lncRNA MEG3靶向调节miR-27a-3p抑制胃癌细胞增殖、迁移和侵袭的实验研究.pdf

基 础 研 究Experimental Research中国现代2023年9 月第2 6 卷第9 期Sep.2023Vo1.26No.9普通外科进展Chin J Curr Adv Gen Surg678IncRNAMEG3靶向调节miR-27a-3p抑制胃癌细胞增殖、迁移和侵袭的实验研究张新如1胡广军宗殿亮孖沧州市人民医院肿瘤内科+2胃肠外科+普外科（河北沧州0061000)孙清森2 赵俊卿3谷斌【摘要】目的：探究IncRNAMEG3对胃癌(GC)细胞增殖、迁移和侵袭的影响及机制。方法：qRT-PCR检测人正常胃上皮细胞GES-1和GC细胞（BGC823、M G C8 0 3、SG C7 90 1、A G S、M K N2 8、M K N451)中MEG3及miR-27a-3p表达差异；将AGS细胞分为对照组、GV144组、GV144-MEG3组、GV144-MEG3+miR-27a-3pNC组、GV144-MEG3+miR-27a-3pmimics组；比较各组AGS细胞中MEG3及miR-27a-3p表达情况;检测细胞增殖、迁移和侵袭能力,Western blot 检测ki-67、M M P-2、MMP-9蛋白表达,双荧光素酶报告实验验证MEG3与miR-27a-3p的靶向关系。结果：与 GES-1细胞相比,GC细胞中MEG3水平降低,miR-27a-3p水平升高(P0.05)；与对照组相比,GV144-MEG3组MEG3水平升高,miR-27a-3p、k i-6 7、M M P-2、M M P-9水平及增殖、迁移和侵袭能力降低（P0.05)；上调miR-27a-3p表达可减弱MEG3过表达对 AGS细胞恶性行为的抑制作用。miR-27a-3p是MEG3的靶基因。结论：过表达MEG3可能通过靶向下调miRNA-27a-3p表达抑制AGS细胞增殖、迁移及侵袭。【关键词】长链非编码RNA人母系表达基因3miR-27a-3p胃癌增殖迁移侵袭【中图分类号】R735.2【文献标识码】A，d o i:10.39 6 9/j.is s n.10 0 9-9 9 0 5.2 0 2 3.0 9.0 0 2【文章编号】10 0 9-990 5(2 0 2 3)0 9-0 6 7 8-0 4Experimental study on lncRNA MEG3 targeted regulation ofmiR-27a-3p inhibiting the proliferation,migration and in-vasion of gastric cancer cellsZHANG Xin-ru,HU Guang-jun,ZONG Dian-liang,SUN Qing-sen,ZHAOJun-qing,GU bin?Department of Oncology,Department of Gastroenterology,Department of GeneralSurgery,Cangzhou Peoples Hospital(Cangzhou 061000,China)ABSTRACT Objective:To explore the effect and mechanism of IncRNA MEG3 on the prolifera-tion,migration and invasion of gastric cancer(GC)cells.Methods:Expression of MEG3 and【基金项目】2 0 2 2 年度河北省医学科学研究课题（2 0 2 2 0 314）作者简介张新如（198 1-12 ），男，主治医师，研究方向：肿瘤内科。E-mail:张新如，等.lncRNAMEG3靶向调节miR-27a-3p抑制胃癌细胞增殖、迁移和侵袭的实验研究miR-27a-3p in human normal gastric epithelial cell line GES-1and GC cells(BGC823,MGC803,SGC7901,AGS,MKN28,MKN451)detected by qRT PCR;the AGS cells Were divided into controlgroup,GV144 group,GV144-MEG3 group,GV144-MEG3+miR-27a-3p NC group,andGV144-MEG3+miR-27a-3p mimics group;the expression of MEG3 and miR-27a-3p in AGS cells ineach group were compared;detectde the cell proliferation,migration and invasion,Western blot wasused to detect the expression of ki-67,MMP-2 and MMP-9 proteins,and double luciferase reporttest was used to verify the targeting relationship between MEG3 and miR-27a-3p.Results:Com-pared with GES-1 cells,the level of MEG3 in the GC cells decreased and the level of miR-27a-3p in-creased(P0.05);compared with the control group,the level of MEG3 in GV144-MEG3 group in-creased,and the levels of miR-27a-3p,ki-67,MMP-2,MMP-9,as well as the ability of proliferation,migration and invasion decreased(P0.05);upregulation of miR-27a-3p expression can weaken theinhibition of MEG3 overexpression on the malignant behavior of AGS cells.MiR-27a-3p is the targetgene of MEG3.Conclusion:Overexpression of MEG3 may inhibit the proliferation,migration andinvasion of AGS cells by targeting down regulation of miRNA-27a-3p expression.KEY WORDS Long non-coding RNA.Human maternally expressed gene 3.MiR-27a-3pGastriccancerProliferationMigrationInvasion679胃癌（gastric cancer,GC)是一种侵袭性强的胃肠道恶性肿瘤 1。由于早期症状不明显,约8 0%的GC 患者确诊时已为晚期 2 。长链非编码RNA(longnon-codingRNA,lncRNA）在GC进展中具有重要作用 3-4。人母系表达基因 3（human maternally ex-pressed gene 3,MEG3)是一种具有抑制癌细胞迁移和增殖能力的lncRNA，其过表达可抑制胰腺癌细胞迁移和生长 5,并抑制喉癌细胞增殖,诱导其凋亡 6 。微小RNA-27a-3(microRNA-27a-3,miR-27a-3)是一种致癌非编码RNA，上调miR-27a-3p表达可提高乳腺瘤细胞MCF-7的增殖、迁移及侵袭能力)。本实验探究MEC3与miR-27a-3p关系及其调控GC细胞进展的机制。1材料和方法1.1细胞系人 GC 细胞 BGC823、M G C8 0 3、SG C7 90 1、AGS、M K N2 8 和MKN451，正常胃上皮细胞GES-1来源于北京市细胞遗传学系癌症研究所。1.2主要试剂及仪器CCK-8细胞计数试剂盒（C A 12 10,北京索莱宝科技有限公司）;Ki-67、M M P-2、M M P-9、G A PD H 兔源抗体、羊抗兔IgG(GTX16667,GTX104577,GTX100458,GTX102111、GTX100118、G T X 2 13110-0 1，美国GeneTex公司）。荧光多模式酶标仪（型号：GM2000,北京普洛麦格生物技术有限公司）；显微镜（型号：CX23，日本奥林巴斯公司）。1.3检测GC细胞中MEG3与miR-27a-3p表达Tri-zol试剂提取细胞总RNA,将RNA进行逆转录制备cDNA后，行qRT-PCR。根据2-ACT法计算MEG3与miR-27a-3p表达水平。引物序列见表1。表1各基因的引物序列基因引物序列MEG3正义反义-actin正义反义miR-27a-3p正义反义U6正义反义1.4分组与转染AGS细胞(2.510 5)接种于6 孔板上，分为对照组（正常培养的AGS细胞）、GV144组（转染阴性GV144）、G V 144-M EG 3组（转染GV144-MEG3)、G V144-M EG 3+m i R-2 7 a-3p NC 组(共转染GV144-MEG3与miRNA-27a-3pNC)、GV144-MEG3+miR-27a-3p mimics 组（共转染GV144-MEG3 与 miR-27a-3p mimics)。培养 2 4 h后，收集细胞进行后续实验。1.5细胞增殖能力将AGS细胞接种到96 孔板中，每孔2 0 0 0 个细胞，培养2 4h、48 h 后加入10 LCCK-8溶液,继续培养2 h。在450 nm处测定吸光度(absorbance,A)值。1.6细胞迁移能力将每孔1x106个AGS细胞接种到6 孔培养板中，培养过夜，伤口是用尖头划伤细胞单层造成。在无血清培养基中培养,分别于0 h和24 h拍照,测量划痕区域距离。细胞愈合率(%)=(0 h5-CGTTTGTAAGCCTGGGATGT-35-CTAAAGGGGACTCACCATGA-35-GGCATCCTCACCCTGAAGTA-35-GGGGTGTTGAAGGTCTCAAA-35-CACGCAATCTCTCTGGCG-35-CCAGTGCAGGGTCCGAGGT-35-CTCGTTCGGCAGCACA-35-AACGCTTCACGAATTTGCGT-3中国现代普通外科进展2 0 2 3年9月第2 6 卷第9期划痕距离-2 4h划痕距离)/0 h划痕距离10 0%。表3各组AGS细胞中MEG3及miR-27a-3p1.7细胞侵袭能力将悬浮在1%血清培养基中的5104个细胞添加到Transwell小室的上室（基质胶包被）。下室加人6 0 0 L含10%胎牛血清的F12培680养基。孵育48 h后固定在4%多聚甲醛中，用0.1%结晶紫染色。显微镜下进行计数。1.8Westernblot检测蛋白表达提取AGS细胞总蛋白并检测蛋白浓度。以30 g每孔上样进行凝胶电泳并转膜。4下与一抗Ki-67、M M P-2、M M P-9、GAPDH(1:500)孵育过夜，二抗（1:50 0 0)常温下孵育2 h。评估目的蛋白的相对表达量。1.9双荧光素酶报告实验TargetScan数据库(http:/www.targetscan.org/vert_71/）预测MEC3与miR-27a-3p的靶向作用位点,构建野生型（WT)和突变型(MUT)MEG3双荧光素酶载体,分别与miR-27a-3pNC和miR-27a-3pmimics转染至AGS细胞中,转染48 h后测定荧光素酶活性。1.10统计学处理采用SPSS25.0软件。t检验比较组间差异，单因素方差分析用于多组间比较，以xs表示。P0.05表示差异有统计学意义。2结果2.1CC细胞中MEG3与miR-27a-3p表达与GES-1细胞比,BGC823、M G C 8 0 3、SG C 7 9 0 1、A G S、MKN28和MKN451细胞中MEG3水平降低（P0.05),miR-27a-3p水平升高（P0.05),其中AGS细胞中MEG3水平最低，miR-27a-3p水平较高（表2)。选取AGS细胞进行后续实验。表2 GC细胞中MEG3与miR-27a-3p表达情况(sn=3)细胞GES-1BGC823MGC803SGC7901AGSMKN28MKN451a:与GES-1细胞比较，P0.052.2各组ACS细胞中MEC3及miR-27a-3p表达与对照组比,GV144-MEG3组MEG3水平升高（P0.05),miR-27a-3p水平降低(P0.05);与GV144-MEG3组比,GV144-MEG3+miR-27a-3p mimics 组miR-27a-3p水平升高(P0.05)。见表3。2.3各组AGS细胞增殖情况与对照组比,GV144-MEG3组AGS细胞增殖能力降低（P0.05)。见表4。2.4各组AGS细胞迁移及侵袭情况与对照组表达情况（xs,n=3)组别对照GV144GV144-MECG3GV144-MEC3+miR-27a-3pNCGV144-MEG3+miR-27a-3pmimicsF值P值a:与对照组比较,P0.05;b:与GV144-MEG3组比较,P0.05表4各组AGS细胞增殖情况（xs,n=3组别对照GV144GV144-MEG3GV144-MEG3+miR-27a-3pNCGV144-MEG3+miR-27a-3p mimicsF值P值a:与对照组比较，P0.05;b:与GV144-MEG3组比较，P0.05比,GV144-MEG3组划痕愈合率与侵袭细胞数降低(P0.05)；与GV144-MEG3组比,GV144-MEG3+miR-27a-3p mimics 组划痕愈合率与侵袭细胞数升高(P0.05)。见表5,图1、2。表5各组AGS细胞迁移及侵袭情况（xs,n=3)组别（个）对照21.661.37GV14422.121.43GV144-MEC38.081.124GV144-MEC3+miR-27a-3pNC8.101.15GV144-MEC3+miR-27a-3pmimics14.111.31b值87.266P值0.001a：与对照组比较，P0.05；b：与GV144-MEG3组比较，P0.05MEG3miR-27a-3p0.950.111.010.120.510.064.420.1340.600.0842.380.1140.550.083.400.1240.230.02a4.630.210.470.064.480.150.440.053.520.14aMEG30.960.110.980.121.990.201.940.241.780.1626.7940.00124hA值1.110.130.980.110.650.070.580.090.810.07b15.6810.001划痕愈合率侵袭细胞数(%）132.6015.02143.0015.1356.689.11458.249.31497.4213.60b30.1700.0012.5各组AGS细胞中相关蛋白表达水平与对照组比,GV144-MEG3组Ki-67、M M P-2、M M P-9蛋白水平降低（P0.05)；与GV144-MEG3组比,GV144-MEG3+miR-27a-3p mimics 组 Ki-67、M M P-2、MMP-9蛋白水平升高（P0.05）。见表6、图2。表6 各组AGS细胞中相关蛋白因子表达水平（xs,n=3)MMP-9/组别GAPDHGAPDHGAPDH对照1.260.171.090.183.040.26GV1441.290.131.170.163.150.25GV144-MEG30.480.05（0.560.021.400.13aGV144-MEG3+miR-27a-3p NC0.510.05*0.480.03a1.780.12aGV144-MEC3+miR-27a-3p mimics 0.710.13=b 0.79 0.16b 2.590.17bF值47.142P值0.001a：与对照组比较，P0.05；b:与CV144-MEC3组比较，P0.052.6MEG3与miR-27a-3p靶向关系预测及验证TargetScan数据库结果显示,MEG3与miR-27a-3p存在靶向关系(图3);双荧光素酶实验结果显示,与miR-27a-3p1.050.121.080.110.530.040.560.05k1.680.14b66.0210.00148hA值1.340.121.320.110.570.080.490.090.960.10bh47.3620.001Ki-67/MMP-2135.10716.7610.0010.001张新如，等.lncRNAMEG3靶向调节miR-27a-3p抑制胃癌细胞增殖、迁移和侵袭的实验研究0h68124h侵袭对照组AKi-67MMP-2MMP-9GAPDH图2 各组AGS细胞相关蛋白表达A：对照组;B:GV144组;C:GV144-MEC3组;D:CV144-MEC3+miR-27a-3p NC组;E:GV144-MEC3+miR-27a-3p mimics 组MEG3WT+阴性对照组相比,MEG3WT+miR-27a-3p组细胞荧光素酶活性降低（P0.05)。见表7。3讨论据报道，MEG3在GC组织中低表达，过表达MEG3可抑制GC细胞系SGC7901和BGC823的增殖和迁移 8 。本研究发现GC细胞MEG3水平低于ENSG00000214548.8(MEG3)(ncRNA,overlapping)microRNA familymiR-27abc/27a-3pGV144组图1各组AGS细胞迁移、侵袭情况BCGV144-MEG3 组DE图3MEG3与miR-27a-3p靶向关系GV144-MEG3+miR-27a-3p NC 组表7 双荧光素酶实验结果（s,n=3)组别相对荧光素酶活性MEC3WT+阴性对照1.150.22MEG3WT+miR-27a-3p0.370.02MEG3MUT+阴性对照1.020.21MEC3MUT+miR-27a-3p0.950.20F值10.753P值0.004a：与MEC3WT+阴性对照组比较，P0.05正常胃上皮细胞，过表达MEG3能抑制AGS增殖、迁移及侵袭，与以往的研究结果一致。此外,West-ernblot结果显示,MEG3过表达时,Ki-67及MMP-2、M M P-9表达下调，这证明了MEG3抑制AGS细胞增殖、迁移和侵袭的能力。本研究通过线上数据库检索及双荧光素酶报告实验发现MEC3可靶向下调miR-27a-3p表达。miR-27a-3p已被证实在GC细胞和组织中高表达，可促进GC细胞增殖，导致GC恶化 9-10 。本研究发现:GC细胞中miR-27a-3p水平升高，与上述研究一致，证实了miR-27a-3p在GC中促进恶性进展。进一步研究显示，上调miR-27a-3p能减弱MEG3过表达对AGS细胞恶性行为的抑制作用。提示Seed positionSeedtypeTranscriptregionRepeatchr14:1013128738-mercRNAGV144-MEG3+miR-27a-3p mimics 组ConservationPrimatesMammalsothervertno67%17%(下转第6 8 7 页）0%韩国雄，等.lncRNA SNHG1靶向miR-7对结直肠癌细胞放疗抵抗的影响lated chitooligosaccharide-induced radiosensitivity in colon cancercells by sponging miR-181a-5pJ.Adv Clin Exp Med,2021,30(1):55-65.7 Bai JH,Xu J,Zhao J,et al.IncRNA SNHG1 cooperated with miR-497/miR-195-5p to modify epithelial-mesenchymal transition un-derlying colorectal cancer exacerbationJJ.J Cell 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