LncRNA CBR3-AS1 promotes the malignant biological behaviors of hepatocellular carcinoma cells by targeting the miR-409-3p/hnRNPA2B1 axis

CHEN Shi; LI Huijuan; XU Ruosi

doi:10.3969/j.issn.1002-7386.2023.24.001

Abstract：Objective To investigate the regulatory effects of long non-coding RNA CBR3-AS1(lncRNA CBR3-AS1)on the malignant biological behaviors of hepatocellular carcinoma(HCC)cells by targeting the microRNA-409-3p(miR-409-3p)/heteronuclear ribonucleoprotein A2B1(hnRNPA2B1)axis.Methods Expression levels of lncRNA CBR3-AS1 and miR-409-3p in HCC tissues were detected by quantitative real-time polymerase chain reaction(qRT-PCR).The binding relationship between lncRNA CBR3-AS1 and miR-409-3p,and that between miR-409-3p and hnRNPA2B1 were assessed by dual-luciferase reporter assay.HepG2 cells were transfected with si-NC,si-CBR3-AS1,si-CBR3-AS1+anti-miR-NC,si-CBR3-AS1+anti-miR-409-3p,miR-NC,miR-409-3p mimics,miR-409-3p mimics+pcDNA,miR-409-3p mimics + pcDNA-hnRNPA2B1.Cell proliferation,apoptosis,migration,and invasion were detected by colony formation assay,Annexin V-fluorescein isothiocyanate/propidium iodide(Annexin V-FITC/PI)staining,wound healing assay and Transwell assay,respectively.Protein expressions of nuclear proliferation antigen marker(Ki67),cell cycle negative regulator(P21),B-cell lymphoblastoma(Bcl-2),Bcl-2 associated X protein(Bax),matrix metalloproteinase-2(MMP-2),and MMP-9 were detected by Western blot.A xenograft model in nude mice was created to validate the in vivo influence of lncRNA CBR3-AS1 on HCC growth and the regulatory effect on the miR-409-3p/hnRNPA2B1 axis.Results Significantly upregulated lncRNA CBR3-AS1 and downregulated miR-409-3p were detected in HCC tissues than those of paracancerous tissues(P<0.05).The binding site in the promoter regions of lncRNA CBR3-AS1 and miR-409-3p was predicted in StarBase.Dual-luciferase reporter assay showed significantly decreased relative luciferase activity in HepG2 cells co-transfected with lncRNA CBR3-AS1 wild-type plasmid and miR-409-3p mimics than that of other groups(P<0.05).LncRNA CBR3-AS1 was significantly downregulated in HepG2 cells transfected with si-CBR3-AS1 than that in cells transfected with si-NC,while miR-409-3p was significantly upregulated(P<0.05).MiR-409-3p was significantly downregulated in HepG2 cells transfected with si-CBR3-AS1+anti-miR-409-3p than that in cells transfected with si-CBR3-AS1+anti-miR-NC(P<0.05).Compared with those in cells transfected with si-NC,HepG2 cells transfected with si-CBR3-AS1 presented significantly less colonies,lower wound healing rate and invasive cell number,downregulated Ki67,Bcl-2,MMP-2 and MMP-9,higher apoptotic rate and upregulated P21 and Bax(P<0.05).Compared with those in cells transfected with si-CBR3-AS1+anti-miR-NC,HepG2 cells transfected with si-CBR3-AS1+anti-miR-409-3p presented significantly more colonies,higher wound healing rate and invasive cell number,upregulated Ki67,Bcl-2,MMP-2 and MMP-9,lower apoptotic rate and downregulated P21 and Bax(P<0.05).Bioinformatic analysis revealed the binding site in the promoter regions of miR-409-3p and hnRNPA2B1.Dual-luciferase reporter assay showed significantly decreased relative luciferase activity in HepG2 cells co-transfected with hnRNPA2B1 wild-type plasmid and miR-409-3p mimics than that of other groups(P<0.05).MiR-409-3p was significantly upregulated in HepG2 cells transfected with miR-409-3p mimics than that in cells transfected with miR-NC,while hnRNPA2B1 was significantly downregulated(P<0.05).hnRNPA2B1 was significantly upregulated in HepG2 cells transfected with miR-409-3p mimics+pcDNA than that in cells transfected with miR-409-3p mimics+pcDNA-hnRNPA2B1(P<0.05).Compared with those in cells transfected with miR-NC,HepG2 cells transfected with miR-409-3p mimics presented significantly less colonies,lower wound healing rate and invasive cell number,downregulated Ki67,Bcl-2,MMP-2 and MMP-9,higher apoptotic rate and upregulated P21 and Bax(P<0.05).Compared with those in cells transfected with miR-409-3p mimics+pcDNA,HepG2 cells transfected with miR-409-3p mimics+ hnRNPA2B1 presented significantly more colonies,higher wound healing rate and invasive cell number,upregulated Ki67,Bcl-2,MMP-2 and MMP-9,lower apoptotic rate and downregulated P21 and Bax(P<0.05).In nude mice implanted with HepG2 cells transfected with si-CBR3-AS1,they presented significantly lower tumor growth,downregulated lncRNA CBR3-AS1,upregulated miR-409-3p,and downregulated positive expression of hnRNPA2B1(immunohistochemical staining)in tumor tissues than those implanted with cells transfected with si-NC(all P<0.005).Conclusion LncRNA CBR3-AS1 is overexpressed in HCC.Knockdown of lncRNA CBR3-AS1 inhibits the proliferation,migration and invasion of HCC cells,and promotes cell apoptosis via upregulating miR-409-3p,thus downregulating hnRNPA2B1.