associated with histological type, lymph node metastasis, distant metastasis and TNM stage (P values were0.027,0.000,0.000and0.000, respectively), but it did’t found a significant relation with other clinical pathologic characteristics in non-small cell lung cancer (P>0.05).The semi-quantitative analysis showed that the rates of high PBK/TOPK expression in normal lung tissue, lung benign lesion and non-small cell primary lung cancer and lymph node metastases were0%,0%,44.8%and75%, respectively, with a significant statistical significance in the group of comparison (P=0.000). Kaplan-Meier single factor analysis showed that the expression of PBK/TOPK protein, lymph node metastasis, TNM stage and distant metastasis were closely associated with the prognosis of patients with non-small cell lung cancer (all P value is0.000). Multivariate cox proportional hazards model analysis showed that the expression of PBK/TOPK, lymph node metastasis and TNM stage could be served as independent prognostic factors in non-small cell lung cancer (P values were0.000,0.009and0.022, respectively).2. The expression relationship and prognostic significance of PBK/TOPK with Ki-67and p53in non-small cell lung cancerThe results of semi-quantitative analysis showed the high expression rates of PBK/TOPK, Ki-67and p53in279cases of non-small cell primary lung cancer were44.8%,64.9%,49.1%, respectively. There was a significant positive correlation of PBK/TOPK expression with Ki-67and p53(r=0.249, P=0.000; r=0.153, P=0.000, respectively).In this section, the results showed that the5-year survival rates of patients with high expression of PBK/TOPK, Ki-67and p53were12.4%,18.2%and21.2%, respectively. Single factor survival analysis showed that the expression of PBK/TOPK, Ki-67and p53was closely related to the prognosis of patients with non-small cell lung cancer (P values were0.000,0.000and0.003, respectively). The median survival of patients with low expression for both PBK/TOPK and Ki-67was significantly higher than those with PBK/TOPK low and Ki-67high, and those with high expression of PBK/TOPK and Ki-67(P=0.029, P=0.000). The median survival of patients with low expression for both PBK/TOPK and p53was also significantly higher than those with PBK/TOPK high and p53low, and those with high expression of PBK/TOPK and p53(P=0.001, P=0.000). Multivariate Cox proportional hazards model analysis showed that the expression of PBK/TOPK, Ki-67and p53could be used as independent prognostic factors in non-small cell lung cancer (P values were0.000,0.015and0.034, respectively).3. Effect of PBK/TOPK down-regulation on the migration, proliferation, and survival of non-small cell lung cancer cellsThe expression level of PBK/TOPK mRNA was decreased by83.6%and69.9%in GLC82and A549cells by siRNA interference, respectively. The results of scratch test and transwell migration assay suggested that PBK/TOPK could promote cell migration. The cell migration rate of interference group in A549and GLC-82cells was significantly lower than those in the control group,(P values were0.006and0.000, respectively). Transwell migration assay showed the number of cell migration in the interference group was significantly less than those in the control group (P=0.000).The results of MTT assay suggested that PBK/TOPK down-regulation could inhibit cell proliferation. In A549and GLC-82cells, the proliferation ability of cells in interference group was significantly lower than those in control group, with a significant difference after interfering for48hours (P=0.000). The results of trypan blue staining showed that the expression of PBK/TOPK down-regulation was unfavorable for the survival of cells. Compared with control group in A549and GLC-82cells, the survival rates were significantly lower in interference group (P values were0.002and0.005, respectively).4. The relationship of PBK/TOPK with Ki-67and p53in non-small cell lung cancer cellsThe results of QRT-PCR detection showed that PBK/TOPK mRNA expressed in the four types of lung cancer cells, specially was high in H358and GLC-82cells and low in A549and H460cells.The results of western blot and cell drop immunohistochemistry showed that PBK/TOPK protein highly expressed in H358and GLC-82cells but with low expression in A549and H460cells. Tne PU value expressed by PBK/TOPK protein in the four cell lines of group was compared with a statistical significance (P=0.000).The results of QRT-PCR detection showed that p53mRNA expressed in four types of lung cancer cells, specially was high in H358, H460and A549cells but was low in GLC-82cell. The results of western blot and cell drop immunohistochemistry showed that the p53protein highly expressed in H358, H460and A549cells while was low in GLC-82cell. And the results of cell drop immunohistochemistry showed there is a significant difference about the PU value in the four cells (P=0.000).The results of cell drop immunohistochemistry showed that Ki-67protein highly expressed in the four cells without a statistical significance (P>0.05).The expression of PBK/TOPK in A549and GLC-82cells was interfered and declined by69.9%and83.6%, respectively. PBK/TOPK down-regulation significantly inhibited the expression of mutant p53mRNA, declining by74.5%and68.5%in A549and GLC-82cells respectively, and it also down-regulated the expression of p53protein. Otherwise, PBK/TOPK down-regulation inhibited the expression of Ki-67protein in549and GLC-82cell, with a significant difference of PU value (P=0.000).CONCLUSIONS:1. PBK/TOPK expression is correlated with the formation and metastasis of non-small cell lung cancer. And it is also correlated with the histological type, lymph node metastasis, distant metastasis and TNM stage, and can be served as an independent factor for evaluating the prognosis in non-small cell lung cancer.2. PBK/TOPK expression is positively correlated with the expression of Ki-67and p53, and the patients with high expression for both PBK/TOPK and Ki-67or for both PBK/TOPK and p53would have unfavorable prognosis.3. The high expression of Ki-67and p53was closely related to the bad prognosis of patients, both of them can be served as independent prognostic factor in non-small cell lung cancer.4. PBK/TOPK can promote the migration, proliferation and survival of non-small cell lung cancer cells.5. The molecular mechanism of PBK/TOPK is closely related with Ki-67and p53in non-small cell lung cancer. PBK/TOPK expression can regulate the mutant p53, and PBK/TOPK down-regulation can down-regulated the expression of mutant p53mRNA and protein. Otherwise, PBK/TOPK down-regulation can inhibit the expression of Ki-67protein. |
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