Page 46 - 《中国药科大学学报》2025年第5期

578 学报 Journal of China Pharmaceutical University 2025, 56(5): 572 − 582 第 56 卷
A Metformin c(metformin)/(mmol/L) Metformin c(metformin)/(mmol/L)

t/h 0 6 12 24 36 48 0 1 2 4 8 16 t/h 0 6 12 24 36 48 0 1 2 4 8 16
AKR1C3 AKR1C3 AKR1C3 AKR1C3
β-actin β-actin β-actin β-actin
HepG2 HepG2 HCC-LM3 HCC-LM3
150 ** 150 ** 150 ** * *** 150 ** ***
Relative AKR1C3 level /(% of control) 100 Relative AKR1C3 level /(% of control) 100 Relative AKR1C3 level /(% of control) 100 Relative AKR1C3 level /(% of control) 100

50
50
50
50
0
0 6 12 24 36 48 0 0 1 2 4 8 16 0 0 6 12 24 36 48 0 0 1 2 4 8 16
t/h c(metformin)/(mmol/L) t/h c(metformin)/(mmol/L)
B 2.0 n.s. 3 HCC-LM3
HepG2
n.s.
Relative AKR1C3 expression 1.5 Relative AKR1C3 expression 2 1 D ASP PRO TYR
A:26
1.0
0.5
A:24
ARG
TRP
0
0 1 2 4 8 16 0 0 1 2 4 8 16 GLN A:224 A:227 A:226
c(metformin)/(mmol/L) c(metformin)/(mmol/L) ARG A:222 H
A:223
+N N
C PBS+CHX Metformin+CHX PBS+CHX Metformin+CHX VAL
t/h 0 2 5 7 10 0 2 5 7 10 t/h 0 2 5 7 10 0 2 5 7 10 A:228 N N
AKR1C3 AKR1C3 SER PHE
A:221 H +N A:306
β-actin β-actin
TYR A:217 TYR A:192
GLU
A:216
HepG2
HCC-LM3
SER
150
Relative AKR1C3 level /(% of control) 100 PBS+CHX Relative AKR1C3 level /(% of control) 100 PBS+CHX Interactions A:305
150
Conventional hydrogen bond
Van der waals
Attractive charge
Carbon hydrogen bond
50
50
Metformin+CHX
0
0
5
t/h 5 48 0 0 Metformin+CHX 48
t/h
E 37 °C 64 °C 150 37 °C 64 °C 150
Flag-AKR1C3 + + + + + + + + PBS AKR1C3 PBS
Metformin
Flag
PBS β-actin Relative AKR1C3 level /(% of control) 100 Metformin PBS β-actin Relative AKR1C3 level /(% of control) 100
Metformin β-actin 50 0 Metformin AKR1C3 50 0
Flag
β-actin
HEK-293T
40
50
50
T/°C 60 70 HepG2 40 T/°C 60 70
Figure 3 MET promotes the degradation of AKR1C3 in HCC cells ( x ± s)
A: Effect of MET on the amount of AKR1C3 protein was detected using Western blot assay; B: Effect of MET on the level of AKR1C3
mRNA was detected using RT-qPCR; C: Effects of MET on degradation of the AKR1C3 protein was detected after the treatment of
cycloheximide (CHX) at the indicated time points by Western blot assays; D: Molecular docking simulations of MET with AKR1C3; E:
Detection of MET interaction with AKR1C3 using cellular thermal shift assay
*P < 0.05, **P < 0.01, ***P < 0.001; n.s.:P > 0.05
别加入蛋白酶体抑制剂 MG132（10 μmol/L）或自 通过检测自噬标志物发现，MET 处理诱导了
噬抑制剂氯喹（CQ，50 μmol/L）干预 4 h 后，检测 胞内 LC3-Ⅱ/Ⅰ的上调，表明自噬被激活。此外，作
AKR1C3 蛋白的降解情况，结果如图 4-A 所示，蛋 为自噬受体蛋白的 p62，其表达在 MET 处理后受到
白酶体抑制剂 MG132 预处理 4 h 对 MET 介导的 抑制（图 4-B）。上述结果证明 MET 可以诱导肝癌
AKR1C3 降解无显著影响，而自噬抑制剂氯喹 CQ 细胞自噬增加。接着观察自噬对 AKR1C3 的调控
处理可显著逆转 MET 对 AKR1C3 的降解效应。综 作用，HBSS 饥饿诱导的自噬显著降低了 AKR1C3
上所述，MET 主要通过自噬-溶酶体途径而非泛素- 蛋白水平，而 CQ 处理引起的溶酶体阻断则以剂量
蛋白酶体系统调控 AKR1C3 蛋白稳定性。 依赖的方式上调 AKR1C3 的蛋白水平（图 4-C，4-