Study On The Mechanism Of HMGB1Cytopiasmic Location In Clear Cell Renal Cell Carcinoma

BackgroundThe high mobility group box1(HMGB1) protein is a widespread and evolutional conservative nuclear protein that presents in most cell types. The protein typically locates in nucleus and functioins as a nuclear cofactor in transcription regulation. HMGB1protein also localizes in the cytoplasm and released to extracellular matrix. Extracellular HMGB1protein plays a critical role in carcinogenesis and inflammation. Recent evidence suggests that post-translational modifications affect the location HMGB1in subcellular department, which involve glycosylation, oxidation, hyperacetylation, methylation and phosphorylation. However, it remains elusive whether HMGB1is relocated to cytoplasm in clear cell renal cell carcinoma (ccRCC). The object of this project is to investigate the subcellular distribution of HMGB1and the possible mechanism of nucleocytoplasmic translocation in ccRCC.MethodsIn the first part of the project,14clear cell renal cell carcinoma samples and corresponding14adjacent renal tissue samples were collected. RT-PCR (Reverse Transcription-Polymerase Chain Reaction) was used to detect the HMGB1mRNA expression at transcriptional level. Western bloting was used to discover the protein expression level of HMGB1. Immunohistochemical staining was applied to analyze the expression of HMGB1in112clear cell renal cell carcinoma cases. In the second part, nuclear proteins and cytoplasma proteins were extracted by different protocols from20ccRCC samples and corresponding adjacent renal tissues. Western blotting and immunohistochemistry were used to identify the expression of HMGB1in ccRCC. To elucidate the potential mechanism of HMGB1cytoplasmic translocation, HMGB1proteins were enriched by immunoprecipitation and analyzed by high performance liquid chromatography(HPLC) and mass spectrometry (MS).ResultsIn the first part of the project the HMGB1mRNA was overexpressed in10(10/14,71.4%) renal cancer cases in comparision to corresponding adjacent tissues. And HMGB1protein expression was upregulated in9(9/14,64.3%rena) cancer cases. The immunohistostaining indicated that HMGB1was overexpressed in59.8%(67/112) clear cell renal cell carcinoma cases. The overexpression was significantly related to tumor size and nuclear grade(P<0.05). In the second part, the HMGB1protein was overexpressed and partially localized in cytoplasm in ccRCC samples(12/20,60%,P<0.05). Immunohistochemistry results indicated that ccRCC of high nuclear grade possessed more HMGB1relocation than the low grade did(P <0.05). Methylation of HMGB1at lysine112in ccRCC was detected by HPLC-MS. Bioinformatics analysis showed that the post-translational modification might affect the binding ability to DNA and mediate its translocation.ConclusionHMGB1is overexpressed in clear cell renal cell carcinoma, which may play a role in carcinogenesis and progression of the tumor. Translocation of HMGB1to cytoplasm was confirmed in ccRCC and methylation of HMGB1at lysine112might mediate the redistribution of the protein.