MiR-505 Modulate Hepatic Cancer Cell Proliferation,Apoptosis And Doxorubicin-induced Cytotoxicity Through Repressing The Akt Pathway By Directly Targeting HMGB1

Objective: HCC is the sixth most common malignancy and the third fatal cancer worldwide,system chemotherapy is a common strategy in advanced hepatocellular therapy.To discover the molecular biology mechanisms may find new marker in early diagnosis,to evaluate the prognosis,to be the therapy target,to improve the chemosensitivity and decrease the chemotherapy doses.Adriamycin is a key chemotherapy agent in advanced hepatocellular cancer therapy and resistance is the main reason which affect the chemotherapy outcome.Mi RNAs have been shown to participate in the regulation of cancer cell proliferation,apoptosis and invasion et al.and modulate the chemotherapy resistance.In the serum of the HCC patients,mi R-505 has been reported to be altered and HMGB1 may induce chemotherapy resistance in HCC.We have predicted the target regulation between mi R-505 and HMBG1 by biological information analysis.The effect and mechanisms of mi R-505 in HCC remain pooly understand.In the study,we observed the effect of mi R-505 and HMGB1 in HCC proliferation,invasion,EMT and apoptosis.We also detected the effect and mechanism in ADM induced HCC cytotoxicity.The aim was to observe the function and mechanisms of mi R-505 modulated ADM induced HCC cytotoxicity via HMGB1 and provided new target gene in HCC early diagnosis,prognosis evaluation,therapeutic target and reversed chemotherapy resistance.Methods: 1.QRT-PCR was used to detect the mi R-505 and HMGB1 m RNA expression in human hepatocellular tissues,paired normal live tissues,four human hepatocellular cell line(QGY-7703,SMMC-7721,MHCC97,Hep G2)and normal human liver cell lines(LO2).Western blot assay was used to detect the HMGB1 protein in above samples.2.To verify whether mi R-505 and HMGB1 modulated the MHCC97 cell proliferation,apoptosis and invasion,mi R-505 mimics or pc DNA3.1-HMGB1 was used to up-regulate mi R-505 or HMGB1 expression,anti-mi R-505 or HMGB1-si R was used to down-regulated mi R-505 or HMGB1 expression,and transfected into MHCC97 cells.Transinfection efficiency was detected by q RT-PCR or Western blot assay.MTT assay was used to detect proliferation,Western blot was used to detect Ki67 protein expression in MHCC97 cells,flowcytometer assay was used to detect apoptosis and Transwell assay was used to detect invasion.3.We observed the chemo-sensitivity of ADM interfered hepatocellular cells(QGY-7703,SMMC-7721,MHCC97,Hep G2),MTT assay was used to detect ADM induced cytotoxicity.MHCC97 and Hep G2 cells were selected to be transfected with mi R-505 or HMGB1 up or down regulated and MTT was used to detect ADM induced cytotoxicity.4.To observe wether mi R-505 modulated TGF-β induced EMT,MHCC97 cells was transfected with mi R-505 mimics or anti-mi R-505,EMT related genes(N-cadherin、fibronectin、vimentin、E-cadherin)proteins and m RNA were detected at m RNA and protein levels by q RT-PCR and Western blot.5.Luciferase report assay was used to identify HMGB1 was the direct target of mi R-505.6.To indetify if mi R-505 inhibited MHCC97 cell proliferation and invasion by down-regulating HMGB1 and accelated ADM induced hepatocellular cell apoptosis,si RNA was transfected into MHCC97 cells,HMGB1 was down-regulated and MTT assay,Transwell assay and flowcytometer assay were used to detect proliferation,invasion and apoptosis.7.To prove mi R-505 overexpress down-regulated HMGB1 affect the caspase-3 activity in ADM interfered hepatocellular cell,Caspase-3 activity was detected in HMGB1-si R or Con-si R transinfected MHCC97 and Hep G2 cells dealt with 0.5 μM or 0.75 μM ADM.8.To indentify the effect of HMGB1 down-regulated on ADM induced hepatocellular cell DNA damage,γH2AX protein was detected with Western blot assay in MHCC97 and Hep G2 cells under different intereferences.9.We detected the γH2AX expression by Western blot in MHCC97 and Hep G2 which infected with Con-mi R,mi R-505,mi R-505+Vector,mi R-505+HMGB1 and dealt with 0.5 μM or 0.75 μM ADM to observe if mi R-505 down regulted HMGB1 to improve ADM induced HCC DNA damage.10.Western blot assay was used to detect Akt signal pathway protein(p-Akt,Akt,p-GSK-3β,GSK-3β)expression to observe whether mi R-505/HMGB1 axis targeted Akt signal pathway to modulate ADM induced HCC cytotoxicity.Results: 1.In human HCC tissues and HCC cell lines(QGY-7703、SMMC-7721、MHCC97、Hep G2),mi R-505 expression was lower than in normal tissue and normal live cell line,HMGB1 expression was contrary to mi R-505.2.Mi R-505 depressed MHCC97 cell proliferation,invasion and ki67 protein expression,promoted apoptosis,but HMGB1 was contrary to mi R-505.3.Exposed to different concentration grade ADM,MHCC97 was the most sensitive,and then Hep G2,mi R-505 improved ADM induced HCC cell cytotoxicity,but HMGB1 depressed the cytoxicity.4.Mi R-505 inhibited TGF-β induced EMT in MHCC97 cells.5.Mi R-505 inhibited HMGB1 expression directly by post-transcriptional manner.6.Mi R-505 inhibited MHCC97 cell proliferation,invasion and improved ADM induced apoptosis by depressing HMGB1.Over expression of HMGB1 at least partially reversed the effect of mi R-505 on MHCC97 cell proliferation,invasion and apoptosis.Down regulating HMGB1 improved ADM induced HCC cell apoptosis.Over expression of mi R-505 improved ADM induced cytotoxicity and apoptosis by depressing HMGB1.7.Mi R-505 down-regulating HMGB1 increased ADM induced HCC cell caspase-3 activity.8.Down-regulating HMGB1 caused ADM induced HCC cell DNA damage.9.Over expression of mi R-505 down regulating HMGB1 accelerated ADM induced HCC cell DNA damage.10.Down regulating HMGB1 reduced p-Akt and p-GSK-3β expression and inhibited ADM induced HCC cell Akt signal pathway activity,up-regulating mi R-505 inhibited Akt and GSK-3βphosphorylation in ADM induced HCC cell by depressing HMGB1.Conclusion: 1.Mi R-505 is down-regulated in HCC tissue and cell,inhibits HCCcell proliferation,invasion and EMT,promotes apoptosis and improves ADM induced HCC cell cytotoxicity.HMGB1 is up-regulated in HCC tissue and cell,accelerates HCCcell proliferation,invasion,inhibits apoptosis and improve HCC cell ADM resistance.2.Mi R-505 modulates HCC cell proliferation,apoptosis and invasion by targeting HMGB13’-UTR directly and improves ADM induced HCC cell cytotoxicity by depressing HMGB1.3.Mi R-505 improves ADM induced HCC cell cytotoxicity,accelerates apoptosis and caspase-3 activity by target down-regulating HMGB1 via depressing Akt signal pathway.