Sema3d Low Expression In HCC Promotes Progression And Epithelial-Mesenchymal Transition And Predicts Poor Prognosis

Background:Hepatocellular carcinoma(HCC)is one of the most common lethal malignancies,which ranks the sixth most popular cancers and the third leading cause of cancer-related death worldwide.About 50%of HCC cancer cases and deaths occurred in China,which have the most abundant new cases and deaths of HCC.Up to now,surgical resection or liver transplantation may provide an opportunity for a cure only when patients are diagnosed at an early stage.For patients with advanced or recurrence HCC who lost the opportunity for surgical resection,the introduction of Sorafenib or Lenvatinib may offer but only limited survival benefits.Despite recent progresses in prevention,diagnosis and treatment of HCC,the prognosis remains unsatisfactory due to the high incidence of postoperative recurrence and extrahepatic metastasis,and the 5-year overall survival after resection remains low.Therefore,it’s crucial to investigate the mechanism of recurrence and metastasis,and to identify novel molecular markers for improving the long term survival of patients with HCC,and means a lot to precision treatment and obtain a better prognosis.Semaphorin 3d is a member of class 3 semaphorins(Sema3 proteins),a family of secreted and transmembrane signaling molecules.Sema3d was originally founded to be an axon guidance protein,and in succession has also been shown to be particularly important in numerous and diverse biological processes,such as cardiovascular development,immune cell regulation,and tumor progression.In previous studies,Sema3d and other Semaphorins exhibited complex functionality through multi-receptors or signal pathways,but its role in HCC has not been investigated.Sema3d involvement in proliferation,migration,cytoskeleton remodeling and activity of signaling pathways gives rise to a hypothesis that Sema3d might play an important role in HCC progression.Purpose:The expression level of Sema3d in HCC and the correlation with prognosis of HCC patients were detected.Furtherly,the function and molecular mechanism of Sema3d in HCC were analyzed.Method:Our research will be studied from the following three aspects.Firstly,we examined the expression of Sema3d in HCC tissues,corresponding adjacent nontumor liver tissues(ANLTs)and normal liver tissues using quantitative real-time PCR(qRT-PCR)and western blot.We also established training and validation cohorts for reporting prognostic biomarkers according to the Reporting of Prognostic Studies of Tumor Markers(REMARK)guideline through randomly selecting paired HCC samples from Xiangya Hospital and Hunan Cancer Hospital respectively.Then we detected Sema3d protein level by immunohistochemistry(IHC)and executed statistical analysis for the correlation of Sema3d expression with clinicopathologic features and clinical prognosis.Secondly,we detected Sema3d expression in HCC cell lines and primary human hepatocytes(PHH).Then we established Sema3d ectopic expression and knockdown HCC cell lines to detect the effects of Sema3d on cell proliferation,migration and invasion in vitro by performing MTT,clone formation,cell cycle analysis,wound healing and transwell invasion assays.Meanwhile,we examined the role of Sema3d in HCC growth and metastasis in vivo using subcutaneous xenograft tumor model and liver orthotopic transplantation model in nude mice.Tumor formation and metastatic progression was monitored periodically and quantified using the In Vivo Imaging Instruments(IVIS).Thirdly,we combined the RNA-seq,bioinformatics analysis,literature retrieval,co-IP,LC/MS,series of in vitro and in vivo experiments together to clarify the molecular mechanisms of Sema3d regulating HCC growth and metastasis.Result:The main experimental results are listed below:Firstly,qRTPCR and western blot assays showed that Sema3d was downregulated in HCC tissues when compared with ANLTs and normal liver tissues,which was further confirmed by the results from GEO and TCGA databases.The IHC results in both training and validation cohorts showed that Sema3d protein was lowly expressed in HCC tissues compared to ANLT(P<0.001).In training and validation cohort,low expression of Sema3d in HCC tissues was significantly correlated with aggressive clinic pathological features,such as tumor size,tumor nodule number,capsulation formation,Edmondson-Steiner grade,BCLC stage,CCLC stage and TNM stage.Kaplan-Meier survival analysis showed that HCC patients with low Sema3d had shorter OS and DFS time and higher early recurrence rate than those with high Sema3d.In addition,uni-and multivariate cox regression analysis proved low Sema3d as an independent risk factor for both OS and DFS in HCC patients.Moreover,the correlations of Sema3d expression with clinic pathological features and clinical prognosis were further verified in validation cohort.Of note,the prognostic significance of Sema3d was also existed in the subgroup analysis.Second,we manipulated Sema3d expression in HCC cells by ectopic expression of Sema3d in HCCLM3 cells and knockdown of Sema3d in PLC/PRF/5 cells.Results showed that overexpression of Sema3d in HCCLM3 cells significantly suppressed cell proliferation rate and activity,decreased the wound closure rate and the invasive cell number.However,knockdown of Sema3d in PLC/PRF/5 cells obtained the opposite results.The subcutaneous xenograft tumor model and liver orthotopic transplantation model combined in vivo imaging instruments(IVIS)in nude mice showed that ectopic expression of Sema3d in HCCLM3 cells markedly inhibited tumor growth and metastasis,while silencing Sema3d in PLC/PRF/5 cells obviously promoted HCC progression in vivo.These results indicated that Sema3d functions as a tumor suppressor in HCC.Thirdly,we screened the potential signaling manipulated by RNA-seq and GSEA in TCGA,and found that Sema3d significantly attenuated the activity of Pi3k/Akt signaling and decreased the phosphorylation level of key members,the Pi3k and Akt.Then Sema3d-interfered cells were treated with Pi3k/Akt signaling inhibitor Wortmannin,and the in vitro assays confirmed that Sema3d inhibits cell proliferation,migration and invasion through Pi3k/Akt signaling in HCC.Subsequently,we performed literature retrieval,bioinformatics techniques,immuno-fluorescence,immunoprecipitation(Co-IP)combined LC-MS assay,which together confirmed that FLNA is a direct target of Sema3d for inactivating Pi3k/Akt signaling.Sema3d could interact and decreased phosphorylation level of FLNA,reducing the protein stability of FLNA and promoting cleaved to 90 kDa fragments.Moreover,results from gain-and loss-of-function assays showed that the suppressive effect of Sema3d on the Pi3k/Akt signaling and cell proliferation,migration and invasion was significantly abrogated by FLNA overexpression in HCCLM3Sema3d cells,while the promotion effect of Sema3d knockdown on the above phenotypes was greatly inhibited by silencing FLNA in PLC/PRF/5shSema3d cellsConclusion:In summary,our study demonstrated that Sema3d expression is downregulated in HCC tissues,which was significantly correlated with aggressive clinicopathological characteristics and poor clinical outcome of HCC patients.Furthermore,overexpression of Sema3d significantly inhibited HCC cell proliferation,migration and invasion in vitro,and suppressed HCC growth and metastasis in vivo,while knockdown of Sema3d produced the opposite results.Mechanism study indicated that Sema3d could interact with FLNA protein and inactivate Pi3k/Akt signaling to restrain HCC progression.Thus,our findings defined Sema3d as a tumor suppressor in HCC,which may be a novel prognostic biomarker and potential therapeutic target for HCC patients.