Study Of The Effect Of Phospholipid Scramblase 1 And Midkine On Primary Liver Cancer

Objection:The aim of this study was to investigate the expression of PLSCR1 and MK in hepatocarcinoma and paracarcinoma tissues and analyze the correlation of PLSCR1 and MK expression with clinicopathologic characteristics of the 40 samples,and to use Cox regession model analysis to analyze the correlation of clinical initial value and patient's overall survial,and to explore the mechanisms of interaction between PLSCR1 and MK in the development of hepatocarcinoma.Methods:1)Twenty pairs of hepatocarcinoma and corresponding adjacent normal tissues were obtained from patients who underwent primary surgical treatment at the Huzhou Central Hospital from 2009 to 2013.All samples were histologically confirmed by staining with hematoxylin-eosin and possessed complete clinical information.The specimens were frozen in liquid nitrogen within 30 min after isolation from patients and store at-80?.Real-time PCR was performed to detect the expression of PLSCR1 and MK between hepatocarcinoma and paracarcinoma tissues,and statistical analyses were carried out to assess the relationship between PLSCR1 and MK expression and clinical parameters,and to use Cox regession model analysis to analyze the correlation of clinical initial value and patient's overall survial with liver cancer,meantime analized the relationship between PLSCR1 and MK expression levels.2)GST pull-down assay was performed to confirm the interaction between PLSCR1 and MK in vitro:The full-length of PLSCR1 was cloned into the pE28a(+)vector and the full-length MK was introduced into the pGEX4T1 vector,then these two combinant plasimds were transformed into BL21 bacterial strain to produce PLSCR1 protein and MK-GST fusion protein induced by IPTG.And prepare the protein complex for SDS-PAGE and Western blot with PLSCR1 and MK antibodies to analysis the interaction between PLSCR1 and MK in vitro.The coimmunoprecipitation assay was carried out to confirm the interaction between PLSCR1 and MK in vivo:The recombinant plasmids pcDNA3.1/myc-His(-)A-PLSCR1 and pcDNA3.1-MK were constructed,Protein complexes bound to the conjugated antibody were separated by denaturing polyacrylamide gel electrophoresis and subjected to immunoblotting.Anti-MK polyclonal antibody and anti-PLSCRl polyclonal antibody were used for immunoprecipitation.Detection was by enhanced chemluminescence.3)The HepG2 cells were transfected or cotransfected of MK siRNA or/and PLSCR1 siRNA,and the absorbance of this colored solution can be quantified by measuring at 450 nm by a spectrophotometer.4)The migration analysis was also performed according to the QCMTM 24-Well Colorimetric Cell Migration Assay manufacturer's procedure HepG2 cells(5x105 cells/mL)were seeded in Boyden chambers(upper chamber),300?l per chamber.Lower chambers contained 500 ?l of serum free medium.After 24 hours,the cells in the upper chambers were removed,cells were treated with or without MK siRNA and PLSCR1 siRNA as previously,and the detection was performed 48 hours later.Whereas the cells that migrated to the lower chamber were counted after fixation and stained in cell stain solution for 20 minutes,then the insert was dipped into a breaker of water several times to rinse.The non-migratory cells layer was removed from the interior of the insert.The cells were photographed with the inverted microscope.The migrated cells were then lysed in 200 ?l of extraction buffer for 15 minutes at room temperature and then 100 ? of the dye mixture was transfected to a 96-cell microtiter plate suitable for colorimetric measurement at 560 nm.Results:1)The results of Real-Time PCR indicated PLSCR1 and MK expression in tumor tissue was much higher than paracarcinoma tissue,The PLSCR1's??Ct was-2.36 ± 1.49,2-??Ct(The value of RQ)was 8.55±7.67;The value of MK's??CCt-2.97±0.86,The value of RQ was 9.05±3.97,as the P value was<0.05,and statistical analysis demonstrated that the expression of PLSCR1 and MK was associated with differentiation degree and clinical stage of the specimens.In accordance with the value of RQ,40 patients with liver cancer were divided into two groups,RQ>10 was high expression,RQ<10 was low expression,Though the study of 40 patients with clinical parameters,the results showed that there were not relevant to age,Liver cirrhosis,AFP,tumor scale and capsular invasion(P>0.05),but were associated to gender,tumor differentiation,tumor number,vascular invation and clinical stages(P<0.05).2)Though the Kaplan-meier study of follow-up 40 patients with liver cancer,to compare difference between survival time of PLSCR1 and MK's RQ,the results showed that there were significant difference between survival time of PLSCR1 and MK in different expression level(P<0.05),the survival time of the low expression group overtop the high expression group.Using Cox regession model analysis to analyze the correlation of clinical initial value and the RQ of PLSCR1 and MK and patient's overall survial with liver cancer,The results showed that the risk factor of overall survival time were capsular invasion,vascular invasion,Ki-67,gender,liver cirrhosis and high RQ of PLSCR1 and MK(P<0.05),using Spearman analized the relationship between PLSCR1 and MK expression levels,the result was that they'er positive correlation property(r=0.30,P=0.001).3)GST pull-down experiment and communoprecipitation assay were performed to verify the interaction of PLSCR1 with MK.When PLSCR1 protein was coincubated with bacteria-expressed and bead-bound GST-MK or GST,it could be specifically pulled down by GST-MK but not by GST,indicating a directly physical interaction between MK and PLSCR1 in vitro.We then coexpressed PLSCR1 and MK proteins in HepG2 cells.The cells were harvested 48 hours after transfection,lysed,and subjected to coimmunoprecipitation with an anti-MK antibody.The subsequent immunoblotting analysis revealed that PLSCR1 was precipitated together with MK,indicating that PLSCR1 interacts with MK in the cellular compartments.4)To elucidate the physiological effect of MK and PLSCR1 interaction in tumorigenesis,HepG2 cells was used as a model system.We initially performed an in vitro proliferation assay in 96-well plates.Cells were transinfected with or without MK and PLSCR1 siRNA.We observed a notable reduction of HepG2 cells showing interference by both MK and PLSCR1.To investigate whether the interaction of MK and PLSCR1 plays any role in invasion of HepG2 cells,we performed the transwell migration analysis assay.The results showed that PLSCR1 and MK both evoked a substantial migration of the cells into the denuded area,and the cotreatment with siRNA of MK and PLSCR1 indicated a significant reduction of invasion,that may suggest the interaction of MK and PLSCR1 have effectively promoted migration of HepG2 cells.Conclusions:1)The real-time PCR results showed that PLSCR1 expression in hepatocarcinoma was much higher than corresponding adjacent normal tissues,the statistical analysis demonstrated that the expression of PLSCRl and MK was associated with differentiation degree and clinical stage of the specimens,there were significant difference between survival time of PLSCR1 and MK in different expression level;Though study of 40 patients with clinical parameters,the results showed that there were associated to gender,tumor differentiation,tumor number,vascular invation and clinical stages.2)the Kaplan-meier results showed that there were significant difference between survival time of PLSCR1 and MK in different expression level.3)GST pull-down experiment and coimmunoprecipitation assay were performed to verify the interaction of PLSCR1 with MK.4)Inhibition of PLSCR1 by RNA interference showed reduced growth and migration of HepG2 cells,and a notable suppression of proliferation and migration was observed when both PLSCR1 and MK were interfered simultaneously.All the results indicated that PLSCR1 may play an important role in the development of hepatocarcinoma and the detailed mechanism needs further study.