| Objective: p190B RhoGAP (p190-B) is a protein encoded by the ARHGAP5 gene, which is localized to the conserved region of human chromosome 14q12. The amplification at 14q12 has been reported in non-small cell lung cancer (NSCLC). Some researches suggested that p190-B, which is a member of RhoGAP family, could regulate the activity of the Rho family of small GTPases (such as Rac, Rho, and Cdc42). Accordingly, it could affect actin cytoskeletal organization, cell adhesion, polarity, proliferation and migration, which are all important processes during cancer progression. In addition, p190-B may regulate the activity of matrix metalloproteinases (MMPs), promoting the degradation of extracellular matrix and plays an important role in angiogenesis. These findings suggested that the expression of p190-B was associated with the cell motility, especially with the process of invasion and metastasis of tumor. By now, the related studys of p190-B were mainly seen in the breast cancer, no detailed data provided as yet on the effects of p190 RhoGAP in human lung cancer.In the present study, we aimed to define the expression of p190-B in human primary NSCLC, and to further address the relationship between p190-B expression and the clinical pathological features. Furthermore, the possible founction of p190-B on cell proliferation, cell motility and migration were further investigated in A549 cell in vitro. Our results may provide theoretical foundation for the etiology of NSCLC.Methods:1 The expression of p190-B was detected in paraffin-embedded sections of the tumor tissues and the corresponding normal lung tissues from 54 patients with non-small cell lung cancer by Immunohistochemistry, and the relationship between p190-B expression and clinical pathological features was analyzed further.2 The level of p190-B protein and ARHGAP5 mRNA was detected in 23 cases of human non-small cell lung cancer and the matched normal lung tissue by Western blot and Real-time PCR, respectively.3 RNA interference (RNAi) was used to knockdown p190-B expression in A549 cells using lipofectamin transfection. Then, the efficiency of siRNA interference was evaluated by Real-time PCR and Western blot. Next, the effects of ARHGAP5 siRNA transfection on cell proliferation were investigated by Colony formation assay and MTT assay, and the effects on cell migration and movement ability were also evaluated by Wound healing assay and Boyden cabin assay, respectively.Results:1 The expression of p190-B in human primary non-small cell lung cancer1.1 The results of p190-B expression in 54 patients with non-small cell lung cancer by immunohistochemistry1.1.1 The expression of p190-B in tumor tissues and the corresponding normal lung tissuesIn normal lung tissue, p190-B expression could be seen in the macrophages in the alveolar wall or alveolar space, whereas it showed negative expression in alveolar epithelial cells and bronchial epithelial cells. In the tumor tissue, the positive staining of p190-B protein was identified as brownish-yellow granules in cytoplasm of the tumor cells. The expression of p190-B showed positive staining in all the 54 tumor tissues at different intensity. 25.93% of cases were expressed as"+", 27.78% and 46.30% cases were expressed as"++"and"+++", respectively. Among the 54 cases, 40 out of 54(74.07%) cases showed high p190-B expression. All these data suggested that the expression of p190B protein is frequently overexpressed in NSCLC tissue than that in normal lung tissue.1.1.2 The relationship between p190-B expression and clinical pathological parametersAccording to the histological types, p190-B is highly expressed in 89.29% cases (25/28) of adenocarcinoma and 57.69% cases (15/26) squamous cell carcinoma, respectively. It indicated that the expression of p190-B in adenocarcinoma was significantly higher than that in squamous cell carcinoma (P<0.05). According to the status of lymph node metastasis, it showed that p190-B expression was enhanced in the cases with lymph node metastasis compared to the cases without lymph node metastasis. Spearman correlation analysis confirmed that the expression of p190-B protein is getting prominent with the increasing of the lymph node metastasis from N0 to N2 stage, indicating that the p190-B protein expression was positively correlated with lymph node metastasis (r=0.704, P=0.000). In addition, p190-B protein expression was positively correlated with TNM stage of the patients (r=0.713,P=0.000). No relationship was found between p190-B expression with age or gender (P> 0.05).1.2 The level of p190-B protein and ARHGAP5 mRNA in 23 cases of human non-small cell lung cancer and the matched normal lung tissueThe whole cell protein and total RNA were extracted from 23 cases of human NSCLC tumor tissue and the matched normal lung tissue, respectively. Then, the different expression of p190-B protein and ARHGAP5 mRNA (encoding p190-B protein) between them was detected by Western bolt and Real-time PCR method. Western bolt results showed that p190-B protein was upregulated obviously in tumor tissue than that in the paired normal lung tissue. Simultaneously, the level of ARHGAP5 mRNA was overexpressed significantly in 65.2% tumor tissues (15 out of 23 cases) than that in the paired normal lung tissues by Real-time PCR assay. Further analysis showed that the average abundance of ARHGAP5 mRNA was statistically higher in tumor tissues than that in normal lung tissues (P <0.05).All the results demonstrated that p190-B expression was significantly higher in tumor tissue than the corresponding normal lung tissues in human primary NSCLC, especially in the adenocarcinoma. Moreover, p190-B expression was positively correlated with lymph node metastasis and TNM stage of the patients. 2 The effects of p190-B in lung cancer was evaluated using siRNA interference techniqueTo further investigate the biological effects of p190-B in lung cancer, we used RNA interference (RNAi) to knockdown the p190-B expression in A549 cells to observe the role of p190 RhoGAP on cell proliferation, cell motility and migration.2.1 The efficiency of siRNA interfereThe expression of p190-B protein and ARHGAP5 mRNA in A549 cells were detected 48hs after transfected with ARHGAP5 siRNA1, siRNA2 or negative control siRNA by Western Blot and Real-time PCR method. The results proved that the abundance of ARHGAP5 mRNA was significantly inhibited by ARHGAP5 siRNA transfection. In comparison with control group, ARHGAP5 mRNA expression was decreased by 75% and 64% in ARHGAP5 siRNA1 and ARHGAP5 siRNA2 group, respectively. Consistant with Real-time PCR result, p190-B protein was inhibited obviously by ARHGAP5 siRNA1 and ARHGAP5 siRNA2 compared to the control group. All these results indicated that both of the two siRNA sequences could knockdown the expression of p190-B in A549 cell, particularly the siRNA1 sequence. So ARHGAP5 siRNA1 was selected to be used in the subsequent experiments.2.2 Effects of ARHGAP5 siRNA transfection on cell proliferation in A549 cellsWe evaluated the ARHGAP5 siRNA1 impact on the growth of A549 cells by MTT on the cells transfected 24h, 48h, 72h and 96h. The MTT results showed that the growth of A549 cells with specific siRNA transfection was significantly inhibited compared with the control group. The inhibition rates in siRNA1 transfected cells were 10.30%, 19.29%, 21.94% and 21.82% at 24h, 48h, 72h and 96h, respectively. We further observed the effects of ARHGAP5 siRNA transfection on cell proliferation by colony formation assay, and also found that the ARHGAP5 siRNA1 reduced the cell colony number compared with the nonsense siRNA group. The results revealed that ARHGAP5 siRNA transfection could inhibit the proliferation of A549 cells, and these provide further support that the important role of ARHGAP5 expression on the maintenance of cell proliferation activity.2.3 Effects of ARHGAP5 siRNA transfection on cell migration in A549 cellsIn this study, we applied two methods to evaluate the effects on cell migration of A549 cells with ARHGAP5 siRNA1 transfection. The first method is Boyden chamber assay. Cells which have acrossed the membrane with ECM were counted and photographed at 24 hour. In ARHGAP5 siRNA1 group, cells in lower chamber were much less than that in control group. Statistical analysis further confirmed the result above (P<0.05). The second method is"monolayer wound healing"assay. The results showed that the speed which cells migrated towards the scratch was lower in ARHGAP5 siRNA1 transfected cells when compared with the control cells. The difference could be detected 24 hour after the scratch. The wound almost trend to heal in control cell at 48hs, while it only covered 40% of the wound in ARHGAP5 siRNA1 transfected cells. These two experimental results showed that the ability of cell migration was reduced by ARHGAP5 siRNA1 transfection in A549 cells. It might suggest that p190-B plays crucial roles in the invasion and metastasis during tumor progression.Conclusion:1 p190-B expression was significantly higher in tumor tissue than that in the corresponding normal lung tissue in human primary NSCLC, especially in the adenocarcinoma.2 The expression of p190-B was correlated with lymph node metastasis and the TNM stage of the patients positively, suggested that the expression of p190-B was associated with the invasion and metastasis of NSCLC.3 p190-B expression was successfully knockdown by specific ARHGAP5 siRNA transfection.4 ARHGAP5 siRNA transfection could inhibit cell proliferation, indicated that ARHGAP5 expression is critical on the maintenance of cell proliferation activity. 5 ARHGAP5 siRNA1 could reduce the ability of cell migration, suggested that p190-B plays important roles in the invasion and metastasis during tumor progression.
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