| Colorectal carcinoma(CRC)is mainly caused by unhealthy lifestyle and poor awareness,which makes CRC rank third and second in morbidity and mortality among tumors.The development of CRC is often accompanied by metastasis,which is the main cause of death from CRC.Epithelial-mesenchymal transition(EMT)is a typical transformation process in which tumor cells acquire metastasis and drug resistance.It is characterized by down-regulation of epithelial markers(E-cadherin,etc.)and up-regulation of mesenchymal markers(vimentin,etc.)in EMT process.WNT/β-catenin is a classic EMT regulatory signaling pathway in which β-catenin proteins enter the nucleus and up-regulate the expression of EMT-related transcription factors such as Snail and ZEB-1.Inhibition or reversal of EMT is an important therapeutic strategy for many metastatic tumors.Retinoid X receptor alpha(RXRα)is a member of nuclear receptor(NR)superfamily which regulate many important physiological functions such as differentiation,proliferation and apoptosis.Recent studies have shown that RXRα expression is abnormal in some tumor tissues.However,the correlation between RXRα expression and EMT-induced tumor metastasis in human CRC remains unclear.20(S)-protopanaxadiol [20(S)-PPD] is the aglylate of panaxadiol saponins prepared by acid-base catalysis.Numerous studies have shown that panaxadiol saponins,have the inhibitory effect on EMT such as Rb1,Rb2,Rb3,Rg3,etc.Previous studies in our laboratory have proved that 20(S)-PPD plays anti-tumor roles by inducing apoptosis,and inhibiting EMT,but its effect on EMT of CRC cells remains unclear.Based on the above study,we will explore the mechanism of RXRαin EMT process in CRC cells,and explore the intervention effect and mechanism of20(S)-PPD in EMT process of CRC cells.We first found abnormal expression of RXRα in colon and rectal cancer patients through UALCAN database.Immunohistochemical staining was performed on tumor tissues and paracancer tissues of 20 CRC patients who did not receive other treatment methods.The expression difference and correlation of RXRα protein in tumor and paracancer tissues under various clinicopathological features were analyzed.The expressions of RXRα protein and EMT related protein in tumor tissues were also compared.The results showed that the expression of RXRα in tumor tissues was significantly lower than that in paracancer tissues.The expression of RXRα in tumor tissues was significantly different in N stage(N0 vs N1-N2).The expression of RXRαprotein was significantly correlated with N stage,and the correlation was moderately negative.The expression of E-cadherin in tumor tissues was lower than that in paracancer tissues,and the expression of vimentin,Snail and ZEB-1 in tumor tissues were significantly higher than that in paracancer tissues,suggesting that RXRα may be involved in tumor metastasis.To prove the role of RXRα in EMT,SW480 and SW620 cells were selected from4 CRC cell lines for experiment.Wound-healing assay and Transwell assay was used to assess the migration and invasion capacities of SW480 and SW620 cells.Western Blot was performed to detect the expression of EMT-related factors in SW480 and SW620 cells.CRC liver metastasis model was prepared in vivo.The results showed that SW620 cells had higher migration and invasion capabilities,lower E-cadherin and higher of vimentin,Snail,Slug and ZEB-1 levels than SW480 cells with lower RXRα protein level.SW620 cells were more prone to liver metastasis than SW480 cells in vivo.Subsequently,SW620 and SW480 cells were treated with RXRα overexpressed plasmid and SiRNA,respectively.Wound-healing assay and Transwell assay was used to assess the migration and invasion capabilities of SW620 and SW480 cells after the intervention of RXRα expression.Western Blot and q RT-PCR were used to detect the expression of RXRα and EMT-related factors in protein and mRNA levels.Finally,the interaction between RXRα and β-catenin protein was verified using Int Act database combined with immunoprecipitation assay.The results showed that overexpression of RXRα inhibited the migration and invasion capabilities of SW620 cells,significantly increased the expression of E-cadherin and significantly decreased the expression of vimentin,Snail,ZEB-1 and β-catenin in protein and mRNA levels.Meanwhile,overexpression of RXRα inhibited the expression of β-catenin in the nucleus.Silencing RXRα promoted the migration and invasion capabilities of SW480 cells,significantly decreased the expression of E-cadherin and significantly increased the expression of vimentin,Snail,ZEB-1 and β-catenin,including the expression ofβ-catenin in the nucleus.Database and immunoprecipitation results showed that RXRα may interact with β-catenin in CRC cells,suggesting that RXRα may inhibit EMT by inhibiting nuclear β-catenin expression in CRC cells.To investigate the effect and mechanism of 20(S)-PPD on CRC cells,SW620 cells were treated with 20(S)-PPD(10,20,and 30μM).Wound-healing assay and Transwell assay was used to assess the effects of 20(S)-PPD on the migration and invasion capabilities of SW620 cells.Western Blot and q RT-PCR were used to detect the effects of 20(S)-PPD on the expression of RXRα and EMT-related factors in SW620 cells.CRC liver metastasis model was prepared in vivo,and the effects of20(S)-PPD(50 and 100 mg/Kg)were observed.The crystal structure of RXRα was simulated with 20(S)-PPD molecular structure by molecular simulation docking technique,and the binding ability of 20(S)-PPD to RXRα was predicted and verified in cell experiments.The results showed that 20(S)-PPD inhibited the migration and invasion capabilities of SW620 cells in a dose-dependent manner,and significantly increased the expression of E-cadherin,significantly decreased the expression of vimentin,Snail,ZEB-1 and β-catenin while upregulating the expression of RXRα in protein and mRNA level.Both low and high doses of 20(S)-PPD significantly inhibited the number of liver nodules and the incidence of liver metastasis in vivo with a dose-dependent manner.In molecular docking simulation,the docking position of20(S)-PPD with RXRα was the same as 9-cis-retinoic acid,which is the endogenous ligand of RXRα,and it has a certain strength of binding energy.The inhibitory effect of 20(S)-PPD on EMT of CRC cells was antagonized by RXRα silencing intervention.It is suggested that 20(S)-PPD has a certain binding effect on RXRα protein,and RXRα may be one of the targets of 20(S)-PPD.In conclusion,this study preliminatively confirmed the relationship between RXRα and tumor metastasis in clinical CRC.The role of RXRα in EMT was demonstrated in vitro and in vivo.In addition,this study found a new role and mechanism of 20(S)-PPD,providing a new theoretical basis and new perspective for the development of 20(S)-PPD into class A new drug of Traditional Chinese medicine. |
PDF Full Text Request