H226细胞和肺癌及其淋巴结转移组织CPA4表达以及生物学功能研究

Expression of CPA4 in lung cancer tissues and the corresponding metastatic lymph node tissues and its functional roles

目的肺癌的发病率和死亡率均居恶性肿瘤的首位。淋巴结转移是肺癌最常见的转移方式,也是制定肺癌治疗策略与判断预后的重要指标。大量研究发现,羧肽酶家族在肿瘤的转移中发挥着重要作用。本研究探讨羧肽酶A4(carboxypeptidase A4,CPA4)在肺癌细胞中的生物学功能,及其在肺癌组织及其配对淋巴结组织中的表达及其临床意义。方法采用实时荧光定量PCR和蛋白质印迹法检测siRNA干扰人肺癌细胞H226中CPA4的表达情况。采用Transwell小室法检测细胞的体外侵袭和迁移能力。采用免疫组化的方法检测购于西安艾丽娜生物科技有限公司的8例正常肺、46例肺癌及其配对淋巴结转移组织中CPA4表达水平。结果 RT-PCR以及蛋白质印迹检测结果显示,siRNA-CPA4较对照组能显著抑制肺癌细胞CPA4的蛋白和mRNA水平。侵袭实验结果显示,siRNA-CPA4组穿膜细胞数(43.14±7.82)显著低于对照组(114.57±27.64),差异有统计学意义,t=6.579,P<0.001。迁移实验结果显示,CPA4siRNA组细胞迁移数目为143.67±14.42,与阴性对照组(276.78±8.07)相比显著降低,差异有统计学意义,t=24.160,P<0.001。免疫组织化学结果显示,CPA4在8例肺、46例肺癌及其配对淋巴结组织中的阳性表达率分别为1/8、50.0%(23/46)和76.1%(35/46)。进一步分析发现,CPA4在肺癌淋巴结转移组织中的表达水平显著高于原发癌组织,t=5.167,P<0.001。CPA4阳性表达与肿瘤病理类型、分化程度和肿瘤分期密切关联,差异有统计学意义,均P<0.05;但与患者的年龄、性别和浸润深度差异无统计学意义,均P>0.05。结论 CPA4促进人肺癌细胞H226的体外侵袭和迁移,且人肺癌原发瘤组织及其配对淋巴结组织中显著高表达且呈递增趋势,可能成为抑制肺癌淋巴结转移的潜在靶点。

OBJECTIVE The incidence and mortality of lung cancer rank first in all malignant tumors.Lymph node metastasis is the most common mode of metastasis in lung cancer,and it is also an important indicator for treatment strategies and prognosis.Most studies demonstrated that the Carboxypeptidase family play important roles in cancer metastasis.In this study,we investigated the effect of CPA4 on human lung cancer cell,and clinical significance of its levels in lung cancer tissues and corresponding lymph node metastasis tissues.METHODS Real-time quantitative PCR and Western Blotting were used to detect the expression of CPA4 in lung cancer cells after transfecting CPA4 interference sequence(siRNA-CPA4)and the negative control sequence.Transwell Assay was used to detect the effect of CPA4 on cell invasion and migration.CPA4 expression was detected in 8 normal lung tissues and 46 lung cancer tissues and corresponding lymph node metastasis tissues by immunohistochemisty.RESULTS RT-PCR and Western Blotting demonstrated that siRNA-CPA4 significantly decreased CPA4 mRNA level in H226 cells compared with the control cells.Invasion assay showed that the number of transmembrane cells(43.14±7.82)in the CPA4 siRNA group was significantly lower than that in siRNAscramble group(114.57±27.64).Similarly,the number of cell migration in CPA4 siRNA group was 143.67±14.42,which was significantly lower than that in negative control group(276.78±8.07).Immunohistochemistry results demonstrated that the positive rate of CPA4 in lung normal tissues,cancer tissues and lymph node metastatic tissues was 1/8,50.0%(23/46)and 76.1%(35/46),respectively.Importantly,the levels of CPA4 in the lymph node metastatic tissues was significantly higher than that of corresponding primary lung cancer tissues(t=5.167,P<0.001).The clinical correlation analysis found that high levels of CPA4 was closely associated with the tumor pathological type,differentiation and stage,but there was no significant difference with the age,sex and the depth of invasion.CONCLUSIONS CPA4 could promote the invasion and migration of human lung cancer cell H226 in vitro.The levels of CPA4 were gradually elevated in primary lung cancer and corresponding lymph node metastasis tissues,which could be the candidate the target for lung cancer treatment.