Liver disease is a serious hazard to human health. However, the current situation of clinical intervention of liver disease is not ideal, and there is a lack of effective drugs and methods to intervene liver disease. The liver transplantation is a conventional and effective intervention method. However, transplantation cannot meet the clinical needs due to the shortage of high-quality liver cells, allograft rejection, and other problems10. Therefore, there is an urgent need to develop new methods for liver disease intervention. Currently, stem cell-based cell replacement therapy has attracted worldwide attention. BMSC transplantation provides a new way to intervene liver disease. Several studies have shown that transplanted BMSCs can differentiate into hepatocytes to replace the function of damaged hepatocytes and tissues in liver due to their directional differentiation ability, promoting the recovery of liver injury. However, the mechanism of differentiation of BMSCs into hepatocytes remains unclear. Therefore, it is of great significance to study the key genes and downstream regulatory mechanisms associated with the differentiation of BMSCs into liver cells, which can provide potential targets for BMSC-based treatment of liver failure.
Transcriptome Analysis Console software was used to compare the differential gene expression data of BMSCs and hepatocytes. A total of 4402 differential genes were identified, of which 1896 genes were upregulated and 2506 genes were downregulated. GO enrichment analysis showed that among the upregulated genes, 11.44% were related to oxidation-reduction process, 7.16% to liver metabolism process, and 1.92% were to liver development process. Among the downregulated genes, 5.04% were related to cell proliferation. Oxidation-reduction process, metabolism process, liver development process, and cell proliferation are closely related to differentiation of hepatocytes.
The STRING search tool was used to build PPI network, and hub genes were screened out. The hub genes Cat and Cyp2e1 are related to the oxidation-reduction process of hepatocytes; Cat and FOXO3 are positively correlated with the differentiation of BMSCs into cells of the osteogenic lineage11. Cyp2e1 is expressed in the later stage of the differentiation process of mouse embryonic stem cells to hepatocytes12. The hub genes Pah, Ugt2a3, Acss2, Aldh6a1, Hmgcs2, H6pd, and Aldh1a7 are related to liver metabolism. The mRNA level of Hmgcs2 increase during the differentiation of human embryonic stem cells into hepatocellular-like cells13. The role of Pah, Ugt2a3, Acss2, Aldh6a1, H6pd, or Aldh1a7 in hepatocyte cells is currently unclear. The hub genes Hmgcl, Ugt1a1, Arg1, Otc, Baat, Slco1b2, Onecut1, Hhex, and Proc are related to liver development. Ugt1a1 is a marker of hepatocytes, and its significant expression during the differentiation of human hematopoietic stem cells (HSCs) into hepatocytes indicates that HSCs have successfully differentiated into normal hepatocytes14. Arg1 expression is observed during hepatic-like phenotype differentiation of unrestricted somatic stem cells from human umbilical cord blood in vitro15. In a study, the 201B7 cells were cultured in Hepatocyte differentiation initiating medium (HDI) for 2 days, and it was observed that the expression of hepatocellular specific transcription factors was upregulated and that of Otc was increased16. Onecut interacts with Lmx1a to promote the differentiation of ventral midbrain neural stem cells into dopamine neurons through the Wnt1-Lmx1a pathway17. Hhex regulates endoderm differentiation of embryonic stem cell derived cells into hepatocytes18. The role of Hmgcl, Baat, Slco1b2, or Proc in hepatocyte differentiation and development is unclear. The hub genes Cdk4, Il6, Fn1, Erbb2, Ccnd1, and Bmp4 regulate cell proliferation networks and are downregulated in hepatocytes compared with untreated BMSCs, suggesting inhibitory effects on liver differentiation. Studies have shown that expression of Cyclin B1 and Cdk4 during the hepatic differentiation of liver epithelial progenitor cells (LEPCs) induced by sodium butyrate may be related to the growth arrest of LEPCs shortly after treatment19. Transplantation of MSCs promotes cell proliferation and organ repair, and activation of Il-6/gp130-mediated STAT3 signaling pathway via soluble IL-6 receptor is crucial in hepatic differentiation of MSCs20. Ccnd1 has been reported to be associated with liver regeneration, and it is speculated that they play a key role in mouse hepatocytes21. Ccnd1 silencing suppresses liver cancer stem cells (LCSCs) differentiation22. Bmp4 is an important regulator of cell proliferation and differentiation. Studies have shown that Bmp4 is a key cytokine for the development of mouse embryonic stem cells into hepatocytes23. Fn1 and Erbb2 have not been reported to regulate the differentiation of BMSCs into hepatocytes.
We used miRWalk to predict upstream target miRNAs that regulate gene expression. In our study, we observed 11 miRNAs that target minimum 2 genes and are involved in the differentiation of BMSCs into hepatocytes. Previous studies have reported that lncRNAs upstream of miRNAs play an important role in BMSC differentiation, so we used StarBase 2.0 for prediction. However, only miRNA-23a, miRNA-23b, miR-137, miRNA-186, miRNA-466l, and miRNA-539 can predict the corresponding upstream lncRNAs among the selected 11 miRNAs. Six lncRNAs, Zfp469, 1700020I14Rik, Gm42418, Zfas1, Dubr, and Peg13 were predicted by StarBase v2.0. Therefore, we introduced the above 6 miRNAs and their predicted lncRNAs in the regulation of BMSC differentiation. Studies have shown that CXCL13 promotes osteogenic differentiation of BMSCs by inhibiting miR-23a expression24. MiR-23b-3p upregulation facilitates the hepatic trans-differentiation of MSCs25. MiR-23b-3p overexpression promoted the differentiation of BMSCs into Type II pneumocytes and alleviated ALI in lung tissues26. In addition, silencing of miR-137-3p was found to facilitate osteogenic differentiation of BMSCs27. Current research shows that the relationship between miRNA-186-5p, miRNA-466l-3p, miRNA-539-5p, and BMSCs differentiation is not very clear. Among the 6 lncRNAs, only Zfas1 was found to be associated with BMSC differentiation, while the relationship between Zfp469, 1700020I14Rik, Gm42418, Dubr, Peg13, and BMSC differentiation remains unclear. Zfas1 has been reported to affect the osteogenic and adipogenic differentiation of BMSCs by sponging miR-499 thereby upregulating ephrin type-A receptor 5 (EPHA5). However, further research is needed to confirm these results28.
Taken together, through bioinformatics analysis, we identified key genes that regulate the differentiation of BMSCs into hepatocytes and their upstream miRNAs and lncRNAs, providing potential targets for stem-cell replacement therapy for liver diseases. Therefore, it is of great significance to clarify the key genes and downstream regulation mechanism of the differentiation of BMSCs into hepatocytes.