Molecular mechanism of latent transforming growth factor beta binding protein 4 inhibiting metastasis of colorectal cancer by affecting pseudopod formation in tumor cells
Objective To investigate the molecular mechanism of latent transforming growth factor beta binding protein 4(LTBP4)in-hibiting the metastasis of colorectal cancer cells by influencing pseudopod formation in tumor cells.Methods In colorectal cancer DiFi cells,si-LTBP4 was used to knock down LTBP4,while in colorectal cancer HCT15 cells,LTBP4 was overexpressed.Stable cell lines shLTBP4 and shNC were constructed using LTBP4-knockdown and empty lentiviral vectors,respectively.Western blot was used to detect the expression of LTBP4,tropomyosin 4(TPM4),matrix metalloproteinase-14(MMP14),tyrosine kinase substrate 5(TKS5),etc.Quanti-tative real-time PCR(qPCR)was used to measure LTBP4 and TPM4 expressions.Transwell migration assay was performed to assess the in vitro migration ability of colorectal cancer cells with altered LTBP4 expression.A lung metastasis mouse model was constructed by in-jecting shLTBP4 cells into the tail vein of nude mice to detect the in vivo metastasis ability of colorectal cancer cells after knocking down LTBP4,and mice injected with shNC cells was used as control.Transcriptome sequencing was conducted to analyze the changes in gene expression.Immunofluorescence staining was used to observe cytoskeleton changes and invadopodia formation in LTBP4-knockdown cells.Results qPCR results showed that LTBP4 was highly expressed in the DiFi,HCT8,and KM12C cell lines(all P＜0.05),and was expressed at low levels in the HCT15 and KM12SM cell lines(both P＜0.01).Western blot assay revealed high expression of LTBP4 in the DiFi,HCT8,and KM12C cell lines(all P＜0.01),and low expression in the HCT15,KM12SM,and RKO cell lines(all P＜0.05).Transfec-tion of small interfering si-LTBP4 in the DiFi cell line which had high LTBP4 expression resulted in the knockdown of LTBP4.Transwell migration assay conducted 72 hours post-transfection showed enhanced migration ability of cells(P＜0.01).Conversely,LTBP4 overex-pression in the HCT15 cell line which had low LTBP4 expression led to weakened migration 72 hours after transfection(P＜0.01).For the lung metastasis mouse model,in vivo fluorescence imaging in small animals performed 5 weeks after injecting shLTBP4 or shNC cells via the tail vein revealed increased fluorescence in the lungs of the mice with LTBP4 knockdown(P＜0.01).High-throughput transcriptome sequencing indicated that LTBP4 knockdown in the DiFi cell line increased the expression of the cytoskeletal protein TPM4(P＜0.01)and significantly enriched pathways related to the maintenance of cell motility functions,such as the contraction of actin filament bundles,regulation of actin filament polymerization and alteration of stress fibers(all P＜0.05),and pathways related to the alteration of cellular morphology,including the regulation of axon stretch,presynaptic membranes,nuclear pore and transmembrane transport of active ions(all P＜0.05).Western blot,qPCR,and immunofluorescence experiments confirmed that LTBP4 knockdown in the DiFi cell line promoted TPM4 expression(all P＜0.01).Conclusions LTBP4 inhibits colorectal cancer cell metastasis by regulating TPM4 and suppressing inva-dopodia formation.
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维普
