| BackgroundAs a common malignant digestive tumor,colorectal cancer(CRC)is characterized with high morbidity and mortality,which seriously threatens human health.La ribonucleoprotein domain family member 6(LARP6)was identified as a new candidate gene associated with CRC metastasis,but its specific role and mechanism in CRC are still unclear.Method1.GEPIA,real-time PCR,western blot and immunohistochemistry were used to analyze the LARP6 expression and its clinical significance in CRC.2.With stable LARP6-overexpressed or interfered CRC cell lines constructed,CCK8,colony formation,transwell,and orthotopic CRC mice model were used to detect the effect of LARP6 on the biological behavior of CRC.3.RNA immunoprecipitation,mRNA stability assay,polysome analysis and transwell assay were used to determine the role of ZNF267 in the inhibition of CRC invasion and metastasis by LARP6.4.Chromatin immunoprecipitation,dual-luciferase assay,enzyme-linked immunosorbent assay,and sphingomyelin synthase inhibitor Ly93 were used to analyze the role of SGMS2 and its mediated sphingomyelin synthesis in LARP6/ZNF267 axis regulation of CRC invasion and metastasis.5.Western blot,electron microscopy and confocal microscopy were used to observe the effect of LARP6 on the autophagy activity of CRC cells.Sphingomyelin synthase inhibitor Ly93 and autophagy activator Rapa were used to analyze the relationship between autophagy and LARP6/ZNF267/SGMS2 axis-regulated CRC invasion and metastasis.6.The effect of LARP6/ZNF267/SGMS2 axis on CRC invasion and metastasis was verified in vivo using orthotopic CRC mice model and clinical CRC tissues.Result1.LARP6 is down-regulated in CRC,and its expression level is associated with T stage,lymph node metastasis,distant metastasis,clinical stage and prognosis of CRC patients.2.LARP6 inhibits CRC cells migration,invasion and metastasis,but has no significant effect on CRC cells proliferation.3.Binding to ZNF267 mRNA,LARP6 enhances its stability and translational activity,thus promoting its expression.ZNF267 overexpression inhibits the migration and invasion of CRC cells,and rescue experiments showed that down-regulation of LARP6 expression promotes the invasion of CRC cells by inhibiting ZNF267 expression.4.ZNF267 binds to the SGMS2 promoter and represses its expression.By up-regulating ZNF267 expression,LARP6 inhibits SGMS2 expression,thereby increasing ceramide accumulation and reducing sphingomyelin content in CRC cells.After rescue experiments with sphingomyelin synthase inhibitor Ly93,the promotion effect of LARP6 interference on CRC cell invasion was abolished,suggesting that sphingomyelin metabolism pathway regulation is an important way for LARP6 to regulate CRC cell invasion through ZNF267/SGMS2.5.LARP6 enhances autophagy activity in CRC cells.The effects of LARP6 interference on the autophagy activity and invasion ability of CRC cells were seperately cancelled by sphingomyelin synthase inhibitor Ly93 and autophagy activator Rapa,suggesting that LARP6 interferes with the autophagy pathway by regulating sphingomyelin metabolism to regulate the invasion and metastasis of CRC cells.6.In orthotopic CRC mice model,interference of LARP6 inhibited ZNF267 expression but promoted SGMS2 expression,with ceramide accumulation reduced,sphingolipid content increased,and autophagy activity attenuated in CRC tissues.The promotion effect of LARP6 interference on the metastasis of CRC cells in vivo was offset by the use of sphingomyelin synthase inhibitor Ly93 and autophagy activator Rapa.In clinical CRC tissue samples,LARP6 expression positively associates with ZNF267 expression,but negatively with SGMS2 expression.ConclusionLARP6 expression is down-regulated in CRC and low expression is associated with poor prognosis.With the mediation of ZNF267/SGMS2,LARP6 down-regulated expression decreases ceramide accumulation,increases sphingomyelin content and inhibits autophagy activity in CRC cells,thereby promoting CRC invasion and metastasis. |
