| Objectives:Hepatocellular carcinoma (HCC), one of the most common malignant tumors, is the second 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 HCC remains unclear. In this study, we investigated the expression of HMGB1in human HCC and analyzed the relationship between HMGB1mRNA and HCC clinicopathologic significance. We also examined the effects of RNA interference (RNAi) HMGB1on the bioactivity of HCC cell line HCCLM3and explored possible downstream mechanism.Methods:(1)11cases of normal liver tissues,32cases of HCC and the corresponding liver tissue just around the tumor (LAT) 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-1,HMGB1-siRNA-2and HMGB1-siRNA-3) and transfected these into HCCLM3cells 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.(3) RT-PCR and Western blot were performed to detect NF-κB/p65and VEGF-C expression after transfection of HMGB1-siRNA-1into HCCLM3. Indirect immunofluorescence assay was used to detect the location of NF-κB/p65. Then, we used enzyme-linked immunosorbent assay (ELISA) to detect NF-KB/p65activity.Results:(1) RT-PCR demonstrated that the expression of relative HMGB1mRNA was0.859±0.159; the highest in the tissue of HCC, significantly up-regulated compared with that of0.506±0.131in LAT and of0.413±0.086in normal liver tissues (P<0.001). HMGB1mRNA overexpression was significantly associated with Edmondson stage, TNM stage, vascular invasion and capsule invasion. Western-blot showed the expression of HMGB1protein in HCC 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 (Person correlation analysis, R=0.833, P<0.01). Furthermore this overexpression revealed by immunostaining was predominantly localized in the nuclei of HCC; whereas, none of the stains were seen in normal liver tissues and only a trace of it was detected in the cytoplasm of LAT 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 HCCLM3cells transfected with HMGB1-siRNA-1had much lower migratory ability and invasive activity than the negative group or blank group cells (P<0.01).(3) Real-Time PCR and Western-blot analysis showed that the mRNA and protein levels of NF-κB/p65and VEGF-C of HCCLM3cells transfected with HMGB1-siRNA-1were significantly decreased compared with the negative group or blank group cells (P<0.01). Indirect immunofluorescence assay revealed that NF-κB/p65nuclear translocation was decreased after HCCLM3cells transfected with HMGB1-siRNA-1. At the same time, ELISA showed that NF-κB/p65activity was also decreased. Conclusions:(1) HMGB1levels in HCC were significantly elevated. HMGB1overexpression was significantly associated with Edmondson stage, TNM stage, vascular invasion and capsule invasion. Overexpression of HMGB1might associated with HCC development and progress.(2) Downregulation of HMGB1by specific small interfering RNA could obviously inhibit the growth and of promotes the apoptosis of HCCLM3cells. It also obviously inhibits their migration and invasion ability. HMGB1may serve as a potential target for treatment of HCC.(3) Downregulation of HMGB1can inhibit the expression of NF-κB/p65and VEGF-C in HCCLM3cells. Meanwhile, NF-κB/p65nuclear translocation and activity were also decreased. HMGB1possibly via the NF-κB signaling pathway that regulates VEGF-C expression.
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