Mechanisms Of Cancer Progenitor Cell Formation In Aristolochic Acid I-induced Acute Inflammatory Liver Of Beagle Canines

Part â… . cancer progenitor cell formation in AA I-induced acute inflammatory liver of beagle caninesObjective:Determining activation of inflammatory cytokine interleukin 6 (IL-6) and inflammatory pathway signaling transducer and activator of transcription-3 (STAT3), transcription factors nuclear factor-KB (NF-κB) which are involved in aristolochic acid I (AA I) induced hepatic acute inflammation. Exploring whether cancer progenitor cells appear or not in AA I induced hepatic acute inflammation.Methods:Canines were orally administrated with AA I capsules of 3 mg/kg/day, while control group were given accessory capsules (n=4/group). All canines were continuously administrated for 10 days, and sacrificed for collection of hepatic samples 2 days after drug withdrawal. Hematoxylin & eosin (H&E) staining was performed to evaluate liver injury and TUNEL staining was used to detect apoptosis of hepatocytes. ELISA and RT-PCR were used to detect the content of IL-6 and gene expression of Il-6, respectively. Western-blot, immunofluorescence and immunohistochemistry were applied to detect IL-6 related inflammatory pathway interleukin 6 receptor a (IL-6Ra)/ janus tyrosine kinases 2 (JAK2)/STAT3, NF-KB/activator protein 1 (AP-1) and transforming growth factor β (TGF-β) pathway. Hepatic progenitor cells were identified by immunohistochemistry, Western-blot and co-immunoprecipitation. Phenotypes of cancer progenitor cells in liver sections were identified by immunofluorescence with confocal microscope. Meanwhile, oncogenic transcription factor Myc (c-Myc) and oncofetal RNA-binding protein Lin28 homolog B (Lin28B) were also detected by immunofluorescence with confocal microscope.Results:H&E staining showed the hepatic lobules were disorganized and TUNEL staining showed a large number of positive staining cells appeared which implied apoptosis of hepatocytes emerged. ELISA, RT-PCR and Western-blot confirmed that IL-6 was up-regulated in liver tissues and its downstream pathway IL-6Rα/JAK2/ STAT3 was activated. Meanwhile, NF-κB was activated, which illustrated that AA I induced hepatic acute inflammation of beagle canines were mainly mediated by IL-6 and NF-κB. Immunohistochemistry, Western-blot and co-immunoprecipitation identified that hepatic progenitor cells with phenotype of octamer-binding transcription factor-4 (Oct4) positive and STAT3 positive emerged in inflammatory microenvironment with TGF-β1 pathway activation. The results above implied that TGF-β1 pathway activation was in favor of proliferation and differentiation of hepatic progenitor cells. In summary, immunofluorescence combined with confocal microscope identified that cancer progenitor cells with phenotype of Oct4 positive, STAT3 positive, TGF-β1 receptor â…¡ (TBRâ…¡)negative, embryonic liver fodrin (ELF) negative emerged in acute inflammatory microenvironment induced by AA I.Conclusions:Cancer progenitor cells appeared in hepatic acute inflammation of beagle canines induced by AA I. The feature of cancer progenitor cells was "Oct4~+, STAT3~+, TBRâ…¡", ELF" with activation of TGF-β1 and IL-6/STAT3/NF-κB signaling pathways and hepatocytes apoptosis.Part â…¡ MiRNome differentially expressing in the AA I-induced inflammatory malignant transformation of beagle canine liver with correlation of cancer progenitor cells formationObjective:To explore the expression pattern of microRNAs (miRNAs) related to cancer progenitor cells formation in AA I induced hepatic acute inflammation, validating target molecular events posttranscriptionally regulated by miRNAs.Methods:Exiqon miRCURY LNATM Chip platform was applied to detect miRNAs expression pattern of canines, and qRT-PCR was used to confirm the differentially expressed miRNAs (fold>1.5, P<0.05). To speculate possible target genes regulated by miRNAs through several data bases (www.targetscan.org), (www.mirbase.org), (www.pubmed.com) and validate the results again by Western-blot. Meanwhile, confocal microscope combined with immunofluorescence was applied to detect the expressions of malignant transformation associated proteins c-Myc and Lin28B.Results:73 miRNAs were detected to alter more than 1.5-fold in AAI group, including cfa-miR-200c, cfa-miR-34a, cfa-miR-378, cfa-miR-223, cfa-miR-24 and cfa-let-7b (P <0.05).6 miRNAs above and the member let-7a-1 of let-7a-l-let-7b cluster, the member miR-27a of miR-27a-miR-24 cluster were detected via qRT-PCR, proving the differential expression of the above 8 miRNAs (fold> 1.5,P<0.05).10 target proteins regulated by miRNAs above were proved to alter at different levels via data base analysis and Western-blot, of which 7 proteins altered more than 1.5-fold (P<0.05). It was noteworthy that the expression of phospho-forkhead box 01 (p-FOXO1) increased as much as 22-fold, together with high expressions of c-Myc and Lin28B, indicating that malignant transformation was possibly associated with development of cancer progenitor cells.Conclusion:Related miRNAs appeared to differentially express in hepatic inflammatory malignant transformation of beagle canines. Results of qRT-PCR indicated that miRNAs let-7a-1, let-7b, miR-27a, miR-223, miR-200c, miR-24, miR-34a and miR-378 showed remarkable differential expression patterns, and the explicitly regulated target proteins were IL-6, NF-κB, Lin28B, c-Myc and p-FOXO1.Part â…¢ Let-7b and miR-27a involving in regulation of inflammatory malignant transformation induced by IL-6 in HepG2 cells and related molecular mechanisms in vitroObjective:Exploring the relationship between miRNAs let-7b, miR-27a, IL-6, NF-κB and p-FOXO1 in inflammatory malignant transformation.Methods:In order to investigate colony formation of HepG2, let-7b mimics and inhibitor were transfected in HepG2 cells with IL-6 treatment. Western-blot was performed to detect expression of Lin28B. Immunofluorescence was used to detect co-localization of STAT3 and p-FOXO1 in cytoplasm. HepG2 cells were transfected with let-7b or miR-27a mimics or inhibitor with IL-6 treatment to analyze the expression of p-STAT3, FOXO1, p-FOXO1 and NF-â'˜B (P50, P65).Results:Colony formation assay in HepG2 showed let-7b mimics could repress colony formation and let-7b inhibitor could promote colony formation inversely. Meanwhile, IL-6 was able to promote colony formation of HepG2 cells. Western-blot analysis demonstrated that IL-6 activated downstream signaling p-STAT3 and NF-κB. On one hand, IL-6 coupled with NF-κB up-regulated Lin28B. Furthermore, up-regulated Lin28B inhibited let-7 which induced IL-6/NF-κB signaling pathway. On the other hand, FOXO1 was regulated by miR-27a. All the results implied that there must be a relationship between cancer progenitor cells and signaling loop IL-6/STAT3/NF-κB/ Lin28B/let-7 and IL-6/NF-KB/miR-27a/FOXO1.Conclusions:Signaling loop IL-6/STAT3/NF-κB/Lin28B/let-7b coupled with another signaling loop IL-6/NF-κB/miR-27a/FOXO1 were related to cancer progenitor cells formation in AA I induced malignant transformation of liver. Let-7b and miR-27a were related to IL-6 induced acute malignant transformation of HepG2, which regulated the expression of p-STAT3, c-Myc, Lin28B, NF-κB (P50, P65), FOXO1 and p-FOXO1.