Data_Sheet_1_Non-alcoholic fatty liver disease causally affects the brain cortical structure: a Mendelian randomization study.docx

BackgroundReduced brain volume, impaired cognition, and possibly a range of psychoneurological disorders have been reported in patients with non-alcoholic fatty liver disease (NAFLD); however, no underlying cause has been specified. Here, Mendelian randomization (MR) was employed to determine the causative NAFLD effects on cortical structure.MethodsWe used pooled-level data from FinnGen’s published genome-wide association study (GWAS) of NAFLD (1908 cases and 340,591 healthy controls), as well as published GWAS with NAFLD activity score (NAS) and fibrosis stage-associated SNPs as genetic tools, in addition to the Enigma Consortium data from 51,665 patients, were used to assess genetic susceptibility in relation to changes with cortical thickness (TH) and surface area (SA). A main estimate was made by means of inverse variance weighted (IVW), while heterogeneity and pleiotropy were detected using MR-Egger, weighted median, and MR Pleiotropy RESidual Sum and Outlier to perform a two-sample MR analysis.ResultsAt the global level, NAFLD reduced SA (beta = −586.72 mm2, se = 217.73, p = 0.007) and several changes in the cortical structure of the cerebral gyrus were found, with no detectable pleiotropy.ConclusionNAFLD causally affects cortical structures, which supports the presence of an intricate liver–brain axis.</p

Data_Sheet_2_Non-alcoholic fatty liver disease causally affects the brain cortical structure: a Mendelian randomization study.xlsx

BackgroundReduced brain volume, impaired cognition, and possibly a range of psychoneurological disorders have been reported in patients with non-alcoholic fatty liver disease (NAFLD); however, no underlying cause has been specified. Here, Mendelian randomization (MR) was employed to determine the causative NAFLD effects on cortical structure.MethodsWe used pooled-level data from FinnGen’s published genome-wide association study (GWAS) of NAFLD (1908 cases and 340,591 healthy controls), as well as published GWAS with NAFLD activity score (NAS) and fibrosis stage-associated SNPs as genetic tools, in addition to the Enigma Consortium data from 51,665 patients, were used to assess genetic susceptibility in relation to changes with cortical thickness (TH) and surface area (SA). A main estimate was made by means of inverse variance weighted (IVW), while heterogeneity and pleiotropy were detected using MR-Egger, weighted median, and MR Pleiotropy RESidual Sum and Outlier to perform a two-sample MR analysis.ResultsAt the global level, NAFLD reduced SA (beta = −586.72 mm2, se = 217.73, p = 0.007) and several changes in the cortical structure of the cerebral gyrus were found, with no detectable pleiotropy.ConclusionNAFLD causally affects cortical structures, which supports the presence of an intricate liver–brain axis.</p

Pyruvate Kinase M2 Plays a Dual Role on Regulation of the EGF/EGFR Signaling via E-Cadherin-Dependent Manner in Gastric Cancer Cells

<div><p>Background and Aims</p><p>EGFR activation and PKM2 expression are instrumental in tumorigenesis. EGFR activation regulates PKM2 functions in a subcellular compartment-dependent manner and promotes gene transcription and tumor growth. In addition, PKM2 is upregulated in EGFR-induced pathways in glioma malignancies. However, we found that PKM2 could also regulate the activity of the EGF/EGFR signaling pathway in gastric cancer cells. We aimed to define the biological mechanisms for PKM2 in regulating the cell motility and invasion.</p><p>Methods</p><p>We employed stable transfection with short hairpin RNA to stably silence the expression of PKM2 in the BGC823, SGC7901 and AGS gastric cancer cell lines. The effects of PKM2 in vitro were determined by assessing cell migration and invasion. Immunohistochemical analysis was used to explore the relationship among PKM2 and other proteins.</p><p>Results</p><p>Our results indicate that the knockdown of PKM2 decreased the activity of E-cadherin and enhanced the EGF/EGFR signaling pathway in the gastric cell lines BGC823 and SGC7901 that were positive for E-cadherin expression. However, in the undifferentiated gastric carcinoma cell line AGS, which lacks E-cadherin expression, PKM2 promoted cell migration and invasion. Immunohistochemical analyses showed that the levels of E-cadherin expression, ERK1/2 phosphorylation, and cytoplasmic PKM2 expression were correlated with each other.</p><p>Conclusion:</p><p>PKM2 may play different roles in differently differentiated gastric cancer cell types, and this finding would be consistent with the previous clinical research. The results of our study reveal an important link between PKM2 and E-cadherin during EGFR-stimulated gastric cancer cell motility and invasion.</p></div

Depletion of PKM2 attenuated the motility of AGS cells and the functional changes after rescuing PKM2 in gastric cancer cell lines.

<p>(A) E-cadherin expression levels were detected by immunoblot analysis in BGC823, SGC7901 and AGS cells. (B) A cross-shaped wound was created in the monolayer, and the AGS stable cells were cultured for an additional 24 h with EGF (100 ng/ml). Representative images of the wounded region are shown. The results of the migration assay are also shown as graphs (*p<0.05). (C) The invasion potential of AGS stable cells was assessed using the BD transwell chamber assay with 100 ng/ml EGF in the lower chamber for 24 hours. The cells that migrated to the lower side of the filter were stained, photographed, and counted. (D) The expression of p-EGFR, E-cadherin in the PKM2 rescuing experiments with stably transfected method by using over-expression plasmid vector pcDNA6.0-mock and pcDNA6.0-PKM2 in BGC823 and AGS cells which stable knockdown PKM2. (E) The functional changes of cell migration and invasion after PKM2 rescuing. The data are expressed as the mean ± SD from three independent experiments (*p<0.05).</p

PKM2 enhanced the activities of EGF/EGFR downstream signaling pathways in AGS cells and was correlated with ERK activity in gastric cancer specimens.

<p>(A) AGS stable cells were exposed to EGF (100 ng/ml) for different times. Western blots of cell lysates were performed. The phospho-EGFR (Tyr1068), phospho-PLCγ1 (Tyr783), phospho-Gab1 (Tyr627), phosphor-c-cbl (Tyr700), and phospho-ERK1/2 (Thr202/Tyr204) protein levels are shown as indicated. (B) MMP7 expression levels were analyzed by quantitative real-time PCR in AGS stable cells. The data are expressed as the mean ± SD from three independent experiments (*p<0.05). (C) IHC staining with the indicated antibodies was performed on 15 human gastric cancer specimens. Representative photos of the tumor, which were taken in the same part of the same piece of tissue, are shown. (D) The correlation analysis among PKM2, E-cadherin and P-ERK1/2 was completed using the Image-pro Plus software. The mean density (IOD/area) was detected in different positive areas of 15 human gastric cancer specimens. The correlation analysis between PKM2 and E-cadherin was determined in an E-cadherin positive area. The correlation analysis between PKM2 and p-ERK1/2 was determined in an E-cadherin negative area.</p

Knockdown of PKM2 promoted the migration and invasion of BGC823 and SGC7901 cells.

<p>(A) BGC823, SGC7901 and AGS cells were stably transfected with shRNA directed against PKM2. The specific knockdown of PKM2 was monitored by immunoblot (bottom). Cells stably transfected with pcPUR+U6-siPKM2 are referred to as siPK, and those transfected with pcPUR+U6-siRenilla are referred to as pu6. (B) The proliferation of the stably transfected cells. The cell number was determined with the CCK-8 assay, and the relative number of cells is shown. (C, D) A cross-shaped wound was created in the monolayer, and the BGC823 and SGC7901 stably transfected cells were cultured for an additional 24 hours with EGF (100 ng/ml). Representative images of the wounded region are shown. The results of the migration assay are also shown as graphs (*p<0.05). (E, F) The invasion potential of the BGC823 and SGC7901 stable cells was assessed using the BD transwell chamber assay with 100 ng/ml EGF in the lower chamber for 36 hours. The cells that migrated to the lower side of the filter were stained, photographed, and counted. The data are expressed as the mean ± SD from three independent experiments (*p<0.05).</p

Depletion of PKM2 decreased the expression of E-cadherin and enhanced the activities of the EGF/EGFR downstream signaling pathways.

<p>(A) E-cadherin, phospho-E-cadherin and N-cadherin expression levels were analyzed by immunoblot analysis in BGC823 and SGC7901 stable cells. (B) E-cadherin and N-cadherin expression levels were analyzed by quantitative real-time PCR in BGC823 and SGC7901 stable cells. (C) BGC823 and SGC7901 stable cells were exposed to EGF (100 ng/ml) for different times. The Western blots of cell lysates are shown. The phospho-EGFR (Tyr1068), phospho-PLCγ1 (Tyr783), phospho-AKT (ser473), and phospho-ERK1/2 (Thr202/Tyr204) protein levels are shown as indicated. (D) MMP7 expression levels were analyzed by quantitative real-time PCR in BGC823 and SGC7901 stable cells. Error bars represent the mean ± SD of triplicate experiments (*p<0.05).</p