| OBJECTIVE:Gastric carcinoma (GC), one of the most common malignant tumors, is the third cause of cancer mortality in China. High-mobility group box1(HMGB1), as a non-histone nuclear protein, has been associated with many human cancers, but the role of HMGB1in GC remains unclear. In this study, we investigated the expression of HMGB1in human GC and analyzed the relationship between HMGB1mRNAand GC clinicopathologic significance. We also examined the effects of RNA interference (RNAi) HMGB1on the bioactivity of GC cell line MGC-803.METHODS:(1)20cases of normal gastric tissues,112cases of GC and the corresponding gastric tissue just around the tumor (GAT) were collected, Then, all the samples were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western-blot to analysis the expression of HMGB1. At the same time, immunohistochemisty was used to detect the location of HMGB1. The relationships between HMGB1mRNA expression and clinicopathologic parameters were analyzed.(2) We designed and synthesized three specific small interfering RNA of HMGB1(HMGB1-siRNA-1HMGB1-siRNA-2and HMGB1-siRNA-3) and transfected these into MGC-803cells by use of LipofectamineTM2000. Cells transfected with no significant homology with human gene sequences (negative group) and without transfection treatment (blank group) were treated in parallel. Real time-PCR and Western blot were performed to determine the effects of HMGB1-siRNAs on HMGB1expression. In vitro proliferation was assessed by MTT assay. Apoptosis was demonstrated by flow cytometry. Migration and invasive ability were determined by use of the Transwell assay.RESULTS:(1) RT-PCR demonstrated that the expression of relative HMGB1mRNA was0.759±0.170; the highest in the tissue of GC, significantly up-regulated compared with that of0.473±0.126in GAT and of0.407±0.078in normal gastric tissues (P<0.001). HMGB1mRNA overexpression was significantly associated with lymphonodes metastasis,histological type and the differentiation of tumor. Western-blot showed the expression of HMGB1protein in GC also as the highest among all the groups. Correlation analysis between HMGB1mRNA and protein indicated that the expression of HMGB1mRNA had a positive correlation with HMGB1protein (P<0.01). Furthermore this overexpression’revealed by immunostaining was predominantly localized in the nuclei of GC; whereas, none of the stains were seen in normal gastric tissues and only a trace of it was detected in the cytoplasm of GAT cells.(2) Real-time PCR and Western-blot showed that all three specific HMGB1-siRNAs significantly inhibited HMGB1expression, with inhibition by HMGB1-siRNA-1being highest. MTT assay revealed that the HMGB1-siRNA-1group significantly reduced cell proliferation compared with the negative group or blank group cells (P<0.01). FCM revealed that apoptosis was significantly increased in HMGB1-siRNA-1-transfected cells compared with negative group or blank group cells (P<0.01). The Transwell assay revealed that MGC-803cells transfected with HMGB1-siRNA-1had much lower migratory ability and invasive activity than the negative group or blank group cells (P<0.01).CONCLUSIONS:(1) HMGB1levels in GC were significantly elevated. HMGB1overexpression was significantly associated with lymphonodes metastasis, histological type and the differentiation of tumor. Overexpression of HMGB1might be associated with GC development and progress.(2) Downregulation of HMGB1by specific small interfering RNA could obviously inhibit the growth and of promoting the apoptosis of MGC-803cells. It also obviously inhibits their migration and invasion ability. HMGB1may serve as a potential target for treatment of GC. |
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