Study Of The Effect And Mechanism Of MAGEC2 In The Malignant Biological Behavior Of Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is the sixth most common malignant tumor worldwide and the fourth leading cause of cancer-related deaths;it is the main cause of cancer-related deaths globally.There are 906,000 new HCC cases and over 830,000 deaths annually.Due to the poor specificity of early symptoms and the lack of effective early diagnostic methods,only 30-40% of patients can receive radical surgery.Despite the development of surgical therapy,interventional therapy,radiotherapy and chemotherapy,targeted therapy and immunotherapy in recent years,the overall treatment effectiveness is unsatisfactory because the 5-year recurrence rate is as high as 70%.The 5-year survival rate is approximately 20% in the United States,while the figure is 5% in developing countries.It is necessary to further clarify the potential molecular mechanisms of HCC development,to identify more effective targets for the diagnosis and treatment of HCC.Melanoma-associated antigen C2(MAGEC2,CT10,HCA587,MAGEE1)is a tumor testis antigen(cancer-testis antigen,CTA),which is a member of the melanoma antigen gene(MAGE)family.This molecule shows a unique expression pattern in human tissues.Normally,MAGEC2 is expressed in various histological types of tumors but not in normal tissues,except for the testis and placenta.In recent years,the role of MAGEC2 in tumor development,invasion and metastasis has been studied.Studies have shown that MAGEC2 is highly expressed in different tumors,including melanoma,multiple myeloma,lung cancer,breast cancer,prostate cancer and bladder urothelial cancer.High MAGEC2 expression is significantly related to the poor clinical prognosis of tumors.However,there are few studies on the prognostic value of MAGEC2 in HCC.The malignant behaviors of MAGEC2 in HCC development as well as the detailed mechanism of MAGEC2 in HCC invasion and metastasis are not fully understood.Therefore,this study examined the following three elements: the expression and clinical significance of MAGEC2 in HCC,the influence of MAGEC2 on malignant behavior in HCC development,and the mechanism by which MAGEC2 promotes HCC invasion and metastasis.Part One: MAGEC2 expression and prognosis research in hepatocellular carcinomaObjective: To investigate the expression of MAGEC2 in hepatocellular carcinoma(HCC)and explore its relationship with the clinicopathological characteristics and prognosis of HCC patients.Methods: Through bioinformatics analyses in The Cancer Genome Atlas(TCGA),the International Cancer Genome Consortium(ICGC),the Gene Expression Omnibus(GEO)and the Clinical Proteomic Tumor Analysis Consortium(CPTAC)databases,the expression and prognostic significance of MAGEC2 m RNA and protein in HCC were investigated.Then,20 HCC tissue and corresponding noncancerous tissue samples were collected.Real-time fluorescence quantitative polymerase chain reaction(qPCR)was performed to detect the MAGEC2 m RNA expression.A customized tissue microarray(TMA)containing 95 HCC specimens and 85 adjacent noncancerous specimens was used to examine the MAGEC2 protein expression using immunohistochemistry(IHC)analysis,and the relationship between MAGEC2 expression and the clinicopathological characteristics of HCC patients was evaluated.Kaplan–Meier curve and Cox regression analyses were performed to assess the prognostic value of MAGEC2 protein in HCC patients.Results: The data of bioinformatic analyses indicated that MAGEC2 was highly expressed in HCC tissues,and the expression of MAGEC2 in HCC tissues was significantly higher than that in non-cancerous tissues.Receiver operating characteristic curve(ROC)analysis showed that in the TCGA database,the AUC of MAGEC2 m RNA expression was 0.704(95% CI:0.653-0.755,P<0.001),the sensitivity was 96.0%,and the specificity was 52.3%.The above three parameters were 0.789(95% CI:0.746-0.832,P<0.001),90.6%,65.0% in ICGC database while the data was 0.611-0.846,30.0%-95.6% and 38.9%-92.7% in GEO database.Furthermore,the information of ROC analysis in CPTAC database confirmed that the AUC of MAGEC2 protein expression is 0.818(95% CI:0.770-0.865,P<0.001),the sensitivity was 94.5% and the specificity was 64.2%.High expression of MAGEC2 m RNA was associated with poor relapse-free survival(RFS)of HCC patients(P=0.036).The results of qPCR of clinical samples showed that MAGEC2 m RNA was also highly expressed in the HCC tissues compared with the adjacent noncancerous tissues.The AUC of MAGEC2 m RNA expression was 0.802(95% CI:0.669-0.936,P=0.001),the sensitivity was 55.0%,and the specificity was 95.0%.IHC analysis in TMA also showed that MAGEC2 protein was highly expressed in the HCC tissues and was higher than that in the adjacent noncancerous tissues.The expression of MAGEC2 was critically related to tumor size(P=0.017)and survival status(P=0.009).Survival analysis demonstrated that high expression of MAGEC2 was significantly associated with poor prognosis of HCC patients(P=0.005).MAGEC2 was identified as an independent prognostic indicator of the overall survival of HCC patients.Conclusion: The specific high expression of MAGEC2 could be witnessed in HCC tissues,and is related to the tumor size,survival status and poor prognosis of HCC patients.MAGEC2 expression can be identified as an independent prognostic factor of HCC patients overall survival.Part Two: The characteristics of MAGEC2 in the development of malignant biological behaviors of HCCObjective: To explore the function of MAGEC2 on the malignant biological behaviors of MHCC97 H,including cell proliferation,migration,invasion,inhibition of apoptosis and cell cycle transformation.Methods: The relative expression levels of MAGEC2 in the human liver cancer cell lines MHCC97 H,SK-H-1,and Hep G2 and the human normal liver cell line L02 were detected by qPCR and Western blot analyses.A MAGEC2 lentivirus interference vector was successfully constructed.After transfection of the HCC cell line,qPCR and Western blot were used to detect the expression of MAGEC2 m RNA and protein in stably transfected HCC cells.Western blot analysis was performed to detect the expression of MAGE-I family members,including MAGEC1,MAGEC3,MAGEA1,MAGEA3,MAGEA6,MAGEA9 and MAGEA10 in MAGEC2-knockdown cell clones.In vitro,CCK-8 analysis detected the proliferation of HCC cells,wound healing and transwell invasion assays explored the migration of HCC cells,and flow cytometry(FCM)analysis examined the apoptosis and cell cycle of HCC cells.Moreover,Western blot analysis was used to detect the expression of apoptosis-and cell cycle-related proteins.Results: qPCR and Western blot analyses showed that MAGEC2 was significantly upregulated in MHCC97 H cells but rarely expressed in SK-H-1,Hep G2 or L02 cells.After transfection,the MAGEC2 m RNA and protein levels were lower than those of the untransfected MHCC97 H group and the negative control group.There were no significant changes of the protein expression of MAGE-I family members MAGEC1,MAGEC3,MAGEA1,MAGEA3,MAGEA6,MAGEA9 and MAGEA10 compared with that of in control groups.CCK-8 experiments confirmed that silencing MAGEC2 could inhibit the proliferation of MHCC97 H tumor cells.Wound healing and transwell invasion assays indicated that the migration and invasion of MHCC97 H cells in the silencing MAGEC2 group was dramatically decreased.FCM detection illustrated that silencing MAGEC2 could induce apoptosis of MHCC97 H tumor cells and inhibit the transformation of G1/S phase.Moreover,silencing MAGEC2 could increase the content of cleaved caspase-3 and promote the down-regulation of Cyclin E.Conclusion: MAGEC2 could promote the proliferation,migration and invasion of the HCC cell line MHCC97 H in vitro and inhibit cell apoptosis,promote cell cycle transformation.The mechanism of inhibiting tumor cell apoptosis and inducing the cell cycle transition of MAGEC2 primarily involved the downregulation of cleaved caspase-3 and upregulation of cyclin E.Part Three: Mechanistic study of MAGEC2 in promoting the invasion and metastasis of HCCObjective: To explore the potential mechanism of MAGEC2 in promoting the invasion and metastasis of HCC.Methods: Thirty-nine formaldehyde-fixed and paraffin-embedded HCC xenograft and clinical tissue samples were collected.Clinical HCC tissue samples were divided into the primary HCC group(13 patients),the invasive group(13 patients)and the metastatic(vascular tumour thrombus)group(13 patients).In vivo,HCC xenografts were established to reveal the role of MAGEC2 in HCC development in nude mice.IHC was performed to detect the expression of the proliferation marker gene Ki67 in the HCC xenograft samples.IHC and Western blot analyses were conducted to detect the expression of MAGEC2 and epithelial mesenchymal transition(EMT)marker genes,including E-cadherin,N-cadherin,fibronectin,and slug,in xenograft samples from nude mice.Furthermore,IHC staining of MAGEC2 and EMT-related biomarkers was performed on 39 clinical HCC tissue samples.We also conducted Western blot analysis to detect the expression of TGF-β/Smad2/3 signaling pathway related genes(Smad2/3 and p-Smad2/3)in MHCC97 H cells and EMT related genes in MHCC97 H cells treating with Smad2/3 activator TGF-β1.Results: Tumor formation experiments in nude mice further confirmed that silencing MAGEC2 could inhibit the growth of MHCC97 H xenografts in vivo.Silencing MAGEC2 inhibited the expression of the Ki67 protein.Moreover,the expression of the epithelial marker E-cadherin was increased,while the expression of MAGEC2,the mesenchymal markers N-cadherin,fibronectin,and slug was decreased.In clinical HCC samples,compared with those of primary HCC tissues,the expression of E-cadherin in invasive and metastatic(vascular tumour thrombus)HCC samples was significantly decreased,and the expression of MAGEC2,N-cadherin,fibronectin,and slug was significantly increased.Furthermore,silencing of MAGEC2 significantly suppressed the expression of phosphorylated Smad2 and Smad3.Knockdown of MAGEC2 partially reversed transforming growth factor-β1(TGF-β1)-induced changes in epithelial-mesenchymal transition(EMT)markers.Conclusion: Our research suggests that MAGEC2 could increase the tumorigenic ability in vivo and promote the up-regulation of Ki67.MAGEC2 suspended the expression of epithelial markers,increased the expression of mesenchymal markers,induced HCC EMT process in MHCC97 H cell line and promoted the invasion and metastasis of HCC.MAGEC2 might promote EMT by modulating TGF-β/Smad2/3 signaling pathway.