The Role And Molecular Mechanism Of SDC2 In The Occurrence And Development Of Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma(HCC)is one of the most common malignant tumors in the digestive tract and seriously threatens people’s health and life.Because the onset of HCC is hidden and the early symptoms are not obvious,most patients are diagnosed in the middle and late stages,often accompanied by local tumor invasion or distant metastasis,thus losing the opportunity for surgery.Moreover,most HCC patients who receive surgical treatment relapse in a short period of time,and the five-year survival rate is only 18%.As a result,there is an increasing need for effective alternative treatment strategies for patients with advanced or relapsed HCC.In recent years,molecular targeted therapy based on tumor carcinogenic mechanisms has become a novel and promising therapeutic approach.Further understanding of the molecular mechanism of HCC formation and identification of target molecules and signaling pathways regulating tumor phenotypes have important clinical significance for screening potential biomarkers and therapeutic targets for inhibiting the occurrence and development of HCC and improving the therapeutic effect and prognosis of HCC patients.Syndecan 2(SDC2),also known as fibrinoglycans,belongs to the type 1 transmembrane heparan sulfate proteoglycan(HSPG)family.SDC2 can bind to a range of ligands through its heparin sulfate side chain,including matrix components,growth factors,cell adhesion molecules,and enzyme inhibitors,and regulate cell-microenvironment interactions through coreceptors.Under physiological conditions,SDC2 is widely involved in tissue differentiation,organ development,angiogenesis and other processes.Recent studies have shown that SDC2 is abnormally expressed in a variety of tumors and plays a key role in tumor formation,metastasis,adhesion,and angiogenesis.SDC2 is upregulated in breast cancer and has been shown to induce cell migration by altering cell morphology and promoting local cell adhesion and diffusion.In addition,the role of SDC2 in melanoma has been studied.SDC2 is highly expressed in malignant melanoma compared with normal melanocytes,and its expression correlates with cell migration.However,the role of SDC2 in HCC and its upstream and downstream regulatory mechanisms remain unclear.In this study,we first screened out the HCC-related molecule SDC2 through proteomics analysis and then verified its expression and analyzed the correlation between its expression and clinicopathological features through public databases and clinical HCC samples.Subsequently,in vitro and in vivo experiments were conducted to explore the differences in the biological behaviors of HCC cells with different SDC2 expression levels,such as proliferation,migration and invasion,and to clarify the specific role of SDC2 in the occurrence and development of HCC.Finally,through bioinformatics and functional experiments,the specific mechanism by which SDC2 promotes HCC migration and invasion was preliminarily revealed,which laid a theoretical foundation for the potential clinical application of SDC2 molecular changes in HCC.Part Ⅰ Proteomic analysis and key gene acquisition of HCC tissue samplesObjective1.To analyze the differentially expressed proteins between HCC tissues and normal tissues and explore their biological functions by proteomics.2.To screen key genes in the occurrence and development of HCC.Methods1.After collecting HCC tissues,protein expression was analyzed by proteomics,and the differentially expressed proteins were screened by Student’s t test.2.Biological functions of differentially expressed proteins were investigated by GO and KEGG functional enrichment analyses.3.The key gene in the occurrence and development of HCC was screened by differential expression quantitative analysis and single gene functional enrichment analysis.Results1.Compared with normal tissues,there were a large number of differentially expressed proteins in HCC tissues,including 703 upregulated proteins and 368 downregulated proteins.2.These differentially expressed proteins regulated the occurrence and development of HCC mainly by participating in biological processes such as mitochondrial electron transfer,mRNA splicing,RNA polymerase II transcriptional termination,and mitochondrial respiratory chain complex I assembly.3.SDC2 was highly expressed in HCC tissues(P<0.05),and functional enrichment analysis showed that SDC2 was involved in multiple tumor-related pathways and biological processes,such as the PPAR signaling pathway,cAMP signaling pathway and cell migration process,suggesting that SDC2 may be an oncogenic gene and play an important role in the occurrence and development of HCC.ConclusionsThere are a large number of differentially expressed proteins between HCC tissues and normal tissues,among which SDC2 is significantly highly expressed in HCC tissues and is involved in a variety of tumor-related pathways and biological processes;SDC2 may be a key gene in the occurrence and development of HCC.Part Ⅱ:The expression and clinical significance of SDC2 in HCCObjective1.To analyze the expression of SDC2 between HCC tissues and normal tissues.2.To explore the correlation between SDC2 expression and the clinicopathologic features of HCC patients.Methods1.Data from public databases such as TCGA,ICGC and GEO were used to analyze the expression of SDC2 mRNA between HCC tissues and normal tissues.2.Data from public databases such as UALCAN and HPA were used to analyze the expression of SDC2 protein between HCC tissues and normal tissues.3.qRT-PCR assays were used to detect the expression of SDC2 mRNA in 28 HCC tissues and normal tissues.4.IHC assays were used to detect the expression of SDC2 protein between HCC tissues and normal tissues,and the chi-square test was used to explore the correlation between SDC2 expression and the clinicopathologic features of HCC patients.Results1.The analysis of data from public databases such as TCGA,ICGC and GEO confirmed that the expression of SDC2 mRNA was significantly increased in HCC tissues(P<0.05).2.The analysis of data from public databases such as UALCAN and HPA confirmed that the expression of SDC2 protein was significantly increased in HCC tissues(P<0.05).3.qRT-PCR and IHC assays confirmed that the mRNA and protein levels of SDC2 were significantly increased in HCC tissues(P<0.05).4.Chi-square tests showed that the expression of SDC2 in male patients was higher than that in female patients(P=0.024),and its expression level was positively correlated with tumor size(P=0.006),pathological grade(P=0.021)and AJCC stage(P=0.004).However,there was no significant correlation with age(P=0.362),cirrhosis background(P=0.506),HBV background(P=0.378),AFP level(P=0.832),or the presence of vascular cancer embolus(P=0.561).ConclusionsSDC2 expression is increased in HCC tissues,and its expression level is positively correlated with tumor size,pathological grade and AJCC stage of patients but not significantly correlated with patient age,cirrhosis background,HBV background,AFP level and the presence of vascular cancer embolus.Part Ⅲ Effects of SDC2 on the proliferation,migration and invasion of HCC cellsObjective1.To explore the changes in the proliferation,migration,invasion and other biological behaviors of HCC cells through in vitro cytofunctional experiments after targeted treatment with SDC2.2.To explore the effect of SDC2 on the formation of HCC through in vivo experiments.Methods1.Lentivirus was used to construct SDC2 overexpression and knockdown hepatoma cell lines,and the transfection efficiency was determined by qRT-PCR and Western blotting assays.2.The CCK-8 and EdU assays were used to explore the effect of SDC2 on the proliferation ability of HCC cells.3.A flow cycle assay was used to explore the effect of SDC2 on the cell cycle of HCC cells.4.Cell scratch and Transwell migration assays were used to explore the effect of SDC2 on the migration ability of HCC cells.5.Transwell invasion assays were conducted to explore the effect of SDC2 on the invasion ability of HCC cells.6.A subcutaneous transplantation tumor model in nude mice was constructed to investigate the effect of SDC2 on the growth of HCC in vivo.Results1.SDC2 knockdown could inhibit the proliferation,migration and invasion of HCC cellsHepG2 cells were selected to establish the SDC2-knockdown hepatoma cell line.qRT-PCR and Western blotting assays showed that SDC2 expression in HepG2 cells was significantly decreased after lentivirus transfection(P<0.05).CCK-8 and EdU assays showed that SDC2 knockdown inhibited the proliferation ability of HepG2 cells(P<0.05).Flow cycle assays showed that SDC2 knockdown inhibited the transformation of HepG2 cells from G1 phase to S phase(P<0.05).The results of cell scratch and Transwell assays showed that SDC2 knockdown inhibited the migration and invasion ability of HepG2 cells(P<0.05).2.SDC2 overexpression could promote the proliferation,migration and invasion of HCC cellsHuh7 cells were selected to establish an SDC2-overexpressing hepatoma cell line.qRT-PCR and Western blotting assays showed that SDC2 expression in Huh7 cells was significantly increased after lentivirus transfection(P<0.05).CCK-8 and EdU assays showed that SDC2 overexpression significantly enhanced the proliferation ability of Huh7 cells(P<0.05).Flow cycle assays showed that SDC2 overexpression promoted the transformation of Huh7 cells from G1 phase to S phase(P<0.05).The results of cell scratch and Transwell assays showed that SDC2 overexpression enhanced the migration and invasion ability of Huh7 cells(P<0.05).3.The results of subcutaneous tumor transplantation in nude mice showed that SDC2 knockdown could inhibit the growth of HCC in vivo.Conclusions1.SDC2 promotes the proliferation,migration and invasion of HCC cells and promotes the transformation of HCC cells from G1 phase to S phase.2.SDC2 promotes HCC growth in vivo.Part Ⅳ Study on the mechanism of SDC2 in HCCObjective1.To explore the upstream regulatory molecules of SDC2 in HCC.2.To explore the molecular mechanism by which SDC2 regulates the migration and invasion of HCC cells.Methods1.ChIP,dual-luciferase reporter and siRNA transfection assays were used to confirm that TBX19 could bind to the SDC2 promoter and positively regulate SDC2 expression.2.Co-IP and siRNA transfection assays were used to confirm that SDC2 could interact with FN1 and positively regulate FN1 expression.3.FN1 overexpression and knockdown hepatoma cell lines were constructed,and cell experiments were used to confirm that FN1 could promote the migration and invasion of HCC cells by activating the PI3K/AKT/mTOR signaling pathway.4.A rescue assay was used to further confirm that SDC2 can activate the PI3K/AKT/mTOR signaling pathway by targeting FN1 to promote the migration and invasion of HCC cells.Results1.TBX19 could bind to the SDC2 promoter region and directly promote its expressionChIP assays confirmed that TBX19 could bind to the SDC2 promoter.After site-specific mutation of the binding site,SDC2 was identified as the direct target gene of TBX19 by a dual-luciferase reporter assay.In addition,the expression of SDC2 was downregulated by TBX19 knockdown in HepG2 and Huh7 cells(P<0.05).2.SDC2 could interact with FN1 and positively regulate its expressionCo-IP assays confirmed that SDC2 could interact with FN1.Moreover,qRT-PCR and Western blotting assays were used to detect the expression of FN1 in SDC2-overexpressing and SDC2-knockdown hepatoma cell lines.The results showed that in HepG2 cells,SDC2 knockdown significantly downregulated FN1 expression(P<0.05);in Huh7 cells,SDC2 overexpression significantly upregulated FN1 expression(P<0.05).3.FN1 could promote the migration and invasion of HCC cells by activating the PI3K/AKT/mTOR signaling pathwayAfter overexpression and knockdown of FN1 by using an overexpression plasmid and siRNA,we detected the protein expression changes of various molecules in the PI3K/AKT/mTOR signaling pathway.The results showed that the expression levels of p-PI3K,p-AKT and p-mTOR were significantly upregulated after overexpression of FN1(P<0.05);however,the expression levels of p-PI3K,p-AKT and p-mTOR were significantly decreased after FN1 knockdown(P<0.05).Meanwhile,a Transwell assay was conducted to explore the effects of FN1 expression changes on the migration and invasion of HCC cells.The results showed that SDC2 overexpression enhanced the migration and invasion of HepG2 cells(P<0.05);however,SDC2 knockdown inhibited the migration and invasion of Huh7 cells(P<0.05).4.SDC2 activated the PI3K/AKT/mTOR signaling pathway by targeting FN1 to promote the migration and invasion of HCC cellsTo further clarify the mechanism of SDC2 in HCC,we designed a rescue assay.The results showed that the migration and invasion abilities of SDC2 knockdown HepG2 cells were significantly recovered after overexpression of FN1(P<0.05);however,the migration and invasion abilities of SDC2-overexpressing Huh7 cells were significantly decreased after knockdown of FN1(P<0.05).Meanwhile,Western blotting assays were used to detect the protein expression changes of various molecules in the PI3K/AKT/mTOR signaling pathway.The results showed that the expression levels of p-PI3K,p-AKT and p-mTOR,which were downregulated due to SDC2 knockdown,were partially restored after overexpression of FN1 in HepG2 cells(P<0.05);however,the expression levels of p-PI3K,p-AKT and p-mTOR,which were upregulated due to SDC2 overexpression,were partially restored after knockdown of FN1 in Huh7 cells(P<0.05).The above results indicated that SDC2 could activate the PI3K/AKT/mTOR signaling pathway by targeting FN1 to promote the migration and invasion of HCC cells.Conclusions1.TBX19 can bind to the promoter region of SDC2 and directly promote its expression.2.SDC2 can activate the PI3K/AKT/mTOR signaling pathway by targeting FN1 to promote the migration and invasion of HCC cells.