| Introduction:Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in adults. HCC patients present with advanced disease stage that are not amenable to surgical resection or transplantation, and thus have a poor prognosis. Although clinical research on HCC has taken a great progress for the past few decades, the mechanism on pathogenesis was still unclear. It is known that accumulation of the disorders on gene expression and protein modulation, results in liver cancer. Thus, to discover and clarify the reasons for these disorders will be benefit for illuminating the mechanism on pathogenesis of HCC and for developing effective treatment.Zinc finger proteins are an important class of regulatory factors involved in many important biological processes, including cancer development. RING finger domain is a protein interaction region, previous research suggests that the protein with the RING domain and generally has two functions:(1) may be involved in transcriptional regulation, DNA repair and recombination, cell transformation and tumor inhibition, and variety of biological processes;(2) Play a pivotal role in the ubiquitin-proteasome pathway, as RING finger domain is a E3ubiquitin-protein ligase activity area.RN181is a C3HC4type zinc finger protein with a gene length of716bp encoding153amino acid protein, containing one RING finger domain. In2008, Brophy was the first to confirm RN181with E3activity. However, the physiological functions of RN181, especially in tumors are unknown.Therefore, the aim of the research was to discover the physiological function of RN181and its role in the growth of liver cancer, using modern experimental methods such as viral expression technology, RNA interference, proteomics technology and mass spectrometry.Methods:1. Prokaryotic expression and purification of RN181recombinant protein, and then immunized mice to prepare polyclonal antibodies against RN181.2. The expression of RN181in the tumors of12different organs analyzed by immunohistochemistry using tissue microarray.3. Compared the expression of RN181with139pairs of clinical hepatocellular carcinoma sections. Discovered the relationship of RN181with the development and progression of liver cancer.4. The RN181gene was amplified from pET28a/RN181recombinant plasmid by PCR. After PCR, restriction, and then connected to the retroviral vector pLNCX2, pLNCX2-Flag/RN181was constructed. After identified by sequence, the pLNCX2-Flag/RN181vector and pVSV-G were cotransfected into the GP2-293packaging cell in order to recombinant virus and then infected the SMMC-7721cells. Infected cells screened by G418for three weeks, carried out to expand the training, for subsequent experiments. SMMC7721, HepG2and Chang cell lines, infected with lentivirus containing the RN181interference fragment, screened by Flow Cytometry and then expanded and used for subsequent experiments.5. Analysis on proliferation of recombinant cells by MTT assay; analysis on colony formation of recombinant cells by soft agar assay; analysis on metastasis of recombinant cells by Transwell assay. Analysis on growth of recombinant cells in vivo by inoculating subcutaneously in nude mice. And analysis on expression of PCNA and actived-Caspase3in tumors from nude mouse by immunochemical staining.6. Differential expression proteins were identified by2D/MALDI-TOF using high expression RN181SMMC7721cell line and control cell line. And then the cell signal pathway was analyzed by Osprey software. On the other hand, the differential expression proteins between RN181-downregulated SMMC7721cell line and control cell line were identified by SILAC-LTQ proteomic methods.7. In vitro and in vivo, verification the RN181whether to participate in the regulation of these signaling pathways.8. Interaction network was identified by co-immunoprecipitation.Results:1. A successful preparation of polyclonal antibody specific against RN181.2. Expression analysis by tissue microarray method to study the RN181in the tumors of different organs. The results show that the RN181was low expression in tumor tissues but highly expressed in the corresponding adjacent tissues.3. Immunohistochemical analysis of liver tissue sample corrected from139cases of liver cancer, showed that RN181was low expression in hepatoma tissues but highly expressed in the corresponding adjacent tissues(t=32.844, P<0.001) and The expression of RN181was positively correlated with the differentiation of clinical samples(F=132.783, P＜0.001).4. Successfully constructed a retroviral expression RN181, and infection of hepatoma cell line SMMC-7721. Cells screened with G418, and then Western Blot testing. Results showed that the cells infected by retroviral-RN181have significantly increased the expression level of RN181, Which suggested to have already built a stable expression RN181hepatoma cell successfully.5. Successfully constructed stable down-regulated RN181cell lines. Infection with recombinant lentiviral expressing the RN181interference fragment, Chang, HepG2and SMMC7721cells, positive cells then separated by flow cytometry sorting. Real-Time Quantitative PCR (P＜0.001) and Western Blot analysis showed that the expression of RN181decreased significantly in the infected shRNA against RN181 cell lines, suggesting that to successfully build stable down-regulated RN181cells.6. MTT assay was used to detect the growth curve of SMMC7721cells with ectopic expression of RN181, stable down regulation of RN181and the corresponding control cells and results show that over expression of RN181in SMMC7721cells can inhibit proliferation (F=786.92, P<0.001)but down regulation the RN181on the contrary(F=255.26, P＜0.001). Soft agar colony formation experiments showed that, with highly expression of RN181, colony formation ability was significantly weaker than the control group(t=6.742, P=0.001), while such an ability of the down regulated RN181was more than the control group, obviously (t=11.031, P＜0.001). This confirmed that RN181can inhibit the colony formation ability of liver cancer cells, and may reduce the degree of malignancy of the liver cancer cells. However, Transwell experiments showed that both increasing and decreasing expression of RN181could inhibit the migration of liver cancer cells in vitro.7. Groups of recombinant cell lines injected the nude mice into armpit of the ministry by subcutaneously and successfully built xenograft tumors in nude mice. Tumor growth curve was observed and found that the injection of liver cancer cells highly expressing RN181in nude mice compared with the control nude mice, tumor growth is slow. And injection of those cells stable decreasing RN181in nude mice has opposite result. Immunohistochemical analysis showed that, PCNA was higher expression in the stable interference group than the control group, on the contrary, the expression of active-caspase3significantly weaker than the control one8. Two-dimensional electrophoresis combined with MALDI-TOF MS analysis for the ectopic expression of RN181cell group,39kinds of differentially expressed protein were identified. Obviously, multiple oncogenic proteins are down-regulated in SMMC7721cells which are high expression of RN181Interacting protein network analysis of these differentially expressed proteins found concentrated in the MAPK-ERK pathway.9. In vitro Western blot detection of the phosphorylation level of ERK found that over expression of RN181was able to inhibit the phosphorylation of ERK, but opposte to the RN181down-regulated cells. In nude mice tumor, immunohistochemical analysis also showed the similar results. On xenograft tumor sections highly expressed RN181, the expression level of phosphorylated ERK was lower than the control group, while the RN181-downregulated group was on the contrary. The expression of RN181and p-ERK were analyzed in40clinical samples then found that there is a negative correlation between the expression of RN181and p-ERK. The experiment in vitro showed that the inhibition of phosphorylation of ERK in the stable RN181-downregulated cells would suppress their ability of the proliferation and colony forming.10. Either upregulation or downregulation of RN181in hepatocellular carcinoma cell lines, inhibited the invasion and migration through Transwell assay.11. Many functional pivotal pathways including cellular assembly and organization and MAPK-ERK signal pathway were identified by SILAC-proteomics and IPA analysis.12. F-actin was detected by Rhodamine-phalloidin under fluorescent microscope and results showed that F-actin was clustered in RN181-downregulated cell lines but not in the control cell lines.13. Analysis of the vital proteins within actin reorganization. Results showed that the phosphorylation level of cofilin but not the protein expression level was increased in RN181-downregulated cell lines compared with the negative control cell lines. Expression level of another important actin regulated protein named VASP was increased too.14. Immunoprecipitation assay showed that RN181interacted with many tumor associated proteins. Functions of these proteins were summarized into apoptosis, actin reorganization and transcription/translation regulation such as HSP90a, HSP90b and RPS3. The interaction and signal network analyzed by IPA included MAPK-ERK pathway and actin reorganization and rearrangement. Western blot assay showed that not only HSP90a and HSP90b but also RPS3were stable compared between SMMC7721/NC and SMMC7721/KD. These results were consistent with SILAC-proteomics analysis. Conclusions:RN181weakly expressed in clinical cancer tissues but highly expressed in the corresponding normal tissues and the expression of RN181was associated with the degree of differentiation of HCC-The expression of RN181is positively correlated with the differentiation of clinical samples. High expression of RN181inhibits liver cancer cell proliferation and reduces its colony forming ability and nude mouse tumorigenicity and decreasing RN181was on the contrary. The expression of RN181in clinical HCC samples and the phosphorylation level of ERK were inversely proportional to the degree. The experiments in vitro showed that inhibition of the phosphorylation of ERK in stable RN181-downregulated cells would inhibit liver cancer cell proliferation and colony formation ability. Thus, RN181inhibit the growth of liver cancer cells through regulating the MAPK-ERK pathway. On the other hand, RN181can mediate the metastasis of liver cancer cell lines in vitro and the proteins interacted with RN181will not be degraded. In generally, RN181plays a multi-functional role in liver cancer cells and its function maybe result from interaction and modification on its substrate proteins but not from proteasome-degradation pathway. |
