Laminin γ2 Mediates Epithelial to mesenchymal Transition in Hepatocytes AML12 Cell Line

doi:10.13523/j.cb.2305043

China Biotechnology

2023, Vol. 43

Issue (11): 1-7 DOI: 10.13523/j.cb.2305043

Laminin γ2 Mediates Epithelial to mesenchymal Transition in Hepatocytes AML12 Cell Line

CHEN Hui^*(

),ZHANG Ji-shun

Digestive Department, Beijing Chaoyang Hospital, Capital Medical University,Beijing 100043,China

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Abstract Objective: To investigate the effect of TGF-β1 on laminin γ2 (LAMC2) expression in mouse normal hepatocyte lines AML12, and the role of LAMC2 on the differentiation of AML12 cells. Methods: Lentiviral vector of LAMC2 (Lent-LAMC2) was transfected with AML12 cells, Lenti-NC was used as the control group, the transfection efficiency was detected by Western blot, and the role of LAMC2 on the AML12 differentiation was detected by qPCR analysis. The AML12 cells were stimulated with TGF-β1 (5 ng/mL) from 6 h to 24 h, and the mRNA expression of TGF-β1 and LAMC2 was detected by real-time PCR (qPCR). AML12 cells were stimulated with TGF-β1 or TGF-β1 + TGF-β inhibitor (SB431542) for 24 h, and the mRNA of LAMC2 was detected by qPCR analysis. Finally, small interfering RNA (siRNA) was used to inhibit the expression of LAMC2 in AML12 cells, and the differentiation of AML12 cells was detected by Western blot followed by stimulation of TGF-β1 at dose of 5 ng/mL. Results: Compared with the Lenti-NC vector group, the protein level of LAMC2 was significantly increased in the LAMC2 overexpressed cells, the expressions of α-SMA and Col1a1 mRNA were up-regulated (all P<0.05), and ALB expressions were significantly down-regulated (P< 0.05). With the prolongation of the AML12 cell time of TGF-β1, the mRNA expression of LAMC2 gradually increased, which was positively correlated with the stimulation time of TGF-β1 (P<0.05). The transcription level of LAMC2 in TGF-β1+SB431542 group was significantly lower than that in TGF-β1 group (P<0.05). Importantly, compared with the siNC group, the siLAMC2 group can effectively block the effect of TGF-β1 on the upregulation of expression in AML12 cells α-SMA and Col1a1. Conclusion: Laminin γ2 overexpression induces mesenchymal-like cell changes in AML12 cells. TGF-β1 promotes the expression of LAMC2 in AML12 cells, and LAMC2 specific siRNAs block the fibrogenic effect of TGF-β1 on AML12 cells.

Key words： Hepatocyte TGF-β1 Laminin Liver fibrosis Epithelial-mesenchymal transition (EMT)

Received: 28 May 2023 Published: 01 December 2023

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Corresponding Authors: *Hui CHEN E-mail: chenhui_0516@126.com

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CHEN Hui, ZHANG Ji-shun. Laminin γ2 Mediates Epithelial to mesenchymal Transition in Hepatocytes AML12 Cell Line. China Biotechnology, 2023, 43(11): 1-7.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2305043 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I11/1

Fig.1 Effects of TGF-β1 on AML12 differentiation and the expression levels of LAMC2

Fig.2 Effects of TGF-β1 on Tgfbr1 and LAMC2 expression in AML12 cells Analysis of Tgfbr1(A) and LAMC2(B) transcripts in AML12 cells by qPCR Data are represented as mean ± SEM. ^* P<0.05 by 2-tailed Student’s t test

Fig.3 Pharmacological inhibition of TGF-β signaling blocks LAMC2 expression A. Cell morphological phenotype was observed under an inverted phases-contrast microscope Tgfbr1(B) and LAMC2(C) expression of AML12 cell by qPCR Data are represented as mean ± SEM. ^**** P<0.000 1 by 1-way ANOVA

Fig.4 LAMC2 overexpression promotes the EMT process in AML12 cells A.LAMC2 expression of AML12 cell transfected with Lenti-LAMC2 or negative control (Lenti- NC) by Western blot B.Col1a1 and ALB expression of AML12 cell transfected with Lenti-LAMC2 or negative control (Lenti- NC) by qPCR Data are represented as mean ± SEM. ^* P<0.05 by 2-tailed Student’s t test

Fig.5 LAMC2 knockdown inhibits the EMT process in AML12 cells LAMC2, ALB, and α-SMA expression of AML12 cell transfected with LAMC2 siRNA or negative control (NC) by Western blot


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