长链非编码RNA SNHG1靶向miR-340-5p/CCND1轴调控食管癌细胞增殖、迁移和侵袭

Long non-coding RNA SNHG1 regulating proliferation,migration and invasion of esophageal cancer cells by targeting miR-340-5p/CCND1 axis

目的:分析长链非编码RNA小核仁RNA宿主基因1(LncRNA SNHG1)靶向miR-340-5p/细胞周期蛋白1(CCND1)轴调控食管癌细胞增殖、迁移和侵袭。方法:体外培养人食管上皮细胞、食管癌细胞KYSE-30、TE-1、NEC、Eca109,qRT-PCR法测定细胞中LncRNA SNHG1、miR-340-5p、CCND1 mRNA水平。将对数期NEC细胞分为对照组、sh NC1组、sh LncRNA SNHG1组、sh NC2组、sh CCND1组、sh LncRNA SNHG1+miR-340-5p inhibitor组、sh CCND1+miR-340-5p inhibitor组。CCK-8法测定细胞增殖能力,Transwell小室法测定细胞侵袭、迁移能力,Western blot法检测CCND1、Ki-67、MMP-2蛋白表达,双荧光素酶验证miR-340-5p与LncRNA SNHG1、CCND1的靶向关系,通过裸鼠瘤内注射转染试剂进行体内试验。结果:与人食管上皮细胞相比,食管癌细胞KYSE-30、TE-1、NEC、Eca109中LncRNA SNHG1、CCND1 mRNA表达升高,miR-340-5p表达降低(P<0.05),其中NEC细胞变化最显著,所以使用NEC作为下续研究菌株。与对照组、sh NC组相比,sh LncRNA SNHG1组NEC细胞LncRNA SNHG1、OD_(450)、侵袭细胞数、迁移细胞数、Ki-67、MMP-2降低(P<0.05);与对照组、sh NC组相比,sh CCND1组CCND1 mRNA与蛋白表达、OD_(450)、侵袭细胞数、迁移细胞数、Ki-67、MMP-2表达降低(P<0.05)。miR-340-5p与LncRNA SNHG1、CCND1均靶向结合,与sh LncRNA SNHG1组相比,sh LncRNA SNHG1+miR-340-5p inhibitor组OD_(450)、侵袭细胞数、迁移细胞数、Ki-67、MMP-2蛋白表达升高(P<0.05);与sh CCND1组相比,sh CCND1+miR-340-5p inhibitor组OD_(450)、侵袭细胞数、迁移细胞数、Ki-67、MMP-2蛋白表达升高(P<0.05);裸鼠移植瘤实验进行了体内验证。结论:LncRNA SNHG1沉默可能通过调控miR-340-5p/CCND1表达抑制食管癌NEC细胞增殖、侵袭与迁移,裸鼠体内也验证了这一结果。

Objective:To analyze the influences of long non-coding RNA small nucleolar RNA host gene 1(LncRNA SNHG1)on the proliferation,migration and invasion of esophageal cancer cells by targeting miR-340-5p/cyclin 1(CCND1)axis.Methods:Human esophageal epithelial cells and esophageal cancer cells KYSE-30,TE-1,NEC,Eca109 were cultured in vitro.The levels of LncRNA SNHG1,miR-340-5p and CCND1 mRNA in cells were measured by qRT-PCR.NEC cells in logarithmic phase were divided into control group,sh NC1 group,sh LncRNA SNHG1 group,sh NC2 group,sh CCND1 group,sh LncRNA SNHG1+miR-340-5p inhibitor group,and sh CCND1+miR-340-5p inhibitor group.CCK-8 method was used to determine the cell proliferation ability.Transwell chamber method was used to measure the invasion and migration of cells.The protein expressions of CCND1,Ki-67 and MMP-2 were detected by Western blot.Double luciferase was applied to verify the targeting relationship between miR-340-5p and LncRNA SNHG1,CCND1,and the transfection reagent was injected into the tumor of nude mice for in vivo test.Results:Compared with human esophageal epithelial cells,the expression of LncRNA SNHG1 and CCND1 mRNA in the esophageal cancer cells KYSE-30,TE-1,NEC,and Eca109 increased,and the expression of miR-340-5p decreased(P<0.05),in which,NEC cells had the most obvious changes,so NEC cells were used as the strain for next study.Compared with the control group and sh NC group,the number of LncRNA SNHG1,OD_(450),the number of invading cells,the number of migrating cells,the expression of Ki-67 and MMP-2 in NEC cells in sh LncRNA SNHG1 group decreased(P<0.05).Compared with the control group and the sh NC group,the expression of CCND1 mRNA and protein,OD_(450),the number of invading cells,the number of migrating cells,the expression of Ki-67 and MMP-2 decreased in the sh CCND1 group(P<0.05).miR-340-5p bound to LncRNA SNHG1 and CCND1.Compared with sh LncRNA SNHG1 group,the OD_(450),the number of invading cells,the number of migrating cells,the expression of Ki-67 and MMP-2 proteins in sh LncRNA SNHG1+miR-340-5p inhibitor group increased(P<0.05).Compared with sh CCND1 group,the OD_(450),the number of invading cells,the number of migrating cells,Ki-67,MMP-2 proteins in sh CCND1+miR-340-5p inhibitor group increased(P<0.05).The nude mouse tumor transplantation experiment was carried out for in vivo verification.Conclusion:LncRNA SNHG1 silencing may inhibit the proliferation,invasion and migration of esophageal cancer NEC cells by regulating the expression of miR-340-5p/CCND1,which is also verified in nude mice.