Effect Of GATA6 Up - Regulation Of LOXL2 On Invasion And Metastasis Of Cholangiocarcinoma And FXR Up - Regulation Of SOCS3 In Inhibiting Hepatocellular Carcinoma Proliferation

Part I GATA6 promotes invasion and metastasis in cholangiocarcinoma by regulting LOXL2Metastasis is the main factor to infect the surgical manners selection and poor prognosis of patients with cholangiocaicinoma (CCA). Initiation of metastasis requires invasion, which is enabled by Epithelial-mesenchymal transition (EMT).Lysyl oxidase like-2 (LOXL2) belongs to the lysyl oxidase (LOX) family, which is proposed to function similarly to LOX in the extracellular matrix (ECM) by promoting crosslinking of collagen and elastin. Recent studies show that over-expression of LOXL2 in many cancers contributes to the invasiveness and metastasis in tumor progression. While LOXL2 expression is lower in some other cancers. However, the regulation LOXL2 is not well understood.Zinc-finger Transcriptional factor GATA binding protein 6 (GATA6) plays important roles in cell proliferation and differentiation. GATA6 was reported to be aberrantly expressed in pancreatic cancer, colon cancer and esophageal adenocarcinoma. It contributes to malignant progression. However, some other studies showed that low expression of GATA6 was involved in invasion and metastasis in some cancers such as astrocytoma and ovarian cancer. Therefore, LOXL2 and GATA6 act as both tumor promoters and suppressors.In our previous study, we found that LOXL2 and GATA6 expression levels are positively correlated in human CCA specimens, and were both closely associated with migratory capacity of CCA. Sequence analysis of the LOXL2 promoter region predicted a potential GATA6 binding site, indicating that GATA6 has the possibility to bind to the promoter of LOXL2 gene. Based on the observations, we hypothesized GATA6 might impact expression of LOXL2 and migratory capacity of cholangiocarcinoma.In order to identify the relationship between GATA6 and LOXL2, researches were carried out as follow:1. The expression profile of LOXL2 and GATA6 were examined in 90 CCA samples by using IHC assay. Statistical analysis showed that there was a positive correlation between LOXL2 and GATA6 expression. RT-qPCR and Western blot showed the expressions of LOXL2 and GATA6 were significantly higher in QBC939 cells at transprtion levels and protein levels compared with in RBE cells.2. Both of GATA6 and LOXL2 are closely associated with enhanced metastatic behavior and poor prognosis in CCA patients. Transwell assay and wound-healing assay revealed that the aberrant expression of LOXL2 and GATA6 were associated with invasive and metastatic ability of CCA cells.3. RT-qPCR and Western blot showed the expression of LOXL2 were significantly decreased by disruption of GATA6 by siRNA in QBC939 cells, and increased by transfected with GATA6 expression plasimd in RBE cells, indicating GATA6 can regulate LOXL2 expression in CCA cells.4. Over-expression of LOXL2 in QBC939 cells remarkably attenuated the effect of promotion of invasion and metastasis by si-GATA6. Knckdown of LOXL2 in RBE cells remarkably enhanced the effect of inhibition of invasion and metastasis by GATA6 over-expression. These data suggest LOXL2 plays the key role in GATA6-EMT pathway.5. Statistical analysis showed that the expressions of EMT Markers E-cadherin and Vimentin were closely associated with LOXL2 and GATA6 expression. Over-expression of LOXL2 in QBC939 cells remarkably attenuated the effect of si-GATA6 mediated E-cadherin down-regulation. Knckdown of LOXL2 in RBE cells remarkably enhanced the effect of GATA6 over-expression mediated E-cadherin up-regulation. These data suggest GAT6-LOXL2 plays an important role in EMT pathway.In summary, our data illustrate that GATA6 promoted invasive and metastatic potentials in CCA by regulating LOXL2 expression. This study will advance our understanding of the molecular mechanism of malignant progression in cholangiocaicinoma targeting GATA6 and LOXL2.Part â…¡ Nuclear receptor FXR suppresses hepatocellular carcinoma proliferation by up-regulating SOCS3Hyperactivation of the signal transducer and activator of transcription 3 (STAT3) plays an important role in diverse types of human cancers including hepatocellular carcinoma (HCC). Suppressors of cytokine signaling 3 (SOCS3), which is a physiologic negative regulator of STAT3, has received increasing attention. Increasing evidences support a role for SOCS3 as a tumor suppressor because inhibition of SOCS3 expression increased proliferation and promoted tumor aggressiveness. Up-regulation of SOCS3 should be a potential therapeutic strategy for HCC prevention and treatment.Farnesoid X receptor (FXR; NR1H4), a member of the nuclear receptor superfamily, is highly expressed in the gut-liver axist. In recent years, the understanding of the role of FXR in the liver has developed from that as a metabolic regulator to the novel function as a cell protector required for participating in carcinogenesis including promoting liver regeneration, suppressing hepatic inflammation, and regulating cell growth and differentiation. Thus FXR may be a promising target for the prevention/treatment of liver cancer. However, it is not well known whether the anti-tumor effect of FXR involves the regulation of SOCS3 or STAT3.In order to identify the relationship between FXR and SOCS3, researches were carried out as follow:1. FXR agonist GW4064 inhibits HCC cells growth by up-regulating SOCS3.MTS assay revealed that FXR specific agonist GW4064 drastically decreased the rate of cell proliferation of HCC cells (HepG2 and Huh7). GW4064-mediated growth inhibition of HCC cells was associated with suppressing entry into the S phase by Cell cycle assay. RT-qPCR and Western blot showed an up-regulation in p21 expression and an inhibition in STAT3 activation. And we also observed that these events accompanied by an increased expression of SOCS3. The above anti-HCC effects of GW4064 were markedly relieved by disruption of the SOCS3 gene by siRN A. FXR agonist also can suppress HCC xenograft and represses STAT3 activation in vivo.2. FXR directly bounds to a FXRE/IR9 in the human SOCS3 promoter region.The effect of GW4064-mediated SOCS3 induction was decreased in the presence of the specific FXR siRNA, further supporting the direct involvement of this nuclear receptor. As a classical transcriptional factor, FXR usually regulates the transcription of target genes via directly binding to FXR-responsive element (FXRE). Reporter assay revealed that FXR activation enhanced the transcriptional activity of SOCS3 promoter. EMSA and ChIP assay displayed that FXR directly bound to a FXRE/IR9 (an inverted repeat spaced by nine nucleotides,-1878 to-1858) in the human SOCS3 promoter region.3. FXR and SOCS3 expression levels are positively correlated in human HCC specimens.Statistical analysis showed that there was a positive correlation between FXR and SOCS3 expression in 66 HCC samples by IHC assay. Additionally, tumoral FXR and SOCS3 expression were significantly lower than that of the peritumoral tissue, whereas STAT3 was over-activated in HCC lesions, indicating that the dysexpression of FXR and SOCS3 might be involved in the development and/or progression of HCC.In summary, our data illustrate that FXR may serve as a key transcriptional regulator of cell proliferation in HCC by stimulating SOCS3 expression, and FXR-SOCS3 pathway may be a novel target for the treatment/prevention of HCC.