Proteome Analysis Of Hepatocellular Carcinoma And Biological Function Analysis Of HSP27

Hepatocellular Carcinoma (HCC) is a common malignancy worldwide and is a leading cause of death. Metastasis and recurrence is the major cause for lower post-operation survival rate of HCC patients. Like other cancers, the development and migration of HCC is a multifactorial and multistage pathogenesis. Because of the limitations in conventional techniques, researchers have been allowed to focus on a few biological target molecules at a time, and little has been known regarding the specific alterations responsible for the development or progression of this aggressive malignancy. Recent technological advances in proteomics allow us for the first time to examine the expression profiles at protein level on a genome-wide scale. In our study, we used 2-DE and MALDI-TOF MS to identify the proteins with different expressing level between metastatic HCC and nonmetastatic HCC tissues, also between HCC tissues and adjacent tissues. We got a group of significant differential proteins including HSP27 protein which may be related to hepatocarcinogenesis and HCC metastasis. We also drew a positive conclusion through further analysis and validation of the biological function of HSP27 with RNA interference technique and some following experiments related to cell properties of proliferation, apoptosis, migration and invasion.Part OneProteomic Analysis on Metastasis-associated Proteins of Human Hepatocellular Carcinoma TissuesBy a comparative proteomic approach we compared protein expressing pattern in metastatic HCC and nonmetastatic HCC issues, in order to screen key molecules related to hepatocellular carcinoma metastasis and recurrence.Proteins extracted from 12 HCC tissue specimens (6 with metastases and 6 without) were separated by two-dimensional gel electrophoresis (2-DE). IPG stripe (24cm, pH3-10 NL) was used in the first dimensional electrophoresis, and in the second dimensional electrophoresis 12. 5% SDS-PAG was used. Analytical gels were stained with silver nitrate and preparative gels with Coomassie brilliant blue. The stained gels were then scanned using ImageScanner and analyzed with ImageMaster 2D Platinum 5. 0 software. Average 1102 + 56 andl230±73 spots had been detected in protein profile of metastatic HCC and nonmetastatic HCC tissue. The protein spots exhibiting statistically alternations between the two groups through computerized image analysis. 31 spots showing 3 or more than 3 fold difference and showing more than 50% frequency in patients were screened.Selected differential protein spots were excised from preparative gels and digested into peptides. These peptides were analyzed by MALDI-TOF MS. Data were then searched with the search engine MASCOT (Matrix Science) against a NCBI nonredundant protein sequence database. 16 spots were identified by MALDI-TOF MS as described in Table 1. There are 10 proteins including HSP27, S100 calcium-binding protein A10 and All (S100A10, S100A11), cytokeratin 18 (CK18), Aldo-keto reductase exerting up-regulated expression in metastatic HCC tissues and 6 down regulated. As to other 4 unidentified spots, database research returned only low quality candidates. To ensure taking spots exactly, each spot was excised from two gels of different samples and digested respectively and the identified results were coincident. To our best knowledge, some of identified proteins were firstly discovered between non metastasis HCC tissues and metastasis tissues. The protein profile of two groups displayed obviously difference. The results implied that various distinct different proteins may join together in HCC metastasis.Part TwoProteome Analysis of Human Hepatocellular Carcinoma Tissue for Identification of Disease-related ProteinsBy using 2-DE and MALDI-TOF MS to identify the proteins with different expressing level between HCC tissues and adjacent tissues, the object of this part is to screen key molecules related to tumorigenesis and tumor growth of hepatocellular carcinoma.Proteins sequentially extracted from 6 pair of HCC tumor tissue specimens and the corresponding adjacent tissues and at last we got 2 part of protein from each specimen. Proteins were separated by 2-DE. In the first dimensional electrophoresis IPG stripes (24cm, pH3~10 NL) and stripes (13cm, pH3~10 NL) were used for 2 different part of protein. In the second dimensional electrophoresis 12. 5% SDS-PAG was used. Analytical gels were stained with silver nitrate and preparative gels with Coomassie brilliant blue. The stained gels were then scanned using ImageScanner and analyzed with ImageMaster 2D Platinum 5. 0 software. Average 1205 + 62 and 1197 + 41 spots had been detected in protein profile of HCC tissues and adjacent tissues. The protein spots exhibiting statistically alternations between the two groups went through computerized image analysis. In all, 74 spots showing 4 or more than 4 fold difference and showing more than 50% frequency in patients were screened.Selected differential protein spots were excised from preparative gels and digested into peptides. These peptides were analyzed by MALDI-TOF MS. Data were then searched with the search engine MASCOT (Matrix Science) against a NCBI nonredundant protein sequence database. 31 kind of protein were identified by MALDI-TOF MS. There are 20 proteins including HSP27 exerting up-regulated expression in HCC tissues and 11 down regulated. To ensure taking spots exactly, each spot was excised from two gels of different samples and digested respectively and the identified results were coincident. The protein profile of two groups displayed obviously difference. The results implied that various distinct different proteins may be relative with HCC.Part Three Verification and Biological Function Analysis of HSP27Because of obvious expressing difference of HSP27 between metastatic HCC and nonmetastatic HCC tissues, also between HCC tissues and adjacent tissues, as well as the broad biological function reported, we chose this molecule as the target for following study. The object of this part is to validate expressing level in HCC tissues and in HCC cell lines, and explore the function of HSP27 in hepatocarcinogenesis and HCC metastasis.Validation of different proteins again is essential for next analysis. The results from RT-PCR, real-time PCR, western blot and immunohistochemistry, all confirmed the upregulation of HSP27 in metastatic HCC tissues than in non metastatic HCC tissues, in HCC tumor tissues than adjacent normal tissues, and in HCC cell lines with metastasis potential than cell lines without metastasis potential.Using RNA interference technique, we can specifically reduce expressing level of HSP27 in MHCC97H cell line, then observed alteration of biology characters of MHCC97H (motility, invasion, extracellular matrix metalloproteinase, apoptosis, etc).We set up 3 groups using MHCC97H cell as experimental model: blank group ( MHCC97H cell without transfection), control group (transfection of nonspecific small dsRNA), HSP27 RNAi group (transfection of HSP27 specific siRNA).Cell motility test showed the average cell numbers per field were 66±5, 63+8, 36 + 6 (blank, control, RNAi) respectively. Cell invasion assay in vitro showed the average invading cell numbers per field were 41 ±8, 38 + 5.1, 17 + 3.2 (blank, control, RNAi) respectively. This result demonstrated the significant difference of cell migration and invasion potential. Such biological behavior changes in MHCC97H with down-regulation protein expression of HSP27 by RNAi indicated that HSP27 may be an important contribution in HCC cell lines invasion and metastasis potential.MHCC97H cell apoptosis ration was 16. 12%, 16. 56%, 27.04% (blank, control, RNAi) respectively by FCM analysis. The significant changes in apoptosis of MHCC97H implied HSP27 was involved in cell apoptosis.The ratio of MHCC97H cell in each phase during cell cycle displayed GO~G1 phase (%): 60.24, 59.61, 60.01; S phase: 29.93, 33.35, 31.99; G2~M phase: 9.83, 7.04, 8.00 (blank, control, RNAi). No significant changesin cell cycles can be observed in cells of 3 groups.Average clone formation ration was 17.2+1.61% , 15.8+1.82% , 9. 3+1. 31% (blank, control, RNAi) respectively. This result showed there was distinct change in clone formation between MHCC97H with HSP27 RNAi and cells of control group.The quantitative analysis of MMP9 in serum free culture media by ABC-ELISA were: 69. 774,70. 736,33. 794 (MHCC97H, Ctrl, RNAi) respectively , unit is ng/ml. The lowest amount of MMP9 in RNAi group indicated HSP27 may regulate MMP9 secretion and further affected HCC cell invasion and metastasis.MMP2 in serum free culture media detected displayed: 31.163, 31.825, 37.402 (blank, control, RNAi) respectively , unit is ng/ml. No great changes in MMP2 of these samples were found in our experiment.MMPs in serum free culture media of MHCC97H were analysed by Gelatin zymography. A visible band at about 92KDa (MMP9) was detected in all groups, whereas MMP9 band in culture media of MHCC97H RNAi group showed lower density than control group, which was consistent with that in MMP9 quantitative assay (ABC-ELISA).Conclusion1. The protein profile of metastatic HCC tissues displayed obviouslydifference compared with non metastatic HCC tissues. The results implied that various distinct different proteins may lead to HCC metastasis together. To our best knowledge, some of 16 identified proteins were firstly reported between non metastatic HCC tissues and metastatic ones.2. The protein profile of HCC tissues displayed obviously difference compared with adjacent tissues. The results implied that various distinct different proteins may lead to HCC tumorigenesis together.3. HSP27 was validated to be a potential and valuable biomarker for HCCdiagnosis and prediction of metastasis.4. HSP27 can inhibit HCC cell apoptosis, enhance proliferation and...