When injury occurs, a series of signaling pathways in the liver are activated to initiate liver regeneration (LR), and the body’s metabolic needs are satisfied by the rapid expansion of the remaining liver tissue. In 1931, Higgins et al. [1] first demonstrated its unique characteristics by performing animal experiments. After approximately 70% partial hepatectomy (PH), the remaining liver in rats could basically recover to the weight, volume and function before operation within 5–7 days, and then the cells returned to static state. It is considered that the whole process of liver regeneration includes three phases including initiation, proliferation and termination [2]. At the initiation phase, a series of cytokines including TNF-$\alpha$ and IL-6 act on hepatocytes to enhance their sensitivity to growth factors. Stimulation of various growth factors makes hepatocytes re-enter G1 phase and then enter proliferation phase [3]. When the function of liver tissue is sufficient to maintain the normal metabolic needs of the body, hepatocytes re-enter G0 phase and stop proliferation [4]. Currently, most of the researches on liver regeneration concentrate on the initiation signal in the early phase of regeneration, while the research on the termination signal in the late phase of regeneration remains relatively few. Therefore, an in-depth study of the negative regulatory signals and key genes involved in the late phase of liver regeneration will not only help to deepen the understanding of the process of liver regeneration, but also provide great significance in explaining the regulatory mechanism of liver tumor growth and screening potential therapeutic targets. In recent years, with the unprecedentedly rapid development of high-throughput sequencing and computer science and technology, it is possible to comprehensively utilize life science, computer and information technology to analyze the potential significance behind massive and complex biological data. In the current work, bioinformatics analysis was used to select the microarrays related to the termination of liver regeneration. Differentially expressed genes (DEGs) with statistical significance were selected and analyzed to find out the key genes and pathways that exert an important role in the process.

Before extracting data, it is of necessity to verify the chip quality at first and then find and reject the problematic chips to ensure the reliability of subsequent analysis. RLE and NUES can reflect the consistency of parallel experiments. An RLE plot exhibits the difference between a gene’s expression level and the median for that gene, assuming that most genes are not differentially expressed. The boxes should be close to 0. A NUSE plot however is scaled so that the median standard error across arrays is 1 for each gene and thus boxes should be close to 1 [13]. If there is a large deviation in the box plot of a chip, it indicates that there is a problem with the chip [14]. RNA degradation is another important factor which can affect chip quality. Since RNA degradation starts from the 5’ end, the fluorescence intensity at the 5’ end is much lower than that at the 3’ end [15]. To assess the viability of the microarray using the RNA degradation plot, we look at the parallelism and similarities between samples slopes in which an extreme difference in one or more of the samples slopes means that the sample is not viable for use. The detection results of the above three indicators show that the chips included in this analysis are reliable in quality, and the batch difference of detection results is small, thus providing credible original data for performing subsequent analysis.

In the proliferation phase, both the regenerated liver and liver tumors show the strong proliferation ability of liver cells. However, because tumor cells lack the negative regulation mechanism for termination, their cells can divide indefinitely. Liver regeneration is a complex and delicate process regulated by multiple factors. When the regeneration level can satisfy the body’s metabolic needs, it will trigger a termination signal to stop cell proliferation. In the present study, altogether 74 DEGs were selected from the rat liver microarray at the termination phase of liver regeneration, further validated by Western blotting assay, and analyzed by KEGG and GO enrichment analyses, so as to reveal the roles of these genes at the termination phase of liver regeneration. Among the seven pathways screened by KEGG analysis, MAPK and TGF-$\beta$ signal pathways were the most noteworthy. MAPK family is a group of serine-threonine protein kinases which can be activated by various extracellular stimuli, and is mainly involved in a lot of important physiological/pathological processes such as cell growth, differentiation, stress and inflammatory reaction. However, it has been reported that p38, as a MAPK subfamily, exert an important role in proliferation, which is closely related to the pathophysiological environment in which hepatocytes live. Studies conducted by Campbell et al. [16] showed that p38$\alpha$ is a negative regulator of hepatocyte proliferation. In the liver regeneration model of mice after PH operation with specific liver cell knockout, the early phase of hepatocyte regeneration exhibits enhanced proliferation activity, and its mechanism may be related to the antagonism of p38$\alpha$ against JNK-c-Jun pathway activity. In the model of chronic biliary cirrhosis, the specific knocking out of liver cells p38$\alpha$ will lead to mitotic retardation and cytokinesis failure, thus reducing the proliferation of liver cells and ultimately lowering the life span of mice [17].

Fortier et al. [18] confirmed through the CCl4-induced acute liver injury model that specific knockout of p38$\alpha$ of hepatocytes altered the immune microenvironment after injury, enhanced the infiltration of inflammatory cells, and mediated the formation of immune microenvironment conducive to liver tissue repair through the chemotaxis of CCL2/CCL5 cytokines. Studies have proved that TGF-$\beta$ signaling pathway is an important signal to inhibit the proliferation of hepatocytes for the reason that it is closely related to the activation of hepatic stellate cells (HSCs). As the most important non parenchymal cells in the liver microenvironment, HSCs are involved in the pathophysiological processes of liver fibrosis, hepatocellular carcinoma and liver regeneration after transdifferentiate into myofibroblasts (MFS). Inhibition of TGF-$\beta$ pathway interferes with epithelial mesenchymal transition of HSCs during liver regeneration and reduces the nuclear aggregation of $\beta$-Catenin and the expression of cytochrome P450, resulting in delayed proliferation. The activation of this pathway can directly promote liver fibrosis [19, 20]. A transcriptome analysis of HCC indicated that 40% of samples have TGF-$\beta$ signaling pathway gene mutation, and up-regulation of this pathway will promote inflammation and fibrosis, while down-regulation will reduce tumor inhibitory activity and significantly shorten the survival time of patients [21]. In addition, recent research results also confirm our GO enrichment analysis results. Notch signaling pathway can promote the proliferation of hepatocytes. Overexpression of Notch-1 can up-regulate the expression of cyclin A1, D1, E and other cyclins [22]. The activation of Notch pathway in hepatocytes is the key condition of liver homeostasis, metabolism, regeneration, vascular physiology and biliary tract morphogenesis [23]. Notch pathway in liver is significantly activated after PH operation, and inhibition of this pathway can lead to the imbalance of cell cycle and its related proteins, and the mechanism involved may be in association with NICD/Akt/HIF-1$\alpha$ pathway [24]. Exosomes derived from hepatocytes can promote intercellular communication during regeneration, and exosomes derived from bone marrow mesenchymal stem cells can inhibit HSCs activation through Wnt/$\beta$-catenin pathway, consequently alleviating CCl4-induced liver fibrosis [25, 26]. At the end of liver regeneration, the molecular function of DEGs is enriched in the activities of various metabolic related enzymes including aromatase and steroid hydroxylase, also indicating that the most important metabolic function of liver has been restored.

In comparison with the control group, the top 10 hub genes related to the termination phase of liver regeneration screened from PPI network were all up-regulated. Ste2 gene regulates the expression of estrogen sulfotransferase 2, maintaining the steady state of estrogen by sulfonating and inactivating estrogen. It has been reported that this enzyme is the direct transcription target of Nrf2. The latest research shows that Nrf2 is activated in the early phase of tumorigenesis in rat model of fatty hepatitis with fibrosis, and knockout of Nrf2 gene can inhibit the amplification of initial cell clone, which can thus prevent the occurrence of HCC [27, 28]. This may be the reason for the increased expression of the gene in the late phase of liver regeneration. A study conducted by Zhao et al. [29] showed that tumor-like growth in the early phase of liver development is positively and negatively coordinated by $\alpha$-2u globulin (extracellular domain)/ppp2r2a-pik3c3 (MAPK signaling pathway)/Hsd3b5 (metabolic pathway), evidently showing that Hsd3b5 gene plays an important role in the biosynthesis of all steroid hormones. In addition, another article by Xiu et al. [30] also showed a significant expression decrease in Hsd3b5 after lowering serum testosterone and estrogen. It is of note that the expression level of Myc, as a oncogene, is still up-regulated at the termination phase of liver regeneration. The sustained overexpression of c-Myc caused by the deletion of hepatocyte nuclear factor 4$\alpha$ (HNF4 $\alpha$) will eventually lead to the non-functional continuous proliferation of liver after PH operation [31]. Moreover, it also indirectly confirmed that there must be a series of negative modulation signals against its proliferation-promoting effect at this phase. In liver cells, Btg2 as a cell cycle inhibitor can prevent FoxM1 activation and inhibit DNA synthesis [32]. MiR-6875-3p can directly inhibit Btg2 expression and up-regulate FAK/Akt pathway activity, which can thus promote HCC invasion and metastasis [33]. PRMT5 can promote tumor proliferation by down-regulating Btg2 expression through ERK pathway [34]. The above results confirm the positive and negative effects of some hub genes in the termination phase of liver regeneration. However, there are no related reports concerning the roles of Mup5, Rup2 and UST4r genes in liver regeneration and HCC, which need to be further explored.

To conclude, liver regeneration is a complex biological process regulated by multiple factors. The main feature of liver regeneration is that it can stop proliferation and produce functional hepatocytes in accordance with the needs of the body at the end phase. In this study, we successfully screened and analyzed the DEGs in rat liver tissue at the end phase of liver regeneration, and preliminarily understood its related functions and signal pathways. In the meanwhile, we also analyzed the hub genes with negative regulatory effect, such as Ste2, Btg2, and Hsd3b5, thus providing a new idea for the screening of liver tumor research targets and the exploration of pathological mechanism.

DEGs, Differentially expressed genes; GEO, Gene Expression Omnibus; HCC, Hepatocellular carcinoma; HNF4 $\alpha$, Hepatocyte nuclear factor 4$\alpha$; HSCs, Hepatic stellate cells; LR, Liver regeneration; MAPK, Mitogen-activated protein kinase; MFS, Myofibroblasts; NUSE, Normalized unscaled standard errors; PH, Partial hepatectomy; PPI, Protein-protein interaction; RLE, Relative log expression; STRING, Search Tool for the Retrieval of Interacting proteins; TGF-$\beta$, transforming growth factor-$\beta$.

JG designed and supervised the study. HS. and MW. contributed to the implementation of the research, analysis of the results and writing of the manuscript. All authors have read and approved the manuscript.

All animals’ care and experimental protocols were complied with the Animal Management Rules of the Ministry of Health of the People’s Republic of China and all experimental designs were approved by the Animal Care and Use Committee of Second affiliated Hospital of Chongqing Medical university.

We thank the Experimental Animal Center of Chongqing Medical university (Chongqing, China) for providing us with animals required and we would also like to thank all the peer reviewers for their opinions and suggestions.

This study was supported by grants from nature science foundation of Chongqing (no: cstc2019jcyj-zdxmX0027).

The authors declare no conflict of interest.