敲低STOML2基因对前列腺癌细胞PC3增殖和迁移以及线粒体膜电位的影响

Effects of knockdown STOML2 gene on proliferation, migration and mitochondrial membrane potential of prostate cancer cells PC3

目的研究发现头颈鳞癌、甲状腺乳头状癌、胃癌、结肠癌等多种恶性肿瘤中Stomatin样蛋白2(STOML2)基因高表达。通过慢病毒技术敲低PC3细胞STOML2基因,观测其对列腺癌细胞增殖、迁移和线粒体膜电位及Erk和Akt通路的影响。方法用PEI法转染HEK293T细胞制作能敲低STOML2基因(shDNA-STOML2)及其随机序列对照(SHCON)的慢病毒,将其分别感染前列腺癌细胞PC3。以未转染前列腺癌细胞PC3为空白对照组,以转染随机序列为阴性对照组,以转染shDNA-STOML2慢病毒载体为敲低组。用免疫荧光显微镜和Western blot检测感染效率和STOML2蛋白表达水平。MTT实验、细胞划痕愈合实验检测对PC3细胞增殖和迁移的影响。流式细胞术及Western blot检测敲低STOML2基因后对PC3细胞线粒体膜电位以及Erk、Akt通路的影响。结果成功构建了shDNA-STOML2和随机序列SHCON两种慢病毒质粒。包装的慢病毒感染PC3细胞感染效率约100%,阴性对照组较敲低组的STOML2/β-actin相对表达水平明显升高(P<0.01),提示模型制作成功。与阴性对照组相比,敲低组在24、48、72 h的细胞活力(1.313±0.028、1.434±0.023、1.558±0.063)较阴性对照组(1.252±0.049、1.306±0.028、1.373±0.032)增加(P<0.05),24、48 h细胞体外迁移能力下降(P<0.05)。提示敲低STOML2基因可以让细胞增殖加速,但迁移能力降低。与阴性对照组相比,细胞线粒体膜电位升高(P<0.05);P-erk蛋白降低;Akt表达虽然降低,但P-Akt的增加(P<0.05)。结论敲低STOML2基因可以使PC3细胞增殖速度加快、体外迁移能力降低,可能与其增加PC3细胞膜电位、Akt通路被激活和Erk通路降低有关。STOML2基因可能在前列腺癌发生和转移过程中起着双重角色。

Objective The high expression of Stomatin-like protein 2(STOML2) gene in head and neck squamous cell carcinoma, thyroid papillary carcinoma, gastric cancer and colon cancer was found. The effects of knocking down STOML2 gene in PC3 cells on the proliferation, migration, mitochondrial membrane potential and Erk and Akt pathways of adenocarcinoma cells were observed by lentivirus technique. Methods HEK293 T cells were transfected with PEI method to produce lentiviruses that can knock down STOML2 gene(shDNA-STOML2) and random sequence control(SHCON), and they were respectively infected with prostate cancer cells PC3. Non-transfected prostate cancer cell PC3 was used as the blank control group, the transfection random sequence was used as the negative control group, and the transfection of shDNA-STOML2 lentivirus vector was used as the knockdown group. The efficiency of infection and STOML2 protein expression were measured by immunofluorescence microscopy and Western blot. The effects of MTT assay and cell scratch healing assay on proliferation and migration of PC3 cells were investigated. Effects of knockdown STOML2 gene on mitochondrial membrane potential and Erk and Akt pathways in PC3 cells were detected by flow cytometry and Western blot. Results Two lentivirus plasmids, shDNA-STOML2 and random sequence SHCON, were successfully constructed. The infection efficiency of packaging PC3 cells infected with slow virus infection was closed to 100%. The relative expression level of STOML2/β actin in negative control group was increased significantly compared with those of knockdown group(P<0.01), which indicated that the model was successfully constructed. Compared with the negative control group, the cell viability(1.313±0.028, 1.434±0.023, 1.558±0.063) of the knockdown group at 24, 48, and 72 h was increased(1.252±0.049, 1.306±0.028, 1.373±0.032)(P<0.05), and the cell migration ability in vitro at 24 and 48 h was decreased(P<0.05). The results suggest that knockdown STOML2 gene can accelerate cell proliferation but reduce migration ability. Compared with the negative control group, the cell mitochondrial membrane potential was increased(P<0.05), Perk protein was reduced, Akt expression was decreased, and PAkt was increased(P<0.05). Conclusion Knockdown of STOML2 gene can accelerate the proliferation and decrease the migration ability in vitro of PC3 cells, which may be related to the increased PC3 cell membrane potential, activation of Akt and reduction of Erk pathway. STOML2 gene may play a dual role in the occurrence and metastasis of prostate cancer.