| BackgroundHepatocellular carcinoma(HCC)is a highly aggressive primary liver cancer.Surgical resection is still the best therapeutic strategy for patients with early disease,but many HCC patients develop postsurgical recurrence or metastasis with poor 5-year survival rates.The high rates of tumor recurrence and distant metastasis after surgical resection are the major reason for the poor prognosis of patients with HCC.Therefore,exploring the molecular mechanism underlying HCC metastasis is still eagerly needed.SORBS2(sorbin and SH3 domain containing 2),also known as ArgBP2(Arg/c-Abl kinase binding protein 2),is a scaffolding protein associated with Abl/Arg non-receptor tyrosine kinase pathways and is known to interact with actin and several other cytoskeletal proteins in various cell types.Reconstitution of SORBS2 expression in cervical cancer cell lines significantly inhibits cell proliferation,colony formation,and anchorage-independent growth,indicating that SORBS2 plays a role as a tumor suppressor in cervical carcinogenesis.SORBS2 expression is repressed during pancreatic oncogenic transformation,and the tumorsuppressing function of SORB S2 in pancreatic cancer occurs through the regulation of cell adhesion and migration,and at least partly via by controlling the formation of the WAVE/PTP-PEST/c-Abl signaling complex.In addition,decreased SORBS2 levels are seen in gastric and breast cancers.More recently,Zhao et al.reported that SORBS2 stabilizes the tumor-suppressive immunomodulatory transcripts of WFDC1 or IL-17D to suppress metastatic colonization of ovarian cancer.These findings point to the involvement of SORBS2 in HCC progression.Nevertheless,the expression levels and biological roles of SORBS2 in HCC remain unclear.Exploring the molecular mechanisms involved in HCC metastasis is of great significance for the early diagnosis and effective therapy of HCC.ObjectiveThe aim of this study is to detect SORBS2 expression in HCC cells and tissue samples,investigate the potential function and the underlying mechanisms.verify the correlation between SORBS2 and HCC,including SORBS2 expression and tumor stage,pathology,survival time and so on.To explore the influence of SORBS2 on the biological characteristics of liver cancer cells,through cell and animal experiments,and to explore the changes in the biological characteristics of HCC after the expression or silence of the SORBS2 gene,so as to confirm the role of SORBS2 in the developmentclarify SORBS2,to regulate the development of HCC molecular mechanism.The purpose of this study is to better and further explore the pathogenesis of HCC and provide a new direction for the early diagnosis and clinical treatment of HCC.Methods(1)The expression level of SORBS2 mRNA in HCC tissues and its correlation with survival rate of HCC patients were analyzed using oncomine database;(2)Twelve fresh HCC tissue samples and matched normal tissue samples were collected.In addition,paraffin-embedded pathological samples from 102 patients with HCC and matched normal tissue samples were obtained between 2012 and 2016.qRT-PCR and Western blot were used to detect the mRNA and protein expressions of SORBS2 in clinical samples.The expression of SORBS2 in tissue chip was detected by immunohistochemistry.The correlation between SORBS2 expression level in 102 HCC tissues and the clinicopathological features of the patients and the prognosis of patients with HCC were analyzed;(3)The protein expression of SORBS2 in 5 HCC cell lines and L02 cell lines was detected by western blot;(4)SMMC-7721 and HCC-LM3 cells were infected with SORBS2-overexpressing lentivirus or controllentivirus,while PLC and Huh-7 cells were infected with a lentivirus expressing SORBS2 shRNA or control shRNA.Then,transwell migration and invasion experiments and western blot were used to analyze the changes of cell migration,invasion and EMT ability;(5)The nude mice were exposed to a 12-hour light/12-hour dark cycle in an animal chamber at 21-24℃.The mice were randomly divided into several groups.After intrasplenal injection,5×105 lentivirus-infected HCC cells from 20 μL PBS were injected into the spleen of nude mice.After three weeks,the mice were euthanized to extract livers.The number of tumors on the liver surface was determined and the liver tissue was immobilized for hematoxylin and eosin(HE)staining.(6)In silico analysis was performed using the JASPAR and PROMO databases to determine possible upstream transcription factors associated with the downregulation of SORBS2 in HCC;(7)The expression of MEF2D in 4 HCC cell lines was detected by western blot,and qRT-PCR was used to detect the expression of MEF2D in HCC tissues,and its correlation with SORBS2 was analyzed.(8)The luciferase reporting assay and Chromatin Immunoprecipitation were used to explore the effect of MEF2D on SORBS2 expression level;(9)SMMC-7721 and HCC-LM3 cells were infected with a lentivirus expressing MEF2D shRNA or control shRNA,and then the transwell migration and invasion experiment and western blot were used to analyze the changes of cell migration,invasion and EMT ability;(10)SMMC-7721 and HCC-LM3 cells were infected with SORBS2-overexpressing lentivirus or controllentivirus,while PLC and Huh-7 cells were infected with a lentivirus expressing SORBS2 shRNA or control shRNA.Then,western blot was used to detect the expression of C-Abl and the phosphorylation levels of ERK1/2(p-ERK1/2);(11)The PLC-shSORBS2 and HUH7-shSORBS2 cells was co-transfected with si-cAbl or tretmented with ERK inhibitor U0126,the expression changes of c-Abl,ERK1/2 and p-ERK1/2 were detected by western blot,and migration and invasion were determined using transwell assays.Results(1)The Oncomine database observed that SORBS2 mRNA levels were significantly lower in HCC tissues than those in matched normal liver tissues,and the low-expression of SORBS2 was strongly associated with lower survival rates of patients with HCC;(2)Showed by the western blot and qRT-PCR analysis,the expression of SORBS2 in HCC tissues was significantly lower than that in normal tissues.Strong positive SORBS2 expression was observed in normal tissues,while only SORBS2 expression was detected in HCC.SORBS2 down-regulation was related to patient gender and tumor stage,but not to other clinical parameters.In addition,the overall survival rate of HCC patients with low SORBS2 expression was lower;(3)The expression of SORBS2 protein level in HCC cell lines was significantly downregulated compared with that in human liver cell line L02 showed by Western blot results.(4)Over-expression of SORBS2 significantly inhibited the invasion,migration and EMT of HCC cells.On the contrary,SORBS2 silencing significantly promoted the invasion,migration and EMT of HCC cells;(5)Over-expression of SORBS2 inhibited liver metastasis in vivo experiments;(6)The JASPAR and PROMO database analysis confirmed that MEF2D was the upstream regulator of SORBS2 in HCC;(7)Down-regulation of MEF2D expression in SMMC-7721 and HCC-LM3 cells increased SORBS2 expression at mRNA and protein levels,while up-regulation of MEF2D inhibited SORBS2 expression in PLC and Huh7 cells;(8)The expression of MEF2D in HCC cells and tissues was significantly higher than that in normal cells and tissues,and the expression levels of SORB S2 and MEF2D were negatively correlated;(9)The luciferase reporting assay and Chromatin Immunoprecipitation confirmed the relationship between SORBS2 and MEF2D;(10)Down-regulation of MEF2D inhibited the migration,invasion and EMT of SMMC-7721 and HCC-LM3 cells;(11)Western blot results showed that the over-expression of SORBS2 reduced the cAbl and phosphorylation of ERK1/2 level,while the reduction of SORBS2 promoted the cAbl and phosphorylation of ERK1/2 level in HCC cells;(12)SORBS2 inhibited the migration,invasion and EMT of HCC cells by regulating the c-Abl-ERK signaling pathway.ConclusionsThis study confirmed that expression of SORBS2 was significantly down-regulated in HCC tissues and cells,and was related to the tumor stage of patients;over-expression of SORBS2 could inhibit HCC cell migration,invasion and EMT;at the same time,overexpression of SORBS2 in nude mice inhibited tumor metastasis;expression of MEF2D was significantly up-regulated in HCC tissues and cells,and was negatively correlated with the expression of SORBS2;reducing the expression of MEF2D could inhibit HCC cell migration,invasion and EMT;MEF2D was the upstream regulator of SORBS2,inhibited the expression of SORBS2,and promoted the invasion and metastasis of HCC through cAbl/ERK signaling pathway.In summary,SORBS2 was down-regulated by MEF2D,and suppressed HCC metastasis through the c-Abl/ERK signaling pathway,had the potential to serve as a novel prognostic marker or therapeutic target for HCC. |
PDF Full Text Request