Liver Transplantation Of Tumor Metastasis After Recurrence Of Molecules Related To Quantitative Proteomics

Primary liver cancer is the second most frequent neoplasm in China and more than half of the new cases happen in our country every year throughout the world. The prognosis of hepatocellular carcinoma(HCC) patients remains very poor. Surgical resection is usually considered to be the first choice for treatment.HCC nearly always develops in the setting of chronic hepatitis virus infection or liver cirrhosis.But many HCC patients with cirrhosis lose the opportunity to undergo surgery,due to their limited liver function and extent of tumor.Liver transplantation (LT),the only potentially curative therapeutic modality,allows radical extirpation of cancer and restores normal liver function.There is increasing evidence that,in the long term,LT will be the best therapeutic option for cirrhosis-associated HCC. Unfortunately,some HCC patients succumb to the disease as a result of metastasis and recurrence after LT.Although,Clinical features,such as the number and size of nodules,micro/macroscopic vascular invasion,and high serum alpha feto-protein (AFP) levels,are considered to be related to recurrence after LT;however,they are still insufficient for recognizing patients at high risk for recurrence and selecting those at low risk.In order to improve the diagnosis and prognosis of HCC,there is an urgent need to identify tumor molecular markers predictive of recurrence that can define a subset of HCC patients that stand to benefit from LT.Recent technological advances in proteomics allow us for the first time to examine the expression profiles at protein level on a genome-wide scale.High throughput genomics and proteomics techniques have facilitated a better understanding of diseases such as cancer by deciphering the unique molecular signature that predicts clinical outcomes and therapeutics.Development of new technology for identification of tumor-related molecular markers is currently in progress.The recent development of proteomic technology,including protein profiling,coupling laser capture microdissection(LCM) with cleavable isotope-coded affinity tag(ICAT) technology and two-dimensional LC tandem MS (2D-LC-MS/MS),provides a potentially powerful tool for accurate qualitative and quantitative proteomic analysis of clinical samples.This procedure can result in the characterization of protein expression patterns from microdissected HCC tissues,and can be used for the discovery of proteins associated with recurrence after LT for HCC.In our study,Samples were obtained from HCC patients at our institute with recurrence or disease-free survival(DFS) after LT.We got a group of significant differential proteins including Capn4 protein which may be related to recurrence after LT for HCC.We also drew a positive conclusion through further analysis and validation of the biological function of Capn4 with RNA interference technique and some following experiments related to cell properties of proliferation,apoptosis, migration and invasion.By using tissue microarray(TMA) technique in clinical validation,we found the expression of Capn4 was related to the invasive potential of HCC and recurrence after LT for HCC.The positive expression of Capn4 might be a potential application in poor prognosis and therapeutic target for HCC after LT.Part One Quantitative proteomics to screen the key molecule associated with metastasis and recurrence of hepotacellular carcinoma after liver transplantationThe purpose of this study was to identify the proteins that play a pivotal role in metastasis and recurrence of HCC after LT.Samples were obtained from HCC patients undergoing LT in our hospital,complete clinicopathological and follow-up data were obtained in all patients,who met the criteria of UCSF and had a similar disease background,which were categorized into two groups:recurrence and disease-free survival(DFS) groups.We extracted the total protein from the acquired homogeneous tumor cells and applied a cleavable isotope-coded affinity tag(cICAT) technology to quantitate relative changes in protein levels between the recurrence and DFS groups.The 2-folds up-regulation of thioredoxin like 1 and calpain small subunit 1(Capn4) and the 2-folds down-regulation of plastin 3 were confirmed by real-time PCR,immunohistochemistry and Western blotting in another cohort of HCC patients treated by LT at our institution.As a result,the quantitative differential expression of 149 proteins,includingβ-actin,was identified,Fifty-two of these(34.9%) proteins were displayed differentially(＞2-folds) in the recurrent group,compared to the DFS group,after calibration ofβ-actin.Among these 52 proteins,29 were found to be up-regulated,23 were down-regulated,all these proteins were classified according to their molecular function,using the tools at www.geneontology.org.Which were classified into nine groups:enzyme activity(49%),binding activity(13%),motor activity(11%),signal transduction(6%),transcription regulation(5%),enzyme regulation(5%),transporter activity(3%),apoptosis regulator activity(3%),and others(5%).The up-regulation of thioredoxin like 1 and the down-regulation of plastin 3 were confirmed by real-time PCR,and the up-regulation of calpain small subunit 1(Capn4) were validated by immunohistochemistry and Western blotting in another cohort of HCC patients treated by LT at our institution.The results implied that LCM combined quantitative proteomics have provided a special protein expression patterns in metastatic and recurrent states of HCC patients undergoing LT.These differential proteins maybe serve as new prognostic biomarker and potential therapeutic targets particularly for HCC patients undergoing LT,which warrants further investigation.Part Two Biological function analysis of differential displayed protein Capn4 in human hepatocellular carcinoma cell lines with different invasive and metastatic potentials in vitro and vivoThe objective of this part of research was to explore biological function of differential displayed protein Capn4,in order to verify its effect in HCC invasion, metastasis and recurrence.Firstly we chose 4 human HCC cell lines with different invasive and metastatic potentials such as Hep3B,MHCC97L,MHCC97H,and HCCLM6 as well,using immunocytofluroence,real-time PCR and Western blotting to validate different expression of Capn4 in both mRNA and protein levels among these cell lines.After that,Using RNA interference technique,we constructed small interfering RNA(siRNA) targeting the mRNA of human Capn4 and non-silencing siRNA mismatching with Capn4 mRNA.Then we packaged these siRNAs with Lipofectamine and transfected the mixture into human HCCLM6 cells with high invasive and metastatic potential.We used real-time PCR and Western blotting to qualify the mRNA and protein levels of Capn4,respectively.We also analyzed the malignant phenotypes of transfected HCCLM6 cells including invasive,motile and migration activities by cell invasion model,cell motility model,and cell scratched wound model,and cell proliferation activities by cell clone formation,MTT essay, cell cycle phase examination,and apoptosis activities in vitro,in order to observe the alteration of HCCLM6 cell biological behavior with Capn4 down-expression. Furthermore,we want to explore the inhibitory effect of silencing Capn4 expression on the in vivo tumor growth and lung metastasis of human HCC cells.We constructed a lentiviral vector of miRNA mediated RNA interference(RNAi) of Capn4 gene,and which were transfected into human HCCLM6 cells with high metastatic potentials, and then subcutaneously inoculated into nude mice.We directly confirm the effect of Capn4 knockdown in vivo tumor growth and lung metastasis of human HCC cells.These results showed that there was significant differentia in the 4 human HCC cell lines with different invasive and metastatic potentials,according to the validation of Capn4 expression in both mRNA and protein level by various methods.We confirmed that Capn4 expressing level increasing from low to high was related to the invasive and metastatic potentials of different cell lines.With the condition of 40～50%of cell fusion,150nM of siRNA concentration, using real-time PCR,the inhibiting rate of Capn4 expression among 3 types or siRNA was 63.1%,66.6%and 81.6%,respectively.Compared with non-silencing siRNA,the inhibiting rate had significant difference(P＜0.01).We used the Capn4-s3 siRNA to evaluate the inhibitory efficiency at different times,with the result that mRNA and protein levels of Capn4 were maximally inhibited at 48 and 72 h after transfection.In addition,and 48 hours of transfecting time,The cell invasion and motile model showed that the number of HCCLM6 cells migrating from artificial basement membrane was 10.8±2.75 and 16.3±3.4 in 2 RNAi group,while 25.3±4.34 and 33.3±4.65 in non-silencing group and 28.3±5.56 and 34.8±5.91 in blank control.Cell migrating number of RNAi group decreased significantly(P＜0.05).The cell scratched wound model showed that an apparent decrease in migration ability of HCCLM6 cells transfected with Capn4-s3 siRNA.Meanwhile,representative photography showed accelerated closure in Capn4-neg siRNA and mock-transfected cells.Cells wounded in negative control and blank control groups cells almost grown to confluence in two days.Cell proliferation of HCCLM6 after Capn4 RNAi was measured by cellular uptake of MTT(3,-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). Each assay was performed three times.Compared with blank and negative control groups,Cell proliferation of RNAi group was markedly inhibited(P＜0.05).The result of cell clone formation assay showed that cell clone formation of RNAi group was variously decreased(P＜0.05).But,no significant changes in cell cycles and apoptosis can be observed in cells of 3 groups by flow cytometry analysis.In addition,HCCLM6 cells(5×10~6) were transfected with the lentiviral vectors of blank control,negative control(Lenti.Capn4-neg) and RNAi group(Lenti.Capn4-s3), and then subcutaneously inoculated into nude mice.Tumor growth and pulmonary metastatic lesions were monitored.At 6 week after the injection,the tumors originating from the cells transfected with 3 groups were measurable.At 6 weeks,the animals were killed for ethical reasons given the tumor volume,and then the tumors were excised from mice.HCCLM6 cells mock-transfected or transfected with Lenti.Capn4-neg developed large tumors 4543.8±1351.8mm~3 and 3889.6±2407.6 mm~3 within 6 weeks.However,growth of HCCLM6 cells transfected with Lenti.Capn4-s3 was markedly suppressed(P＜0.001),suggesting that knockdown of Capn4 leads to significant inhibition of tumor growth in vivo.In the sixth week, pulmonary metastatic lesions were detected in every mouse in blank control group, with mainly gradeⅠ-Ⅱand some gradeⅢ-Ⅳtumor clusters.The mice of blank control group and negative control groups had an average of 13±1.4 and 11.5±3.4 tumor clusters per lung.However,only 33.3%mice infected with the Lenti.Capn4-s3-treated cells developed lung metastasis with mostly gradeⅠtumor clusters with a combined average of 1.3±1.6 tumor clusters per lung.The effect was statistically significant(P＜0.01).The results of this study indicate that Capn4 expressing level increasing following the invasive and metastatic potentials of HCC cell lines.In vitro,the knockdown of Capn4 can suppress the ability of cell invasion,motile,migration and proliferation, but no significant effect on cell cycle phase and apoptosis.In vivo,the down-regulation of Capn4 can inhibit the tumor formation and lung metastasis.Part Three The study of the mechanism of inhibition of invasive and mstastatic ability of HCC by knockdown of Capn4In the second part of research,we found that the knockdown of Capn4 can suppress the ability of cell invasion,motile,migration and proliferation.The purpose of the present study was to investigate the underline mechanism in vitro,Gelatin zymogrphy assay showed that transfection of HCCLM6 cells with Capn4-s3 reduced MMP-2 activities but had little effect on MMP-9 activity. Furthermore,the MMP-2 activity of HCCLM6 cells transfected with Capn4-s3 was significantly lower than those of Capn4-neg and mock-transfected cells(P＜0.001). Scanning electron microscope demonstrated that cell pseudopod formation of Capn4-s3 group was markedly suppressed and decreased.Focal adhesion plaques play a pivotal role in the cell migration.Using the co-immunoprecipitation assay,we found interaction of FAK and Talin with Capn4 in HCC cell lines,while decreasing of Capn4 and rising expression of Talin and inhibition of FAK phosphorylation were found in HCCLM6 cells after Capn4 RNAi.Together,these findings revealed mediated roles of Capn4 in clearage of Talin and FAK phosphorylation in HCC cell lines involving in cellular migration activation.The finding indicated that Capn4 may mediate the secretion of MMP-2 and clearage of Talin and FAK phosphorylation in HCC cell lines,which suppressed the invasive ability and motility of HCC in vitro and vivo.Part Four The clinical significance of Capn4 expression in primary hepatoma tissues undergoing LTIn this part of research,we analyzed the correlation between Capn4 expression in primary tumor tissue from HCC after LT and other clinical-pathological characters from patients suffered from HCC after LT and the impact on the prognosis to evaluate the potential value of Capn4 in judging of survival and recurrence for HCC after LT.We detected 192 cases of Capn4 expression in primary tumor tissue from HCC after LT by tissue micro array(TMA) and immunohistochemical techniques,and then made correlation analysis between Capn4 and other clinical-pathological characters. We also compared expression of Capn4 in predicting invasive potential of HCC after LT by receiver operating characteristic(ROC) curve,and evaluated the significance of Capn4 as a new marker of HCC after LT prognosis.There was differential expression of Capn4 between the primary tumor tissue and Para-tumor tissue,the increase of Capn4 had significant correlation with some clinical-pathological characters such as multiple tumor,single tumor maximum diameter＞5cm,not well-capsule,vascular invasion,and late TNM stage(P＜0.05). There was significant correlation between Capn4 negative and positive group in DFS, but not in Overall survival.The ROC curve indicated the higher Capn4 in tumor expressed,the stronger the tumor recurrent potential would be.The finding implied that the expression of Capn4 was associated with recurrence of HCC after LT,and may serve as a new prognostic marker for metastasis and recurrence of HCC after LT.CONCLUSIONS1.LCM combined quantitative proteomics have provided a special protein expression patterns in metastatic and recurrent states of HCC patients undergoing LT.These differential proteins maybe serve as new prognostic biomarker and potential therapeutic targets particularly for HCC patients undergoing LT.2.Capn4 expression level was related to the invasive and metastatic potential of different cell lines.The motile and invasive ability of high metastatic cell line decreased significantly when Capn4 was down-regulated.This implied Capn4 may play an important role in HCC cell invasion and metastasis through motility and invasion potential strengthening.3.Capn4 may mediate the secretion of MMP-2 and clearage of Talin and inhibition of FAK phosphorylation in HCC cell lines,which suppressed the invasive ability and motility of HCC in vitro and vivo.4.Capn4 had some clinical value in predicting invasive potential of HCC.Positive expression of Capn4 was a signal for poor prognosis for HCC after LT. Combining Capn4 with other clinical pathological characters;we could predict recurrence and metastasis of HCC after LT.POTENTIAL APPLICATION1.Capn4 may become a biomarker for predicting recurrence and metastasis for HCC after LT and a potential therapeutic target.2.Further research for forward and backward control of Capn4 may help elucidate the molecular mechanism of recurrence and metastasis for HCC after LT. NOVELTY1.We obtained quantitative protein expression profiles of primary tumor by 2D-LC-MS/MS combined with ICAT,which may overcome the difficulties by traditional techniques.Furthermore,only HCC patients who underwent LT were included in the present study,due to the fact that recurrence after resection of HCC could be confused by multicentric carcinogenesis.2.First time of verifying the function of protein Capn4 in human HCC invasion and recurrence after LT.It is helpful to elucidate pathological mechanism of HCC after LT and find new therapeutic target through exploring Capn4 function in HCC invasion and metastasis.3.Capn4 may serve as a molecular biomarker in predicting recurrence and metastasis for HCC after LT.