The Expression Of HMGB1in Human Hepatocellular Cell Lines With Different Metastatic Potentials And Its Underlying Functions

Backround:Hepatocellular carcinoma (HCC), is one of the commonest malignant tumors in China with high degree of malignancy and mortality. Recurrence and metastasis of hepatocellular carcinoma is one of the main reasons leading to treatment failure and death,from which about66.7%to77.2%of the HCC patients died.The cumulative study found that the occurrence and development of HCC is multifactorial, and multiple steps are involved in this complex pathological process, which includes the abnormal activation and expression of numerous growth factors, oncogenes and tumor suppressor genes.Therefore, launching in-depth study on the molecular mechanism of the HCC recurrence and metastasis and exploring effective measures of prevention and new, effective therapeutic methods are of great significance all over the world.Highly mobility group box1protein (HMGB1) is a non-histone chromosomal protein rich in the nucleus of eukaryotic. Numerous studies indicate that, HMGB1can be actively released to the extracellular environment and involved in tumor development and metastasis.However, little research has been conducted on the relationship between HMGB1and the invasion and metastasis of HCC. We aim to provide new ideas for the pathogenesis and treatment of HCC by detecting the expression of HMGB1in three HCC cell lines with different transferring potentials, and elucidate the relationship between HMGB1and proliferation, migration, invasion of HCC cells.Objective:(1)Detecting the expression of HMGB1in three HCC cell lines with different metastatic potential and cell culture supernatants at different time points in vitro;(2)Construct three pairs of small interfering RNA (siRNA) specificly targeting HMGB1and transiently transfect them into cells in vitro,study the effects of siRNA on the biological behaviors of hepatocellular carcinoma cells (3) Obeserve the effects of recombinant human HMGB1(rhHMGB1) and anti-HMGB1antibody(HMGB1-Ab) on the biological behaviors of hepatocellular carcinoma cells in vitro;(4) Observation of HMGB1on the expression of NF-κB in HCC cells.Methods:(1) Extract the total mRNA and total protein and collect the cell culture supernatants at Oh,12h,24h,48h respectively,then detect the expression of HMGB1using RT-PCR,Westerning blotting and ELISA respectively;(2) Design and synthesize three pairs of siRNA fragments targeting HMGB1(siRNA1, siRNA2, siRNA3), and transiently transfect them into HCC cells in vitro. The levels of target gene expression were validated by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting.Then we select the best silencing sequence to inhibit the expression of HMGB1,after that,MTT assay, migration assay and Matrigel Assay were performed to assess cell proliferation, migration, invasion;(3) MTT assay, migration assay and Matrigel Assay were performed to measure cell proliferation, migration, invasion on the administration of rhHMGB1or anti-HMGB1antibody;(4) NF-κB p50and p65mRNA and protein level were detected by RT-PCR and western blotting;(5)Statistical analysis was performed with SPSS16.0software.Values are expressed as means and standard deviations.The student-t test and one-way analysis of variance were used for comparisons between groups. P<0.05is statistically significant.Results:(1) The expression of HMGB1increased consistently according to cell metastatic potentials.The relative expression of HMGB1mRNA is0.912±0.004,1.289±0.008,1.400±0.007in Hep3B,MHCC97L and HCCLM3respectively (P<0.05);The relative level of HMGB1protein in the same three cells is0.841±0.033,0.963±0.022,1.141±0.027respectively (P<0.05); The concentration of HMGB1is high in all the cell culture supernatants,but it is time-indepent and transferring potential-disassociated;(2) After the transfection of three pairs of small interfering RNA into HCCLM3cells,RT-PCR is performed to detect the expression of HMGB1mRNA1; the relative expression are0.195±0.003,0.358±0.037,0.744±0.019,0.996±0.012and0.823±0.037respectively in siRNA1group,siRNA2group,siRNA3group,NC group and BLANK group.RNA interference technology remarkably reduced the expression of HMGB1,in comparison with the control group,and the t value is4.302,5.885and2.776, P<0.05;Among the three pairs of siRNA,siRNA1 presents the strongest inhibitory effect with an inhibition rate at around60%.(3) rhHMGB1promotes cellular proliferation,migration and invasion, whereas knockdown of HMGB1by specific siRNA and administration of anti-HMGB1antibody inhibit cellular proliferation, migration and invasion(P<0.05).(4)HMGB1modulates the expression of NF-κB p50and p650Compared to control group,the NF-κB p50and p65mRNA levels are decreased by the transfection of HMGB1-siRNA,while increased on the treamtment of rhHMGB1.The results are statisticly significant.(P<0.05).The same trend holds true for the protein level of NF-κB p50and p65.Conclusion:(1)We demonstrate that the expression of HMGB1is different in HCC cells with different metastatic potential,which indicates that there may be a association between HMGB1and the metastasis and invision of HCC cells;(2) rhHMGB1can promote cell proliferation, migration and invasion and increases the level of NF-κB, whereas knockdown of HMGB1by specific siRNA and administration of HMGB1-specific antibody inhibit cell proliferations migration and invasion,which suggests that HMGB1can become a potential target for therapy.(3)HMGB1modulates the expression of NF-κB in HCC cells/which certificates that the biological function of HMGB1on the proliferation,migration and invasion of HCC cells is tightly associated with the activation of NF-κB signaling pathway.