STRN Expression And Its Effect On Invasion And Migration Of Tumor Cells In Hepatocellular Carcinoma

Objective:To investigate the expression and significance of Striatin(STRN)in hepatocellular carcinoma(HCC),and further explore its effect on tumor cell proliferation,apoptosis,invasion,migration and the possible molecular mechanisms in vitro,thus provide valuable information in understanding the hepatocarcinogenesis,and also potentially novel ideas and strategies for HCC clinical treatment.Methods:A total of 45 pairs of HCC samples and the corresponding adjacent non-tumor(ANT)liver tissue specimens were collected,and immunohistochemical technique was used to detect the expression and localization of STRN in these tissues.Possible association between STRN expression and clinicopathologic parameters of HCC was also analyzed.Human normal hepatic cell line LO2 and different HCC cell lines(SMMC-7721,Huh7,and HepG2)were used in vitro to verify the characteristics of STRN expression pattern.The gene and protein expression of STRN were measured by qRT-PCR and Western blot analysis,respectively.The Huh7 cells with dual-directional regulation of STRN was conduced by either siRNA or cDNA transient transfection technique.MTT assay and Annexin V/PI double staining assay were used to detect the effect of STRN on cell proliferation and apoptosis;transwell invasion assay and wound-healing assay were performed to measure the effect of STRN on cell invasion and migration.During the regulation of STRN in Huh7 cells,the changes of cell morphology were observed by microscopy,and the EMT molecular markers including E-cadherin and Vimentin were determined by Western blot and immunofluorescence assays.Results:1.Expression and clinical significance of STRN in HCCThe results of immunohistochemistry showed that STRN was mainly expressed in cytoplasm,and its protein expression level in HCC tissues was significantly higher than that in ANT tissues.Among 45 specimens,only 1(1/45,2.2%)HCC specimen showed negative STRN expression,while others with either low or high expression of STRN.For ANT tissues,there were 15(15/45,33.3%)specimens showing negative STRN expression,while the remaining specimens were mostly with low levels of the indicator.STRN expression was no significantly associated with patient's age,gender,tumor size,tumor histologic grade,cirrhosis or hepatitis background,intrahepatic vascular embolism,aminotransferase level,alpha-fetoprotein(AFP)level,C-reactive protein level,serum albumin level,or prothrombin time(all P>0.05),but positively correlated with lymph node metastasis and TNM staging(all P<0.05).In addition,STRN expression at both mRNA and protein levels was observed to be markedly up-regulated in LO2 cell line when compared to that of three HCC cell lines SMMC-7721,Huh7,and HepG2,in vitro experiments.Thus,we confirmed an increased STRN expression during hepatocarcinogenesis both histologically and cytologically.2.Down-regulation of STRN had no significant effect on HCC cell proliferation and apoptosis but decreased its invasion and migration capacities in Huh7 cellsThe Huh7 cell line with moderate STRN expression was used in further studies.Using the siRNA interference technology,down-regulation of STRN in Huh7 cells was established and verified by western blot and immunofluorescence assays.In this cell model,no effect of STRN inhibition on the cell proliferation and apoptosis was observed by MTT assay and Annexin V/PI double staining assay.However,a decrease in tumor cell invasion and migration capacities was detected by transwell invasion and wound-healing assays.3.Up-regulation of STRN had no significant effect on HCC proliferation and apoptosis but increased its invasion and migration in Huh7 cellsThe Huh7 cell model with STRN over-expression was successfully established and verified by cDNA transient transfection technology.In this cell model,no effect of STRN inhibition on the cell proliferation and apoptosis was observed by MTT assay and Annexin V/PI double staining assay.However,an increase in tumor cell invasion and migration capacities was detected by transwell invasion and wound-healing assays.4.STRN affected HCC cell invasion and migration ability via its effect on EMTDuring the process of dual-directional regulation of STRN in Huh7 cells,cell morphologic changes displaying an epithelioid and cobblestone appearance with little pseudopodia could be observed by microscopy upon STRN down-regulation.On the contrary,STRN over-expressed Huh7 cells showed loss of cell polarity and increased formation of pseudopodia,leading to elongated,irregular fibroblastoid morphology.Moreover,both western blot and immunofluorescence assays confirmed an increase in expression of epithelial marker E-cadherin but a decreased in expression of mesenchymal marker Vimentin by STRN suppression.However,an opposite result was observed in protein expression of EMT markers by STRN up-regulation.These results suggested that STRN might affect HCC cell invasion and migration capacities throuth EMT regulation.Conclusion:STRN was up-regulated in hepatocarcinogenesis,and STRN expression in HCC tissues was positively associated with lymph node metastasis and clinical stage;Regulation of STRN in Huh7 cells in vitro had no significant effect on tumor cell proliferation and apoptosis,but positively correlated to tumor cell invasion and migration capacities,in which process modulation of EMT could be involved.