Molecular Mechanism Of Curcumin Targeting SPAG5 To Regulate Wnt/β-catenin Signaling Pathway And Inhibit Liver Cance

Objectives:To verify the expression of SPAG5 in hepatoma cell lines and cancer tissues of liver cancer patients,and to explore the effects of curcumin on SPAG5 expression,liver cancer cell function,and Wnt/β-catenin pathway.Methods:TCGA database was used to analyze the expression of SPAG5 in human cancer tissues;cell immunofluorescence,immunohistochemistry,Western Blot,RT-qPCR experiments were used to verify the expression of SPAG5 in liver cancer cell lines Huh7 and HCCLM3 and cancer tissues of liver cancer patients;The CCK8 experiment was used to prove the effect of curcumin on cell growth,and the effect of curcumin on the migration of liver cancer cells was observed by scratch test;the knockdown and overexpression of SPAG5 plasmids were transfected by cell transfection technology,and the regulation of curcumin on SPAG5 was verified;The effects of curcumin treatment on SPAG5 and Wnt/β-catenin pathway were verified by Western Blot and flow cytometry experiments.Results:1.The TCGA database showed that compared with normal tissues,the expression of SPAG5 in liver cancer tissues was significantly enhanced(P<0.001);2.The results of immunohistochemistry showed that SPAG5 was highly expressed in cancer tissues of patients with liver cancer,and in paracancerous tissues no expression or low expression;3.Cell immunofluorescence results showed that SPAG5 was expressed in Huh7 cells,while SPAG5 was not expressed in normal liver cells MIHA;4.RT-qPCR results showed that the expression of SPAG5 mRNA levels in Huh7 and HCCLM3 were respectively MIHA 5.The results of Western Blot showed that the relative expression of SPAG5 protein in MIHA cells of normal liver cells was(1.546±0.286),and the relative expression of SPAG5 proteins in liver cancer cell lines Huh7 cells and HCCLM3 cells were respectively(3.267 ± 0.546),(4.650 ± 0.375),(P<0.01,P<0.001);6.The results of CCK8 experiments showed that curcumin could significantly inhibit the proliferation of Huh7 and HCCLM3 cells;7.The results of cell scratch experiments showed that,a certain concentration of curcumin acting on Huh7 and HCCLM3 cells can significantly inhibit cell migration(P<0.01,P<0.001);8.Western Blot results show that curcumin inhibits the expression of SPAG5 in Huh7 cells in a concentration-dependent manner;9.Curcumin attenuated the protein expression of CyclinDl in two liver cancer cell lines(P<0.001,P<0.01).Compared with untreated liver cancer cell lines,after overexpression of SPAG5,curcumin had no effect on the expression of SPAG5 and CyclinD1.After knocking down SPAG5,curcumin also inhibited the expression of SPAG5 and CyclinD1(P<0.05,P<0.01),and increased the apoptosis of liver cancer cells(P<0.0001,P<0.0001),the expression of β-catenin protein level was weakened(P<0.05).Conclusions:1.SPAG5 showed high expression in both liver cancer tissues and liver cancer cells;2.Curcumin can inhibit the expression of SPAG5,and can inhibit the proliferation and migration of hepatoma cells,and promote the apoptosis of hepatoma cells;3.Curcumin targets SPAG5 to regulate the Wnt/β-catenin signaling pathway to inhibit liver cancer.