Page 49 - 《中国药科大学学报》2025年第5期

第 56 卷第 5 期 齐 磊，等：二甲双胍通过促进醛酮还原酶 AKR1C3 降解抑制肝细胞癌恶性进展的机制研究 581
A HepG2 HepG2 HCC-LM3 HCC-LM3

HA-Ub K48R + + HA-Ub K63R + + HA-Ub K48R + + HA-Ub K63R + +
Metformin − + Metformin − + Metformin − + Metformin − +
Chloroquine + + Chloroquine + + Chloroquine + + Chloroquine + +

IP:AKR1C3 HA IP:AKR1C3 HA IP:AKR1C3 HA IP:AKR1C3 HA

AKR1C3 AKR1C3 AKR1C3 AKR1C3
AKR1C3 AKR1C3 AKR1C3 AKR1C3
Input β-actin Input β-actin Input β-actin Input β-actin

B IP:AKR1C3 C D
Input lgG IP:p62 Input lgG shNC shp62-1 shp62-2 HCC-LM3
AKR1C3 AKR1C3 1.5 n.s.
p62
p62 p62 1.0
HepG2 HepG2 AKR1C3 Relative AKR1C3 expression
AKR1C3 AKR1C3 0.5
β-actin
p62 p62 HCC-LM3 0
HCC-LM3 HCC-LM3 shNC shp62-1 shp62-2
E 250 n.s. n.s. F
Metformin − + − − + + *** Input IP:p62 Input IP:p62
shp62-1 − − + − + −
shp62-2 − − − + − + *** Metformin − + − + Metformin − + − +
150
AKR1C3 Relative AKR1C3 level /(% of control) 200 ** p62 p62
p62 100 AKR1C3 AKR1C3
β-actin 50 β-actin β-actin
0
HCC-LM3 Metformin − + − − + + HCC-LM3 HepG2
shp62-1 − − + − + −
shp62-2 − − − + − +
G DAPI p62 AKR1C3 Merge H 150
Metformin − + − + ***
Compound C − − + + 100 ***
PBS AKR1C3 Relative AKR1C3 level /(% of control)
P-AMPK 50
AMPK
β-actin 0
Con Compound C
Metformin Combination
HBSS I Chloroquine + + +
Metformin
Compound C − − + − + +
LC3-I
IP:AKR1C3 AKR1C3
LC3-II
Metformin AKR1C3
LC3-I
LC3-II
Input
β-actin
Figure 5 MET promotes p62-mediated AKR1C3 selective autophagic degradation ( x ± s)
A: After exogenous transfer of K48 or K63 mutant ubiquitin, effect of MET on the ubiquitination of AKR1C3 was observed by Co-IP assay;
B: Interaction of AKR1C3 with p62 was determined by Co-IP assay; C: Protein amount of AKR1C3 in HCC-LM3 cells after knockdown of
p62 was observed by Western blot assay; D: mRNA level of AKR1C3 in HCC-LM3 cells after knockdown of p62 was observed by RT-
PCR; E: Effect of MET on AKR1C3 protein content after knockdown of p62 was detected in HCC-LM3 cells by Western blot assay; F:
Effect of MET on interaction of AKR1C3 with p62 was determined by Co-IP assay in HCC-LM3 cells; G: Effect of MET on colocalization of
AKR1C3 with p62 was determined by confocal microscopy in HCC-LM3 cells；H: Effect of MET on AKR1C3 protein content was detected
after the treatment of Compound C by Western blot assays in HCC-LM3 cells; I: Effect of MET on interaction of AKR1C3 with LC3 was
determined after the treatment of Compound C by Co-IP assay in HCC-LM3 cells
*P < 0.05, **P < 0.01, ***P < 0.001; n.s.:P > 0.05