Effects Of Co-culture Of Prostate Mesenchymal Stem Cells And LNCaP Cells On Key Components Of Wnt Pathway

OBJECTIVE:To explore the relationship between the influence of mesenchymal stem cells on prostate cancer cells and the Wnt signaling pathway,as well as between the influence of prostate cancer cells on mesenchymal stem cells and the Wnt signaling pathway,to reveal the molecular mechanism of the interaction between mesenchymal stem cells and prostate cancer cells,and to provide ideas for seeking a new treatment for prostate cancer.METHODS:Using human prostate mesenchymal stem cells(hPMSCs),human prostate cancer cells(LNCaP)and human normal prostate matrix immortalized cells(WPMY-1),four groups of non-contact co-culture system were established.Group A(upper hPMSCs-LNCaP under),group B(upper LNCaP-hPMSCs under),group C(upper WPMY-1-LNCaP under),group D(upper LNCaP-WPMY-1 under),and control group hPMSCs(upper BLANK-hPMSCs under),control group LNCaP(upper BLANK-LNCaP under)and control group WPMY-1(upper BLANK-WPMY-1 under);The proliferation ability of the middle and lower layer cells in groups A,B,C,D and blank control group was detected by CCK-8 method at 0d,2d,4d and 6d,respectively,and the differences of the proliferation ability of the middle and lower layer cells in each system were compared and analyzed.After 6 days of co-culture,qRT-PCR was used to detect and compare the expressions of GSK-3β,β-catenin and CyclinDl genes of key components of Wnt signaling pathway in the middle and lower layer cells of groups A,B,C,D and blank control.After 6 days of co-culture,the expression of GSK-3β,β-catenin and CyclinD1 proteins in the middle and lower layer cells of groups A,B,C and D as well as blank control were extracted and compared by Westernblot method.RESULTS:1.The results of CCK-8 method showed that the cell viability of the lower and middle layers of LNCaP cells in co-culture system A was significantly higher than that of the lower and middle layers of LNCaP cells in co-culture system C at 0d,2d,4d and 6d,with statistical significance(P<0.05).The cell viability rates of hPMSCs in the middle and lower layers of co-culture system B at 0d,2d,4d and 6d were significantly higher than those of blank control group,with statistical significance(P<0.05).The cell viability of WPMY-1 cells in the middle and lower layers of coculture system D at 0d,2d,4d and 6d was slightly lower than that of the blank control group,and the cell viability of the two groups at 2d and 4d was statistically significant(P<0.05).There was no significant difference in cell viability between the two groups at day 0 and day 6(P>0.05).2.The results of qRT-PCR showed that the key components of Wnt signaling pathway expressed by LNCaP in the sublayer cells of co-culture system A were significantly higher than LNCaP in the sublayer cells of co-culture system C,with statistical significance(P<0.05).The expressions of key components of Wnt signaling pathway GSK-3β,β-catenin and CyclinDl genes of hPMSCs in the sublayer of coculture system B were significantly higher than those of blank control hPMSCs,with statistical significance(P<0.05).The key components of the Wnt signaling pathway GSK-3β,β-catenin and CyclinDl genes expressed in the lower layer cells of coculture system D were significantly higher than those in blank control WPMY-1 cells,with statistical significance(P<0.05).3.The results of Westernblot showed that the LNCaP of the underlayer cells of co-culture system A expressed GSK-3β,β-catenin and CyclinD1,the key components of Wnt signaling pathway,were significantly higher than LNCaP of the underlayer cells of co-culture system C,with statistical significance(P<0.05).The expressions of key components of Wnt signaling pathway GSK-3β,β-catenin and CyclinD1 in the sublayer cells of co-culture system B were significantly higher than those of blank control hPMSCs,with statistical significance(P<0.05).The expression of key components of Wnt signaling pathway GSK-3β,β-catenin and CyclinD1 in the lower layer cells of coculture system D was significantly higher than that in blank control WPMY-1 cells,with statistical significance(P<0.05).CONCLUSION:Human prostate mesenchymal stem cells can promote the proliferation of prostate cancer cells,and prostate cancer cells can also promote the proliferation of human prostate mesenchymal stem cells.Human prostate mesenchymal stem cells can increase the expression of genes and proteins of key components of Wnt/β-catenin signaling pathway(GSK-3β,β-catenin,CyclinD1)in prostate cancer cells.Meanwhile,prostate cancer cells can also increase the expression of genes and proteins of key components of Wnt/β-catenin signaling pathway(GSK-3β,β-catenin,CyclinD1)in human prostate mesenchymal stem cells.In addition,prostate cancer cells can also increase the expression of genes and proteins of key components of Wnt/β-catenin signaling pathway(GSK-3β,β-catenin,CyclinD1)in prostate stromal cells WPMY-1.