Abstract：
BACKGROUND: Hepatic stellate cells (HSCs) serve as the crucial accelerating factor in the progression of liver fibrosis (LF). In contrast to HSCs, adult-derived human liver stem/progenitor cells (ADHLSCs) exhibit greater potency in terms of differentiation and proliferation, rendering them highly applicable in LF treatment. The objective of this study is to identify new therapeutic targets for LF by comparing differentially expressed genes (DEGs) between ADHLSCs and HSCs.
METHODS: We investigated DEGs between ADHLSCs and HSCs using the GSE49995 dataset obtained from the Gene Expression Omnibus (GEO) database, aiming to identify new therapeutic targets for LF. Subsequently, we activated HSCs to delve deeper into the mesenchyme homeobox 2 (MEOX2), PH domain Leucine-rich repeat protein phosphatase (PHLPP), and Phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways in LF progression, employing platelet-derived growth factor (PDGF), and conducted infection with Overexpression (OE)-MEOX2 and shRNA-MEOX2 (sh-MEOX2) lentiviruses. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay, while cell proliferation was evaluated through 5-ethynyl-2\'-deoxyuridine (EdU) staining and flow cytometry. Relative mRNA expression levels were determined via qPCR. Western blot analysis was performed to measure protein expression levels, and the regulatory role of MEOX2 was investigated using dual luciferase reporter assays.
RESULTS: We identified 332 DEGs that were down-regulated and 201 DEGs that were up-regulated between ADHLSCs and HSCs. Notably, MEOX2 expression in ADHLSCs was significantly reduced. These DEGs primarily participated in the collagen-containing extracellular matrix and the PI3K/AKT signaling pathway. MEOX2 could inhibit cancer cell proliferation via the PI3K/AKT signaling pathway. Additionally, the JASRPAR2022 database predicted the target gene PHLPP of MEOX2. Our results indicated that OE-MEOX2 significantly inhibited HSCs\' cell vitality and proliferation. Further analysis revealed that MEOX2 binds to PHLPP promoters, thereby up-regulating its transcription. This action led to the inhibition of p-AKT expression, consequently reducing HSC proliferation and slowing the progression of LF.
CONCLUSIONS: MEOX2 up-regulates PHLPP expression and inhibits AKT phosphorylation, thereby reducing the cell activity and proliferation ability of HSCs and inhibiting the progression of LF.
METHODS: We investigated DEGs between ADHLSCs and HSCs using the GSE49995 dataset obtained from the Gene Expression Omnibus (GEO) database, aiming to identify new therapeutic targets for LF. Subsequently, we activated HSCs to delve deeper into the mesenchyme homeobox 2 (MEOX2), PH domain Leucine-rich repeat protein phosphatase (PHLPP), and Phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways in LF progression, employing platelet-derived growth factor (PDGF), and conducted infection with Overexpression (OE)-MEOX2 and shRNA-MEOX2 (sh-MEOX2) lentiviruses. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay, while cell proliferation was evaluated through 5-ethynyl-2\'-deoxyuridine (EdU) staining and flow cytometry. Relative mRNA expression levels were determined via qPCR. Western blot analysis was performed to measure protein expression levels, and the regulatory role of MEOX2 was investigated using dual luciferase reporter assays.
RESULTS: We identified 332 DEGs that were down-regulated and 201 DEGs that were up-regulated between ADHLSCs and HSCs. Notably, MEOX2 expression in ADHLSCs was significantly reduced. These DEGs primarily participated in the collagen-containing extracellular matrix and the PI3K/AKT signaling pathway. MEOX2 could inhibit cancer cell proliferation via the PI3K/AKT signaling pathway. Additionally, the JASRPAR2022 database predicted the target gene PHLPP of MEOX2. Our results indicated that OE-MEOX2 significantly inhibited HSCs\' cell vitality and proliferation. Further analysis revealed that MEOX2 binds to PHLPP promoters, thereby up-regulating its transcription. This action led to the inhibition of p-AKT expression, consequently reducing HSC proliferation and slowing the progression of LF.
CONCLUSIONS: MEOX2 up-regulates PHLPP expression and inhibits AKT phosphorylation, thereby reducing the cell activity and proliferation ability of HSCs and inhibiting the progression of LF.
摘要：
背景：肝星状细胞（HSC）是肝纤维化（LF）进展的关键加速因子。与HSC相比,成人来源的人肝干/祖细胞（ADHLSCs）在分化和增殖方面表现出更大的潜力，使它们在LF治疗中高度适用。这项研究的目的是通过比较ADHLSCs和HSCs之间的差异表达基因（DEGs）来确定LF的新治疗靶标。
方法：我们使用从基因表达综合（GEO）数据库获得的GSE49995数据集研究了ADHLSC和HSC之间的DEG，旨在确定LF的新治疗靶点。随后，我们激活了HSC以深入研究间充质homeobox2（MEOX2），PH结构域富含亮氨酸的重复蛋白磷酸酶（PHLPP），和磷酸肌醇3-激酶（PI3K）/蛋白激酶B（AKT）信号通路在LF进展中，使用血小板衍生生长因子（PDGF），并且用过表达(OE)-MEOX2和shRNA-MEOX2(sh-MEOX2)慢病毒进行感染。使用细胞计数试剂盒-8（CCK-8）测定评估细胞活力，同时通过5-乙炔基-2'-脱氧尿苷（EdU）染色和流式细胞术评估细胞增殖。通过qPCR测定相对mRNA表达水平。进行蛋白质印迹分析以测量蛋白质表达水平,并使用双荧光素酶报告基因测定法研究了MEOX2的调节作用。
结果：我们在ADHLSCs和HSCs之间鉴定了332个DEGs下调和201个DEGs上调。值得注意的是,MEOX2在ADHLSCs中的表达显著降低。这些DEGs主要参与含胶原的细胞外基质和PI3K/AKT信号通路。MEOX2可通过PI3K/AKT信号通路抑制癌细胞增殖。此外,JASRPAR2022数据库预测了MEOX2的靶基因PHLPP。我们的结果表明OE-MEOX2显着抑制HSC的细胞活力和增殖。进一步分析显示MEOX2与PHLPP启动子结合，从而上调其转录。这种作用导致p-AKT表达的抑制，因此减少HSC增殖并减缓LF的进展。
结论：MEOX2上调PHLPP表达并抑制AKT磷酸化，从而降低HSCs的细胞活性和增殖能力，抑制LF的进展。
方法：我们使用从基因表达综合（GEO）数据库获得的GSE49995数据集研究了ADHLSC和HSC之间的DEG，旨在确定LF的新治疗靶点。随后，我们激活了HSC以深入研究间充质homeobox2（MEOX2），PH结构域富含亮氨酸的重复蛋白磷酸酶（PHLPP），和磷酸肌醇3-激酶（PI3K）/蛋白激酶B（AKT）信号通路在LF进展中，使用血小板衍生生长因子（PDGF），并且用过表达(OE)-MEOX2和shRNA-MEOX2(sh-MEOX2)慢病毒进行感染。使用细胞计数试剂盒-8（CCK-8）测定评估细胞活力，同时通过5-乙炔基-2'-脱氧尿苷（EdU）染色和流式细胞术评估细胞增殖。通过qPCR测定相对mRNA表达水平。进行蛋白质印迹分析以测量蛋白质表达水平,并使用双荧光素酶报告基因测定法研究了MEOX2的调节作用。
结果：我们在ADHLSCs和HSCs之间鉴定了332个DEGs下调和201个DEGs上调。值得注意的是,MEOX2在ADHLSCs中的表达显著降低。这些DEGs主要参与含胶原的细胞外基质和PI3K/AKT信号通路。MEOX2可通过PI3K/AKT信号通路抑制癌细胞增殖。此外,JASRPAR2022数据库预测了MEOX2的靶基因PHLPP。我们的结果表明OE-MEOX2显着抑制HSC的细胞活力和增殖。进一步分析显示MEOX2与PHLPP启动子结合，从而上调其转录。这种作用导致p-AKT表达的抑制，因此减少HSC增殖并减缓LF的进展。
结论：MEOX2上调PHLPP表达并抑制AKT磷酸化，从而降低HSCs的细胞活性和增殖能力，抑制LF的进展。
