摘要
背景及目的
近年来许多研究表明,LRIGs基因家族在垂体瘤、神经胶质瘤和皮肤鳞状细胞癌等多种肿瘤中具有抑癌作用,分析其机制可能是通过下调EGFR的表达抑制肿瘤细胞的增殖、侵袭等作用。但是对于其在膀胱癌方面的研究较少,尤其是对于多种膀胱癌细胞株生物学行为的检测分析还未见报道。
本实验的目的在于,建立稳定表达LRIG3基因的多种膀胱癌细胞株,系统观察分析LRIG3对稳定表达LRIG3基因的膀胱癌细胞系EJ、T24和BIU-87在生长、侵袭及凋亡等方面的影响,全面评价LRIG3基因对膀胱癌的作用及可能机制。方法
提取目的基因,通过琼脂糖凝胶电泳、限制性酶切、连接、转化入感受态大肠杆菌DH5a菌株并从中提取并大量制备pLVX-DsRed-LRIG3真核表达质粒,然后再进行限制性内切酶酶切、琼脂糖凝胶电泳法及DNA测序进行鉴定。鉴定正确无误后,转染入EJ、T24和BIU-87膀胱癌细胞中。通过RT-PCR和Western Blotting技术测定各组膀胱癌细胞LRIG3 mRNA和蛋白的表达。使用Transwell小体测定EJ、T24和BIU-87膀胱癌细胞株各组的体外侵袭能力。运用Annexin-V/7-aad双染法检测三种膀胱癌细胞的凋亡情况。采用流式细胞仪观察分析LRIG3对三种膀胱癌细胞的诱导效应。
结果
经过限制性酶切、PCR及DNA测序鉴定,pLVX-DsRed-Monomer-LRIG3构建成功,与对照组相比,三种膀胱癌细胞株EJ、T24和BIU-87实验组LRIG3 mRNA的表达水平分别上升79.2%、68.6%和60.3%,差异具有统计学意义(P<0.05); LRIG3蛋白的表达水平则分别上升90.5%、78.6%和68.2%,差异显著(P<0.05)。实验组作用24h后三种膀胱癌细胞株EJ、T24和BIU-87的增殖和侵袭能力显著减弱。过表达的LRIG3基因诱导EJ和T24细胞S+G2/M期阻滞：结果表明,实验组EJ和T24细胞中S期和G2/M期细胞数之和显著低于对照组(P<0.05)。BIU-87细胞变化未见明显统计学意义。凋亡检测结果显示在EJ、T24和BIU-87细胞中,实验组的凋亡细胞均显著多于对照组。
结论
使用慢病毒载体系统成功构建携带有LRIG3基因的重组质粒并将其成功转染膀胱癌细胞株；转染后的三种膀胱癌细胞株,LRIG3 mRNA和蛋白水平都明显提高,实验结果表明LRIG3可以明显降低膀胱癌细胞的侵袭能力,阻滞膀胱癌细胞生长和促进膀胱癌细胞的凋亡。
BACKGROUND AND OBJECTIVE
Recently, many studies showed that the human LRIG gene family is tumor-suppressor gene in various types of human cancer. Further studies revealed that the function of LRIGs to inhibit proliferation and invasion of tumor is through down-regulate the expression of EGFR. The impact of LRIGS on the biological behaviors of bladder cancer cells in vitro is not very clear. The aim of our study was to analysis the impact of LRIG3 on the biological features of bladder cancer cell lines EJ, T24 and BIU-87 and the possible mechanisms of enhanced apoptosis induced by upregulation of LRIG1.
MATERIALS AND METHODS
Constructed plasmid and transfected into bladder cancer cells, and then stably transfected cell were individually selected and expanded for further experiments. The expression levels of the mRNA and protein of LRIG3 were measured by RT-PCR and Western blot, and the changes in the Cell cycle were arrested by flow cytometry(stained by propidium iodide). The invasiveness was measured by Transwell assay and the cell apoptosis were detected by flow cytometry with ANNEXIN V and 7-aad staining.
RESULTS
Compared with the control group, the mRNA levels of LRIG3 in the three cells with over expressed LRIG3 were raised 79.2%,68.6% and 60.3% respectively(P<0.05), and the protein levels were raised 90.5%,78.6% and 68.2%(p<0.05). The three bladder cancer cells in experiment groups showed low proliferation and invasion potential. The over-expression LRIG3 in EJ and T24 cells showed an arrest in S+G2/M phase by flow cytometry, but not in BIU-87. The apoptosis rates of the three bladder cancer cells in the experiment groups were significantly higher than the control groups.
CONCLUSION
Taken together, the over expression of LRIG3 could influence the cell cycle and invasion, and induce apoptosis in bladder cancer cell lines.
引文
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