Mechanisms Of YAP Promoting Colon Cancer Cell Proliferation Through Inhibiting Autophagic Death

Objective: Colon cancer is one of the most common malignant tumors in the world.Its incidence is the third highest in the world and in China,and it shows an upward trend year by year.Studies have shown that YAP,high expression in colon cancer,is closely related with prognosis.As a downstream effector of hippo signaling pathway,the disregulation of YAP will change the expression of related genes,which will affect the cell proliferation and cell apoptosis,then,will affect the size of organs and the formation of tumors.Recent years,the role of autophagy in tumorigenesis has drawn more and more attention,which plays an important role in tumorigenesis,metastasis and therapeutic resistance.Studies have shown that YAP is involved in autophagy and tumorigenesis,but the mechanism is unclear.Therefore,in-depth study of the molecular mechanism of YAP and autophagy in colon cancer has a positive meaning for us to elucidate the pathogenesis of colon cancer and look for new therapeutic targets.Methods: Immunohistochemistry and Western blot were used to detect the expression of YAP in the cancer tissues of patients with colon cancer or colon cancer cells.The third generation lentivector packaging system was used to establish CRC cell lines(HCT116 and SW620)stably overexpressing YAP or shYAP.Then,the expression of YAP in stable cell lines was dectected through Real-time PCR and western blot.Cell proliferation was detected by Cell counting and Colony formation assays.Western blot was used to detect the expression of autophagy-related marker in different YAP expression levels.Electron microscopy was used to detect the number of autophagosomes under different YAP expression levels.RT-qPCR and Western blot were used to dectect the autophagy-related genes in colon cancer transfected with pcDNA3.1-HAYAP or in stable YAP-overexpressing cell lines.SPSS 17.0 was used for statistical analysis.Differences between data groups were evaluated for significance using Student t test of unpaired data or one way analysis of variance and Bonferroni post-test.P values less than 0.05 indicate statistical significance.Results: Immunohistochemistry showed that YAP highly expressed in colon cancer tissues compared with normal tissues;Western blot also showed that YAP highly expressed in colon cancer cells compared with 293 T.We successfully established CRC cell lines(HCT116 and SW620)stably overexpressing YAP or shYAP.RT-qPCR and Western blot showed that YAP mRNA levels and YAP protein expression were significantly increased or inhibited in stable CRC cell lines(p<0.05).Cell growth assay showed that YAP promoted colon cancer cell growth(p<0.05);Colony formation assays showed that YAP encouraged cell clone formation(p<0.05).Western blot showed that autophagy was inhibited whenYAP highly expressed(p<0.05);furthermore,Autophagy was stimulated after inhibiting the expression of YAP(p<0.05).Moreover,electron microscopy showed that the number of autophagosomes was decreased after overexpressing YAP;however,the number of autophagosomes was significantly increased after inhibiting YAP.RT-qPCR and Western blot showed that Bcl-2 mRNA level and protein level both improved in colon cancer transfected with PCDNA3.1-HA-YAP or in stable YAP-overexpressing cell lines(p<0.05).Conclusion:(1)YAP can promote colon cancer cell proliferation.(2)YAP can inhibit spontaneous autophagy in colon cancer cells.(3)YAP may promote tumor cell proliferation by inhibiting autophagic death.