Effect Of HIF-1?/Ascl2/miR-200b Axis And YAP1 On Cellular Behavior Of Colorectal Cancer

Background and ObjectiveThe hypoxic microenvironment of tumors is one of the characteristics of most solid tumors.Expression of hypoxia-inducible factors?HIFs?is promoted and stabilized under Hypoxia.HIF-1?can activate Epithelial-mesenchymal transition?EMT?-related proteins expression and induce EMT occurrence.The HIF-1?subunit is the major reactive group of HIFs^[1]and is regulated by intracellular oxygen levels.HIF-1a regulates EMT through a variety of pathways.EMT is considered as a key process including morphological changes during embryonic development and tumor formation.The miR-200 family plays critical roles in arresting cell migration and modulating epithelial-mesenchymal transition in a wide range of cancer cells.It has been reported that miR-200/ZEB is involved in multiple pathways to inhibit EMT^[2-5].Our previous study provided the evidence of a link between Achaete Scute-like 2?Ascl2?and miR-200s in the regulation of EMT-MET plasticity in colorectal cancer?CRC?^[6].Ascl2 has been shown to be affected by hypoxia in previous reports.Yes-associated protein1?YAP1?,a downstream effector of the Hippo pathway,which plays an important role in regulating cell growth,apoptosis and self-renewal of stem cells.0ver-expression of YAP1 was found in several types of malignancies including breast,pancreas,liver,gastric,cholangiocarcinoma,and colorectal cancers^[7].AXL,CTGF,and CYR61 play an important role in promoting tumor metastasis as a downstream target molecule of YAP1^[8].We primarily studied how HIF-1?/Ascl2/miR-200b axis regulated EMT occurrence in CRC cells,and the effect of YAP1 on tumor prognosis of CRC patients and biological behavior change of CRC cells.Methods1.HIF-1?affects EMT of CRC cells through Ascl2/miR-200b axis1.1 The real-time quantitative PCR was performed for the quantitation of HIF-1?,Ascl2 mRNAs and miR-200b in 50 CRC and their adjacent tissues from CRC patients.1.2 Correlation analysis between HIF-1?and miR-200b,HIF-1?and Ascl2 expression.1.3 Real-time quantitative PCR and Western blot were used to detect the expression changes of HIF-1?,Ascl2 and EMT markers during anoxia?1%O2?of CRC cells?LS2174T cells and SW480 cells?.1.4 Under the condition of hypoxia?1%O2?,the dynamic changes of EMT process in CRC cells?LS174T cells and SW480 cells?were observed by interfering with the synthesis of Ascl2.1.5 Transfection of sh-Ascl2/LS174T cells and control cells with miR-200b inhibitor.1.6 Real-time quantitative PCR and Western blot were used to detect the dynamic changes of Ascl2,miR-200b and EMT in CRC cells?LS174T cells and SW480 cells?under HIF-1?interference and HIF-1?overexpression.2.YAP1 affects the prognosis of CRC patients and promotes cellular behavior changes through its target genes2.1 YAP1 expression levels in tumor tissues and normal tissues were from TCGA database,and the relationship betweenf YAP1 levels in tumor tissues and distant metastasis of patients was based on SPSS13.0.2.2 Analysis of the correlation between YAP1 and CRC prognosis.2.3 YAP1 enforced expression in CRC cell lines?Lv-YAP1/SW480 cells and Lv-YAP1/HT29 cells?was produced using Lentivirus particles expressing YAP1.2.4 The cellular behaviors of Lv-YAP1/SW480 cells and Lv-YAP1/HT29 cells was based on cell proliferation and cytotoxicity assay using CCK-8 solution,transwell migration assay,transwell invasion assay.2.5 Real-time quantitative PCR and Western blot were used to detect AXL,CTGF and CYR61 expression in Lv-YAP1/SW480 and Lv-YAP1/HT29 cells.The migration and invasion ability changes in AXL,CTGF or CYR61 interfered Lv-YAP1/SW480 or Lv-YAP1/HT29 cells were performed as described previous.Results1.HIF-1?affects EMT of CRC cells through Ascl2/miR-200b axis1.1 HIF-1?and Ascl2 in cancer tissues was significantly higher than normal adjacent tissues?P<0.01?.The miRNA-200b levels in cancer tissues was significantly lower than normal tissues?P<0.01?.1.2 The HIF-1?mRNA level was inversely correlated with miR-200b?R=-0.324,p<0.05?,and the HIF-1?mRNA was correlated with Ascl2 expression?R=0.333,p<0.05?in human CRC samples.1.3 HIF-1?,Ascl2 and mesenchymal markers?N-cadherin and ZEB1?were increased under hypoxia?1%O2?,whereas,E-cadherin expression was repressed.The phenomenon was reversed by Ascl2 interference.1.4 MiR-200b in CRC cells reduced under hypoxia?1%O2?which was also reversed by Ascl2 interference.1.5 MiR-200b inhibitor transfection in Ascl2 interfered CRC cells promoted EMT.1.6 HIF-1 interference promoted miR-200b expression in CRC cells,inhibited Ascl2expression,and inhibited EMT progression.HIF-1?enforced expression in CRC cells exerted opposite changes.2.YAP1 affects the prognosis of CRC patients and promotes cellular behavior changes through its target genes2.1 There was higher YAP1 expressed in CRC tissues than normal adjacent tissues?P=0.002?,YAP1 expression is positively correlated with the distant metastasis of CRC patients?P=0.001?.2.2 YAP1 expression is correlated with disease-free survival of CRC patients?P=0.008?,and correlated with overall survival of CRC patients?P=0.022?.2.3 YAP1 expression was associated with the patient age?P=0.040?and distant metastasis?P=0.024?.2.4 Univariate and multivariate Cox regression analysis indicated that YAP1 was independent prognostic factor of disease-free survival and overall survival.2.5 YAP1 overexpression promoteed the proliferation,invasion and migration of CRC cells.The migration and invasion ability in Lv-YAP1/SW480 and Lv-YAP1/HT29 cells was mediated AXL,CTGF and CYR61.Conclusions1.HIF-1?/Ascl2/miR-200b axis regulated EMT occurrence in CRC cells.2.YAP1 affects CRC prognosis and cellular behaviors of CRC cells via its target genes AXL,CTGF and CYR61.