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[摘要] 目的 探讨miR-122调控LAMC1表达与肝癌细胞迁移侵袭相关性研究。 方法 选取人正常肝细胞株LO2、肝癌细胞株HepG2和高转移能力肝癌细胞株SMMC-7721,构建 miR-122 mimics稳定表达载体并对其进行转染,Real-Time PCR检测各组细胞株内microRNA-122含量;CCK-8法检测细胞存活率,流式细胞仪检测细胞凋亡;划痕实验检测肝癌细胞迁移侵袭能力;生物信息学预测、荧光报告载体实验Real-Time PCR检测层粘连蛋白γ1(Recombinant laminin gamma 1,LAMC1)mRNA水平变化。 结果 与对照组人正常肝细胞株LO2相比,miR-122过表达后,肝癌细胞株HepG2和高转移能力肝癌细胞株SMMC-7721细胞存活率显著降低,细胞凋亡率显著升高,LAMC1 mRNA含量显著降低,肝癌细胞株HepG2和高转移能力肝癌细胞株SMMC-7721细胞侵袭和迁移能力显著下降,差异有统计学意义(P<0.05)。 结论 上调miR-122表达可通过调控LAMC1表达抑制肝癌细胞的增殖、迁移和侵袭。
[关键词] 小分子核糖核酸-122;肝癌;LAMC1;迁移;侵袭
[中图分类号] R735.2 [文献标识码] A [文章编号] 1673-9701(2018)22-0027-05
[Abstract] Objective To investigate the correlation between LAMC1 expression regulated by miR-122 and tumor cell migration and invasion. Methods Human normal hepatocyte cell line LO2, HepG2 cell line and SMMC-7721 cell line with high metastatic potential were selected, and the stable miR-122 mimics expression vector was constructed and transfected. Real-Time PCR was used to detect the expression of microRNA-122; Cell viability was detected by CCK-8 assay; Cell apoptosis was detected by flow cytometry; Scratch assay was used to detect the migration and invasion ability of hepatocellular carcinoma cell; Bioinformatics prediction and fluorescence reporter assay Real-Time PCR detected Protein γ1 (Recombinant Laminin Gamma 1, LAMC1) mRNA level changes. Results Compared with the normal human hepatocyte cell line LO2, the survival rate of HepG2 cells and SMMC-7721 cell lines with high metastatic potential were significantly decreased after miR-122 was overexpressed, and the apoptosis rate was significantly increased. The mRNA levels of HepG2 and SMMC-7721 were significantly decreased. The cell migration and invasion ability of HepG2 and SMMC-7721 were significantly decreased. The difference was statistically significant(P<0.05). Conclusion The up-regulation of miR-122 expression can inhibit the proliferation, migration and invasion of hepatocellular carcinoma cells by regulating the expression of LAMC1.
[Key words] Small molecule RNA-122; Liver cancer; LAMC1; Migration; Invasion
原发性肝癌是一种常见的严重危害人类健康的恶性肿瘤,其发病率近年来在世界范围内逐年上升,严重危害患者的生命健康[1,2]。肝癌起病隐匿,临床早期无明显的症状或临床症状缺乏特异性;同时肝癌发病进展迅速,具有高侵袭性和高复发性,导致其临床治疗难度大,预后极差[3-5]。microRNAs近年来已经发展为一个生物肿瘤治疗领域的新靶点。microRNAs作为基因表达的调节剂,其广泛参与并有效涉及多种生物及细胞的增殖、分化、凋亡代谢等过程[6-8]。miR-122作为肝脏特异性表达的microRNAs,其过表达可有效降低肝癌细胞的特异性,抑制肝癌细胞的分化周期,并能增强肝癌细胞对药物的敏感性,但其对于肝癌细胞的具体调控机制仍未阐明[9-12]。本研究中,我们通过上調肝癌细胞内miR-122表达,分析检测其对肝癌细胞的增殖凋亡和迁移侵袭的影响,旨在探讨miR-122对肝癌细胞迁移侵袭的调节作用和其对肝癌细胞内层粘连蛋白γ1(LAMC1)调控作用。 1 材料与方法
1.1 药品与试剂
美国培养物保藏中心(ATCC,Manassas,VA)购买人正常肝细胞株LO2、肝癌细胞株HepG2和高转移能力肝癌细胞株SMMC-7721。miR-122质粒、LAMC1及Real-Time-PCR配套引物(广州RiboBio公司);CCK-8试剂盒(日本同仁化学研究所);TUNEL凋亡试剂盒(上海碧云天生物有限公司)。
1.2 细胞培养
人正常肝细胞株LO2和肝癌细胞株HepG2用DMEM 10%胎牛血清、37°C、5%CO2细胞培养箱中培养,高转移能力肝癌细胞株SMMC-7721细胞用RMPI 1640 10%胎牛血清、37°C、5%CO2细胞培养箱中培养;3种细胞正常传代后提取细胞样品、PBS洗涤2次后转移入EP管中,冰上裂解15 min后超声破碎细胞,离心15 min,取上清液作为蛋白样品。
1.3 细胞miR-122mimics质粒转染[13]
慢病毒质粒(Lv-miR-122)与对照scarmble RNA的病毒质粒(Lv-Ctrl)连同包装质粒pRRE、pRSV-REV、pCMV-VSVG通过磷酸钙转染法转到293T细胞,48 h收集上清液,冷冻超速离心获得病毒浓缩液。人正常肝细胞株LO2、肝癌细胞株HepG2和高转移能力肝癌细胞株SMMC-7721接种于12孔板加入病毒上清液和polybrene(8 μg/mL) 选择感染16 h后更换为常规培养基继续培养,以绿色荧光蛋白(green fluorescent protein,GFP)为荧光标记慢病毒感染,72 h时用荧光显微镜观察报告基因的表达情况,荧光率即为阳性感染率。
1.4 CCK-8法检测细胞存活率
将转染后的人正常肝细胞株LO2、肝癌细胞株HepG2和高转移能力肝癌细胞株SMMC-7721细胞制备成细胞悬液并接种到96孔板中,37℃、5%CO2条件下培养箱过夜培养。加入10 μL CCK8,按说明书操作进行细胞培养和测定450 nm波长处OD值,计算细胞存活率。
1.5 TUNEL流式细胞术检测细胞凋亡率
将转染后的人正常肝细胞株LO2、肝癌细胞株HepG2和高转移能力肝癌细胞株SMMC-7721细胞制备成浓度为1×106细胞悬液接种于6孔板中培养,分别加入FITc标记的AnnexinV和PI,按试剂盒说明书进行操作和测定激发波长488 nm,收集波长630 nm的细胞的荧光强度,检测肺癌细胞的凋亡百分比。
1.6 Real-Time PCR方法检测miR-122和LAMC1水平变化
将转染后的人正常肝细胞株LO2、肝癌细胞株HepG2和高转移能力肝癌细胞株SMMC-7721细胞制备成浓度为1×106细胞悬液接种于6孔板中培养,Trizol法提取细胞内总RNA。按照试剂盒说明书进行逆转录成cDNA,并用SYBR Premix Ex TaqⅡ试剂盒扩增和实时荧光定量Real-time PCR分析。分析:通过计算miR-122和LAMC1基因的循环阈值(CT值)确定各个基因的表达含量。
1.7 划痕实验检验细胞迁移和侵袭[14]
取人正常肝细胞株LO2、肝癌细胞株HepG2和高转移能力肝癌细胞株SMMC-7721细胞及miR-122病毒转染后的3种细胞接种于96孔板上,第2天更换低浓度血清培养基,将划痕仪对准96孔板的下端中央部位,向上轻推形成划痕。用无血清培养基轻轻漂洗2~3遍,加入低浓度血清培养基(如0.5
[关键词] 小分子核糖核酸-122;肝癌;LAMC1;迁移;侵袭
[中图分类号] R735.2 [文献标识码] A [文章编号] 1673-9701(2018)22-0027-05
[Abstract] Objective To investigate the correlation between LAMC1 expression regulated by miR-122 and tumor cell migration and invasion. Methods Human normal hepatocyte cell line LO2, HepG2 cell line and SMMC-7721 cell line with high metastatic potential were selected, and the stable miR-122 mimics expression vector was constructed and transfected. Real-Time PCR was used to detect the expression of microRNA-122; Cell viability was detected by CCK-8 assay; Cell apoptosis was detected by flow cytometry; Scratch assay was used to detect the migration and invasion ability of hepatocellular carcinoma cell; Bioinformatics prediction and fluorescence reporter assay Real-Time PCR detected Protein γ1 (Recombinant Laminin Gamma 1, LAMC1) mRNA level changes. Results Compared with the normal human hepatocyte cell line LO2, the survival rate of HepG2 cells and SMMC-7721 cell lines with high metastatic potential were significantly decreased after miR-122 was overexpressed, and the apoptosis rate was significantly increased. The mRNA levels of HepG2 and SMMC-7721 were significantly decreased. The cell migration and invasion ability of HepG2 and SMMC-7721 were significantly decreased. The difference was statistically significant(P<0.05). Conclusion The up-regulation of miR-122 expression can inhibit the proliferation, migration and invasion of hepatocellular carcinoma cells by regulating the expression of LAMC1.
[Key words] Small molecule RNA-122; Liver cancer; LAMC1; Migration; Invasion
原发性肝癌是一种常见的严重危害人类健康的恶性肿瘤,其发病率近年来在世界范围内逐年上升,严重危害患者的生命健康[1,2]。肝癌起病隐匿,临床早期无明显的症状或临床症状缺乏特异性;同时肝癌发病进展迅速,具有高侵袭性和高复发性,导致其临床治疗难度大,预后极差[3-5]。microRNAs近年来已经发展为一个生物肿瘤治疗领域的新靶点。microRNAs作为基因表达的调节剂,其广泛参与并有效涉及多种生物及细胞的增殖、分化、凋亡代谢等过程[6-8]。miR-122作为肝脏特异性表达的microRNAs,其过表达可有效降低肝癌细胞的特异性,抑制肝癌细胞的分化周期,并能增强肝癌细胞对药物的敏感性,但其对于肝癌细胞的具体调控机制仍未阐明[9-12]。本研究中,我们通过上調肝癌细胞内miR-122表达,分析检测其对肝癌细胞的增殖凋亡和迁移侵袭的影响,旨在探讨miR-122对肝癌细胞迁移侵袭的调节作用和其对肝癌细胞内层粘连蛋白γ1(LAMC1)调控作用。 1 材料与方法
1.1 药品与试剂
美国培养物保藏中心(ATCC,Manassas,VA)购买人正常肝细胞株LO2、肝癌细胞株HepG2和高转移能力肝癌细胞株SMMC-7721。miR-122质粒、LAMC1及Real-Time-PCR配套引物(广州RiboBio公司);CCK-8试剂盒(日本同仁化学研究所);TUNEL凋亡试剂盒(上海碧云天生物有限公司)。
1.2 细胞培养
人正常肝细胞株LO2和肝癌细胞株HepG2用DMEM 10%胎牛血清、37°C、5%CO2细胞培养箱中培养,高转移能力肝癌细胞株SMMC-7721细胞用RMPI 1640 10%胎牛血清、37°C、5%CO2细胞培养箱中培养;3种细胞正常传代后提取细胞样品、PBS洗涤2次后转移入EP管中,冰上裂解15 min后超声破碎细胞,离心15 min,取上清液作为蛋白样品。
1.3 细胞miR-122mimics质粒转染[13]
慢病毒质粒(Lv-miR-122)与对照scarmble RNA的病毒质粒(Lv-Ctrl)连同包装质粒pRRE、pRSV-REV、pCMV-VSVG通过磷酸钙转染法转到293T细胞,48 h收集上清液,冷冻超速离心获得病毒浓缩液。人正常肝细胞株LO2、肝癌细胞株HepG2和高转移能力肝癌细胞株SMMC-7721接种于12孔板加入病毒上清液和polybrene(8 μg/mL) 选择感染16 h后更换为常规培养基继续培养,以绿色荧光蛋白(green fluorescent protein,GFP)为荧光标记慢病毒感染,72 h时用荧光显微镜观察报告基因的表达情况,荧光率即为阳性感染率。
1.4 CCK-8法检测细胞存活率
将转染后的人正常肝细胞株LO2、肝癌细胞株HepG2和高转移能力肝癌细胞株SMMC-7721细胞制备成细胞悬液并接种到96孔板中,37℃、5%CO2条件下培养箱过夜培养。加入10 μL CCK8,按说明书操作进行细胞培养和测定450 nm波长处OD值,计算细胞存活率。
1.5 TUNEL流式细胞术检测细胞凋亡率
将转染后的人正常肝细胞株LO2、肝癌细胞株HepG2和高转移能力肝癌细胞株SMMC-7721细胞制备成浓度为1×106细胞悬液接种于6孔板中培养,分别加入FITc标记的AnnexinV和PI,按试剂盒说明书进行操作和测定激发波长488 nm,收集波长630 nm的细胞的荧光强度,检测肺癌细胞的凋亡百分比。
1.6 Real-Time PCR方法检测miR-122和LAMC1水平变化
将转染后的人正常肝细胞株LO2、肝癌细胞株HepG2和高转移能力肝癌细胞株SMMC-7721细胞制备成浓度为1×106细胞悬液接种于6孔板中培养,Trizol法提取细胞内总RNA。按照试剂盒说明书进行逆转录成cDNA,并用SYBR Premix Ex TaqⅡ试剂盒扩增和实时荧光定量Real-time PCR分析。分析:通过计算miR-122和LAMC1基因的循环阈值(CT值)确定各个基因的表达含量。
1.7 划痕实验检验细胞迁移和侵袭[14]
取人正常肝细胞株LO2、肝癌细胞株HepG2和高转移能力肝癌细胞株SMMC-7721细胞及miR-122病毒转染后的3种细胞接种于96孔板上,第2天更换低浓度血清培养基,将划痕仪对准96孔板的下端中央部位,向上轻推形成划痕。用无血清培养基轻轻漂洗2~3遍,加入低浓度血清培养基(如0.5