The Role Of HIF-1α And Galectin-3 In Carcinogenesis And Progression Of Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC),which accounts for 85%～90% of primary liver cancer,remains the fourth common cancer and the third most cause of cancer mortality in China.HCC is a one of the highly vascularized tumors,and its microcirculation patterns are complex.Vasculogenic mimicry(VM)is essential for HCC malignancy,correlating with short survival in patients with HCC.The effect of currently available anti-angiogenic drugs is composed of inhibiting endothelial cell proliferation,inducing endothelial cell apoptosis,and decreasing vascular density.However,vasculogenic networks of VM cannot be restrained by these drugs.Previous studies found that hypoxia was observed in HCC tumor tissue.As one of the major mediators of hypoxic response,hypoxia-inducible factor-1α(HIF-1α)has been shown to activate hypoxiaresponsive genes,which contributes to cellular proliferation,apoptosis,angiogenesis,and VM formation.Previous data reported that that Galectin-3 is over-expressed in several cancers,and higher expression of Galectin-3 can facilitate tumor metastasis by promoting the formation of vascular and is closely related to a poor prognosis.Hypoxia is critical for Galectin-3 expression in renal carcinoma cells through HIF-1α.However,the mechanisms underlying the regulation of Galectin-3 in HCC under hypoxia remains largely unknow.Therefore,it is important to identify the role and mechanisms of Galectin-3 and HIF-1α in the tumor carcinogenesis and progression and long-term prognosis in HCC under hypoxia microenvironment.Objective:The objective of this study was to examine the expression of Galectin-3 and HIF-1α in HCC patient samples in order to evaluate their relation to HCC related clinical features and their prognostic values.The role of Galection-3 and HIF-1α in tumor growth and metastasis were also investigated in HCC cell lines to reveal the mechanism of Galection-3 and HIF-1α in HCC occurrence and progression.Thus,our study will provide insight on the pathogenesis of HCC and clinical diagnosis and treatment.Methods:The patients with HCC who underwent surgery at the First Hospital of Jilin University were enrolled in this study.We evaluated Galectin-3,Galectin-9,HIF-1αand CD31/PAS expression in HCC patients by immunohistochemistry method,then analyzed the relationship between expression levels of Galectin-3,Galectin-9,HIF-1αprotein and tumor parameters or clinical outcomes.The relationship of Galectin-3,Galectin-9,and HIF-1α in HCC tumor tissues was also accessed.We utilized small interfering RNA(si RNA)to silence Galectin-3,MTS cell proliferation assay,woundhealing assay,cell transwell assay,flow cytometry assay and three-dimensional culture were used to investigate the biological function of Galectin-3 in HCC cells.SMMC7721 HCC cells were cultures under normal oxgen and hypoxia for 24 or 48 hours,respectively.Real-time PCR and Western blot were used to confirm the expression level of HIF-1α and Galectin-3 in HCC cells both in m RNA level and protein level.The role of HIF-1α and Galectin-3 in the biological function of HCC cells under hypoxia microenvironment was investigated using MTS cell proliferation assay,wound-healing assay,cell transwell assay,flow cytometry assay and three-dimensional culture.Slicencing HIF-1α downregulate the expression of Galectin-3 and Ch IP-q PCR were used to further determine the target genes of HIF-1α transcription factor.Results:1.Expression of HIF-1α in HCC patients and clinical significanceThe expression of HIF-1α and CD31/PAS was determined among 202 patients with HCC.In non-cirrhosis patients,the HIF-1α expression was significantly higher in tumor tissues compared with adjacent non-tumor tissues(P=0.023).A higher HIF-1αpositive rate was significantly associated with VM presentation(P=0.021).Survival analysis showed that patients with positive HIF-1α expression had worse overall survival(P=0.002).Multivariate analysis showed that multiple tumor(HR=1.83,95%CI [1.24-2.72]),tumor size≥5cm(HR=2.23,95% CI [1.48-3.37])and HIF-1α positive expression(HR=2.16,95% CI [1.42-3.30])were independent influencing factors of prognosis in patients with hepatocellular carcinoma.2.Expression of Galectin-3 and Galectin-9 in HCC patients and their relationship with prognosisThe Galectin-3 expression was significantly higher in tumor tissues compared with adjacent non-tumor tissues(P<0.001),while no significant differences of Galectin-9was detected(P=0.222).A higher Galectin-3 expression was significantly associated with infection with HCV,AFP≥20ng/ml,lymph-vascular invasion,poor histological differentiation,no cirrhosis,and VM presentation(P<0.05).Survival analysis showed that patients with higher Galectin-3 expression had worse overall survival(P=0.012),however no correlation was found between Galectin-9 expression and survival(P=0.185).Multivariate analysis showed that multiple tumor(HR=2.02,95% CI [1.41-2.90),tumor size≥5cm(HR=1.12,95% CI [1.07-1.19])and Galectin-3 expression(HR=1.50,95% CI [1.01-2.24])were independent influencing factors of prognosis in patients with hepatocellular carcinoma.In HCC tumor tissues,the expression level of HIF-1α was positively correlated with that of Galectin-3(R=0.167,P=0.043).3.Effect of Galectin-3 on the function of HCC cellsMTS cell proliferation assay showed that the average value of survival HCC cells in Galectin-3-knockdown group was lower than that in control group(P<0.05).Flow cytometry assay showed that the number of apoptotic cells in Galectin-3-knockdown group was higher than that in control group in Hep G2 cells and SMMC7721 cells with a statistical significance(P<0.05).Cell scratch assay showed that after 48 h,the cell migration rate in Galectin-3-knockdown group was significantly smaller than that in control group(P<0.05).Cell transwell assay showed that the number of invaded cells in control group was statistically higher than that in Galectin-3-knockdown group(P<0.05).Three-dimensional cultures were used to examine the influence of Galectin-3 on VM.The results showed that after 6h tube-like strictures area in Galectin-3-knockdown group was as 0.94 times as that in control group in Hep G2 cells(P=0.038)and as 0.88 times as that in control group in SMMC7721 cells with a statistical significance(P=0.001).4.Effect of hypoxia microenvironment on the function of HCC cellsAfter SMMC7721 cells were cultured under hypoxia for 24 hours,we found that hypoxia triggered proliferation of SMMC7721 cells significantly(P<0.001).As expected,hypoxia decreased apoptosis of SMMC7721 cells(P=0.006).Cell scratch assay showed that hypoxia accelerated cell migration remarkably(P=0.166).Similarly,cell transwell assay also revealed that hypoxia induced invasion of SMMC7721 cells significantly(P=0.001).Three-dimensional cultures of SMMC7721 cells showed that hypoxia promoted the formation of tube-like strictures area significantly(P=0.003).5.Effect of HIF-1α and Galectin-3 on the function of HCC cells under hypoxia microenvironmentWe investigated the effects of hypoxia on expressions of HIF-1α and Galectin-3 in SMMC7721 cells,showing that 48 hours of hypoxia enhanced the transcription and translation of HIF-1α and Galectin-3 significantly(P<0.05).To explore connectivity of HIF-1α and Galectin-3 with the effect of hypoxia microenvironment on the function of HCC cells,we utilized si RNA to silence HIF-1α or Galectin-3,parsing that the silencing HIF-1α or Galectin-3 reduced proliferation(P<0.001);Silencing HIF-1αincreased apoptosis with no statistical significance(P=0.352),while the number of apoptotic cells was significantly increased in Galectin-3-silencing group(P=0.008).Indeed,silencing HIF-1α or Galectin-3 decreased migration and invasion of SMMC7721 cells(both P<0.05).Indeed,the formation of tube-like strictures area was reduced by silencing Galectin-3 or HIF-1α under hypoxia significantly(P<0.001).6.HIF-1α modulates Galectin-3 expression in HCC cell lines under hypoxia microenvironmentThe SMMC7721 cells were cultures under hypoxia.Real-time PCR and Western blot confirmed that the expression levels of Galectin-3 and HIF-1α were lower in HIF-1α-knockdown group than that in control group(P<0.05).Relation between Galectin-3 and HIF-1α validated using Ch IP-q PCR assay.Conclusions:1.In non-cirrhosis patients,the HIF-1α expression was significantly higher in tumor tissues compared with adjacent non-tumor tissues.The patients with positive HIF-1α expression had worse overall survival.2.The Galectin-3 expression was significantly higher in tumor tissues compared with adjacent non-tumor tissues.A higher Galectin-3 expression was related to VM presentation and worse overall survival.3.In HCC tumor tissues,the expression level of HIF-1α was positively correlated with that of Galectin-3.4.Galectin-3 promotes cell proliferation,migration,invasion and VM formation of HCC cells,but supressed apoptosis of HCC cells.5.Galectin-3 is the target genes of HIF-1α transcription factor.HIF-1α-regulated Galectin-3 contributes to cell proliferation,apoptosis,migration,invasion and VM formation of HCC cells under hypoxia.