| Objective: According to the reports of Global Cancer Figures in 2011,the incidence of liver cancer ranks the fifth of all malignancies in the world.About 700,000 people worldwide died from liver cancer each year.China is the country with the highest incidence of hepatic carcinoma in the world,which alone accounts for more than 50% of the total.More than 90% of hepatic carcinoma is hepatocellular carcinoma(HCC).So far,many problems remain unsolved concerning HCC treatment,including low resection rate,limited source of liver,significant toxicity and high recurrence rate.The 5-year relative survival rate of hepatic carcinoma is less than 15% even in developed countries,which is a serious threat to the public health.Therefore,professionals in life science have been focused on to the clarification of the pathogenesis of HCC and seeking effective methods of treatment.In the early 1970 s,Pierce et al proposed the theory of "Tumor was an issue of developmental biology".Numerous studies demonstrated that early embryonic development and tumorigenesis share striking similarities in many respects,including the similarities between early embryonic cellular differentiation and proliferation mechanisms and the mechanism of tumor cell differentiation and proliferation;the process of early embryoic implantation and invasive metastasis of the tumor;the immune escape in both conditions and so on.Expression of embryonic genes such as alpha-fetoprotein(AFP),carcinoembryonic antigen(CEA)and pancreatic oncotetal antigen(POA)has been confirmed by Misirlioglu et al;Dvorak et al found that large amount of oncogenes expressed druing the formation of tumor are also expressed in the implantation of embryos into endometrium.Hochedlinger et al also confirmed that the embryonic microenvironment may modulate the differentiation of tumor cells to and their change of malignant phenotype.According to these,Professor Jinsheng Qi proposed that embryonic cells may induce the redifferentiation of tumor cells with spatial and temporal specificity.It has been proved that co-culture of Hep G2 cells with specific embryonic hepatocytes can induce the redifferentiation of the tumor cells.The purpose of this experiment is to further explore the molecular mechanisms of the induced redifferentiation on this basis.Young et al found that HNF4?,HNF1?,HNF6 and USF1 formed a highly connected core transcriptional regulation circuitry by cross regulation and self-regulation in the hepatocytes.Related studies also proved that liver enriched transcription factors(LETFs)including C/EBP,DBP,HNF-3,HNF-1,HNF-4 and HNF6 play a crucial role in the development and differentiation of embryonic hepatocytes.HNF4? is the key factor that regulates the differentiation and functional specificity of the hepatocytes,and its normal expression is essential for the normal state of differentiation and function.The absence of its expression may stimulate the proliferation of hepatocytes and lead to HCC.Therefore,HNF4? is considered an HCC suppressor gene,which becomes a new target for the treatment of HCC.Preliminary experiments of our group showed that,after the co-culture of embryo hepatocytes(13.5-day)and Hep G2 cells(14.5-day)for 48 h,the growth of Hep G2 cells was significantly inhibited,with some of the cells retracting,turning round and eventually disintegrating;The Mrna and protein levels of differentiation marker gene including HNF4?,HNF1?,HNF6 and USF1 in Hep G2 cell were up-regulated,while the expression of protooncogene c-Myc was down-regulated in both transcriptional and translational levels.AFP expression declined significantly in Hep G2 cells.These suggested that the embryonic hepatocytes may spatial and temporal specifically induce the redifferentiation of hepatic tumor cells.However,further study is needed to prove whether the redifferentiation of Hep G2 cells rebuilds the core transcriptional regulation circuitry of HNF4?,HNF1?,HNF6,USF1 and HNF4? as the core factor of the regulation circuitry.This study was designed on the basis of preliminary experimental results to further reveal the major molecular mechanisms of embryoic hepatocytes induced hepatocellular carcinoma cells redifferentiation.First,13.5d embryo hepatocytes are co-cultured with Hep G2 cells for 48 h,the re-establishment of HNF4?,HNF1?,HNF6 and USF1 core transcriptional regulation circuitry was detected with Chromatin Immunoprecipitation(Ch IP)technology in Hep G2 cells.Next,after the knockdown of HNF4? expression with transfected si RNA-HNF4?(si-HNF4?)in the Hep G2 cells,the variation of HNF4?,HNF1?,HNF6 and USF1 in transcriptional and translational levels in Hep G2 cells co-cultured with embryo hepatocytes were detected.The cross regulation among HNF4?,HNF1?,HNF6 and USF1 was further demonstrated.We then use immunofluorescence assay to detect the change of AFP expression in Hep G2 cells.The results show that blocking of the regulational loop among HNF4?,HNF1?,HNF6 and USF1 can affect the process of embryo hepatocytes induced hepatocellular carcinoma cell redifferentiation.Accordingly,exploring the molecular mechanisms of embryo hepatocytes induced hepatocellular carcinoma cell redifferentiation provide experimental basis for the treatment of HCC.Methods:1 The cell cultureHep G2 cells: We recovered Hep G2 cells with 1% non-essential amino acids and DMEM high glucose culture medium containing 10% fetal bovine serum,0.25% trypsin digestion passaged and then inoculated in 100 ml flasks.We cultured Hep G2 cells in incubator with 37? and 5% CO2.The isolation and culture of the primary embryo hepatocytes: With the collagenase IV digestion solution,at first,we removed the fetal mouse from pregnant mice belly and isolated embryo liver;next,we cut the embryo liver tissue into the tissue blocks;third,we successfully isolated the tissue blocks into a single embryo hepatocyte following digested by collagenase IV.In order to remove fibroblasts,we used selective plating technique repeatedly to purify the embryo hepatocytes.Embryo hepatocytes cultured with high glucoseDMEM.2 The Experimental Design and Grouping(1)To detect the regulatory effects among the HNF4?,HNF1?,HNF6 and USF1 in the redifferentiation of Hep G2 cells induced by embryo hepatocytes,experiments were divided into 2 groups: untreated group(pure Hep G2 cells);co-culture group(the Hep G2 cells co-cultured with 13.5 days embryo hepatocytes for 48h).(2)To estimate the transfection efficiency of Hep G2 cells transfected with FAM-si RNA by Lipofectamine 2000(Lipo2000),experiments were divided into 2 groups: transfection reagent control(Mock)group;FAM-si RNA group.(3)To estimate the inhibition rate of the positive control in Hep G2 cells transfected with si-GAPDH,experiments were divided into 2 groups: Mock group;si-GAPDH group.(4)To detect the inhibition rates of different targets of si-HNF4? in Hep G2 cells transfected with si-HNF4?,according to the same proportion between the si RNA(pmol)and Lipo2000(?l)(1:0.05)with the different targets,experiments were divided into five groups: Mock group;negative control(si-NC)group;si-HNF4?-273 group(si-T1);si-HNF4?-297 group(si-T2);si-HNF4?-699 group(si-T3).(5)To detect the inhibition rates of different concentrations of si-HNF4? in Hep G2 cells transfected with si-HNF4?,according to the same target(HNF4?-273)with the different proportion between the si RNA(pmol)and Lipo2000(?l),experiments were divided into five groups: Mock group;si-NC group;1:0.04 group;1:0.05 group;1:0.06 group.(6)To detect the impaction of Hep G2 cells transfected with si-HNF4? in the redifferentiation of Hep G2 cells induced by embryo hepatocytes,experiments were divided into four groups: untreated group(pure Hep G2 cells);co-culture group;co-culture+si-NC group;co-culture+si-HNF4? group.3 The detection of the regulatory effects among HNF4?,HNF1?,HNF6 and USF1 in the redifferentiation of Hep G2 cells induced by embryo hepatocytes.Collecting the HepG2 cells,the ChIP assay was performed to detect the mutual regulation among HNF4?,HNF1?,HNF6 and USF1.4 The estimation for the transfection efficiency,the inhibition rates of si-GAPDH and si-HNF4? in Hep G2 cells transfected with the si RNA.The transfection efficiency was detected by fluorescence microscopy and flow cytometry.Total RNA was isolated using a one-step Trizol reagent in Hep G2 cells.Real-time PCR analysis for the expression of m RNA for HNF4? and GAPDH genes and ?-actin as a control reference were carried out.5 Hep G2 cells were co-cultured with 13.5d embryo hepatocytes for 48 h after transfected with si-HNF4? for 24 h,and then the changes of HNF4?,HNF1?,HNF6,USF1 and c-Myc genes transcriptional levels were detected in Hep G2 cells.Total RNA was isolated using a one-step Trizol reagent in Hep G2 cells.Real-time PCR analysis for the expression of m RNA for HNF4?,HNF1?,HNF6,USF1 and c-Myc genes and ?-actin as a control reference were carried out.6 Hep G2 cells were co-cultured with 13.5d embryo hepatocytes for 48 h after transfected with si-HNF4? for 24 h,and then the detection of HNF4?,HNF1?,HNF6,USF1 and c-Myc proteins was carried out.Whole proteins from Hep G2 cells were extracted by RIPA and quantified by Lowry method.Western-blotting analysis of HNF4?,HNF1?,HNF6,USF1 and c-Myc protein levels was performed.7 Hep G2 cells were co-cultured with 13.5d embryo hepatocytes for 48 h after transfected with si-HNF4? for 24 h,and then the detection of AFP protein content was assessed,The expression amount of the AFP was examined by immunofluorescence in Hep G2 cells.Results:1 The purification and culture of primary embryonic hepatocytesWith the collagenase IV digestion solution,at first,we removed the fetal mouse from pregnant mice belly and isolated embryo liver;next,we cut the embryo liver tissue into the tissue blocks;third,we successfully isolated the tissue blocks into a single embryo hepatocyte following digested by collagenase IV.In order to remove fibroblasts,we used selective plating technique repeatedly to purify the embryo hepatocytes.Embryo hepatocytes cultured with high glucose DMEM.AFP was expressed in the embryo hepatocytes via immunofluorescence assay,indicating that the primary embryo hepatocytes cultured successfully.2 Ch IP assay was performed to detect the changes of the regulatory effects among HNF4?,HNF1?,HNF6 and USF1 in Hep G2 cells co-cultured with 13.5d embryo hepatocytes for 48 h.2.1 The binding effect of HNF4? on HNF1? promoter region was significantly higher in Hep G2 cells co-cultured with 13.5d embryo hepatocytes for 48h(6.366±0.099,P<0.05)than that in the untreated group.2.2 The binding effect of HNF4? on the USF1 proximal promoter region was significantly higher in Hep G2 cells co-cultured with 13.5d embryo hepatocytes for 48h(28.485±1.080,P<0.05)than that in the untreated group.2.3 The binding effect of HNF4? on the USF1 distal promoter region was significantly higher in Hep G2 cells co-cultured with 13.5d embryo hepatocytes for 48h(19.924±0.622,P<0.05)than that in the untreated group.2.4 The binding effect of USF1 on HNF6 promoter region was significantly higher in Hep G2 cells co-cultured with 13.5d embryo hepatocytes for 48h(5.606±0.475,P<0.05)than that in the untreated group.2.5 The binding effect of HNF6 on the proximal of HNF4? promoter region was significantly higher in Hep G2 cells co-cultured with 13.5d embryo hepatocytes for 48h(2.445±0.181,P<0.05)than that in the untreated group.2.6 The binding effect of HNF6 on the distal of HNF4? promoter region was significantly higher in Hep G2 cells co-cultured with 13.5d embryo hepatocytes for 48h(2.767±0.187,P<0.05)than that in the untreated group.2.7 The binding effect of HNF1? on HNF4? promoter region was significantly higher in Hep G2 cells co-cultured with 13.5d embryo hepatocytes for 48h(2.623±0.079,P<0.05)than that in the untreated group.3 Hep G2 cells were transfected with si-HNF4? to knockdown HNF4? expression,then co-cultured with 13.5d embryo hepatocytes for 48 h,and then the changes of the transcription levels of HNF4?,HNF1?,HNF6,USF1 and c-Myc were detected in Hep G2 cells.3.1 The transfection efficiency of FAM-si RNA was higher than 90% in Hep G2 cells by fluorescence microscopy and flow cytometry.3.2 The relative expression level of GAPDH m RNA was significantly descended in Hep G2 cells transfected with si-GAPDH(0.437±0.022,P<0.01)compared to untreated group.3.3 After Hep G2 cells transfected with different targets of the si-HNF4?,the HNF4? m RNA relative expression in Hep G2 cells was compared to Mock group.There was no significance in si-NC group(1.057±0.030,P>0.05),but it was significantly decreased in the si-T1 group(0.366±0.022,P<0.01),si-T2 group(0.552±0.037,P<0.01),and si-T3 group(0.692±0.027,P<0.01).3.4 After Hep G2 cells transfected with different concentrations of the siHNF4?,the HNF4? m RNA relative expression in Hep G2 cells was compared to Mock group.There was no significance in si-NC group(1.063±0.040,P>0.05),but it was significantly decreased in 1:0.04 group(0.318±0.033,P<0.01),1:0.05 group(0.391±0.035,P<0.01)and 1:0.06 group(0.514±0.021,P<0.01).3.5 After transfected with si-HNF4? for 24 h,Hep G2 cells were co-cultured with 13.5d embryo hepatocytes for 48 h,the HNF4? m RNA relative expression in Hep G2 cells was significantly increased in co-culture group compared to the untreated group(0.481±0.015,P<0.01);the HNF4? m RNA relative expression in Hep G2 cells compared to co-culture group was no significance in co-culture+si-NC group(1.029±0.093,P>0.05),but it was significantly decreased in co-culture+si-HNF4? group compared with co-cultured group(0.319±0.008,P<0.05).3.6 After transfected with si-HNF4? for 24 h,Hep G2 cells were co-cultured with 13.5d embryo hepatocytes for 48 h,the HNF1? m RNA relative expression in Hep G2 cells was significantly increased in co-culture group compared to the untreated group(0.252±0.049,P<0.01);the HNF1? m RNA relative expression level in Hep G2 cells compared to co-culture group was no significant change in co-culture+si-NC group(0.980±0.063,P>0.05),but it was significantly decreased in co-culture+si-HNF4? group compared with co-cultured group(0.203±0.039,P<0.05).3.7 After transfected with si-HNF4? for 24 h,Hep G2 cells were co-cultured with 13.5d embryo hepatocytes for 48 h,the HNF6 m RNA relative expression in Hep G2 cells was significantly increased in co-culture group compared to the untreated group(0.365±0.010,P<0.01);the HNF6 m RNA relative expression level in Hep G2 cells compared to co-culture group was no significant change in co-culture+si-NC group(0.933±0.062,P>0.05),but it was significantly decreased in co-culture+si-HNF4? group compared with co-cultured group(0.286±0.019,P<0.05).3.8 After transfected with si-HNF4? for 24 h,Hep G2 cells were co-cultured with 13.5d embryo hepatocytes for 48 h,the USF1 m RNA relative expression in Hep G2 cells was significantly increased in co-culture group compared to the untreated group(0.529±0.038,P<0.01);the USF1 m RNA relative expression in Hep G2 cells compared to co-culture group was no significant change in co-culture+si-NC group(0.974±0.058,P>0.05),but it was significantly decreased in co-culture+si-HNF4? group compared with co-cultured group(0.429±0.003,P<0.05).3.9 After transfected with si-HNF4? for 24 h,Hep G2 cells were co-cultured with 13.5d embryo hepatocytes for 48 h,the c-Myc m RNA relative expression in Hep G2 cells was significantly decreased in co-culture group compared to the untreated group(2.133±0.173,P<0.05);the c-Myc m RNA relative expression level in Hep G2 cells compared with Mock group was no significant change in co-culture+si-NC group(1.104±0.074,P>0.05),but significantly increased in co-culture+si-HNF4? group compared with co-cultured group(1.622±0.178,P<0.05).4 Hep G2 cells were transfected with si-HNF4? for 24 h to knockdown HNF4? expression,then co-cultured with 13.5d embryo hepatocytes for 48 h,and then the changes of the protein levels of HNF4?,HNF1?,HNF6,USF1 and c-Myc were detected in Hep G2 cells.4.1 Before co-cultured with 13.5d embryo hepatocytes for 48 h,Hep G2 cells were transfected with si-HNF4? for 24 h.The HNF4? protein content in Hep G2 cells was significantly higher in co-culture group than that in untreated group(0.440±0.017,P<0.01);the HNF4? protein content in Hep G2 cells was no significant change in co-culture+si-NC group(1.857±0.016,P>0.05),but significantly lower in co-culture+si-HNF4? group(0.971±0.015,P<0.01)than that in co-culture group(1.920±0.052).4.2 Before co-cultured with 13.5d embryo hepatocytes for 48 h,Hep G2 cells were transfected with si-HNF4? for 24 h.The HNF1? protein content in Hep G2 cells was significantly higher in co-culture group than that in untreated group(1.168±0.050,P<0.01);The HNF1? protein content in Hep G2 cells was no significant change in co-culture+si-NC group(2.005±0.069,P>0.05),but significantly lower in co-culture+si-HNF4? group(0.558±0.017,P<0.01)than that in co-culture group(1.750±0.056).4.3 Before co-cultured with 13.5d embryo hepatocytes for 48 h,Hep G2 cells were transfected with si-HNF4? for 24 h.The HNF6 protein content in Hep G2 cells was significantly higher in co-culture group than that in untreated group(0.615±0.032,P<0.01);The HNF6 protein content in Hep G2 cells was no significant change in co-culture+si-NC group(1.008±0.065,P>0.05),but significantly lower in co-culture+si-HNF4? group(0.677±0.049,P<0.05)than that in co-culture group(1.004±0.068).4.4 Before co-cultured with 13.5d embryo hepatocytes for 48 h,Hep G2 cells were transfected with si-HNF4? for 24 h.The USF1 protein content in Hep G2 cells was significantly higher in co-culture group than that in untreated group(0.688±0.023,P<0.01);The USF1 protein content in Hep G2 cells was no significant change in co-culture+si-NC group(1.219±0.043,P>0.05),but significantly lower in co-culture+si-HNF4? group(0.659±0.018,P<0.01)than that in co-culture group(1.293±0.033).4.5 Before co-cultured with 13.5d embryo hepatocytes for 48 h,Hep G2 cells were transfected with si-HNF4? for 24 h.The c-Myc protein content in Hep G2 cells was significantly lower in co-culture group than that in untreated group(0.344±0.013,P<0.01);the c-Myc protein content in Hep G2 cells was significantly higher in co-culture+si-NC group(0.531±0.037,P<0.01)and co-culture+si-HNF4? group(0.317±0.016,P<0.05)than that in co-culture group(0.210±0.014).5 After transfected with si-HNF4? to knockdown the expression of HNF4?,Hep G2 cells co-cultured with 13.5d embryo hepatocytes for 48 h.The change of AFP expression was examined by immunofluorescence in Hep G2 cells.Before co-cultured with 13.5d embryo hepatocytes for 48 h,Hep G2 cells were transfected with si-HNF4? for 24 h.The AFP protein content in Hep G2 cells was significantly lower in co-culture group than that in untreated group;the AFP protein content in Hep G2 cells was no significant change in co-culture+si-NC group,but significantly higher in co-culture+si-HNF4? group than that in co-culture group.Conclusion:The results of the experiments reveal that rebuilding of the core transcriptional regulatory circuitry among HNF4?,HNF1?,HNF6 and USF1 factors in Hep G2 cells contributes to the redifferentiation of Hep G2 cells induced by embryonic hepatocytes in the mouse.The effect of induced redifferentiation declines after the loop is blocked. |
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