| Background & AimsHepatocellular carcinoma(HCC)is one of the most prevalent malignancies in the world,and approximately 700,000 new cases are diagnosed annually,about half of them in China.The complicated etiologies and complex pathogenesis lead to the extremely high heterogeneity of HCC,which renders it difficult to eliminate cancer cells with precision treatment or targeted drugs.Therefore,deepening the understanding of hepatocarcinogenesis and developing more effective intervention strategies for HCC is urgently needed.Hepatocyte nuclear factor FOXA is a transcription factor specifically expressed in hepatocytes,whose members include FOXA1,FOXA2 and FOXA3.Okumura et al.reported that FOXA1 and FOXA2 dramatically repressed hepatitis B virus S protein expression in HCC cells.Kaestner et al.found that FOXA1 and FOXA2 protected female mice from HCC but promoted HCC in male mice.In contrast,a recent study showed that FOXA3 was increased in lung cancer and high FOXA3 levels indicate poor patient survival.Nevertheless,the expression of FOXA3 in liver cancer and its role in HCC development remain unclear,which remains to be explored.To sum up,this study intends to explore the value of FOXA3 in the treatment of liver cancer by studying its role in the differentiation of HCC cells.Combined with the clinicopathological data,the correlation between FOXA3 and the prognosis of patients with liver cancer was analyzed to evaluate its feasibility as a prognostic marker for HCC.We analyzed the correlation between the expression of FOXA3 and the reactivity of HCC cells to sorafenib to explore the guiding value of FOXA3 in the individualized use of sorafenib.Methods:1.The expression of FOXA3 in HCC was analyzed to determine its correlation with the prognosis of patients(1)Realtime PCR,immunohistochemical staining(IHC)or Western blot were used to detect the differential expression of FOXA3 in paracancer tissues,HCC tissues,HCC metastasis or recurrence.(2)Kaplan-Meier was used to analyze the relationship between the expression of FOXA3 in HCC and the prognosis of patients,and COX regression was used to analyze independent prognostic risk factors.2.A study on the downregulation mechanism of FOXA3 expression in liver cancer(1)Si-RNA was used to interfere the expression of m6 A methylase and demethylase in HCC cell lines HCCLM3 and Huh7,and the expression of FOXA3 was detected by Real-Ttime PCR.(2)The expressions of FOXA3,METTL3,METTL14,WTAP and FTO in liver cancer were detected by Realtime PCR or IHC,and the correlation of molecular expression was statistically analyzed.(3)m6A-RIP-q PCR assay was used to determine whether the m RNA level of FOXA3 was regulated by m6 A methylation.3.Study on the regulation of FOXA3 on HCC differentiation(1)Realtime PCR was performed to detect the expression of FOXA3 in hepatocytes and the effect of FOXA3 on the functional indexes of HCC cell lines or THLE3 cell lines(2)The expression of FOXA3 in fetal liver,adult liver and HCC tissues was detected by Realtime PCR and IHC,and the relationship between FOXA3 and the degree of differentiation of HCC in patients was analyzed.(3)Changes in liver function indexes were detected by Realtime PCR,while PAS and oil red staining,urea and albumin secretion and other experiments were used to detect the improvement of liver function of HCC cell line infected with Ad-FOXA3.Then RNA-seq detection and bioinformatics analysis were performed.(4)The effect of FOXA3 on the self-renewal ability of liver cancer stem cells was analyzed by cell pelletizing test and flow analysis,and the in vivo and in vitro limited dilution test verified the effect of FOXA3 on the pelletizing ability of HCC cell lines.(5)Cell flow analysis and Ed U detection of the changes in the growth cycle of liver cancer cell lines after overexpression of FOXA3.(6)HCC-PDX intratumoral injection of AAV-FOXA3 was used to investigate the therapeutic effect in vivo.4.Mechanism and clinical study of FOXA3 sensitized sorafenib(1)Flow cytometry was used to detect the apoptosis of HCC cell lines induced by sorafenib after overexpression of FOXA3.(2)Ch IP-PCR experiment was used to explore the transcriptional regulation of FOXA3 with OATP1B1 and OATP1B3.(3)LC-MS/MS assay was used to detect the sorafenib uptake in HCC cell lines transfected with si-OATP1B1 and si-OATP1B3.(4)FOXA3 expression was detected by IHC in HCC patients using sorafenib therapy,and Kaplan-Meier analyzation was used to evaluate the guiding value of FOXA3 in treatment of sorafenib.(5)To test the response of FOXA3 with different levels of HCC-PDX expression to sorafenib treatment.Results:1.FOXA3 expression was significantly decreased in HCC tissues compared with paracancer tissues,and further decreased in HCC metastasis and recurrence.Low FOXA3 expression is associated with poor prognosis,which is an independent prognostic risk factor.2.FOXA3 is associated with METTL14 expression in HCC,and FOXA3 is regulated by METTL14-mediated m6 A methylation at m RNA level.3.FOXA3 is gradually increased during hepatocyte differentiation,and its expression in fetal liver and HCC is significantly lower than that in adult liver.4.After overexpression of FOXA3 in HCC cell lines,hepatocyte-related functions,such as albumin secretion,urea synthesis and glycogen synthesis,were significantly enhanced.5.RNA-seq combined with bioinformatics analysis showed that FOXA3 may regulate HCC cell differentiation by up-regulating a series of molecules with differentiation functions.6.Overexpression of FOXA3 significantly down-regulated the proportion of liver cancer stem cells in HCC cell lines and inhibited the self-renewal of liver cancer stem cells.7.Overexpression of FOXA3 could significantly inhibit the proliferation of HCC cells,and intratumoral injection of AAV-FOXA3 could effectively inhibit the growth of PDX.8.Apoptosis detection showed that the overexpression of FOXA3 could promote the apoptosis of HCC cells induced by sorafenib.9.The results of Ch IP-PCR and LC-MS/MS showed that FOXA3 promoted OATP1B1 and OATP1B3 transcription,thereby increasing sorafenib uptake in HCC cells.10.Kaplan-Meier analysis of postoperative HCC patients taking sorafenib showed that only patients with high FOXA3 expression could benefit from sorafenib treatment.11.Compared with PDX with low FOXA3 expression,PDX with high FOXA3 expression was more reactive to sorafenib.Conclusion:FOXA3 expression was significantly decreased in HCC tissues and correlated with the prognosis of patients.The expression of FOXA3 in HCC is regulated by METTL14-mediated m6 A methylation.The high expression of FOXA3 was associated with better differentiation of HCC.Only patients with high FOXA3 expression in HCC benefited from sorafenib therapy.These results provide a new idea for the differentiation treatment of HCC and a potential marker for the individualized treatment of HCC patients with sorafenib. |
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