磷酸钙纳米颗粒介导干扰LMO4对皮肤鳞癌细胞增殖的影响  

The effect of calcium phosphate nanoparticles-mediated interference of LMO4 on skin squamous cells

作　　者：项明华 郭立钰 涂珍珍 王月 周海胜 Xiang Minghua;Guo Liyu;Tu Zhenzhen;Wang Yue;Zhou Haisheng(Dept of Applied Physics,Anhui Medical University,Hefei 230032;Shenzhen Bay Laboratory,Shenzhen 518107;Dept of Biochemistry and Molecular Biology,Anhui Medical University,Hefei 230032;School of Clinical Medicine,Anhui Medical University,Hefei 230031)

机构地区：[1]安徽医科大学生物医学工程学院应用物理学系,合肥230032 [2]深圳湾实验室,深圳518107 [3]安徽医科大学基础医学院生物化学教研室,合肥230032 [4]安徽医科大学临床医学院,合肥230031

出　　处：《安徽医科大学学报》2023年第11期1807-1812,共6页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81772909);安徽高校自然科学研究项目(编号:KJ2019A0946)。

摘　　要：目的运用磷酸钙纳米颗粒包裹DNA转染皮肤鳞癌细胞,以靶向干扰LIM结构域蛋白4(LMO4)表达,探讨转录因子LMO4在皮肤鳞癌中作用和机制。方法采用逆转录-定量PCR技术(RT-qPCR)、免疫组织化学和蛋白质免疫印迹技术检测目的分子表达。利用经典方法制备磷酸钙纳米颗粒,包裹含有靶向干扰LMO4的shRNA表达载体,转染人皮肤鳞癌细胞A431。采用噻唑蓝(MTT)方法检测细胞增殖能力;运用流式细胞分析技术检测细胞周期变化。结果LMO4在皮肤鳞癌组织和A431细胞中高水平表达。磷酸钙纳米颗粒与DNA的最佳包裹比例为10∶1。此纳米颗粒包裹的DNA可以有效转染A431细胞并有效干扰LMO4表达,与脂质体转染后的干扰效率相比,差异无统计学意义(P=0.21)。在A431细胞中干扰LMO4后的24、36和48 h,细胞的增殖能力较对照组均显著降低。细胞周期分析显示:干扰组G_(0)/G_(1)期细胞为39.82%±1.86%,与对照组G_(0)/G_(1)期细胞(30.76%±1.96%)相比,差异有统计学意义(P=0.003);干扰组的S期细胞为39.56%±0.65%,与对照组的S期细胞(50.65%±0.62%)比较,差异有统计学意义(P=0.002)。干扰LMO4表达可以抑制细胞周期素蛋白E和周期素蛋白激酶2(CDK2)的表达。结论磷酸钙纳米颗粒能有效包裹DNA,并具有较高的转染效率;干扰LMO4表达可抑制细胞周期素蛋白E和CDK2的表达,抑制细胞增殖。Objective To investigate lim domain protein 4(LMO4)functions and mechanisms in regulating proliferation of skin squamous cells(A431),the shRNAs targeted to human LMO4 were coated by calcium phosphate nanoparticles(NP)and transfected into A431 cells to inhibit LMO4 expression.Methods Reverse transcription and quantitative polymerase chain reaction(RT-qPCR),immunohistochemistry analysis and Western blot were used to detect expression of the interest genes.The expression vectors with shRNA targeted to human LMO4(NP/sh-L)were coated by the calcium phosphate nanoparticles,and transfected into A431.The MTT assay was conducted to determine cell proliferation after transfected for 24,36 and 48 h.Cells were stained with propidium iodide and examined cell cycles by using flow cytometry.Results LMO4 expressed at higher levels both in the skin squamous tissues and A431 cells.DNA-binding efficiency of NP/sh-L showed that the better combined ratio between calcium phosphate nanoparticles and DNA was 10∶1.There was no significant difference of transfection efficiency between the NP/sh-L and lipofection approaches.The MTT assay showed that silencing LMO4 inhibited proliferation of cells.RNAi-induced LMO4 inhibition exerted cell cycle arrest in G_(0)/G_(1)phase.Both CDK2 and cyclin E were downregulated in the A431 cells transfected with NP/sh-L comparing to the controls.However,LMO4 knockdown did not alter expression of CDK4 and cyclin D1.Conclusion The calcium phosphate nanoparticles could bind and transfer the foreign DNA into the targeted cells with high efficiency.Silencing LMO4 decreased expression of cyclin E and CDK2 resulted in inhibition of cell proliferation.

关 键 词：磷酸钙纳米颗粒 LMO4 皮肤鳞癌细胞 细胞增殖 细胞周期 

分 类 号：R739.5[医药卫生—肿瘤]