1. Eslam M, Valenti L, Romeo S. Genetics and epigenetics of NAFLD and NASH: Clinical
impact. J Hepatol. 2018;68(2):268-79.
2. Kleiner DE, Brunt EM, Van Natta M, et al. Design and validation of a histological scoring
system for nonalcoholic fatty liver disease. Hepatology. 2005;41(6):1313-21.
3. Angulo P, Kleiner DE, Dam-Larsen, et al. Liver fibrosis, but no other histologic features,
is associated with long-term outcomes of patients with nonalcoholic fatty liver disease.
Gastroenterology. 2015;149(2):389-97.
4. Romeo S, Kozlitina J, Xing C, et al. Genetic variation in PNPLA3 confers susceptibility
to nonalcoholic fatty liver disease. Nat Genet. 2008;40(12):1461-5.
5. Speliotes EK, Yerges-Armstrong LM, Wu J, et al. Genome-wide association analysis
13
identifies variants associated with nonalcoholic fatty liver disease that have distinct effects
on metabolic traits. PLoS Genet. 2011;7(3):e1001324.
6. Singal AG, Manjunath H, Yopp AC, et al. The effect of PNPLA3 on fibrosis progression
and development of hepatocellular carcinoma: a meta-analysis. Am J Gastroenterol.
2014;109(3):325-34.
7. Hotta K, Yoneda M, Hyogo H, et al. Association of the rs738409 polymorphism in
PNPLA3 with liver damage and the development of nonalcoholic fatty liver disease. BMC
Med Genet., 2010;11:172.
8. Sookoian S, Castaño GO, Scian R, et al. Genetic variation in transmembrane 6
superfamily member 2 and the risk of nonalcoholic fatty liver disease and histological
disease severity. Hepatology. 2015;61(2):515-25.
9. Pirola CJ, Sookoian S. The dual and opposite role of the TM6SF2-rs58542926 variant in
protecting against cardiovascular disease and conferring risk for nonalcoholic fatty liver:
A meta-analysis. Hepatology. 2015;62(6):1742-56.
10. Macaluso FS, Maida M, Petta S. Genetic background in nonalcoholic fatty liver disease: a
comprehensive review. World J Gastroenterol. 2015;21(39):11088-111.
11. Goffredo M, Caprio S, Feldstein AE, et al. Role of TM6SF2 rs58542926 in the
pathogenesis of nonalcoholic pediatric fatty liver disease: A multiethnic study. Hepatology.
2016;63(1):117-25.
12. Liu YL, Reeves HL, Burt AD, et al. TM6SF2 rs58542926 influences hepatic fibrosis
progression in patients with non-alcoholic fatty liver disease. Nat Commun. 2014; 5:4309.
13. Dongiovanni P, Petta S, Maglio C, et al. Transmembrane 6 superfamily member 2 gene
variant disentangles nonalcoholic steatohepatitis from cardiovascular disease. Hepatology.
2015;61(2):506-14.
14. Li TT, Li TH, Peng J, et al. TM6SF2: A novel target for plasma lipid regulation.
Atherosclerosis, 2018;268:170-6.
14
15. O'Hare EA, Yang R, Yerges-Armstrong L, et al. TM6SF2 rs58542926 impacts lipid
processing in liver and small intestine. Hepatology. 2017;65(5):1526-42.
16. Luukkonen PK, Zhou Y, Nidhina Haridas PA, et al. Impaired hepatic lipid synthesis from
polyunsaturated fatty acids in TM6SF2 E167K variant carriers with NAFLD. J Hepatol.
2017;67(1):128-36.
17. Prill S, Caddeo A, Baselli G, et al. The TM6SF2 E167K genetic variant induces lipid
biosynthesis and reduces apolipoprotein B secretion in human hepatic 3D spheroids. Sci
Rep. 2019;9(1):11585.
18. Mahdessian H, Taxiarchis A, Popov S, et al. TM6SF2 is a regulator of liver fat
metabolism influencing triglyceride secretion and hepatic lipid droplet content. Proc Natl
Acad Sci U S A. 2014;111(24):8913-8
19. Smagris E, Gilyard S, BusuRay S, et al. Inactivation of TM6SF2, a gene defective in fatty
liver disease, impairs lipidation but not secretion of very low density lipoproteins. J Biol
Chem. 2016;291(20):10659-76.
20. Kozlitina J, Smagris E, Stender S, et al. Exome-wide association study identifies a
TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease. Nat Genet.
2014;46(4):352-6.
21. Li JT, Liao ZX, Ping J, et al. Molecular mechanism of hepatic stellate cell activation and
antifibrotic therapeutic strategies. J Gastroenterol., 2008;43(6):419-28.
22. Dewidar B, Mayer C, Dooley S, et al. TGF-β in hepatic stellate cell activation and liver
fibrogenesis-updated 2019. Cells. 2019;8(11).1419
23. Carpino G, Morini S, Corradini G, et al. Alpha-SMA expression in hepatic stellate cells
and quantitative analysis of hepatic fibrosis in cirrhosis and in recurrent chronic hepatitis
after liver transplantation. Dig Liver Dis. 2005;37(5):349-56.
24. Henao-Mejia J, Elinav E, Jin C, et al. Inflammasome-mediated dysbiosis regulates
progression of NAFLD and obesity. Nature. 2012;482(7384):179-85.
15
25. Eslam M, Mangia A, Berg T, et al. Diverse impacts of the rs58542926 E167K variant in
TM6SF2 on viral and metabolic liver disease phenotypes. Hepatology. 2016;64(1):34-46.
26. Sookoian S, Pirola CJ. Meta-analysis of the influence of TM6SF2 E167K variant on
Plasma Concentration of Aminotransferases across different Populations and Diverse
Liver Phenotypes. Sci Rep. 2016;6:27718. doi: 10.1038/srep27718.
27. Pingitore P, Dongiovanni P, Motta BM, et al. PNPLA3 overexpression results in reduction
of proteins predisposing to fibrosis. Hum Mol Genet. 2016;25(23):5212-22.
28. Bruschi FV, Claudel T, Tardelli M, et al. The PNPLA3 I148M variant modulates the
fibrogenic phenotype of human hepatic stellate cells. Hepatology. 2017:65(6):1875-90.
29. Testerink N, Ajat M, Houweling M, et al. Replacement of retinyl esters by
polyunsaturated triacylglycerol species in lipid droplets of hepatic stellate cells during
activation. PLoS One. 2012;7(4):e34945.
Figure legends
Figure 1. TM6SF2 regulates αSMA expression in LX-2 cells
A) The cloned TM6SF2 expression plasmid (p3FLAG/TM6SF2-WT) and empty vector
(Mock) were transiently transfected into LX-2 cells followed by 24 hours of incubation.
Intracellular αSMA expressions were measured by quantitative PCR. The expression of
GAPDH served as a control. Experiments were performed in triplicate wells. All data are
expressed as the mean ± SD.
B) Non-treated and TM6SF2 knocked-down LX-2 lysates were transferred onto an automated
capillary western blot. Anti-TM6SF2 antibody or anti-GAPDH antibody were applied,
followed by anti-rabbit immunoglobulin. Signal intensity was corrected by GAPDH and is
shown in the bar graph. Experiments were performed in triplicate wells. All data are
expressed as the mean ± SD (*P < 0.05).
C) Intracellular αSMA expression, measured by quantitative PCR, was compared in
16
non-treated and TM6SF2 knocked-down LX-2 cells. GAPDH expression was used as a
control. Experiments were performed in triplicate wells. All data are expressed as the mean ±
SD (*P < 0.05).
D) Non-treated and TM6SF2 knocked-down LX-2 lysates were transferred onto an automated
capillary western blotting system. Anti-αSMA antibody or anti-GAPDH antibody were
applied, followed by anti-rabbit immunoglobulin. Signal intensities were corrected by
GAPDH and are shown in the bar graph. Experiments were performed in duplicate wells. All
data are expressed as the mean ± SD (*P < 0.05).
Figure 2. TM6SF2 suppresses αSMA induction by TGFβ1 in LX-2 cells
A) The cloned TM6SF2 expression plasmid (p3FLAG/TM6SF2-MT) and empty vector
(Mock) were transiently transfected into LX-2 cells followed by 24 hours of incubation. LX-2
cells were stimulated with or without 10 ng/ml of TGFβ1 for 48 hours. Intracellular αSMA
expression was measured by quantitative PCR. The expression of GAPDH served as a control.
Experiments were performed in triplicate wells. All data are expressed as the mean ± SD (*P
< 0.05).
B) Non-treated and TM6SF2 knocked-down LX-2 cells were stimulated with or without
10ng/mL of TGFβ1 for 48 hours and intracellular αSMA expression was compared via
quantitative PCR. The expression of GAPDH served as a control. Experiments were
performed in triplicate wells. All data are expressed as the mean ± SD (*P < 0.05).
Figure 3. The impact of TM6SF2 phenotype on αSMA induction in LX-2 cells
A) The cloned TM6SF2 expression plasmid consisting of p3FLAG/TM6SF2-WT and
p3FLAG/TM6SF2-MT and empty vector (Mock) were transiently transfected into LX-2 cells,
followed by 24 hours of incubation. Intracellular αSMA expression was measured by
quantitative PCR, with the expression of GAPDH serving as a control. Experiments were
17
performed in triplicate wells. All data are expressed as the mean ± SD.
B) Cloned TM6SF2 expression plasmids consisting of p3FLAG/TM6SF2-WT and
p3FLAG/TM6SF2-MT and empty vector (Mock) were transiently transfected into LX-2 cells,
followed by 24 hours of incubation. LX-2 lysates were transferred onto an automated
capillary western blotting system. Anti-TM6SF2 antibody or anti-GAPDH antibody were
applied, followed by anti-rabbit immunoglobulin. Signal intensity was corrected by GAPDH,
as shown in the bar graph. Experiments were performed in triplicate wells. All data are
expressed as the mean ± SD.
C) Cloned TM6SF2 expression plasmids consisting of p3FLAG/TM6SF2-WT and
p3FLAG/TM6SF2-MT and empty vector (Mock) were transiently transfected into LX-2 cells,
followed by 24 hours of incubation. LX-2 cells were stimulated with or without 10 ng/ml of
TGFβ1 for 48 hours. Intracellular αSMA expression was measured by quantitative PCR, with
GAPDH as a control. Experiments were performed in triplicate wells. All data are expressed
as the mean ± SD.
18
19
20
21
22
23
24
Supplementary Figure 1. Fluorescence intensities of TM6SF2 in LX-2 cells
Non-treated and TM6SF2 knockdown LX-2 were stained with anti-TM6SF2 monoclonal
antibody. The bound antibodies were detected with an Alexa 594-conjugated antibody against
rabbit IgG. Fluorescence intensities of TM6SF2 were shown in box-whisker plot (*P < 0.05).
Supplementary Figure 2. Immunostaining of TM6SF2 LX-2 cells
Non-treated and TM6SF2 knockdown LX-2 were stained with anti-TM6SF2 monoclonal
antibody. The bound antibodies were detected with an Alexa 594-conjugated antibody (red)
against rabbit IgG. Nuclei were counterstained with bisbenzimide H 33258 (blue).
Supplementary Figure 3. TM6SF2 regulates COL1A1 expression in LX-2 cells
Intracellular COL1A1 (collagen type 1 alpha 1) expression, measured by quantitative PCR,
was compared in non-treated and TM6SF2 knocked-down LX-2 cells. GAPDH expression
was used as a control. Experiments were performed in triplicate wells. All data are expressed
as the mean ± SD (*P < 0.05).
Supplementary Figure 4. The impact of TM6SF2 phenotype on COL1A1 induction in
LX-2 cells
The cloned TM6SF2 expression plasmid consisting of p3FLAG/TM6SF2-WT and
p3FLAG/TM6SF2-MT were transiently transfected into LX-2 cells, followed by 24 hours of
incubation. TM6SF2 overexpressing or non-treated LX-2 cells were stimulated with or
without 10ng/mL of TGFB for 48 hours and compared by intracellular COL1A1 expressions
measured by quantitative PCR. The expression of GAPDH served as a control. Experiments
were performed in triplicate wells. All data are expressed as the mean ± SD.
25
26
...
論文の公開元へ
