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中华细胞与干细胞杂志(电子版) ›› 2024, Vol. 14 ›› Issue (06) : 336 -343. doi: 10.3877/cma.j.issn.2095-1221.2024.06.003

论著

阻断PI3K/Akt信号通路促进低表达FoxA2肝脏前体细胞对分化诱导剂应答并朝肝细胞方向分化
任江波1, 李丽1,2,3, 王萍1,2,,3   
  1. 1.100050 北京,首都医科大学附属北京友谊医院肝病中心
    2.100069 北京,国家消化系统疾病临床医学研究中心
    3.100069 北京,肝硬化转化医学北京市重点实验室
  • 收稿日期:2024-08-21 出版日期:2024-12-01
  • 通信作者: 王萍
  • 基金资助:
    国家自然科学基金 (81570548)

Blocking PI3K/Akt signal pathway promotes FoxA2 low-expressing liver progenitors to respond to differentiation reagent and differentiate towards hepatocytes

Jiangbo Ren1, Li Li1,2,3, Ping Wang,1,2,,3   

  1. 1.Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China
    2.National Clinical Research Center of Digestive Diseases, Beijing 100069, China
    3.Beijing Key Laboratory of Translational Medicine in Liver Cirrhosis, Beijing 100069, China
  • Received:2024-08-21 Published:2024-12-01
  • Corresponding author: Ping Wang
引用本文:

任江波, 李丽, 王萍. 阻断PI3K/Akt信号通路促进低表达FoxA2肝脏前体细胞对分化诱导剂应答并朝肝细胞方向分化[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(06): 336-343.

Jiangbo Ren, Li Li, Ping Wang. Blocking PI3K/Akt signal pathway promotes FoxA2 low-expressing liver progenitors to respond to differentiation reagent and differentiate towards hepatocytes[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2024, 14(06): 336-343.

目的

探讨低表达叉头蛋白 (Fox)A2的肝脏前体细胞对分化诱导剂的应答反应与促进其朝肝细胞方向分化的机制。

方法

以大鼠肝脏前体细胞为研究对象,将非特异性shRNA和FoxA2 shRNA质粒转染进入细胞,应用分化诱导剂丁酸钠或联合应用丁酸钠与PI3K/ Akt信号通路抑制剂Ly294002进行肝细胞方向分化诱导。分别采用Giemsa染色检测细胞形态,糖原染色检测细胞糖原合成能力。转录组测序分析差异基因表达,KEGG分析关键上调信号通路,Western blot检测FoxA2、白蛋白、Akt和磷酸化Akt表达,RT-qPCR检测细胞中白蛋白、HNF4α、Lama1、IL6、PI3K、Myc mRNA表达水平。两组比较采用独立样本t检验,三组以上比较采用单因素方差分析,两两比较采用LSD-t检验。

结果

与对照相比,敲减FoxA2组细胞中肝细胞相关基因白蛋白 (0.27 ± 0.05比1.01 ± 0.30)、HNF4α mRNA (0.41 ± 0.10比1.08 ±0.30)表达降低 (P < 0.05)。分化诱导剂丁酸钠处理后,与对照相比,敲减FoxA2组细胞糖原合成能力下降、白蛋白表达 (0.59 ± 0.08比1.09 ± 0.08)减少 (P < 0.01)。与对照相比,转录组测序发现敲减FoxA2组细胞中表达显著上调的基因为1 243个,对这些基因进行KEGG分析显示PI3K/Akt通路是上调基因数最多的通路。与对照相比,敲减FoxA2组细胞中PI3K/Akt通路关键基因Lama1 (5.49 ± 0.62比1.01 ± 0.19)、IL6 (2.20 ± 0.10比1.00 ± 0.06)、PI3K (1.44 ± 0.09比1.02 ± 0.23)、Myc (1.44 ± 0.19比1.01 ± 0.14)mRNA表达增加,Akt的磷酸化水平 (0.95 ±0.11比0.71 ± 0.03)升高 (P 均 < 0.05)。与单用丁酸钠处理相比,联合应用丁酸钠与PI3K/Akt通路的抑制剂Ly294002处理敲减FoxA2组细胞白蛋白 (0.97 ± 0.10比0.76 ± 0.03,P < 0.05)及HNF4α mRNA (5.36 ± 0.34比0.41 ± 0.15,P < 0.01)表达升高。与单用丁酸钠处理的敲减FoxA2组相比,联合应用丁酸钠与Ly294002处理的敲减FoxA2组细胞糖原合成能力有所恢复,接近丁酸钠与Ly294002联合处理的对照组细胞。

结论

干预PI3K/Akt信号通路有助于提高低表达FoxA2的肝脏前体细胞对分化诱导剂的应答并促进其朝肝细胞方向分化。

Objective

To explore the mechanism of the Forkhead box protein A2 (FoxA2)low-expressing hepatic progenitor cells response to differentiation reagent and their differentiation towards hepatocytes.

Methods

Rat liver progenitor cells were transfected with non-specific shRNA and FoxA2 shRNA and induced to differentiate towards hepatocytes by sodium butyrate or the combination of sodium butyrate and Ly294002, an inhibitor of PI3K/Akt signal pathway. Giemsa staining was used for morphology analysis, and glycogen staining was used for glycogen storage analysis. RNA sequencing was used to analyze differentially expressed genes between FoxA2 knockdown cells and the control cells, and KEGG was used to find the key signal pathway based on the differentially expressed genes. Western blot was used to detect the expression levels of FoxA2,albumin, Akt and phosphorylated Akt. RT-qPCR was used to detect the expression levels of albumin,HNF4α, Lama1, IL6, PI3K, and Myc mRNA. Independent-sample t-test was used to compared the difference between two groups; and ANOVA analysis was used to compare the differences among groups. LSD-t test was used for further comparison between two groups.

Results

Compared to control group, knocking down FoxA2 reduced the expression of albumin (0.27 ± 0.05 vs 1.01 ±0.30) and HNF4α mRNA (0.41 ± 0.10 vs 1.08 ± 0.30). The glycogen synthesis function and albumin expression (0.59 ± 0.08 vs 1.09 ± 0.08) were reduced (P < 0.01) in FoxA2 knockdown cells after sodium butyrate treatment when compared to those in the control group. RNA sequencing analysis found there were 1 243 upregulated expression genes in the FoxA2 knockdown cells, and KEGG analysis revealed that PI3K/Akt signal pathway is the pathway with the most upregulated expression genes. Compared with control group, the expression levels of Lama1 (5.49 ± 0.62 vs 1.01 ± 0.19), IL6 (2.20 ± 0.10 vs 1.00 ± 0.06), PI3K (1.44 ± 0.09 vs 1.02 ± 0.23) and Myc (1.44 ±0.19 vs 1.01 ± 0.14) and p-Akt (P < 0.05) of Akt (0.95 ± 0.11 vs 0.71 ± 0.03) in FoxA2 knockdown cells were increased (P < 0.05). Furthermore, the combined treatment with Ly294002 and sodium butyrate increased the expression level of albumin (0.97 ± 0.10 vs 0.76 ± 0.03, P < 0.05) and HNF4α mRNA (5.36 ± 0.34 vs 0.41 ± 0.15, P < 0.01) in FoxA2 knockdown cells when compared with sodium butyrate-treated FoxA2 knockdown cells. In addition, glycogen synthesis function in FoxA2 knockdown cells treated with Ly294002 and sodium butyrate was better than that in sodium butyrate- treated FoxA2 knockdown cells, which was similar to that in the control group treated with Ly294002 and sodium butyrate.

Conclusion

Blocking PI3K/Akt signal pathway contributes to enhance the response to differentiation reagent of FoxA2 low-expressing liver progenitor cells to differentiate towards hepatocytes.

表1 大鼠引物序列
图1 敲减FoxA2降低肝脏前体细胞中FoxA2与肝细胞相关基因表达 注:a图为敲减FoxA2组肝脏前体细胞与对照组细胞的制备过程与细胞形态 (×20);b图为Western blot与灰度分析显示与对照相比,敲减FoxA2组细胞FoxA2蛋白表达下降;c图为RT-qPCR结果显示与对照相比,敲减FoxA2组细胞白蛋白与HNF4a基因转录mRNA表达下降;两组相比,aP < 0.05,bP < 0.01 (n = 3)
图2 敲减FoxA2降低肝脏前体细胞对分化诱导剂的应答反应 注:a图为Giemsa染色显示丁酸钠处理对照组与敲减FoxA2组细胞的形态 (箭头指示双核细胞,×10);b图为糖原染色显示丁酸钠处理对照组细胞与敲减FoxA2组细胞的糖原合成情况 (箭头指示具有糖原合成功能的细胞,×10)
图3 Western blot与灰度分析显示丁酸钠处理对照组细胞与敲减FoxA2组细胞白蛋白的相对表达水平 注:两组相比,aP < 0.05,bP < 0.01 (n = 3)
图4 敲减FoxA2激活肝脏前体细胞的PI3K/Akt信号通路 注:a图为火山图展示与对照组细胞相比敲减FoxA2组细胞mRNA表达增加的基因 (红色)与降低的基因 (蓝色);b图为转录组测序结果显示与对照组细胞相比,敲减FoxA2组细胞中成熟肝细胞相关基因mRNA表达降低;c图为对敲减FoxA2后表达增加超过2倍的基因进行KEGG分析得到的上调基因数位列前10的信号通路;d图为转录组测序结果显示与对照组相比,敲减FoxA2组细胞中PI3K/Akt通路关键基因mRNA表达升高;e图为RT-qPCR结果证实与对照组相比,敲减FoxA2组细胞Lama1、IL6、PI3K与Myc基因mRNA表达升高;f图为Western blot结果显示与对照组相比,敲减FoxA2组细胞Akt的磷酸化水平增加。 组间相比,aP < 0.05,bP < 0.01
图5 阻断PI3K/Akt信号通路恢复敲减FoxA2前体细胞对分化诱导剂丁酸钠的应答反应 注:a图为Western blot与灰度分析显示联合应用Ly294002与丁酸钠处理敲减FoxA2肝脏前体细胞的白蛋白相对表达水平;b图为RT-qPCR结果检测单用丁酸钠处理、联合应用Ly294002与丁酸钠处理对照组细胞与敲减FoxA2组细胞后HNF4α基因mRNA表达水平。两组相比,aP < 0.05,bP < 0.01(n = 3);c图为糖原染色显示联合应用Ly294002与丁酸钠处理对照组与敲减FoxA2组细胞的糖原合成情况 (箭头指示具有糖原合成功能的细胞,×20)
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