The Molecular Mechanism Of Cell Adhesion Molecule SVEP1 In Regulating Malignant Progression Of Hepatocellular Carcinoma

Background and ObjectiveSince the 20th century,cancer has become a critical disease threatening human health.The International Agency for Research on Cancer evaluated the morbidity and mortality of 27 major cancers worldwide in 2012,and the results showed that there were 14.1 million new cancer cases and 8.2 million cancer-related deaths,among which about 22%new cases and 27%deaths occurred in China.As it's known,the poor prognosis of malignant tumors is often closely related to their early metastasis.Metastasis is one of the most essential biological characteristics of malignant tumors,and it is the most critical factor of death in most patients in clinical practice.Approximately,90%cancer patients dead of metastasis.It can be recognized that metastasis is the most challenging task in the treatment of cancer,as well as one of the central issues in cancer research.The variation of intercellular adhesion ability plays an important role in the process of metastasis,and cell adhesion molecules(CAMs)are closely related to the variation of the adhesion ability of tumor cells,but the mechanism still needs to be clarified.In the early stage of this project,the whole transcriptome sequencing analysis was performed on the tumor tissues of two groups of hepatocellular carcinoma(HCC)patients.The patients'Barcelona staging(BCLC staging)were B and they had different relapse and metastasis characteristics.We found that the differentially expressed genes were significantly enriched in the adhesion-related pathways,and by extracting and comparing the differentially expressed genes,the Heatmap analysis revealed SVEP1,a CAMs molecule,expressed significantly lower in high postoperative recurrence group compared with the low postoperative recurrence group.By considering relevant domestics and previous research results,SVEP1 may be a key molecule which can mediate recurrence and metastasis of HCC.Therefore,this project aims to clarify the relationship between SVEP1 and HCC recurrence and metastasis,then explore the specific mechanism of SVEP1 in HCC metastasis through several methods such as immunohistochemistry,bioinformatics analysis,western blotting,RT-PCR,a series of cell function assays,dual luciferase reporter assays,whole transcriptome sequencing analysis,functional rescue experiments,and animal experiments.Methods1.207 HCC tissue microarrays and 4 pairs of HCC frozen tissue samples were used to explore the expression of SVEP1 and its effect on the prognosis of HCC through immunohistochemistry staining,Kaplan-Meier analysis,Cox regression,Spearman correlation analysis,subgroup analysis and Western Blotting.2.Based on TCGA and GEO databases,the conclusions that obtained by immunohistochemistry staining and Western Blotting were verified through bioinformatics analysis(differential expression analysis,survival analysis and GSEA analysis).3.Western Blotting were used to detect the differences of SVEP1 expression level in different HCC cell lines.The SVEP1 stably down-regulated group and control group of HCC cell lines were constructed by lentiviral infection.4.Cell proliferation related assay(CCK-8 assay),cell invasion and migration related assays(chemotaxis assay,invasion assay,cell movement assay and wound-healing assay)were conducted by using SVEP1 stably down-regulated group and control of HCC cell lines.The effect of different SVEP1 expression levels on the proliferation,invasion and migration of HCC were explored.5.To investigate potential microRNAs which could regulate SVEP1,we recompared the transcriptome sequencing results of BCLC-B HCC patients with different recurrence risks,combined with five public databases,including miRWalk,RNA22,miRanda,Targetscan,and mirbase.6.TCGA public database was used to perform differential expression analysis and survival analysis on related microRNAs.7.The direct regulation of related microRNA on SVEP1 was verified by dual luciferase reporter assay.HCC cell lines with different microRNA expression levels were constructed and verified,then the expression levels of SVEP1 among them were detected.The effects of different microRNA expression levels on the function of HCC cell line were performed through functional experiments.8.Completely transcriptome sequencing analysis of the SVEP1 down-expressing group and control group of HCC cell lines was used to explore differentially expressed genes and related enrichment pathways.The downstream molecular network of SVEP1 regulating HCC progression was explored.9.HCC xenograft models with low expression of SVEP1 and control cell lines were built to observe the effects of different levels of SVEP1 on the proliferation, invasion and metastasis of HCC cells in vivo.The changes of the expression level of important molecules in related signaling pathways were verified.Results1.By applying high-throughput RNA sequencing in BCLC-B HCC patients with different recurrence risk,we confirmed that the differential genes were significantly enriched in the adhesion pathway.SVEP1 expression was significantly reduced in patients with high recurrence risk.The low expression of SVEP1 was associated with poor prognosis of HCC,which was confirmed by TCGA,GEO and GSEA analysis.2.Through Western Blotting and immunohistochemical staining,we found that the expression level of SVEP1 in HCC adjacent tissues was significantly higher than that in corresponding cancer tissues.Low expression of SVEP1 is an independent risk factor of the overall survival(OS)and disease-free survival(DFS)in HCC. The correlation between the expression level of SVEP1 and clinicopathological factors was analyzed through Spearman method,and it demonstrated that the expression level of SVEP1 was significantly negatively correlated to the tumor size (3cm was defined as the critical value of tumor size)and the incidence of microsatellite lesions of HCC.Surprisingly,in these two subgroups,patients with high-SVEP1 expression levels still had a longer DFS time than patients with low- SVEP1 expression levels,indicating that high-SVEP1 level may be a protect factor for prognosis of HCC.3.Hep3B and HCCLM3 cells were used to establish stably knockdown cell lines,and Western Blotting was performed to confirm the efficiency of SVEP1 deletion.4.We found that SVEP1 expression down-regulation significantly promoted HCC cell proliferation by CCK-8 assay.Functional cell movement assay in Hep3B,as well as the chemotaxis assay,transwell assay and wound-healing assay results in both Hep3B and HCCLM3 cells showed that SVEP1 knockdown promoted migration and invasion in vitro.5.Based on the whole transcriptome sequencing data,Target Scan and other public databases,we found that miR-1269b might potentially regulate the transcription level of SVEP1.6.TCGA database analysis showed that the expression level of miR-1269b in HCC was significantly higher than that in para tumor tissues.High expression of miR-1269b was related to poor prognosis of HCC patients.7.The dual luciferase reporter assay confirmed that miR-1269b can directly regulate the transcription level of SVEP1.Then we constructed and verified the miR-1269b over-expression and low-expression HCC cell lines.The scratch assay confirmed the over-expression of miR-1269b enhanced cell migration ability,while low-expression of miR-1269b led to the reduction of cell migration ability.8.Through the whole transcriptome sequencing analysis of Hep3B-SCR and KD cell lines,we found that the differentially expressed genes were mainly enriched in the PI3K/Akt signaling pathway.Heatmap analysis confirmed that down-regulation of SVEP1 could mediate the upregulation of FGF9,THBS1,NFKB1,CCNE2 and other important gene expression in the PI3K/Akt signaling pathway.RT-PCR were further used to verify the above conclusions in the Hep3B-SCR/KD cell line.9.The expression levels of p-Akt³⁰⁸ in Hep3B-SCR/KD and Hep3B-KD+LY294002 (the inhibitor of PI3K/Akt signaling pathway)was changed,while the expression levels of total AKT and p-Akt⁴⁷³ were not altered.Comparing with Hep3B-KD cell line,the proliferation and invasion ability of Hep3B-KD+LY294002 cells was decreased.10.We found that compared with the control group,the SVEP1-knockdown group had dramatically accelerated tumor growth and markedly formed larger and heavier tumor nodules.The formation of metastatic lung nodules in the SVEP1-knockdown group was also markedly increased compared with that in the control group.And the expression levels of p-Akt³⁰⁸ and PKC?were higher in SVEP1-knockdown group.ConclusionsSVEP1,as an important CAMs for cell adhesion,plays an important role in regulating the stable condition of cells and tissues.There is no research to explain the regulatory mechanism of SVEP1 in the progression of malignant tumors at present.In our previous data,through the transcriptome sequencing analysis on BCLC-B stage HCC patients with different recurrence risk,we selected SVEP1,as one of the DEGs,was significantly enriched in the adhesion signaling pathway.Further immunohistochemical staining analysis with a large sample,as well as TCGA and GEO database analysis initially confirmed that low expression of SVEP1 could be an independent risk factor for predicting the prognosis of HCC.Low expression of SVEP1was closely related to tumor size and metastasis.A series of functional experiments confirmed that down-expression of SVEP1 promoted the proliferation,invasion and metastasis of HCC.Through full transcriptome sequencing and public database analysis,we found that SVEP1 was the direct target gene of miR-1269b.The SVEP1expression regulated the malignant phenotype transformation of HCC through the PI3K/Akt signaling pathway.The series of results were further confirmed by animal models.Low expression of SVEP1 promoted the progression of HCC in vivo.The analysis of large databases of biological samples,molecular biology experiments,cell biology experiments and animal models were performed in our research.SVEP1 may act as the key factor in regulating mechanisms and principles in the transformation of malignant phenotypes of HCC.These results confirmed the role of miR-1269b-SVEP1-PI3K/Akt axis in proliferation,invasion and metastasis of HCC.It will play a vital role as a potential therapeutic target in enriching clinical treatment methods.