Mechanism Of Ursolic Acid Inhibiting Colon Cancer Metastasis Through KLF4/Arl4c Pathway

Objective:The occurrence of colon cancer metastasis is a great challenge for clinical treatment of colon cancer.In this study,we explored the role of ursolic acid in regulating KLF4/Arl4c pathway to inhibit colon cancer metastasis and its molecular mechanism in vitro and vivo,so as to provide a theoretical basis for ursolic acid in the treatment of colon cancer metastasis.Method:1.Human colon cancer HCT-116 and SW480 cells were used as the research objects.CCK-8 assay was used to detect the effect of different concentrations of ursolic acid(5μM,10μM,15μM,20μM,25μM,30μM,40μM)on the proliferation of colon cancer cells.Transwell assay was used to detect the effect of ursolic acid at different concentrations on the metastasis of colon cancer cells.Western blot was used to detect the protein expression of E-cadherin and MMP2.2.The expression levels of KLF4 and Arl4c in human colon cancer tissues and adjacent tissues were detected by immunohistochemistry.TCGA database was used to analyze the relationship between KLF4,Arl4c expression levels and survival rate and lymph node metastasis of patients with colon cancer.3.The colon cancer cell lines with KLF4 interference,Arl4c over-expression and Arl4c knockdown were constructed by lentivirus and siRNA transfection.Transwell assay was used to detect the effects of KLF4 interference and Arl4c over-expression on the migration and invasion of colon cancer cells.The effect of KLF4 interference on the expression of E-cadherin and MMP2 protein was detected by immunofluorescence.Western blot was used to detect the expression of AKT/mTOR,Arl4c,E-cadherin and MMP2.4.Western blot was used to detect the effects of AKT/mTOR signaling pathway inhibitors LY294002,Rapamycin and LY294002 combined with MG-132 on the expression of Arl4c protein.Immunoprecipitation and Western blot were used to measured the effects of LY294002 and ursolic acid on the ubiquitination level of Arl4c protein.5.Transwell assay was used to detect the effects of KLF4 interference and Arl4c overexpression on ursolic acid inhibiting the migration and invasion of colon cancer cells.Western blot was used to detect the effects of KLF4 interference and Arl4c overexpression on ursolic acid inhibiting the metastasis-related protein expression.The lung metastasis model of colon cancer was established by injecting KLF4 knockout HCT-116-luc cells via tail vein,which was divided into KLF4-NC group,KLF4-KD group,KLF4-NC combined with ursolic acid group,KLF4-KD combined with ursolic acid group.The effect of KLF4 knockdown on ursolic acid inhibiting lung metastasis of colon cancer was observed by in vivo imaging.The pathological changes of lung metastases were detected by HE staining.The expression of Arl4c protein in each group was detected by immunohistochemistry.Results:1.With the increase of drug dosage,the inhibitory effect of ursolic acid on the proliferation,invasion and metastasis of colon cancer cells gradually increased.Western blot confirmed that ursolic acid up-regulated E-cadherin expression and down-regulated MMP2 expression.In vivo experiments also confirmed that ursolic acid could reduce the number of lung metastasis in nude mice and alleviate the pathological changes of lung metastases.2.Immunohistochemistry showed that KLF4 was low expressed in human colon cancer,while Arl4c was high expressed in human colon cancer.TCGA database analysis showed that KLF4 expression level was positively correlated with survival rate and lymph node metastasis of colon cancer patients,while arl4c expression level was negatively correlated with survival rate of colon cancer patients.3.KLF4 knockdown could promote the expression of Arl4c protein.KLF4 knockdown or Arl4c over-expression promote the invasion and migration of colon cancer cells.Immunofluorescence showed that the expression of E-cadherin decreased and MMP2 increased after KLF4 knockdown.Western blot showed that KLF4 knockdown or Arl4c over-expression could down-regulate E-cadherin expression and up-regulate MMP2 xpression,while Arl4c knockdown could up-regulate the expression of Ecadherin and down-regulate the expression of MMP2.4.Western blot results showed that KLF4 knockdown could promote the phosphorylation of AKT and mTOR.LY294002 and rapamycin could reduce the protein expression of Arl4c.MG-132 could reduce the inhibitory effect of LY294002 on Arl4c.LY294002 could increase the ubiquitination level of Arl4c.LY294002 could weaken the inhibitory effect of KLF4 knockdown on Arl4c.5.Ursolic acid promote the expression of KLF4,inhibit the activation of AKT/mTOR signaling pathway,and then reduce the expression of Arl4c in colon cancer cells.KLF4 knockdown or Arl4c over-expression weakened the inhibitory effect of ursolic acid on metastasis of colon cancer cells.KLF4 knockdown weakened the inhibitory effect of ursolic acid on the expression of Arl4c.Arl4c over-expression reduced the effect of ursolic acid on up-regulation of E-cadherin expression and down-regulation of MMP2 expression.In vivo experiments also confirmed that the inhibitory effect of ursolic acid on lung metastasis of colon cancer was decreased after KLF4 knockdown.In vivo imaging experiments showed that the inhibitory effect of ursolic acid on lung metastasis of nude mice was decreased after KLF4 knockdown.Immunohistochemistry showed that the inhibitory effect of ursolic acid on the expression of Arl4c was decreased after KLF4 knockdown.Conclusion:1.Ursolic acid inhibit the metastasis of colon cancer in vivo and vitro.2.KLF4 promote the ubiquitination of Arl4c by inhibiting AKT/mTOR signaling pathway,and ultimately inhibit the invasion and migration of colon cancer cells.3.The mechanism of ursolic acid inhibiting the metastasis of colon cancer cells in vitro and vivo is related to promoting the expression of KLF4,inhibiting AKT/mTOR signaling pathway and and then reducing the expression of Arl4c protein.