缺氧条件下沉默Rab11基因的表达对子宫颈癌细胞系SiHa细胞侵袭和迁移能力的影响

Effect of Rab11 gene expression on the invasion and migration of cervical cancer cell line SiHa inhypoxia

目的通过观察缺氧条件下沉默Rabll基因对子宫颈癌细胞系SiHa细胞侵袭和迁移能力的影响，并探讨其可能的作用机制。方法实验分为4组：缺氧Rabll干扰组（缺氧状态下转染siRNA）、缺氧阴性干扰组（缺氧条件下转染空白无干扰意义的siRNA）、缺氧空白组（缺氧条件下培养）、常氧空白组（常氧条件下培养）。蛋白印迹法检测4组SiHa细胞中Ras相关蛋白11（Rabll）、整合素d5、整合素p3、磷酸化局部黏着斑激酶（p-FAK）、磷酸化磷脂酰肌醇3激酶（p-P13K）及Ras相关的c3肉毒素底物1（Racl）的表达水平；实时荧光定量PCR技术检测4组SiHa细胞中RabllmRNA的表达水平；体外侵袭实验检测4组SiHa细胞的侵袭能力；体外迁移实验检测4组SiHa细胞的迁移能力；免疫荧光法检测4组SiHa细胞中Racl蛋白的表达。结果（1）蛋白印迹法检测显示：①缺氧Rabll干扰组中SiHa细胞Rabll、整合素仪5、整合素133、p-FAK、p-P13K、Racl蛋白的表达水平分别为0．44±0．03、0．30±0．03、0．29±0．03、0．30±0．03、0．30±0．03、0．34±0．04；②缺氧阴性干扰组分别为0．72±0．03、0．73±0．03、0．59±0．03、0．61±O．03、0．59±0．03、0．72±0．03；③缺氧空白组分别为0．73±0．03、0．74±0．03、0．61±0．03、0．62±0．03、0．60±0．03、0．73±0．03；④常氧空白组分别为0．56±0．04、0．33±0．03、0．33±O．03、0．36±0．03、0．35±0．03、0．47±0．03。（2）4组SiHa细胞中RabllmRNA的表达水平，缺氧Rabll干扰组为0．570±0．046，缺氧阴性干扰组为1．442±0．101，缺氧空白组为1．454±0．114，常氧空白组为1．000±0．000。（3）4组SiHa细胞侵袭至小室下层的细胞数分别为，缺氧Rabll干扰组（50±11）个，缺氧阴性干扰组（104±17）个，缺氧空白组（106±16）个，常氧空白组（65±12）个。（4）4组SiHa细胞迁移的数量分别为，常氧空白组（127±12）个，缺氧空白组（169±15）个，缺氧阴性干扰组（161±13）个，缺氧Rabll干扰组（77±13）个。（5）免疫荧光法显示4组SiHa细胞核中周围分布有红色荧光，为Racl蛋白的定位表达。以上检测指标的比较结果均为：缺氧Rabll干扰组〈常氧空白组〈缺氧阴性干扰组〈缺氧空白组；缺氧Rabll干扰组的结果较缺氧空白组、缺氧阴性干扰组均明显降低（P〈O．05）；而缺氧空白组的结果与缺氧阴性干扰组比较，差异无统计学意义（P〉O．05）；缺氧空白组的结果较常氧空白组明显升高（P〈O．05）。结论缺氧促进子宫颈癌细胞系SiHa细胞的侵袭、迁移；缺氧时，Rabll蛋白下调能够抑制SiHa细胞的侵袭、迁移，其机制可能与整合素仅5、整合素133、p-FAK、p-P13K及Racl蛋白表达水平下降有关。

Objective To explore the expression of Ras-related protein 11 （Rabll） in hypoxia, the effect of Rabll on the invasion and migration of cervical cancer cell line SiHa and its possible mechanism. Methods SiHa cells were divided into 4 groups, the normoxic blank group （normal culture in normoxia）, the hypoxic blank group （normal culture in hypoxia）, the negative control group [transfection ofnegative control small interfering RNA （siRNA） in hypoxia], the Rabl 1-siRNA group （transfection of Rabl 1 siRNA in hypoxia）. Western blot was used to examine the expression of Rabl 1, integrin ctS, integrin [33, phosphorylated focal adhesion kinase （p-FAK）, phosphorylated phosphatidylinositol 3 kinase （p-PI3K） protein, together with the expression of Ras correlative C3 creotoxin substrate 1 （Racl）, which was critical in regulating cell invasion. The mRNA expression of Rabl 1 in the 4 groups was detected by realtime-qPCR. The cell invasion was detected by matrigel assay, while the cell migration was detected by transwell assay. Immunofluorescence was used to identify intracellular location of Racl in SiHa cell. Results （1） The expression of Rabl 1, intergrin cL5, intergrin 133, p-FAK, p-PI3K and Racl in the normoxic blank group were 0.56±0.04, 0.33±0.03, 0.32±0.03, 0.36±0.03, 0.35±0.03 and 0.47±0.03, respectively. In the hypoxic blank group, they were 0.73±0.03, 0.74±0.03, 0.61±0.03, 0.62±0.03, 0.60±0.03 and 0.73±0.03, respectively. In the negative control group, their expressions were 0.72±0.03, 0.73±0.03, 0.59±0.03, 0.61 ±0.03, 0.59±0.03 and 0.72±0.03, respectively. While in the Rabl 1-siRNA group, they were 0.44±0.03, 0.30±0.03, 0.29±0.03, 0.30±0.03, 0.30±0.03 and 0.34±0.04, respectively. The expressions of Rabl 1, c5, β3, p-FAK, p-PI3K and Racl were significantly higher in the hypoxic blank group than in the normoxic blank group （P〈0.05）, and were significantly lower in the Rabl 1-siRNA group than in the hypoxic blank group and the negative control group （P〈O.05）. （2） The expressions of Rabl 1-mRNA were 1.000±0.000, 1.454±0.114, 1.442±0.101, 0.570± 0.046 in the normoxic blank group, the hypoxic blank group, the negative control group and the Rabl 1- siRNA group, respectively. It was significantly higher in the hypoxic blank group than in the normoxic blank group （P〈0.05）, and was significantly lower in the Rab 11-siRNA group than in the hypoxic blank group and the negative control group （P〈0.05）. （3） By Matrigel, the invasion cell number in the normoxic blank group, the hypoxic blank group,the negative control group and the Rab 11-siRNA group were 65± 12, 106± 16, 104± 17 and 50± 11, respectively. The invasion capacity was significantly higher in the hypoxic blank group than in the normoxic blank group （P〈O.05）, and was significantly lower in the Rab 11-siRNA group than in the hypoxic blank group and the negative control group （P〈O.05）. （4） By transwell assay, the migration cells in the normoxic blank group, the hypoxic blank group, the negative control group and the Rabl 1-siRNA group were 127±12, 169±15, 161±13 and 77±13, respectively. The capacity of invasion was significantly higher in the hypoxic blank group than in the normoxic blank group （P〈0.05）, and was significantly lower in the Rabll-siRNA group than in the hypoxic blank group and the negative control group （P〈0.05）. （5） The immunofluorescence showed that the red fluorescence intensity around nucleus was significantly increased in the normoxic blank group, the hypoxic blank group and the negative control group than in the Rabl 1- siRNA group. Conclusions Hypoxia could promote the invasion and migration of SiHa cells. In hypoxia, the down regulation of Rabl 1 expression could inhibit the invasion and migration of SiHa cells. This might be due to the decreased expression of the intergrin a5, intergrin β3, p-FAK, p-PI3K and Racl protein.