SRPK1基因对膀胱癌细胞生物学行为的影响及机制研究

Effect and mechanism of SRPK1 gene on biological behavior of bladder cancer cells

目的观察丝氨酸/精氨酸蛋白特异性激酶1(SRPK1)对膀胱癌细胞增殖、迁移和侵袭的影响,并进一步探究其分子机制。方法利用Oncomine癌症数据库分析SRPK1基因在膀胱癌组织及正常膀胱组织中的表达;构建SRPK1特异的慢病毒干扰质粒(sh1和sh2)和阴性对照质粒(NC),包装慢病毒颗粒,分别感染T24细胞和5637细胞。设空白对照组(T24和5637)、阴性对照组(T24/NC和5637/NC)、干扰组1(T24/sh1和5637/sh1)和干扰组2(T24/sh2和5637/sh2);采用实时荧光定量PCR(qRT-PCR)和Western blot检测各组细胞中SRPK1 mRNA和蛋白水平的表达变化;采用CCK-8法检测各组细胞增殖能力的变化;采用Transwell细胞迁移和侵袭实验检测各组细胞迁移和侵袭能力的变化;采用Western blot检测各组细胞上皮间质转化(EMT)及AKT通路相关蛋白表达变化。结果Oncomine癌症数据库中SRPK1 mRNA在膀胱癌组织中的表达明显高于正常膀胱组织(P<0.001)。通过qRT-PCR和Western blot检测结果显示病毒感染后的细胞SPRK1在mRNA和蛋白水平均明显下调;在慢病毒感染后的T24细胞中,第3~5天干扰组的吸光度值均低于阴性对照组,差异有统计学意义(均P<0.05);在慢病毒感染后的5637细胞中,第2~5天干扰组的吸光度值均低于阴性对照组,差异有统计学意义(均P<0.05);Transwell细胞迁移和侵袭实验显示:与阴性对照组比较,干扰组的迁移和侵袭的膀胱癌细胞数目均明显减少,差异均有统计学意义(均P<0.05);Western blot结果显示,与阴性对照组比较,干扰组的E-cadherin表达量增加,而N-cadherin和vimentin的表达明显减少,同时AKT通路蛋白p-AKT及p-GSK3β明显下调,差异均有统计学意义(P<0.001)。结论SRPK1基因在膀胱癌组织中高表达,下调SRPK1的表达可抑制膀胱癌细胞的增殖,并降低膀胱癌细胞的侵袭和迁移能力。

Objective To investigate the effect of serine/arginine protein specific kinases 1(SRPK1)on the proliferation,migration and invasion of bladder cancer cell lines,and further study its molecular mechanisms.Methods Oncomine cancer database was used to analyze the difference of SRPK1 expression in bladder cancer tissues and normal bladder tissues.SRPK1-specific lentivirus interference plasmids(sh1 and sh2)and negative control plasmids(NC)were constructed and packaged.The lentivirus particles were infected with T24 and 5637 cells,respectively.The cells were divided into blank control group(T24 and 5637),negative control group(T24/NC and 5637/NC),interference group 1(T24/sh1 and 5637/sh1)and interference group 2(T24/sh2 and 5637/sh2).Real-time quantitative fluorescent PCR(qRT-PCR)and Western blot were used to detect the changes of SRPK1 mRNA and protein expression in each group.CCK-8 assay was used to detect the changes of cell proliferation ability in each group.Transwell cell migration and invasion assay was used to detect the changes of cell migration and invasion ability in each group.Western blot was used to detect the changes of EMT and AKT pathway related proteins in each group.Results The expression of SRPK1 mRNA in bladder cancer tissues was significantly higher than that in normal bladder tissues in Oncomine cancer database(P<0.001).qRT-PCR and Western blot showed that the mRNA and protein levels of SPRK1 were significantly down-regulated after virus infection.In T24 cells infected with lentivirus,optical density values of the interference group were lower than those of the negative control group from the 3rd to 5th day,and the differences were statistically significant(all P<0.05).In 5637 cells infected with lentivirus,optical density values of the interference group were lower than those of the negative control group from day 2 to day 5,and the differences were statistically significant(all P<0.05).Transwell cell migration and invasion assay showed that compared with the negative control group,the number of migration and invasion bladder cancer cells in the interference group was significantly reduced,and the difference was statistically significant(all P<0.05).The results of Western blot showed that compared with the negative control group,the expression of E-cadherin in the interference group was significantly increased,while the expressions of N-cadherin and vimentin were significantly decreased,and the AKT pathway proteins p-Akt and p-GSK3βwere significantly down-regulated(P<0.001).Conclusions SRPK1 is highly expressed in bladder cancer.Down-regulate the SRPK1 expression can inhibit the proliferation,and reduce the invasion and migration ability of bladder cancer cells.