Xu H, Yuan Y, Ji J Y, Jiang Q, Niu L J, Liu N L, Zhang L Z, Wang X
Department of Radiology, the Affiliated Hospital of Xuzhou Medical University, Xuzhou 221002, China.
Zhonghua Fu Chan Ke Za Zhi. 2016 Dec 25;51(12):928-933. doi: 10.3760/cma.j.issn.0529-567X.2016.12.009.
To explore the expression of Ras-related protein 11 (Rab11) in hypoxia, the effect of Rab11 on the invasion and migration of cervical cancer cell line SiHa and its possible mechanism. 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 of negative control small interfering RNA (siRNA) in hypoxia], the Rab11-siRNA group (transfection of Rab11 siRNA in hypoxia). Western blot was used to examine the expression of Rab11, integrin α5, integrin β3, phosphorylated focal adhesion kinase (p-FAK), phosphorylated phosphatidylinositol 3 kinase (p-PI3K) protein, together with the expression of Ras correlative C3 creotoxin substrate 1 (Rac1), which was critical in regulating cell invasion. The mRNA expression of Rab11 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 Rac1 in SiHa cell. (1) The expression of Rab11, intergrin α5, intergrin β3, p-FAK, p-PI3K and Rac1 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 Rab11-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 Rab11, α5, β3, p-FAK, p-PI3K and Rac1 were significantly higher in the hypoxic blank group than in the normoxic blank group (0.05), and were significantly lower in the Rab11-siRNA group than in the hypoxic blank group and the negative control group (0.05). (2) The expressions of Rab11-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 Rab11-siRNA group, respectively. It was significantly higher in the hypoxic blank group than in the normoxic blank group (0.05), and was significantly lower in the Rab11-siRNA group than in the hypoxic blank group and the negative control group (0.05). (3) By Matrigel, the invasion cell number in the normoxic blank group, the hypoxic blank group,the negative control group and the Rab11-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 (0.05), and was significantly lower in the Rab11-siRNA group than in the hypoxic blank group and the negative control group (0.05). (4) By transwell assay, the migration cells in the normoxic blank group, the hypoxic blank group, the negative control group and the Rab11-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 (0.05), and was significantly lower in the Rab11-siRNA group than in the hypoxic blank group and the negative control group (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 Rab11-siRNA group. Hypoxia could promote the invasion and migration of SiHa cells. In hypoxia, the down regulation of Rab11 expression could inhibit the invasion and migration of SiHa cells. This might be due to the decreased expression of the intergrin α5, intergrin β3, p-FAK, p-PI3K and Rac1 protein.
为探讨Ras相关蛋白11（Rab11）在缺氧条件下的表达、Rab11对宫颈癌SiHa细胞侵袭和迁移的影响及其可能机制。将SiHa细胞分为4组，即常氧空白组（常氧下正常培养）、缺氧空白组（缺氧下正常培养）、阴性对照组[缺氧条件下转染阴性对照小干扰RNA（siRNA）]、Rab11-siRNA组（缺氧条件下转染Rab11 siRNA）。采用蛋白质免疫印迹法检测Rab11、整合素α5、整合素β3、磷酸化黏着斑激酶（p-FAK）、磷酸化磷脂酰肌醇3激酶（p-PI3K）蛋白的表达，以及在调节细胞侵袭中起关键作用的Ras相关C3肉毒素底物1（Rac1）的表达。采用实时定量聚合酶链反应检测4组中Rab11的mRNA表达。采用基质胶侵袭实验检测细胞侵袭能力，采用Transwell实验检测细胞迁移能力。采用免疫荧光法鉴定SiHa细胞内Rac1的定位。（1）常氧空白组中Rab11、整合素α5、整合素β3、p-FAK、p-PI3K和Rac1的表达分别为0.56±0.04、0.33±0.03、0.32±0.03、0.36±0.03、0.35±0.03和0.47±0.03。缺氧空白组中，它们分别为0.73±0.03、0.74±0.03、0.61±0.03、0.62±0.03、0.60±0.03和0.73±0.03。阴性对照组中，它们的表达分别为0.72±0.03、0.73±0.03、0.59±0.03、0.61±0.03、0.59±0.03和0.72±0.03。而在Rab11-siRNA组中，它们分别为0.44±0.03、0.30±0.03、0.29±0.03、0.30±0.03、0.30±0.03和0.34±0.04。Rab11、α5、β3、p-FAK、p-PI3K和Rac1在缺氧空白组中的表达显著高于常氧空白组（P<0.05），而在Rab11-siRNA组中显著低于缺氧空白组和阴性对照组（P<0.05）。（2）常氧空白组、缺氧空白组、阴性对照组和Rab11-siRNA组中Rab11-mRNA的表达分别为1.000±0.000、1.454±0.114、1.442±0.101、0.570±0.046。缺氧空白组中的表达显著高于常氧空白组（P<0.05），而在Rab11-siRNA组中显著低于缺氧空白组和阴性对照组（P<0.05）。（3）通过基质胶实验，常氧空白组、缺氧空白组、阴性对照组和Rab11-siRNA组的侵袭细胞数分别为65±12、106±16、104±17和50±11。缺氧空白组的侵袭能力显著高于常氧空白组（P<0.05），而在Rab11-siRNA组中显著低于缺氧空白组和阴性对照组（P<0.05）。（4）通过Transwell实验，常氧空白组、缺氧空白组、阴性对照组和Rab11-siRNA组的迁移细胞数分别为127±12、169±15、161±13和77±13。缺氧空白组的侵袭能力显著高于常氧空白组（P<0.05），而在Rab11-siRNA组中显著低于缺氧空白组和阴性对照组（P<0.05）。（5）免疫荧光显示，与Rab11-siRNA组相比，常氧空白组、缺氧空白组和阴性对照组细胞核周围的红色荧光强度显著增加。缺氧可促进SiHa细胞的侵袭和迁移。在缺氧条件下，Rab11表达下调可抑制SiHa细胞的侵袭和迁移。这可能是由于整合素α5、整合素β3、p-FAK、p-PI3K和Rac1蛋白表达降低所致。
