LINC00662通过调控miR-199a-5p/MAP3K1通路对胃癌侵袭、转移的影响

Influence of LINC00662 on the invasion and metastasis of gastric cancer via miR-199a-5p/MAP3K1 pathway regulation

目的探讨LINC00662通过调控miR-199a-5p/丝裂原活化蛋白激酶激酶激酶1(MAP3K1)通路对胃癌侵袭、转移的影响。方法收集2018年6月至2020年6月期间我院胃癌患者癌组织及癌旁组织,实时定量PCR检测胃癌组织及癌旁组织中LINC00662、miR-199a-5p、MAP3K1 mRNA表达。取对数生长期的SGC-7901细胞,利用LipofectamineTM2000转染试剂盒进行转染,并分为si-NC组、si-LINC00662组、si-LINC00662+anti-miR-NC组、si-LINC00662+anti-miR-199a-5p组、si-LINC00662+miR-NC组、si-LINC00662+miR-199a-5p组、miR-NC组、miR-199a-5p组、空载体组、LINC00662过表达组、anti-miR-NC组、anti-miR-199a-5p组,另取未转染的SGC-7901细胞作为空白组。Transwell实验检测各组细胞的侵袭与迁移;划痕实验检测各组细胞迁移能力;Western blotting检测各组细胞中MAP3K1、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)、神经型钙黏蛋白(N-cadherin)表达;双荧光素酶报告基因实验验证LINC00662与miR-199a-5p、miR-199a-5p与MAP3K1的靶向关系。结果与癌旁组织比较,胃癌组织中LINC00662、MAP3K1 mRNA表达水平显著升高,miR-199a-5p表达水平显著降低(均P<0.05)。与空白组和si-NC组比较,si-LINC00662组SGC-7901细胞中LINC00662表达水平显著降低,侵袭、迁移细胞数目显著减少,划痕愈合率显著降低,MAP3K1、MMP-2、MMP-9、N-cadherin相对表达量显著降低,miR-199a-5p表达水平显著升高(均P<0.05)。与si-LINC00662组和si-LINC00662+anti-miR-NC组比较,si-LINC00662+anti-miR-199a-5p组SGC-7901细胞中miR-199a-5p表达水平显著降低,侵袭、迁移细胞数目显著升高,划痕愈合率显著升高,MAP3K1、MMP-2、MMP-9、N-cadherin相对表达量显著升高(均P<0.05)。与si-LINC00662组和si-LINC00662+miR-NC组比较,si-LINC00662+miR-199a-5p组SGC-7901细胞中miR-199a-5p表达水平显著升高,侵袭、迁移细胞数目显著降低,划痕愈合率显著降低,MAP3K1、MMP-2、MMP-9、N-cadherin相对表达量显著降低(均P<0.05)。双荧光素酶报告基因实验证实LINC00662靶向负调控miR-199a-5p表达,miR-199a-5p靶向负调控MAP3K1表达。结论胃癌组织中LINC00662高表达,si-LINC00662通过调控miR-199a-5p/MAP3K1通路抑制胃癌细胞侵袭、转移。

Objective To investigate the influence of LINC00662 on the invasion and metastasis of gastric cancer cells via miR-199a-5p/mitogen-activated protein kinase kinase kinase 1(MAP3K1)pathway regulation.Methods Cancer and adjacent tissues of patients with gastric cancer collected in our hospital from June 2018 to June 2020 were used for this study.qRT-PCR was used to detect the expression of LINC00662,miR-199a-5p,and MAP3K1 mRNAs in gastric cancer and adjacent tissues;SGC-7901 cells in logarithmic growth phase were transfected using the LipofectamineTM2000 transfection kit,and were subsequently divided into si-NC,si-LINC00662,si-LINC00662+anti-miR-NC,si-LINC00662+anti-miR-199a-5p,si-LINC00662+miR-NC,si-LINC00662+miR-199a-5p,miR-NC,miR-199a-5p,empty vector,LINC00662 overexpression,anti-miR-NC,and anti-miR-199a-5p groups.Untransfected SGC-7901 cells were considered the blank group.Transwell assays were performed to detect the invasion and migration of cells in each group;a scratch test was performed to detect the cell migration ability of each group;Western blotting was performed to detect the protein expression of MAP3K1,matrix metalloproteinase-2(MMP-2),matrix metalloproteinase-9(MMP-9),and neural cadherin(N-cadherin)in cells from each group;and a dual luciferase reporter gene experiment was performed to verify the targeting relationship between LINC00662 and miR-199a-5p,and between miR-199a-5p and MAP3K1.Results Compared with adjacent tissues,the expression levels of LINC00662 and MAP3K1 mRNAs in gastric cancer tissues were significantly increased,whereas the expression level of miR-199a-5p was significantly reduced(P<0.05).Compared with the blank and si-NC groups,the mRNA expression level of LINC00662 in SGC-7901 cells from the si-LINC00662 group was significantly reduced,as were the numbers of invading and migrating cells,the scratch healing rate,and the relative protein expression of MAP3K1,MMP-2,MMP-9,and N-cadherin.In contrast,the mRNA expression level of miR-199a-5p was significantly increased(P<0.05).Compared with the si-LINC00662 and si-LINC00662+anti-miR-NC groups,the mRNA expression level of miR-199a-5p in SGC-7901 cells from the si-LINC00662+anti-miR-199a-5p group was significantly reduced,whereas the numbers of invading and migrating cells,the scratch healing rate,and the relative protein expression of MAP3K1,MMP-2,MMP-9,and N-cadherin were significantly increased(P<0.05).Compared with the si-LINC00662 and si-LINC00662+miR-NC groups,the mRNA expression level of miR-199a-5p in SGC-7901 cells from the si-LINC00662+miR-199a-5p group was significantly increased,whereas the numbers of invading and migrating cells,the scratch healing rate,and the relative protein expression of MAP3K1,MMP-2,MMP-9,and N-cadherin were significantly reduced(P<0.05).The dual luciferase reporter gene experiment confirmed that LINC00662 targeted and negatively regulated the expression of miR-199a-5p,while miR-199a-5p targeted and negatively regulated the expression of MAP3K1.Conclusion LINC00662 is highly expressed in gastric cancer tissues.The inhibition of LINC00662 deters the invasion and metastasis of gastric cancer cells via miR-199a-5p/MAP3K1 pathway regulation.