Preliminary Studies Of CRABP2 Inhibition Of Esophageal Squamous Cell Carcinoma Through The Classical Tretinoin Signaling Pathway Under The Intervention Of ATRA

Objective: By detecting the expression of tretinoin binding protein II in esophageal squamous cell carcinoma and exploring its role and function,it is proposed to preliminarily verify the possible mechanism of CRABP2 in the classical tretinoin signaling pathway through the transport of ATRA to synergistically inhibit the proliferation,migration,invasion and promote apoptosis of ESCC,so as to provide potential therapeutic targets for improving the prognosis of ESCC and supply a theoretical foundation for improving the clinical treatment effect of ESCC.Methods:(1)Isobaric Tagging Reagent for Absolute titration Quantitation(i TRAQ)proteomics was used to screen differentially expressed proteins in normal squamous epithelial tissues and ESCC of 6 patients from Xinjiang,and candidate markers were identified by bioinformatics analysis platform.At the tissue level,240 cases of paraffin-embedded ESCC and paired adjacent normal tissues were selected to make tissue chips,and the expressions of CRABP2、FABP5、RARγ、PPARβ/δ in 172 cases of ESCC and adjacent tissues were detected by immunohistochemistry(IHC),the correlation between the expressions of CRABP2,FABP5,RARγ,PPARβ/δ and clinicopathological parameters and prognosis of ESCC patients were analyzed.The relationship with CRABP2,FABP5,RARγ,PPARβ/δ,CRABP2/FABP5 and RARγ/PPARβ/δ was analyzed.(2)At the cell level,a stable strain of CRABP2 overexpression was established,and the overexpression efficiency of CRABP2 was detected by Western blot and q RT-PCR.The effects of CRABP2 and CRABP2+ATRA on the biological functions of ESCC cells were observed by CCK-8,Transwell and flow cytometry.The localization of CRABP2 with FABP5 and CRABP2 with mitochondria in cells were observed by immunofluorescence,and the relationship between CRABP2 and ATRA was analyzed.Biochemical kits and Western blot were used to detect oxidative stress,mitochondria and key proteins in related signaling pathways,and analyze the possible signaling pathways involved in the synergistic inhibitory effect of CRABP2 on cancer under ATRA intervention.(3)The tumor model of nude mice was established and compared among the four groups.The effect of CRABP2 combined with ATRA on tumor-forming ability in mice was observed by monitoring the volume and weight of tumor-forming tissue,HE staining,immunohistochemistry and TUNEL staining.Results:(1)After proteomics and online database analysis,the differentially expressed protein CRABP2,retinoic acid derivative ATRA and classical retinoic acid signal pathway were selected as the research objects.(2)The low expression of CRABP2 was found in ESCC tissues,and there was no correlation between the low expression of CRABP2 and the clinicopathological features of ESCC.Patients with high expression of CRABP2 had a longer overall survival(P=0.025),and CRABP2 could be used as an independent prognostic factor for OS evaluation in patients with ESCC.(3)Overexpression of CRABP2 inhibited the malignant phenotype of ESCC,and CRABP2 inhibited the proliferation,migration,invasion and promoted apoptosis of ESCC through the classical retinoic acid signaling pathway.Oxidative stress imbalance,mitochondrial pathway,p53 and NF/κB signaling pathway may be involved in the synergistic inhibitory effect of CRABP2 and ATRA.(4)CRABP2 combined with ATRA could significantly inhibit the growth of subcutaneous tumor-forming tissue in tumor-bearing mice,and the combination of CRABP2 and ATRA could affect the early and late stage of apoptosis.Conclusion:CRABP2 is a key differentially expressed protein down-regulated in ESCC.The low expression of CRABP2 is related to poor prognosis of ESCC.Through the three levels of tissue,cell and mouse,it was verified that CRABP2 transported ATRA through the classical tretinoin signaling pathway to inhibit cell proliferation,invasion,migration,and promote apoptosis.CRABP2 combined with ATRA can significantly inhibit tumor growth.CRABP2 combined with ATRA may exert anticancer effects through oxidative stress,mitochondrial pathway,p53 and NF-κB signaling pathway.ATRA is expected to be a candidate for complementary esophageal squamous cancer treatment.CRABP2 may be a new target for adjuvant targeted therapy of ESCC.