Expression And Molecular Mechanism Of Maelstrom In Esophageal Squamous Cell Carcinoma

Background and objective Esophageal carcinoma respectively accounted for the sixth and eighth of the tumor-related deaths and the diagnosis of malignant diseases in China.Although the improvement of diagnosis and treatment methods of esophageal carcinoma,prognosis of patients is still poor,5-year survival rate is only 17%.The main reason for this is that early symptoms of esophageal carcinoma is not obvious,most patients are diagnosed at the advanced stage.In China,Esophageal Squamous Cell Carcinoma(ESCC)is the main pathological type of esophageal carcinoma.So far,the cellular and molecular mechanisms leading to ESCC have not been illuminated.Therefore,it is urgent for us to find a target for the treatment of ESCC.Maelstrom(MAEL)was initially identified on the chromosome of Drosophila,which played an important role in spermatogenesis,meiosis and the silence of Piwi-mediated transcription transposon.In humans,it was first discovered that MAEL was only expressed in male testis germ cells;later studies demonstrated that it was also abnormally expressed in many kinds of tumor cells.There are a lot of evidence demonstrating that MAEL is a novel cancer-testis antigen that could be regulated by methylation.At present,MAEL was identified at 1q24 in human hepatocellular carcinoma,bladder cancer and colorectal cancer,promoting tumor cell invasion,migration and metastasis and being closely related to the poor prognosis of cancer patients.Therefore,MAEL has a number of advantages as targets for immunotherapy.So far,expression,function and the related mechanism of MAEL in ESCC has not been studied.In tumor microenvironment,apart from tumor cells,it is also composed of many immunosuppressive cytokines and cells,they can affect the development of tumor.IL-8(interleukin-8)is a important immunosuppressive chemokine in the tumor microenvironment,functional on CXCR1 and CXCR2,could chemoattract Myeloid-derived suppressor cells(MDSCs)to tumor site.MDSCs are a important immunosuppressive cell in tumor microenvironment,it could secret lots of immunosuppressive cytokines,playing a role in promoting tumor progression.This study was designed to investigate the expression and function of MAEL in ESCC and to analyze its correlation with clinical pathological parameters and prognosis of ESCC patients,and further discuss the related mechanisms involved in the progression of ESCC.Part1 The expression and function analysis of Maelstrom in Esophageal Squamous Cell CarcinomaMethods 1)qRT-PCR were used to detect the expression of MAEL in ESCC and corresponding adjacent normal tissues.2)The relationship of MAEL expression and clinicopathological parameters of ESCC patients was analyzed.3)Immunohistochemistry(IHC)were used to detect the expression of MAEL protein in ESCC and corresponding adjacent normal tissues.4)The relationship of MAEL expression and the prognosis of ESCC patients was analyzed.5)qRT-PCR was performed to detect MAEL expression in immortalized human esophageal cell line Het-1A and esophageal cancer cell lines.6)The lentiviral-mediated MAEL overexpression or knockdown vectors was respectively used to transfect TE7 or TE1 cell lines.qRT-PCR and Western blot was respectively utilized to detect the expression of MAEL.7)CCK-8 kit were used to detect the growth of tumor cells when MAEL overexpression or knockdown.8)Sphere formation assay was used to observe the effect of MAEL overexpression or knockdown on the ability of sphere formation.9)qRT-PCR were used to detect the effect of MAEL overexpression or knockdown on the stemness of tumor cells.10)In-vivo xenograft esophageal tumor model of nude mice was established to observe the effect of MAEL overexpression or knockdown on the tumor growth.Results 1)The expression of MAEL m RNA was higher in ESCC than that in corresponding adjacent normal tissues(P <0.05).2)The expression of MAEL m RNA was correlated with lymph node metastasis,stage and differentiation of ESCC patients(P <0.05).3)The expression of MAEL protein was higher in ESCC than that in corresponding adjacent normal tissues(P <0.05).4)Disease-free survival rate and overall survival rate of MAEL high expression was lower than that of MAEL low expression,P<0.05.5)The expression of MAEL m RNA was detected in the cell lines,which was the lowest in TE7 and the highest in TE1(P <0.05).6)qRT-PCR and Western blot was used to detect the efficiency of MAEL overexpression or knockdown,that is,the expression of MAEL at m RNA and protein level was significantly increased or decreased(P <0.05).7)Overexpression or knockdown of MAEL,the growth of esophageal cancer cell was significantly increased or decreased(P <0.05).8)The ability of sphere formation was promoted or inhibited when MAEL overexpression or knockdown(P <0.05).9)The expression level of stemness-related marker CD133,OCT4,Nanog and Notch1 was significantly increased or decreased when MAEL overexpression or knockdown(P <0.05).10)In in-vivo xenograft esophageal tumor model of nude mice,tumor formation ability,tumor volume and quality was significantly increased or decreased when MAEL overexpression or knockdown(P <0.05).Summary 1)The expression of MAEL was higher in ESCC than that in corresponding adjacent normal tissues,and it was correlated with clinicopathological parameters and survival time of ESCC patients.The results show that MAEL might be a prognostic factor in ESCC.2)Tumor cell proliferation,self-renewal and the stemness was significantly increased or decreased when MAEL overexpression or knockdown,suggesting that MAEL plays a important role in ESCC malignant behavior.3)MAEL overexpression or knockdown could promote or inhibit the tumor formation and development in the xenograft tumor model of nude mice.Part2 Maelstrom could regulate Akt1/Rel A/IL-8 signaling pathway to recruit MDSCsMethods 1)Multi-cytokine kit、qRT-PCR and Elisa assay were utilized to detect the effect of MAEL overexpression or knockdown on the expression of IL-8.2)qRT-PCR was used to detect the expression of IL-8 in ESCC and corresponding adjacent normal tissues,and analyze the correlation with MAEL.3)The expression of IL-8 in ESCC and corresponding adjacent normal tissues was detected by IHC.4)The correlation between IL-8 protein and prognosis of ESCC patients was analyzd. 5)Flow cytometry was used to detect the purity of MDSCs sorted by magnetic beads.6)Transwell assay were used to detect the effect of MAEL overexpression or knockdown and IL-8 neutralizing antibody on recruiting MDSCs by IL-8.7)Xenograft esophageal tumor model of nude mice was established to observe the effect of IL-8 on recruiting MDSCs when MAEL knockdown.8)Western blot and qRT-PCR were used to detect the expression of Akt1,Rel A and IL-8 protein when MAEL overexpression or knockdown and PI3 k,Akt1 and Rel A inhibitors.9)qRT-PCR was used to detect the expression of MAEL,stemness-related gene and EMT marker when the supernatant of MDSCs was incubated with cancer cells.10)Elisa assay was used to detect the expression of cytokines in MDSCs supernatant.11)The expression of MAEL,stemness-related gene and EMT marker were detected by qRT-PCR after TE7 were treated with TGF-β,IL-6 and IL-10.12)Western blot was used to detect the expression of MAEL,Smad2 and Smad3 in cells treated with TGF-β and/or TGF-β inhibitor.Results 1)Expression of IL-8 was increased or decreased accordingly when MAEL overexpression or knockdown(P <0.05).2)At the m RNA level,the expression of IL-8 in cancer tissues was significantly higher than that in adjacent normal tissues,and positively correlated with MAEL(P <0.05).3)The expression of IL-8 protein in ESCC was significantly higher than that in corresponding adjacent normal tissues(P <0.05).4)Disease-free survival rate and overall survival rate of IL-8 protein high expression was lower than that of IL-8 protein low expression(P <0.05).5)The purity of MDSCs was higher than 90% after sorted by magnetic beads.6)IL-8 on the recruitment of MDSCs was significantly increased or decreased when MAEL overexpression or knockdown,and the effect was significantly decreased when cells treated with IL-8 neutralizing antibody(P <0.05).7)In in-vivo xenograft esophageal tumor model of nude mice,the ability to recruit MDSCs was significantly decreased when MAEL knockdown or IL-8 neutralizing antibody-treated cells(P <0.05).8)The expression of p Akt1、p Rel A and IL-8 protein and m RNA was significantly increased or decreased when MAEL overexpression or MAEL knockdown or cells respectively treated with p Akt1、p Rel A and IL-8 inhibitor(P <0.05).9)The expression of MAEL、CD133、Nanog、Notch1、b β-catenin、E-caderin、Fibro and Vimentin was significantly changed when the supernatant of MDSCs was incubated with cancer cells(P <0.05).10)The expression of TGF-β、IL-6、IL-10、IL-8 and TNF-β protein could be detected in the supernatant of MDSCs by Elisa.11)TE7 was respectively treated with TGF-β、IL-6 and IL-10,the expression of MAEL was only significantly increased when cells treated with TGF-β(P <0.05).12)The expression of MAEL,p Smad2 and p Smad3 protein in TE7 and TE1 cells treated with TGF-β was significantly increased(P <0.05);the expression of that was downregulated when cells treated with TGF-β inhibitor(P <0.05).Summary 1)The expression of IL-8 in ESCC was higher than that in adjacent normal tissues,and correlated with the expression of MAEL and the survival time of patients,suggesting that IL-8 may be an indicator of the prognosis of patients with ESCC.2)The capacity of IL-8 on the recruitment of MDSCs was significantly increased or decreased when MAEL overexpression or knockdown(P <0.05).3)MAEL could regulate IL-8 via the activation of Akt1/Rel A signaling pathway in ESCC.4)TGF-β could regulate MAEL via the activation of smad2/smad3 signaling pathway in MDSCs supernatant of ESCC patients.Conclusions 1)The expression of MAEL in ESCC was significantly higher than that in adjacent normal tissues,and correlated with the clinical parameters and prognosis of patients,and affected the proliferation,self-renewal ability and stemness of esophageal cancer cells.2)MAEL regulates IL-8 by activating the Akt1/Rel A signaling pathway,recruiting MDSCs to the tumor site,and TGF-β secreted by MDSCs regulates MAEL by activating smad2/smad3.