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.

Pleckstrin homologous domain leucine-rich repeating protein phosphatases (PHLPPs) were originally identified as protein kinase B (Akt) kinase hydrophobic motif specific phosphatases to maintain the cellular homeostasis. With the continuous expansion of PHLPPs research, imbalanced-PHLPPs were mainly found as a tumor suppressor gene of a variety of solid tumors. In this review, we simply described the history and structures of PHLPPs and summarized the recent achievements in emerging roles of PHLPPs in lung cancer by 1) the signaling pathways affected by PHLPPs including Phosphoinositide 3-kinase (PI3K)/AKT, RAS/RAF/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and Protein kinase C (PKC) signaling cascades. 2) function of PHLPPs regulatory factor USP46 and miR-190/miR-215, 3) the potential roles of PHLPPs in disease prognosis, Epidermal growth factor receptors (EGFR)- tyrosine kinase inhibitor (TKI) resistance and DNA damage, 4) and the possible function of PHLPPs in radiotherapy, ferroptosis and inflammation response. Therefore, PHLPPs can be considered as either biomarker or prognostic marker for lung cancer treatment.

UNASSIGNED: Recently, emerging studies have validated that circular RNAs participate in multiple biological progresses in various human malignant tumors, including hepatocellular carcinoma (HCC). However, until now, the elucidated mechanism of circular RNAs is only the tip of the iceberg. In this study, we firstly identify a novel circular RNA circRASSF5 (the only circular RNA derived from the RASSF5 gene), and attempt to investigate its biological function and underlying mechanism in HCC.
UNASSIGNED: qRT-PCR, Western blotting and IHC were applied to detect the expression of related genes. CCK-8 assay, EdU staining, wound healing and transwell assays were used to investigate HCC proliferation, migration and invasion abilities. Animal model studies were included to investigate the function of circRASSF5 in HCC tumorigenesis and metastasis. RNA pull-down assay, luciferase reporter assay and FISH (fluorescence in situ hybridization) assay were performed to explore the potential biological mechanism underlying circRASSF5 function in HCC.
UNASSIGNED: CircRASSF5 is obviously downregulated in both HCC tissues and cell lines. Low level of circRASSF5 is negatively associated with larger tumor size, severe vascular invasion, more portal vein tumor embolus and unfavorable prognosis. Loss-of-function assay reveals that circRASSF5 remarkably impedes the growth and metastasis of HCC cells in vitro and in vivo. Mechanistically, circRASSF5 directly interacts with miR-331-3p as a sponge, and then enhances the expression of PH domain and leucine-rich repeat protein phosphatase (PHLPP), thus restraining the progression of HCC cells.
UNASSIGNED: Altogether, we validate that circRASSF5 is a tumor suppressor in HCC, which competitively sponges with miR-331-3p and then enhances the tumor inhibitory effect of PHLPP, indicating the potential application value of circRASSF5 for HCC diagnosis and clinical treatment.

UNASSIGNED: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective in advanced EGFR-mutation non-small cell lung cancer (NSCLC) but the magnitude of tumor regression varies, and drug resistance is unavoidable. The pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) levels are reduced or lost and acts as a tumor suppressor in many cancers. Here, we hypothesized that PHLPP is a key regulator of EGFR-TKI sensitivity and a potential treatment target for overcoming resistance to EGFR-TKI in lung cancer.
UNASSIGNED: Cell proliferation and growth inhibition were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assay. PHLPP- knockdown stable cell lines were generated by lentivirus-mediated delivery of PHLPP shRNAs. The expression of PHLPP mRNA and protein levels was detected by real-time quantitative polymerase chain reaction (qPCR) and Western blotting. Immunohistochemical (IHC) staining was performed to detect the PHLPP expression in clinical patient tissue samples. A transcriptomic assay of genome-wide RNA expressions of PHLPP in NSCLC cell lines according to gefitinib sensitivity was obtained from Gene Expression Omnibus (GEO) database. Murine xenograft model was established to verify the function of PHLPP in gefitinib resistance in vivo.
UNASSIGNED: PHLPP highly expressed in gefitinib-sensitive NSCLC cell lines than gefitinib-resistant NSCLC cell lines. In gefitinib-acquired resistance cell line HCC827-GR, PHLPP expression even dramatically reduced. Knockdown of PHLPP in NSCLC cells decreased cell death induced by the EGFR-TKI, while overexpression PHLPP in gefitinib-resistance NSCLC cells can enhance or restore EGFR-TKIs sensitivity. Mechanism study indicated that PHLPP downregulation attenuates the effect of EGFR-TKI on the both AKT and ERK pathway, thereby decreasing the cell death sensitivity to EGFR inhibitors. In xenograft mice, knockdown of PHLPP decreased tumor response to gefitinib and advanced tumor cells re-growth after gefitinib treatment. In clinical, PHLPP expression were reduced in the post-relapse tumor compared to that of pre-treatment, and lower pre-treatment PHLPP levels were significantly correlated with shorter progression-free survival (PFS) in patients with EGFR-mutant lung adenocarcinoma whom treated with EGFR-TKI.
UNASSIGNED: Our data strongly demonstrated that loss of PHLPP function was a key factor of EGFR-TKI resistance in NSCLC. Downregulated PHLPP expression activated PI3K-AKT and MAPK-ERK pathway which strengthened cell survival to EGFR-TKI. Therefore, PHLPP expression level was not only a potential biomarker to predict EGFR-TKIs sensitivity but also as a therapeutic target in EGFR-TKIs therapy, enhancing PHLPP expression may be a valuable strategy for delaying or overcoming EGFR-TKIs drug resistance.

BACKGROUND: Treatment of bortezomib (BTZ) improves the clinical outcomes of patients with multiple myeloma (MM). However, primary resistance and acquired resistance to BTZ frequently develop in patients with MM. PH domain leucine-rich repeat protein phosphatase (PHLPP) plays an important role in chemoresistance in a number of cancers. However, the role of PHLPP on MM remains unclear. In this study, we investigated the role of PHLPP in BTZ-resistant MM cells.
METHODS: BrdU assays, immunoprecipitation, flow cytometry analyses, and immunofluorescence assays were performed.
RESULTS: PHLPP and lysosome-associated membrane protein 2 (LAMP2) levels were downregulated in BTZ-resistant MM cells compared with BTZ-sensitive MM cells, accompanied by inactivation of autophagy pathway evaluated by a reduction in Beclin1, Atg5 and LC3B and increase in p62. Gain- and loss-of-function experiments revealed that PHLPP partially re-sensitized MM cells to BTZ. In addition, PHLPP overexpression increased whereas PHLPP knockdown reduced LAMP2 expression, subsequently regulating the autophagy pathway in MM cells. Further findings demonstrated that LAMP2 knockdown reversed PHLPP-mediated cell apoptosis and autophagy activation in MM cells.
CONCLUSIONS: This study demonstrated that PHLPP is a potential strategy for overcoming BTZ resistance in patients with MM.

OBJECTIVE: Loss of endothelial barrier function plays an important role in the development of ventilator-induced lung injury (VILI). This study aimed to investigate the effects of miR135a on VILI in a model of mechanical stretch (MS)-induced human umbilical vein endothelial cell (HUVEC) injury.
METHODS: HUVECs were randomly assigned to 7 groups: blank, negative control (NC), NC+MS, miR135a over-expression (mi-miR135a), mi-miR135a + MS, miR135a silencing (si-miR135a) and si-miR135a + MS groups. MS was induced by subjecting cells to cyclic stretch at 20% stretch for 4 h. After 24 h, levels of reactive oxygen species (ROS) were measured by DCFH-DA fluorescence intensity. Apoptosis was measured using annexin V-FITC/propidium iodide assay with flow cytometry. Inflammatory cytokine levels were determined by ELISA. Barrier integrity was determined using FITC-conjugated dextran assay. Expression levels of PI3K, p-PI3K, Akt, p-Akt, Bcl-2 and Bax were examined using western blotting. The interaction between miR135a and PHLPP2 was evaluated by dual-luciferase reporter assay.
RESULTS: Our results showed that MS reduced cell numbers, increased the number of apoptotic cells, increased ROS, barrier dysfunction and inflammatory cytokines in HUVECs, and reduced p-PI3K and p-Akt expression; silencing of miR135a worsened MS-induced HUVEC injury. However, miR135a over-expression protected HUVECs against MS-induced increases in apoptotic cells, ROS, barrier dysfunction and inflammatory cytokines, which were accompanied by activation of the PI3K/Akt signaling pathway. Simultaneous silencing of miR135a and PHLPP2 partially salvaged the effects of miR135a silencing, and miR135a was found to interact with PHLPP2.
CONCLUSIONS: miR135a may protect HUVECs from MS-induced injury by inhibiting PHLPP2 to activate PI3k/Akt signaling pathway.

Small nucleolar RNAs (snoRNAs) have been implicated in the development of many cancers. We therefore examined the differential expression of snoRNAs between gallbladder cancer (GBC) tissues and matched adjacent non-tumor tissues using expression microarray analysis with confirmation by quantitative real-time PCR (qRT-PCR). Western blot analysis showed that SNORA74B levels were higher in GBC than non-tumor tissues. SNORA74B expression was positively associated with local invasion, advanced TNM stage, CA19-9 level, and Ki67 expression in patients with GBC, while it was negatively associated with expression of PHLPP, an endogenous Akt inhibitor. Moreover, SNORA74B expression was prognostic for overall survival (OS) and disease-free survival (DFS). Functional studies revealed that silencing SNORA74B in GBC cells using sh-SNORA74B suppressed cell proliferation, induced G1 arrest, and promoted apoptosis. Preliminary molecular investigation revealed that SNORA74B silencing inhibited activation of the AKT/mTOR signaling pathway, while increasing PHLPP expression. PHLPP depletion using shRNA abrogated sh-SNORA74B suppression of GBC cell proliferation, indicating that the antitumor effects of SNORA74B silencing were mediated by PHLPP. These findings define the important role of SNORA74B in cell proliferation, cell cycle, and apoptosis of GBC, and suggest that it may serve as a novel target for GBC treatment.

Recent studies have shown that multiple phosphatases deactivate the PI3K/AKT signaling pathway. Here we demonstrated that, by suppressing multiple phosphatases, miR-3127 promotes growth of hepatocellular carcinoma (HCC). Our study also reveals clinical significance of miR-3127 expression in HCC patients. MiR-3127 expression was markedly upregulated in HCC tissues and cells. Furthermore, high miR-3127 expression was associated with an aggressive phenotype and poor prognosis. MiR-3127 overexpression promoted HCC cell proliferation in vitro and tumor growth in vivo. Also, miR-3127 accelerated G1-S transition by activating AKT/ FOXO1 signaling, by directly targeting the 3\' untranslated regions (3`UTR) of pleckstrin homology domain leucine-rich repeat protein phosphatase 1/2 (PHLPP1/2), inositol polyphosphate phosphatase 4A (INPP4A), and inositol polyphosphate-5-phosphatase J (INPP5J) mRNA, repressing their expression. In agreement, the miRNA antagonist antagomir-3127 suppressed HCC cell proliferation and tumor growth by inhibiting the AKT/FOXO1 signaling. Taken together, these findings suggest that silencing miR-3127 might be a potential therapeutic strategy.