ETV4 Promotes Proliferation And Migration Of Colorectal Cancer Cells Via Transcriptional Activation Of LOXL2

Colorectal cancer is a common malignant tumor of the digestive tract.Screening and identification of new colorectal cancer diagnosis and treatment markers and analyzing the mechanism is of great significance for accurate diagnosis and treatment of colorectal cancer.ETV4 is an important candidate oncogene discovered in our previous screening and closely related to the development of colorectal cancer.Our previous studies have confirmed that ETV4 is highly expressed in colorectal cancer,and its expression level is correlated with the prognosis,grade,and lymph node metastasis of colorectal cancer.In vitro and in vivo animal experiments have also confirmed that ETV4 can significantly promote colorectal cancer cells Proliferation and metastasis.And it was also revealed that LOXL2 may be a potentially important target gene for ETV4.Based on this,the thesis further explores the transcriptional regulation effect of ETV4 on LOXL2 and its role in promoting the proliferation and migration of colorectal cancer cells.The main findings are briefly described below.(1)LOXL2 is highly expressed in colorectal cancer and highly positively correlated with ETV4 expressionFirst,we analyzed the expression of ETV4 in colorectal cancer.The TCGA database analysis showed that LOXL2 is also highly expressed in colorectal cancer compared with normal colorectal tissue.Quantitative RT-PCR technique was used to further verify that the expression of LOXL2 was significantly up-regulated in colorectal cancer tissues compared with adjacent normal tissues.The TCGA database analysis also showed that the overall survival time of patients with high expression of LOXL2 was lower than that of patients with low expression of LOXL2,and the disease-free survival time of patients with high expression of LOXL2 was significantly lower than that of patients with low expression of LOXL2.We further analyzed the expression correlation of LOXL2 and ETV4.The results of TCGA database analysis showed that the expression of ETV4 and LOXL2 was significantly positively correlated in colorectal cancer tissues.Quantitative RT-PCR validation also showed a significant positive correlation between ETV4 and LOXL2 expression in colorectal cancer tissues.This result suggests that ETV4 as a transcription factor may regulate the expression of LOXL2.(2)ETV4 directly activates LOXL2 transcriptionTo further analyze whether ETV4 regulates the expression of LOXL2,we used the colorectal cancer cell line HCT116 to construct the corresponding ETV4 stable overexpressing cell line(ETV4)and its control cell line(Empty),the stable knockdown cell line(ETV4sh)and its control cell line(NCsh).Quantitative RT-PCR and Western Blot analysis showed that over-expression of ETV4 could lead to up-regulation of LOXL2 mRNA and protein expression,while ETV4 knock-down resulted in down-regulation of LOXL2 mRNA and protein expression.This result suggests that ETV4 may contribute to the transcription of endogenous LOXL2.We further analyzed whether ETV4 directly regulates the transcription of LOXL2.Analysis of transcription factor binding sites revealed that the LOXL2 promoter region has multiple ETV4 binding sites.We used PCR-mediated cloning techniques to construct multiple luciferase reporter gene recombinant plasmids with different lengths of the LOXL2 gene promoter and mutants of the ETV4 binding site.Double luciferase reporter assays showed that overexpression of ETV4 significantly enhanced the activity of the LOXL2 promoter containing the ETV4 binding site,but had no activation on the site-directed mutagenesis of the ETV4 binding site.The results of ChIP experiments showed that ETV4 can bind directly to the promoter region of LOXL2.The results in this section indicate that ETV4 directly activates the transcription of LOXL2,a new ETV4 target gene.(3)LOXL2 knockdown inhibits the promotion of ETV4 on cell proliferation,migration,and EMTTo further analyze the role of LOXL2 in ETV4-mediated proliferation,migration,and EMT of colorectal cancer cells,we used RNA interference technology to knock down the expression of endogenous LOXL2 in ETV4 stable overexpressing cell line(ETV4)of HCT116.We set 4 groups,namely Empty group,ETV4 group,ETV4+siNC group and ETV4+siLOXL2 group.Cell proliferation assays and colony formation assays have shown that LOXL2 knockdown can result in significant reduction of ETV4-promoting cell proliferation and colony formation.The results of scratch healing test and Transwell invasion and metastasis assay also showed that LOXL2 knockdown significantly decreased the ETV4 promoted-migration and invasion ability of colorectal cancer cells.The results of Western Blot and indirect immunofluorescence showed that knockdown of LOXL2 could inhibit the EMT markers E-cadherin downregulation and Vimentin upregulation induced by ETV4.The results of this part suggest that LOXL2,as a target gene of ETV4,mediates the promotion of ETV4 on cell proliferation,migration,and EMT.(4)Overexpression of LOXL2 can rescue the knockdown effect of ETV4In order to further confirm the above results,we further constructed the corresponding ETV4 stable knockdown cell line(ETV4sh)and its control cell line(NCsh)respectively using the colorectal cancer cell lines HCT116 and RKO,and further in the ETV4 sh stable cell line the LOXL2 overexpression plasmid was transfected.The results of cell proliferation experiments and scratch healing experiments showed that overexpression of LOXL2 could rescue cell proliferation inhibition and cell migration inhibition caused by ETV4 knockdown.Western Blot results showed that overexpression of LOXL2 also rescued EMT marker E-cadherin upregulation and Vimentin downregulation caused by ETV4 knockdown.This part of the results again showed that LOXL2,as a target gene of ETV4,mediates the promotion of ETV4 on cell proliferation,migration,and EMT.(5)LOXL2 binds to ETV4 and promotes transcriptional activation of downstream target genes by ETV4Since both LOXL2 and ETV4 are mainly located within the nucleus.We analyzed by molecular docking method and found that there may be interaction between ETV4 and LOXL2.Co-immunoprecipitation analysis showed that ETV4 and LOXL2 bind to each other in the cells.Quantitative RT-PCR analysis showed that knockdown of LOXL2 reduced the transcriptional upregulation of ETV4 on its downstream target genes such as NID1 and PDGFRB.This result suggests that the binding of LOXL2 to ETV4 may promote or facilitate the transcriptional activation of downstream target genes by ETV4.In summary,this study found that LOXL2 is a novel ETV4 target gene.ETV4 can directly activate the transcription of LOXL2,and LOXL2 further binds to ETV4 and promotes or contributes to the transcriptional activation of downstream target genes thereby mediating the increasing colorectal cancer cell proliferation,migration and EMT effects induced by ETV4.This study further expands our understanding of the biological function of ETV4 and its promotion of the development of colorectal cancer,and provides a new perspective and experimental basis for the diagnosis and treatment of colorectal cancer based on ETV4 and LOXL2 as targets.