Objective:MicroRNAs ( miRNAs) function as key regulator of gene expression and their dereg-ulation play critical roles in tumorigenesis and metastasis of various cancers. The purpose of this study is to identify miRNAs targeting DAB2IP and to determine their expression and function in bladder cancer (BC). Methods and Results:We first predicted candidate miRNAs targeting Disabled homolog 2- interaction protein ( DAB2IP) and then determine their expression and biological function in BC. We showed that miRNA-556-3p directly regulated DAB2 IP expression by binding to DAB2 IP 3 '-UTR and endogenous miRNA-556-3 p expression was significantly up-regulated in clinical samples of BC patients and BC cell lines in comparison to the controls. Conversely, simul-taneous DAB2IP expression in BC tissues and BC cell lines was remarkably down-regulated. Gain or loss function showed that enhanced miRNA-556-3p expression by Lv-miRNA-556-3p transfection promoted proliferation,in-vasion, migration, and colony formation of BC cells, whereas repressed miRNA-556-3p expression by Lv-sh-miRNA-556-3p transfection resulted inan opposite results. Importantly,restored DAB2IP expression by "rescue"assay could attenuate the promotion effect induced by miRNA-556-3p. Further investigation verified that overex-pressed miRNA-556-3p in BC cells not only decreased DAB2IP expression, but also dramatically increased Ras-and pERK1/2 protein expression. In conclusion, our results suggested that DAB2IP was a direct target of miRNA-556-3p, and endogenous miRNA-556-3p expression was reversely correlated with simultaneous DAB2IP expres-sion in BC tissues and cells. Conclusions: MiRNA-556-3p, as a tumor promoter, functioned in tumorigenesis and metastasis of BC via targeting DAB2IP. Moreover, miRNA-556-3p mediated DAB2IP suppression played an oncogenic role by activation of Ras-ERK pathway partially.

DAB2IP基因慢病毒载体构建及其在前列腺癌PC3细胞中的表达

目的：构建重组慢病毒质粒 pSin-EF2-Puro-DAB2IP并转染前列腺癌PC3细胞，观察其转染效率及其在PC3细胞中的表达。方法：以人前列腺癌PC3细胞基因组cDNA为模板PCR扩增获得目的基因DAB2 IP片段后，应用基因重组技术将目的片段克隆到 PC3-pSin慢病毒表达载体，经PCR、双酶切和测序鉴定后，重组慢病毒表达质粒和包装质粒共转染293 FT细胞，获得携带 DAB2 IP基因的重组慢病毒。取病毒上清感染人前列腺癌PC3细胞，以PC3-pSin-EF2为对照，分别采用 RT-QPCR、Western blotting法检测 DAB2IP在靶细胞中的表达水平。结果：重组慢病毒质粒 pSin-EF2-Puro-DAB2IP经PCR、EcoRⅠ和Nhe Ⅰ双酶切鉴定结果与目的基因条带吻合，克隆测序结果与 NCBI收录的DAB2IP基因序列（NM_138709）完全一致。重组慢病毒质粒转染293FT细胞收获慢病毒上清可高效感染 PC3细胞，对照组细胞株PC3-pSin-EF2及过表达细胞株 PC3-pSin-EF2-DAB2 IP内 DAB2IP 基因的相对拷贝数分别为0.001±0.000和0.158±0.013，与对照组细胞比较，过表达细胞内DAB2IP基因mRNA表达水平上调（179.37±15.89）倍，差异有统计学意义（P＜0.001）；Western blotting 法，对照组PC3-pSin-EF2细胞中DAB2IP蛋白表达水平为内参的（1.002±0.783）倍，PC3-pSin-EF2-DAB2IP细胞组为内参的（2.431±0.892）倍，过表达组DAB2IP蛋白表达水平为对照组的（2.415±0.961）倍，差异有统计学意义（P＜0.01）。结论：成功构建携带DAB2IP基因的慢病毒表达载体pSin-EF2-Puro-DAB2IP，并使目的基因在靶细胞中稳定表达，为进一步研究DAB2 IP基因的相关功能提供了优质的稳定转染载体。