过表达细胞因子信号转导抑制因子2调控JAK2/STAT3通路抑制直肠癌细胞活性和移植瘤生长

Overexpression of SOCS2 on inhibition of rectal cancer cell growth activity and xenograft tumor growth by JAK2/STAT3 pathway

摘要:

目的： 探究细胞因子信号转导抑制因子2（suppressor of cytokine signaling 2，SOCS2）过表达对直肠癌细胞生长、运动和JAK2/STAT3通路的影响。方法： HCT8细胞分为对照组、pcDNA组和pcDNA-SOCS2组，根据组别转染pcDNA空载体或pcDNA-SOCS2重组表达载体，RT-PCR检测SOCS2基因表达，BrdU染色检测细胞增殖，流式细胞术检测细胞凋亡，Transwell检测细胞侵袭，Western blot检测细胞SOCS2、上皮钙黏蛋白、波形蛋白、纤连蛋白表达以及JAK2和STAT3的磷酸化。另设BALB/c nu/nu裸鼠分为对照组、pcDNA-SOCS2组，建立HCT8皮下移植瘤模型，称取移植瘤质量，免疫组化检测Ki-67和血管内皮生长因子（vascular endothelial growth factor，VEGF）平均光密度，Western blot检测瘤组织JAK2和DTAT3磷酸化。结果： 离体实验中，RT-PCR结果显示，与对照组（1.00±0.00）比较，pcDNA-SOCS2组细胞SOCS2 mRNA水平（4.70±0.27）明显升高（P=0.000）。Western blot结果显示，pcDNA-SOCS2组细胞SOCS2蛋白表达（0.130±0.020）较对照组（0.010±0.005）明显上调（P=0.000）。BrdU阳性百分比明显降低（P=0.000）；流式细胞术结果显示，pcDNA-SOCS2组[（35.0±5.0）%]HCT8细胞凋亡率较对照组[（6.0±3.4）%]明显升高（P=0.000）。Transwell分析结果显示，pcDNA-SOCS2组（89.0±10.0）HCT8细胞侵袭数目较对照组（87.0±13.0）明显减少（P=0.000）。Western blot结果显示，pcDNA-SOCS2组HCT8细胞中上皮钙黏蛋白水平（0.120±0.030）较对照组（0.010±0.004）明显升高（P=0.000），同时波形蛋白水平（0.008±0.004）和纤连蛋白水平（0.010±0.005）较对照组（0.170±0.024，0.070±0.020）明显降低（P=0.000），JAK2和STAT3磷酸化水平降低（P=0.000）。在体实验中，pcDNA-SOCS2组瘤体质量（0.14±0.06）较对照组（0.45±0.08）明显减轻（P=0.000）。免疫组化结果显示，pcDNA-SOCS2组Ki-67和VEGF平均光密度（17.0±6.0，7.0±6.0）较对照组（52.0±9.0，43.0±9.0）明显降低（P=0.000），瘤体JAK2和STAT3磷酸化水平明显下调（P=0.000）。结论： SOCS2过表达可抑制直肠癌HCT8细胞的增殖和运动能力并促进其凋亡，这一作用与JAK2/STAT3通路有关。

Abstract:

Objective: To investigate the effect of suppressor of cytokine signaling 2 (SOCS2) overexpression on the growth, movement and JAK2/STAT3 pathway of rectal cancer cells. Methods: HCT8 cells were divided into control group, pcDNA group and pcDNA-SOCS2 group, transfected with pcDNA empty vector or pcDNA-SOCS2 group recombinant expression vector according to different groups. SOCS2 gene expression was detected by RT-PCR, cell proliferation was detected by BrdU staining, flow cytometry was used to detect apoptosis, Transwell method was used to detect cell invasion, and Western blot was used to detect SOCS2, E-cadherin, vimentin, fibronectin protein expression, and phosphorylation of JAK2 and STAT3. In addition, BALB/c nu/nu nude mice were divided into con-trol group and pcDNA-SOCS2 group. HCT8 subcutaneous xenograft model was established, and the weight of xenograft tumor was weighed. The average optical density of Ki-67 and vascular endothelial growth factor (VEGF) was measured by immunohistochemistry, and the Western blot was used to detect the phosphorylation of JAK2 and STAT3 in tumor tissues. Results: In the ex-vivo experiment, the RT-PCR results showed that compared with the control group (1.00±0.00), the SOCS2 mRNA level (4.70±0.27) of the pcDNA-SOCS2 group was significantly increased (P=0.000). Western blot results showed that the expression of SOCS2 protein in the pcDNA-SOCS2 group (0.130±0.020) was significantly up-regulated (P=0.000) compared with that in the control group (0.010±0.005). The percentage of BrdU positive was signifi-cantly reduced (P=0.000); flow cytometry results showed that the apoptosis rate of HCT8 cells in the pcDNA-SOCS2 group [(35.0±5.0) %] was significantly higher than that in the control group [(6.0±3.4) %, P=0.000]. Transwell analysis results showed that the number of HCT8 cell invasions in the pcDNA-SOCS2 group (89.0±10.0) was significantly lower than that of the control group (87.0±13.0, P=0.000). Western blot results showed that the E-cadherin protein level (0.120±0.030) of HCT8 cells in the pcDNA-SOCS2 group was significantly increased compared to the con-trol group (0.010±0.004, P=0.000), while the Vimentin protein level (0.008±0.004) and Fibronectin protein level (0.010±0.005) were significantly reduced compared to the control group (0.170±0.024, 0.070±0.020), P=0.000, the phosphorylation level of JAK2 and STAT3 decreased (P=0.000). In the in-vivo experiment, the tumor weight in the pcDNA-SOCS2 group (0.14±0.06) was signifi-cantly less than that in the control group (0.45±0.08, P=0.000). The results of immunohistochemistry showed that the average optical density of Ki-67 and VEGF in the pcDNA-SOCS2 group (17.0±6.0, 7.0±6.0) was significantly lower than that in the control group (52.0±9.0, 43.0±9.0, P=0.000), and the phosphorylation levels of JAK2 and STAT3 tumor bodies were significantly down-regulated (P=0.000). Conclusion: The overexpression of SOCS2 can inhibit the proliferation and exercise ability of rectal cancer HCT8 cells and promote the apoptosis, which is related to JAK2/STAT3 pathway.