A Study Of Portal Vein Tumor Thrombi-associated Small Molecular Protein Biomarkers In Hepatocellular Carcinoma And Verification Of S100A11

Primary liver cancer is the second most frequent neoplasm in China and morethan half of the new cases happen in our country every year throughout the world.Hepatocellular carcinoma (HCC) tended to invade portal vein to cause tumorthrombus (PVTT) which could cause the intra-hepatic dissemination and metastasis ofHCC. There was no reliable approach to early detect and predict the occurrence ofPVTT till now. As the formation of PVTT was a multi-step and multi-variant process,researches based on uni-variant and single gene level could not elucidate themolecular mechanisms of PVTT totally, which must be based on the research ofhigh-throughput genomics and proteomics.The combination of using techniques of two-dimensional electrophoresis (2-DE)and mass spectrometry (MS) provides a research platform for proteome analysis. Ourprojects focused on screening and identifying some low molecular weight (MW)different proteins between primary tumor tissue with PVTT and those without PVTTby using techniques of low MW 2-DE combined with matrix assisted laser desorptionionization time of flight mass spectrometry (MALDI-TOF MS). We got a group ofsignificant differential proteins including S100A11. We also drew a positiveconclusion through further analysis and validation of the biological function ofS100A11 with RNA interference (RNAi) technique and some experiments related tocell invasion properties. By using tissue microarray (TMA) technique in clinicalvalidation, we found the expression of S 100A11 was related to the invasive potentialof HCC. The positive expression of S 100A11 might be a potential application in poorprognosis of HCC.Part OneComparative proteome analysis of PVTT related smallmoleculesIn this part of research, we established a stable and standard low MW (＜20kD)protein electrophoresis platform by changing both SDS-PAGE gel concentration andcomponents of electrophoretic buffer system in the second step of 2-DE. Usingoptimized 2-DE and MALDI-TOF MS/MS, we compared and studied differential expressed proteomes in primary tumor tissue from human HCC with or without PVTTto screen low MW protein biomarkers which played an important role during thecourse of PVTT formation.RESULTS:1. Compared with 12.5%SDS-PAGE gel, there were more protein bands between3kD and 20kD in 16% SDS-PAGE gel and low molecular weight (MW) proteinspots (＜20kD) clearly showed in the two groups of HCC tissue.2. Protein samples from PVTT group or non-PVTT group ran 2-DE for 3 timesrepeatedly in the same condition. Average 764±56 protein spots (n=3) weredetected in non-PVTT group with Imagemaster(?) 2D Platinum 6.0 Software while799±44 sports were found in PVTT group. Taking the gel which had most andclearest protein spots as an intra-class reference gel in each group, average687±30 matched spots were detected in non-PVTT group while 701±21 matchedspots in PVTT group, and the intra-class matching ratio for non-PVTT group andPVTT group was 89.87% and 87.77%, respectively.3. Taking the intra-class reference gel of PVTT group as the inter-class reference,there were 393±42 matched spots between the two groups, and the intra-classmatching ratio was 51.44%. The average positional deviation of the matched spotsamong different profiles was (2.95±0.95 )mm in IEF direction and (2.87±0.88)mm in SDS-PAGE direction. The inter-class comparison is conducted by adjustedintensity value in the gel of protein spots, the specific values equal to or largerthan two folds being defined as differential spots. 88 differential spots weredetected between primary tumor tissues of the two groups. Compared withnon-PVTT group, there were 41 over expressed spots (including 19 MW lowerthan 20 kD) and 47 down spots in PVTT group (including 23 MW lower than20kD).4. Thirty six spots were picked and redundancy was eliminated. Finally, elevensignificant differential low MW proteins such as S100A11 and fat acid binding(FAB) protein etc were further identified by MALDI-TOF MS/MS.5. The result from Western blotting validation confirmed the differentia of S100A11in primary tumor tissue with PVTT or without PVTT.Part TwoVerification and biological function analysis of differential displayed protein S100A11 in human hepatocellularcarcinoma cell lines with different invasive and metastaticpotentialsThe objective of this part of research was to explore biological function ofdifferential displayed protein S100A11, in order to verify its effect in HCC invasion,metastasis and recurrence. Firstly we chose 4 human HCC cell lines with differentinvasive and metastatic potentials such as Hep3B, MHCC97L, MHCC97H, andHCCLM6 as well, using real-time RT-PCR and Western blotting to validate differentexpression of S100A11 in both mRNA and protein levels among these cell lines. Afterthat we constructed small interfering RNA (siRNA) targeting the mRNA of humanS100A11 and non-silencing siRNA mismatching with S100A11 mRNA. Then wepackaged these siRNAs with Lipofectamine and transfected the mixture into humanHCCLM6 cells with high invasive and metastatic potential. We used real-timeRT-PCR and Western blotting to qualify the mRNA and protein levels of S100A11,respectively. We also analyzed the malignant phenotypes of transfected HCCLM6cells including motile and invasive activities by cell motility model, cell invasionmodel, and cell scratched wound model in vitro in order to observe the alteration ofHCCLM6 cell biological behavior with S100A11 down-expression.RESULTS:1.According to the validation of S100A11 expression in both mRNA and proteinlevel, we confirmed that there was significant differentia in the 4 human HCCcell lines with different invasive and metastatic potentials. S100A11 expressinglevel increasing from low to high was related to the invasive and metastaticpotentials of different cell lines.2.The inhibiting rate of S100A11 expression among 3 types or siRNA was 73.1%,88.6%, and 91.0%, respectively. Compared with non-silencing siRNA, theinhibiting rate had significant difference (P＜0.01). With the condition of 40～50%of cell fusion, 100nM of siRNA concentration, and 48 hours of transfecting time,the effect of S100A11 expression inhibition was the best.3. The cell invasion model showed that the number of HCCLM6 cells migratingfrom artificial basement membrane was 55.0±10.0 and 41.0±5.6 in 2 RNAi group,while 153.0±11.0 in non-silencing group and 165.0±7.2 in blank control. Cellmigrating number of RNAi group decreased significantly (P＜0.05). 4. The cell motility model showed that the number of HCCLM6 cells migratingfrom upper well membrane was 96.0±9.7 and 78.04±14.0 in 2 RNAi group, while211.0±18.1 in non-silencing group and 225.04±15.8 in blank control. Cellmigrating number of RNAi group decreased significantly (P＜0.05).5. The cell scratched wound model showed that the distance of HCCLM6 cellmigration 48 hours after cell division inhibition was 35.00±11.07μm in RNAigroup, while 81.984±12.11μm in non-silencing group and 119.98±8.17μm in blankcontrol. Cell migrating speed of RNAi group decreased significantly (P＜0.05).Part ThreeThe state and clinical meaning of S100A11 expression inprimary tumor tissue from HCCIn this part of research, we analyzed the correlation between S100A11expression in primary tumor tissue and other clinical-pathological characters frompatients suffered from HCC and the impact on the prognoses to evaluate the potentialvalue of S100A11 in judging of survival and recurrence after surgery for liver cancer.We detected 192 cases of S100A11 expression in primary tumor tissue from HCC bytissue microarray (TMA) and immunohistochemical techniques, and then madecorrelation analysis between S100A11 and other clinical-pathological characters. Wealso compared the coherence of S100A11 with AFP in predicting invasive potential ofHCC by receiver operating characteristic (ROC) curve, and evaluated the significanceof S100A1 las an indes of HCC prognosis.RESULTS:1. The positive rate of S100A11 expression in HCC tissue was 43.75%, and the rateincreased in turn in non-PVTT, micro-PVTT and macro-PVTT groups and thedifferentia was significant (P＜0.01).2.The increase of S100A11 had significant correlation with 8 clinical-pathologicalcharacters such as single tumor maximum diameter＞5cm, not well-capsuled,unclear borderline, vascular invasion, presence of satellite nodules, poorEdmondson class, and late TNM stage (P＜0.05).3. The ROC curve indicated the higher S100A11in tumor expressed, the stronger thetumor invasive potential would be. CONCLUSIONS1. The 16% SDS-PAGE/Tris-Tricine electrophoresis system is quite capable ofseparating the protein and polypeptide with MW lower than 20 kD. The low MWprotein profile of HCC primary tumor tissue with PVTT displayed obviouslydifference compared with HCC tissue without PVTT. The result indicated that theinvasive and metastatic characteristic of HCC had relations with many differentialproteins, including S100A11.2. In human HCC cell lines, S100A11 expression level must relate to the invasiveand metastatic potential of different cell. The motile and invasive ability of highmetastatic cell decreased significantly when S100A11 was blocked. This impliedS100A11 play an important role in HCC cell invasion and metastasis throughmotility and invasion potential strengthening and might have some relations withthe formation of PVTT.3. S100A11 had some clinical value in and predicting invasive potential of HCC.Positive expression of S100A11 was a signal for poor prognosis. CombiningS100A11 with other clinical pathological characters, we could find some clinicalpotential in early detecting vascular invasion before operation and predictingpossibility of HCC recurrence and metastasis after operation.POTENTIAL APPLICATION1. S100A11 may become a biomarker for predicting HCC vascular invasion andmetastasis and for prognosis of HCC.2. Further research for forward and backward control of S100A11 may helpelucidate the molecular mechanism of PVTT formation, and HCC invasion andmetastasis.NOVELTY1. Improvement of present proteomic technique to be suitable for the research of lowMW protein and polypeptide biomarkers.2. First time of verifying the function of protein S100A11 in human HCC cellinvasion and metastasis.3. S100A11 may be a molecular biomarker in predicting prognosis of Hcc.