Jie Du Fang Reduced The Stemness Of Hepatocellular Carcinoma Through Wnt/β-catenin Signaling Pathway And The Regulatory Mechanism Of Cripto-1 Under Hypoxia

BackgroundHepatocellular carcinoma（HCC）is one of the most common malignant tumors in the world.With the progress of basic and clinical research,the treatment of HCC has made great progress in the past ten years.Because the early diagnosis rate of HCC is low,and HCC has high drug resistance and is easy to develop distant metastasis,,the 5-year survival rate of HCC is still less than 50%.Therefore,it is crucial to elucidate the underlying mechanisms and new targets of HCC progression in order to develop therapeutic drugs that can effectively combat or delay HCC.Hypoxia is an important feature of solid tumors,especially HCC.Recent studies have found that hypoxia can induce cancer cell invasion,metastasis,epithelial-mesenchymal transition（EMT）,and enhance the stemness of cancer cells.The stemness of liver cancer is the fundamental driving force for the occurrence and development of liver cancer.Hypoxia is the main reason for maintaining and activating the stemness of liver cancer.Therefore,improving the hypoxic microenvironment to suppress the stemness characteristics of HCC is a key step in the treatment of HCC.Traditional Chinese medicine has a long history in the prevention and treatment of liver diseases,and more and more evidences showed that traditional Chinese medicine can delay the progression of HCC and improve the quality of life of HCC patients.Based on years of clinical experience,our unit has summed up the traditional Chinese medicine compound"Jie Du Fang（JDF）"for HCC,which has achieved good clinical results.Through network pharmacology analysis,we found that JDF can be enriched in HIF and Wnt pathway,which was highly related to stemness regulation.Affymetrix gene chip analysis showed that JDF mainly inhibited Wnt/β-catenin signaling pathway.In addition,under hypoxic conditions,the cytokine chip found that the target of JDF may be Cripto-1,which is not expressed or lowly expressed in normal tissues,but is highly expressed in various tumors（including HCC）.And it had been confirmed that Cripto-1 can activate the Wnt/β-catenin signaling pathway in HCC Therefore,this topic put forward the hypothesis that JDF can reduce the stemness of HCC through Wnt/β-catenin signaling pathway in hypoxic microenvironment,and this effect is related to the regulation on Cripto-1.Objective1.To observe the effect of JDF on the stemness properties of HCC in hypoxic microenvironment.2.The role of Wnt/β-catenin signaling pathway in reducing the stemness properties of HCC under the hypoxic microenvironment.3.Preliminary study on the role of JDF in the regulation of Wnt/β-catenin signaling pathway via Cripto-1 in hypoxic microenvironment.MethodsPart I:To observe the effect of JDF on the stemness properties of HCC in hypoxic microenvironment1.To explore the pharmacological mechanism of JDF in the treatment of HCC through network pharmacology,and to collect the main components and corresponding targets of JDF by using the TCMSP database and CNKI literature.The GENECARDS database was used to obtain the corresponding targets of HCC,and the drug prediction targets and disease targets were mapped to each other,and the intersection was obtained.The DAVID online analysis tool was used to analyze the gene function of the intersection targets and the KEGG pathway enrichment analysis.2.The effects of JDF on the proliferation,migration and invasion of HCC cells SMMC-7721（7721）and Huh7 cell lines under hypoxic microenvironment were detected by MTT assay,scratch assay and Transwell chamber invasion assay.3.Western blot and Realtime-PCR（RT-PCR）were used to detect the effect of JDF on EMT-related proteins（E-cadherin,Vimentin,α-SMA）in HCC cells 7721 and Huh7 in hypoxic microenvironment.4.Image-IT^TM Hypoxia Reagent detects that JDF improves hypoxia in HCC cells 7721 and Huh7 under hypoxic microenvironment.5.Western blot and RT-PCR to detect the effect of JDF on the expression of hypoxia key protein HIF-1αin HCC cells 7721 and Huh7 under hypoxic microenvironment.6.Serum-free suspension sphere culture method was used to enrich liver cancer stem cells,and the effect of JDF on the ability of suspension spheres to form spheroids in hypoxic microenvironment was detected.7.RT-PCR to detect the effect of JDF on stemness transcription factor（NANOG,Oct4,Sox2）m RNA in hypoxic microenvironment.8.To establish a subcutaneous tumor model in nude mice to further verify the effects of JDF on hypoxia,EMT and stemness of suvcutaneous tumor in mice in vivo.Part II:The role of Wnt/β-catenin signaling pathway in reducing the stemness properties of HCC under the hypoxic microenvironment1.Affymetrix gene chip to analyze the molecular mechanism of JDF reducing HCC stemness properties.2.The Wnt/β-catenin signaling pathway agonist lithium chloride（Li Cl）was added to 7721 and Huh7 cells,Western blot was used to detect the effect of JDF onβ-catenin and p-GSK3β（Ser9）in the Wnt/β-catenin signaling pathway,the main pathway of activating tumor cell stemness.3.Western blot was used to detect the effect of JDF onβ-catenin and p-GSK3β（Ser9）in Wnt/β-catenin signaling pathway in hypoxic microenvironment.4.Western blot was used to detect the effect of JDF on the hypoxia key protein HIF-1αandβ-catenin in Wnt/β-catenin signaling pathway under hypoxia by adding Wnt/β-catenin signaling pathway inhibitor IWR-1-endo.At the same time,RT-PCR detected the effects on stemness transcription factors（NANOG,Oct4,Sox2）and HIF-1αm RNA.5.To verify the effect of JDF on the Wnt/β-catenin signaling pathway ofβ-catenin in vivo.Part III:Preliminary study on the role of JDF in the regulation of Wnt/β-catenin signaling pathway via Cripto-1 in hypoxicmicroenvironment1.Human XL Cytokine Array Kit was used to detect the effect of JDF on the secretion of cytokines in 7721 HCC cells under hypoxic conditions.2.Western blot detection the expression of Cripto-1 in HCC cells in hypoxic microenvironment and the effect of JDF on Cripto-1.3.Western blot was used to detect the effect of Cripto-1 on Wnt/β-catenin signaling pathway and the effect of JDF intervention.4.Western blot was used to detect the effect of JDF on the hypoxia key protein HIF-1αand Wnt/β-catenin signaling pathway after adding Cripto-1 stimulation in the hypoxic microenvironment.At the same time,RT-PCR detected the effect of stemness transcription factors（NANOG,OCT4,Sox2）.ResultsPart I:To observe the effect of JDF on the stemness properties of HCC in hypoxic microenvironment.1.A total of 6 active ingredients of JDF were obtained through database search,corresponding to 171 targets.GENECARDS retrieved HCC-related targets,matched JDF targets with HCC targets,and obtained a total of 81 common targets.Using the DAVID database to analyze the GO biological process and the KEGG pathway（P<0.05）,it can be found that JDF can be enriched in the HIF and Wnt signaling pathways.2.MTT results showed that JDF could effectively inhibit the proliferation of HCC cells 7721 and Huh7 in a concentration-and dose-dependent manner in hypoxic microenvironment.3.Scratch test and Transwell invasion test showed that hypoxia enhanced the migration and invasion ability of HCC cells,JDF could inhibit the effect.4.Western blot experiments showed that hypoxia could enhance the EMT of liver cancer cells（decreased expression of E-cadherin,increased expression of Vimentin andα-SMA）,JDF could inhibit EMT（increased expression of E-cadherin,decreased expression of Vimentin andα-SMA）.RT-PCR experiments showed that the m RNA level of E-cadherin was decreased in hypoxic microenvironment,while the m RNA levels of Vimentin andα-SMA were increased（P<0.05）.JDF could antagonize EMT in hypoxic microenvironment（P<0.05）,but The inhibitory effect of JDF on Vimentin andα-SMA in 7721 cells was not statistically significant,indicating that JDF inhibited EMT at the protein level in 7721 cells,and at both protein and m RNA levels in Huh7 cells.5.Image-IT^TM Hypoxia Reagent detected aggravation of hypoxia in HCC cells 7721 and Huh7under hypoxic microenvironment（green fluorescence represents cellular hypoxia）,and JDF can antagonize this effect in hypoxic microenvironment.6.Western blot experiments showed that hypoxia can enhance the expression of hypoxia key protein HIF-1α,and JDF can inhibit the effect.RT-PCR experiments showed that in the hypoxic microenvironment,the m RNA level of HIF-1αin 7721 cells was increased compared with that in normoxia（P<0.05）,and the Huh7 cells had no statistical significance,but JDF could increase in the hypoxic microenvironment.The m RNA level of HIF-1αwas inhibited in the environment（P<0.05）.7.The stem cell suspension sphere formation experiment showed that in the hypoxic microenvironment,the number of HCC cells 7721 and Huh7 suspension spheres increased significantly compared with normoxia（P<0.05）.JDF can inhibit the suspension spheres under hypoxia（P<0.05）.8.RT-PCR detection showed that the m RNA level of stemness transcription factors（NANOG,OCT4,Sox2）in HCC cells were significantly increased in hypoxic microenvironment compared with normoxia（P<0.05）.JDF conld inhibited the m RNA level of stemness transcription factors under hypoxia（P<0.05）.8.The HCC 7721 cells was used to establish a nude mouse subcutaneous tumor model.The results showed that the body weight and tumor size of the nude mice in the JDF group were significantly lower than those in the control group（P<0.05）.Western blot,immunohistochemistry and RT-PCR were carried out to verify the protein and m RNA levels,which showed that compared with the control group,JDF inhibited the expression of HIF-1αand could significantly improve EMT（the expression of E-cadherin was increased,the expression of E-cadherin was increased,and the decreased vimentin andα-SMA expression）.Tumor tissue hypoxia probe(Hypoxyprobe^TM-1)showed that JDF can improve intratumoral hypoxia.RT-PCR results showed that compared with the control group,JDF could significantly inhibit stemness transcription factors（NANOG,OCT4,Sox2）（P<0.05）.Part II:The role of Wnt/β-catenin signaling pathway in reducing the stemness properties of HCC under the hypoxic microenvironment.1.Based on Affymetrix gene chip results,KEGG pathway and IPA analysis showed that JDF significantly inhibited Wnt/β-catenin signaling pathway in 7721 suspension sphere cells.2.Western blot detected that adding Li Cl could activate the expression ofβ-catenin and p-GSK3β（Ser9）in the Wnt/β-catenin signaling pathway,while adding JDF could antagonize this effect.3.Western blot experiments showed that adding Li Cl could activate the expression ofβ-catenin（nuclear and cytoplasmic）and p-GSK3β（Ser9）in the Wnt/β-catenin signaling pathway,and JDF could inhibit this effect.RT-PCR experiments showed that after adding Li Cl,the m RNA levels of stemness transcription factors NANOG,OCT4and Sox2 were increased（P<0.05）,and JDF could inhibit this effect（P<0.05）.4.Western blot experiments showed that hypoxia can increase the expression ofβ-catenin（nuclear and cytoplasmic）and p-GSK3β（Ser9）in the Wnt/β-catenin signaling pathway,indicating that hypoxia could activate Wnt/β-catenin signaling pathway,JDF could inhibit the effect.5.Western blot experiments showed that the addition of Wnt/β-catenin signaling pathway inhibitor IWR-1-endo in the hypoxic microenvironment,compared with no inhibitor,the expression ofβ-catenin could be effectively inhibited,The expression ofβ-catenin changed little after the addition of JDF.RT-PCR results showed that the m RNA expression of stemness transcription factors NANOG,Oct4 and Sox2 decreased（P<0.05）with the addition of IWR-1-endo under hypoxic conditions,but not change too much after the addition of JDF.6.Western blot and immunofluorescence results showed that the JDF group could effectively inhibit the expression ofβ-catenin in the subcutaneous tumor of nude mice compared with the control group.Part III:Preliminary study on the role of JDF in the regulation of Wnt/β-catenin signaling pathway via Cripto-1 in hypoxicmicroenvironment1.Human XL Cytokine Array Kit cytokine chip detection results showed that Cripto-1 expression increased in hypoxic microenvironment,and JDF could significantly inhibit Cripto-1 in hypoxic microenvironment.2.Western blot experiment found that the expression of Cripto-1 increased in hypoxic microenvironment,and JDF could inhibit the expression of Cripto-1.In RT-PCR experiments,the m RNA level of Cripto-1 increased in hypoxic microenvironment（P<0.05）,and JDF could reduce the m RNA level of Cripto-1（P<0.05）.3.Western blot experiment found that the expression ofβ-catenin and p-GSK3β（Ser9）increased after the addition of Cripto-1 cytokine.After JDF intervention,the expression ofβ-catenin and p-GSK3β（Ser9）could be inhibited.4.Cripto-1 cytokine was added in hypoxic microenvironment,Western blot results showed that Cripto-1 could increase the expression of HIF-1αandβ-catenin,while the expression of p-GSK3β（Ser9）decreased in 7721 cells and increase in Huh7 cells.The expression of p-GSK3β（Ser9）was increased.Adding JDF can inhibit the expression of HIF-1αandβ-catenin.RT-PCR results showed that adding Cripto-1 in a hypoxic microenvironment,compared with the Huh7 cell control group,hypoxia could activate the m RNA levels of stemness-related regulators NANOG,Oct4,and Sox2（P<0.05）;Compared with the 7721 cell control group,hypoxia could activate the expression of Sox2（P<0.05）.JDF intervention could reduce the m RNA levels of NANOG,Oct4 and Sox2（P<0.05）.Conclusions1.JDF could inhibit the proliferation,migration,invasion and stemness properties of HCC cells through Wnt/β-catenin signaling pathway in hypoxic microenvironment.2.JDF could regulate the Wnt/β-catenin pathway in hypoxic microenvironment to reduce the stemness properties of liver cancer.3.JDF could regulate Wnt/β-catenin signaling pathway via Cripto-1 in hypoxic microenvironment.