circRNF20/miR-766-5p/MTHFR轴对乳腺癌细胞恶性生物学行为的影响

Influence of circRNF20/miR-766-5p/MTHFR axis on malignant biological behaviors of breast cancer cells

目的探究circRNF20在乳腺癌(BC)中的表达及对BC细胞恶性生物学行为的影响,并分析其潜在机制。方法BC细胞系分为:Control组(空白对照)、Si-NC组(阴性对照)、Si-circRNF20组(转染体外靶向敲低circRNF20的siRNA)、Si-circRNF20+in-miR-766-5p组(共转染体外靶向敲低circRNF20的siRNA和miR-766-5p抑制物)和Si-circRNF20+5,10-亚甲基四氢叶酸还原酶(MTHFR)组(共转染体外靶向敲低circRNF20的siRNA和重组pcDNA的MTHFR过表达质粒)。qRT-PCR、Western blot或免疫组化检测circRNF20、miR-766-5p和MTHFR表达。采用CCK-8和集落形成检测细胞增殖;采用划痕愈合和Transwell实验评估细胞迁移和侵袭;采用流式细胞术检测细胞凋亡。双荧光素酶分析circRNF20、miR-766-5p和MTHFR之间的作用关系。建立异种移植小鼠模型,分析circRNF20对体内异种肿瘤生长的影响。结果circRNF20和MTHFR在BC中高表达,miR-766-5p低表达(P<0.05)。敲低circRNF20可明显抑制BC细胞活力、集落形成数量、迁移和侵袭能力(P<0.05),加速细胞凋亡(P<0.05);抑制miR-766-5p表达或上调MTHFR可明显削弱敲低circRNF20对BC细胞恶性行为的抑制作用(P<0.05)。miR-766-5p与circRNF20和MTHFR均存在靶向关系(P<0.05)。circRNF20的敲低抑制了体内异种移植肿瘤的生长(P<0.05)。结论敲低circRNF20通过上调miR-766-5p并抑制MTHFR的表达,从而抑制BC细胞恶性生物学行为。

Objective To explore the expression of circRNF20 in breast cancer(BC)and its effect on the malignant biological behavior of BC cells,and to analyze its potential mechanism.Methods BC cell lines were divided into:Control group(blank control),Si-NC group(negative control),Si-circRNF20 group(transfected with siRNA targeting to knock down circRNF20 in vitro),Si-circRNF20+in-miR-766-5p group(co-transfected with siRNA targeting to knock down circRNF20 in vitro and miR-766-5p inhibitor),and Si-circRNF20+5,10-methylenetetrahydrofolate reductase(MTHFR)group(co-transfected with siRNA targeting circRNF20 and MTHFR overexpression plasmid of recombinant pcDNA in vitro).The expression of circRNF20,miR-766-5p and MTHFR was detected by qRT-PCR,Western blot or immunohistochemical method.Cell proliferation was detected by CCK-8 and colony formation assay,scratch healing and Transwell assays were used to assess cell migration and invasion,apoptosis was detected by flow cytometry.The relationship between circRNF20,miR-766-5p and MTHFR were analyzed by dual-luciferase model.A xenograft mouse model was established to analyze the effect of circRNF20 on the growth of xenografts in vivo.Results circRNF20 and MTHFR were highly expressed in BC,and miR-766-5p was lowly expressed(P<0.05).Knockdown of circRNF20 greatly inhibited BC cell viability,colony formation number,migration and invasion abilities(P<0.05),and accelerated cell apoptosis(P<0.05),inhibiting the expression of miR-766-5p or up-regulating MTHFR greatly weakened the inhibitory effect of knockdown of circRNF20 on the malignant behaviors of BC cells(P<0.05).There was a targeting relationship between miR-766-5p and circRNF20 or MTHFR(P<0.05).Knockdown of circRNF20 inhibited the growth of xenograft tumors in vivo(P<0.05).Conclusion Knockdown of circRNF20inhibits the malignant biological behavior of BC cells by up-regulating miR-766-5p and inhibiting the expression of MTHFR.