Abstract:

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.

Key words: Liver progenitor cells, Hepatocyte differentiation, FoxA2, PI3K/Akt