miR-153-3p通过靶向ACTN4调节食管癌细胞的恶性生物学行为

miR-153-3p regulates malignant biological behaviors of esophageal cancer cells by targeting ACTN4

为探究miR-153-3p靶向调控α-辅肌动蛋白4(α-actinin-4,ACTN4)对食管癌(esophageal cancer,ESCA)细胞增殖、迁移和侵袭的影响。通过qRT-PCR检测不同细胞系中miR-153-3p和ACTN4 mRNA表达,双荧光素酶实验验证miR-153-3p和ACTN4的靶向关系。体外培养ECA109细胞并将其分为6组:空白组、对照模拟物(miR-NC)组、 miR-153-3p模拟物(miR-153-3p)组、空载体组、 ACTN4过表达(ACTN4)组以及miR-153-3p模拟物和ACTN4过表达(miR-153-3p+ACTN4)组。qRT-PCR和Western blotting检测miR-153-3p、 ACTN4表达水平;CCK-8和集落形成实验检测细胞增殖情况;Transwell检测细胞迁移、侵袭情况。Western blotting检测Ki67、 E-钙黏蛋白(E-cadherin)和N-钙黏蛋白(N-cadherin)蛋白水平。构建皮下异种移植肿瘤裸鼠模型,检测上调miR-153-3p对肿瘤生长的影响,免疫组化检测ACTN4、 Ki67、 E-cadherin和N-cadherin表达。结果显示,miR-153-3p在ESCA细胞系中低表达(P<0.05),ACTN4 mRNA高表达(P<0.05)。miR-153-3p可负向调节ACTN4表达。miR-153-3p过表达可降低细胞活力、集落形成能力和迁移、侵袭能力,并抑制Ki67和N-cadherin表达,上调E-cadherin表达(P<0.05)。ACTN4过表达则表现出与之相反的效果(P<0.05);过表达ACTN4可逆转miR-153-3p过表达对ESCA细胞行为的影响(P<0.05)。体内研究表明,miR-153-3p过表达可抑制ACTN4、 Ki67、 N-cadherin表达,升高E-cadherin表达,减缓肿瘤生长。该研究提示,miR-153-3p过表达可能通过靶向负调控ACTN4抑制ESCA细胞增殖、侵袭和迁移。

The study aims to explore the effects of miR-153-3p on the proliferation,migration and invasion of esophageal cancer(ESCA) cells by targeting and regulating α-actinin 4(ACTN4).The expression of miR-153-3p and ACTN4 mRNA in different cell lines was detected by qRT-PCR.The targeting relationship between miR-153-3p and ACTN4 was verified by double luciferase assay.ECA109 cells were cultured in vitro and divided into six groups:blank group,mimic control(miR-NC) group,mimic(miR-153-3p) group,empty vector group,ACTN4 overexpression(ACTN4) group,the mimic and ACTN4 overexpression(miR-153-3p+ACTN4) group.qRT-PCR and Western blotting were performed to measure the expression of miR-153-3p and ACTN4;CCK-8 and colony formation experiment was performed to measure the proliferation of cells;Transwell was performed to measure the migration and invasion of cells.Western blotting was used to detect the protein levels of Ki67,E-cadherin,and N-cadherin.A nude mouse model of subcutaneous xenograft tumors was constructed in vivo and the effect of up-regulation of miR-153-3p on tumor growth was detected.Immunohistochemistry was performed to measure the expression levels of ACTN4,Ki67,E-cadherin and N-cadherin in tumor tissues.The results showed that,the expression of miR-153-3p in the ESCA cell line was low(P<0.05),and ACTN4 mRNA was highly expressed(P<0.05).miR-153-3p was able to negatively regulate ACTN4 expression(P<0.05).Overexpression of miR-153-3p can reduce cell viability,colony formation,migration and invasion,inhibit Ki67 and N-cadherin expression,and up regulate E-cadherin expression(P<0.05).ACTN4 overexpression showed the opposite effects(P<0.05);overexpression of ACTN4 can reverse the effects of miR-153-3p overexpression on ESCA cell behavior(P<0.05).In vivo studies have shown that overexpression of miR-153-3p can inhibit the expression of ACTN4,Ki67,N-cadherin,increase the expression of E-cadherin,and slow down tumor growth.This study suggests that overexpression of miR-153-3p may inhibit the proliferation,invasion and migration of ESCA cells by targeting ACTN4 negatively.