下调MARCH8基因的表达对宫颈癌Siha细胞侵袭、迁移与增殖的影响

目的：研究下调MARCH8基因表达对人宫颈癌Siha细胞侵袭、迁移、增殖以及克隆的影响，并探讨其可能的作用机制。方法：构建特异性针对MARCH8基因的siRNA片段，并将其通过脂质体介导转染至宫颈癌Siha细胞，同时设立转染无义序列的阴性对照组。分别采用Real-time PCR法与蛋白质印迹法检测转染后，细胞中MARCH8在mRNA和蛋白水平上的表达改变。通过Transwell小室实验、划痕实验检测MARCH8表达下调对细胞侵袭、迁移能力的影响，通过CCK-8法、细胞平板克隆实验检测MARCH8表达下调对细胞增殖、克隆形成能力的影响，并采用蛋白质印迹法检测MARCH8基因表达下调对基质金属蛋白酶9（MMP9），波形蛋白（Vimentin）以及增殖细胞核抗原（PCNA）蛋白表达的影响。最后采用蛋白质印迹法检测MARCH8基因表达下调，对Wnt/β-catenin信号通路中β-catenin以及E钙黏蛋白（E-cadherin）表达的影响。结果：与转入siRNA-NC组相比，转入siRNA-MARCH8组细胞MARCH8在 mRNA和蛋白表达水平上均明显降低，细胞的侵袭、迁移以及增殖和克隆形成能力明显被抑制（P<0.05），且侵袭标记蛋白MMP9，Vimentin以及增殖标记蛋白PCNA的表达也明显下降。MARCH8基因表达成功下调后，转入siRNA-MARCH8组细胞中β-catenin蛋白的表达水平较阴性对照组明显下降（P<0.05），而E-cadherin蛋白的表达水平明显升高（P<0.05）。结论：下调MARCH8基因的表达可以抑制宫颈癌Siha细胞的侵袭、迁移、增殖和克隆能力，其机制可能与Wnt/β-catenin通路的抑制有关。

Effects of down regulation of MARCH8 expression on cell invasion,migration and proliferation in cervical cancer Siha cells

Objective: To explore the effects of down regulation of MARCH8 expression on the invasion, migration, proliferation and cell forming clone ability in cervical cancer Siha cells, and investigate the probable mechanism. Methods: The siRNA fragments aiming at MARCH8 were constructed and transfected to cervical cancer Siha cells by liposome. The cervical cancer Siha cells transfected with siRNA-NC were regarded as the negative control group. After down-regulation of MARCH8 expression, the expression of MARCH8 at mRNA level and at protein level were respectively detected using real-time PCR and Western blot. Cell invasion and cell migration were determined by Transwell assay and scratch assay. Cell proliferation and cell forming clone ability were measured by cell counting kit-8 (CCK-8) assay and cell clone assay. Then, Western blot was used to detect the changes of the expression of MMP9,Vimentin and PCNA.The changes of the expression of β-catenin and E-cadherin were detected by Western blot. Results:Compared with the siRNA-NC transfected group, the expression of MARCH8 in the siRNA-MARCH8 transfected group at both mRNA and protein level were obviously decreased（P<0.05).Cell invasion,migration, proliferation and cell forming clone ability were also inhibited（P<0.05).The expression of MMP9,Vimentin and PCNA in the siRNA-MARCH8 transfected group were obviously decreased.The expression of β-catenin in the siRNA-MARCH8 transfected group was obviously reduced, while E-cadherin was increased（P<0.05).Conclusion: Down-regulation of MARCH8 expression can inhibit cell invasion, migration, proliferation and cell forming clone ability in cervical cancer Siha cells, and the inhibition of Wnt/β-catenin pathway may be involved in this process.