SPOP Affects The Biological Behavior Of Gastric Cancer Cells By Regulating Hh/Gli Signaling Pathway

Part I：Expression and relation with clinical pathological features ofSpeckle-type POZ protein (SPOP) in human gastric cancerObjective：To detect the expression of SPOP in gastric cancer (GC) cell lines，GC tissues，and to evaluate its correlation with clinical pathological features．Methods：SPOP expression in101GC tissues and adjacent normal gastric (AG)tissues was detected by immunohistochemistry．Correlations of SPOP with clinicalpathological features were subsequently assessed．Four frensh GC tissues and its AGtissues were detected by Western blot. Four gastric cancer cell lines and one normalgastric mucosa epithelial cell line (GES-1) were detected by Western Blot．Results：1. In101gastric cases，SPOP expressed lower in GC than AG in88cases．Immunohistochemistry showed the expression of SPOP in GC tissue waslowerr than AG tissues (P <0.05)．The statistical results showed the SPOPexpression had a positive correlation with lymphatic metastasis (r=0.225，P <0.05)，but negative correlation with gastric tumor pathological grade (r=－0.232，P <0.05)and TNM stage (r=－0.375, P <0.01)，but no correlation with the age and gender ofpatients．2．SPOP expressed lower in GC tissues than AG in four fresh tissues byWestern blot assy．3．Western blot assay showed that SPOP expressed in GES-1andfour gastric cacer cell lines (AGS，SGC-7901，MKN-28，MKN-45)．Conclusions：1．SPOP was obviously down-regulated in GC tissues comparedwith AG tissues (P <0.05)．2．The SPOP expression was associated with lymphaticmetastasis，gastric tumor pathological grade and TNM stage，but no correlation withthe age and gender of patients． Part II：Construction of SPOP-V5/His, SPOP-ShRNA and screening of gastric cancer cell modelsObjective：In order to explore the function of SPOP in the development ofgastric caner， we constructed SPOP expression plasmid SPOP-V5/His andSPOP-shRNA．We also constructed cell models with up-regulation/down-regulaion ofSPOP in human gastric cancer cell lines．Methods：1．SPOP-V5/His was constructed by use of recombinant DNAtechnique and was demonstrated by restriction endonuclease mapping．SPOP-V5/Hiswere transfected into human gastric cell line AGS by using lipofectamine2000forscreening stable cell lines．2．The miRNA expression vector targeting SPOP wereconstructed using pcDNATM6.2-GW/EmGFP to construct SPOP-shRNA．It wasidentitied by Western blot analysis after recombinant．SPOP-shRNA-1430transfectedthe gastric cancer cell line MKN-45. The protein expression of SPOP was detected bythe Western blot assay．Results：1．We constructed SPOP-V5/His and SPOP-shRNA plasmids．2．Western blot showed the level of protein expression of SPOP in gastric cancer cellline AGS transfected with SPOP-V5/His was significantly increased．3．Western blotidentified that the interference effect of SPOP-shRNA-1430was the best．4．Westernblot showed the level of protein expression of SPOP in gastric cancer cell lineMKN-45transfected with SPOP-shRNA-1430were significantly decreased．Conclusions：We successfully constructed cell models with up-regulation/down-regulaion of SPOP in human gastric cancer cell lines． Part III：The impact of SPOP on the gastric cancercell biological behaviorObjective：To evaluate the effect of SPOP on the proliferation，clone formationand migration of human gastric cancer cell lines．Methods：The cell cultures were measured for cell proliferation levels after transfection using MTT assay and cell cout；scratch assay was used to evaluate themigration of gastric cancer cell；colony forming unit assay was used to evaluate thecolony forming of gastric cancer cell lines AGS and MKN-45after transfection．Results：MTT assay indicated that the proliferation level of AGS cells wereobviously decreased after transfection with SPOP-V5/His as compared with controlgroups．MTT assay indicated that the proliferation level of MKN-45cells wereobviously increased after transfection with SPOP-shRNA as compared with controlgroups．In the scratch test，AGS cells transfected with SPOP-V5/His obviouslyreduced migration， MKN-45cells transfected with SPOP-shRNA obviouslymultiplied migration．In the colony forming unit assay，the colony forming of AGScells transfected with SPOP-V5/His obviously reduced as compared with controlgroups，and the colony forming unit of MKN-45cells transfected with SPOP-shRNAobviously increased as compared with control groups．Conclusions：SPOP may decrease the proliferation，migration and colony forming ofhuman gastric cancer．SPOP is a potential target for treat gastric cancer and diagnosticindicator． Part IV：The mechanism of SPOP affecting the gastriccancer cell biological behavior by regulation Hh/Glisignaling pathwayObjective：To study the regulation of SPOP with Hh/Gli signaling pathway．Methods：We detected the interation of SPOP with Gli2by immunity coprecipi-tation (IP) and Immunofluorescent staining with confocal microscope imaging．HEK-293T cell lines were transfected with Myc-SPOP to detect the impact of Gli2，the impact on thedownstream target genes，and the activity of Hh/Gli signaling pathway by dual Luciferase reporterassay．Results：IP test detected that SPOP interacted with Gli2；under the confocalmicroscopy，intensity of Gli2staining was found to be negatively related to SPOPintensity in cytoplasm；SPOP could downregulate the protein level of Gli2，but no changes with the mRNA level．Increasing expression of SPOP can promote theincrease of apoptosis factors PARP and Caspase-3．Dual Luciferase reporter assayshowed that SPOP decrease the activity of Hh/Gli signaling pathway．Conclusions：SPOP may affect the protein level of Gli2，suppress Hh/Glisignaling pathway and promote cell apoptosis， thus affect the genesis anddevelopment of gastric cancer．...