Upregulation of LncRNA WT1-AS Inhibits Tumor Growth and Promotes Autophagy in Gastric Cancer via Suppression of PI3K/Akt/mTOR Pathway.

Zhang X ，Jin M ，Yao X ，Liu J ，Yang Y ，Huang J ，Jin G ，Liu S ，Zhang B ... -  《-》

Regulation of hsa_circ_0112136 by m6A demethylase FTO can enhance the malignancy of gastric cancer via the regulation of the PI3K/AKT/mTOR pathway.

A growing body of research highlights the role that N6-methyladenosine (m6A) modification and circular RNAs (circRNAs) play in gastric cancer (GC) cases. However, studies elucidating the function and mechanism of the recently discovered circRNA hsa_circ_0112136 in GC are limited. This study aimed to examine the pathophysiology of GC progression due to fat mass and obesity-associated protein (FTO)-mediated N6-methyladenosine (m6A) modification of hsa_circ_0112136. The hsa_circ_0112136 and FTO levels in the GC samples were analyzed using qRT-PCR. The Transwell invasion assay, wound healing assay, and CCK8 assays were employed to assess alterations in GC cell invasiveness, migration, and viability due to the aberrant regulation of hsa_circ_0112136 and FTO. Phosphorylated PI3K, AKT, and mTOR (the key proteins of the PI3K/AKT/mTOR pathway) were detected via western blotting after hsa_circ_0112136 suppression. A tumor transplantation mouse model was constructed to evaluate the suppression of hsa_circ_0112136's function in vivo. The correlation among hsa_circ_0112136 and FTO was identified using the MeRIP assay. Levels of hsa_circ_0112136 and FTO were evidently elevated in GC samples. Suppression of has_circ_0112136 reduced the viability, migration, and invasive ability of GC cells in vitro, as well as delayed tumor growth in vivo via suppression of the activation of the PI3K/AKT/mTOR pathway. FTO decreased hsa_circ_0112136 m6A levels and enhanced hsa_circ_0112136 expression. Furthermore, FTO upregulation enhanced GC cell invasion, migration, and survival, which was reversed by hsa_circ_0112136 suppression. Our study proposes that hsa_circ_0112136 functions as a tumor promoter, facilitating the malignant progression of GC through m6A modification (suppressed by FTO) and activating the PI3K/AKT/mTOR pathway. This suggests that targeting FTO-m6A-hsa_circ_0112136-PI3K/AKT/mTOR may be a novel approach for GC intervention.

Liu J ，Fang X 《-》

MYBL2 promotes proliferation of clear cell renal cell carcinoma by regulating TOP2A and activating AKT/mTOR signaling pathway.

Renal cell carcinoma (RCC) is one of the most common malignancies in the urinary system, and clear cell renal cell carcinoma (ccRCC) is the most common subtype. MYBL2 has been reported to be overexpressed in various tumors and associated with poor prognosis in patients, but its biological role in ccRCC remains unclear. In this study, we investigated the mRNA and protein expression levels of MYBL2 in ccRCC samples and evaluated the prognostic value of MYBL2 using TCGA dataset. In vitro functional assays were performed using CCK-8, EdU, colony formation, cell scratch, and transwell assays, as well as in vivo tumorigenesis assays to investigate the biological functions of MYBL2 in ccRCC. Additionally, gene set enrichment analysis (GSEA) was used to explore the downstream pathways of MYBL2, which were further validated. Finally, we predicted the target genes of MYBL2 using bioinformatics and validated them using ChIP and dual-luciferase reporter gene assays. MYBL2 expression was significantly higher in ccRCC than in adjacent normal tissues and was associated with poor prognosis. MYBL2 expression was positively correlated with the pathological tumor grade and clinical TNM stage of ccRCC patients. Knockdown of MYBL2 significantly inhibited the proliferation of renal cancer cells in vitro and in vivo, and knockdown of MYBL2 could inhibit cell invasion and migration, while overexpression of MYBL2 had the opposite effect. GSEA revealed that MYBL2 was associated with the mTOR signaling pathway and cell cycle pathway, which was confirmed by our study. Finally, we found that TOP2A was a target gene of MYBL2, and MYBL2 could bind to the TOP2A promoter to regulate its transcriptional activity, promoting the proliferation of clear cell renal cell carcinoma cells. MYBL2 emerges as a highly expressed factor that significantly correlates with adverse patient prognosis in ccRCC. Mechanistically, MYBL2 transcriptionally upregulates TOP2A, thereby modulating the proliferation of ccRCC cells. Furthermore, MYBL2 activates the mTOR signaling pathway, a critical node in the progression of ccRCC. Collectively, these findings position MYBL2 as a promising candidate for both a biological marker and a therapeutic target in the management of ccRCC.

Huang Y ，Xi X ，Ye Z ，Zhang C ，Jiang Y ，Yu F ，Huang G ... -  《-》

Non-lethal sonodynamic therapy mitigates hypertensive renal fibrosis through the PI3K/AKT/mTORC1-autophagy pathway.

Hypertension constitutes a significant public health concern, characterized by a high incidence and mortality rate. Hypertensive kidney disease is a prevalent complication associated with hypertension and is the second leading cause of end-stage renal disease (ESRD). Renal fibrosis linked to hypertension has emerged as the third leading cause of disease in dialysis patients. Autophagy activity is crucial for maintaining homeostasis, vitality, and physiological function of kidney cells, while also protecting the kidneys from fibrosis. The deficiency of autophagy will increase the sensitivity of the kidney to the damage, leading to impaired renal function, accumulation of damaged mitochondria and more severe of renal fibrosis. However, enhancing autophagy by activating the PI3K/AKT, AMPK, and mTOR pathways, improves podocyte injury and renal pathological changes, and ameliorates renal function. Current clinical interventions aimed at halting or reversing renal fibrosis in hypertensive patients are notably limited in their efficacy. Here, we present Non-lethal Sonodynamic Therapy (NL-SDT), in which ultrasound is used to activate locally sonosensitizers, thereby stimulating the production of reactive oxygen species for the purpose of modulating cell function or fate, as a novel methodology to inhibit progression of hypertensive renal fibrosis.To confirm whether NL-SDT can reduce hypertensive renal fibrosis and its mechanism. The mice model of hypertensive renal fibrosis was established by using osmotic minipumps (Alzet model 2004, Cupertino, CA) equipped with angiotensin-II (Ang II). The pumps were implanted in mice, ensuring constant infusion of Ang II at a dose of 1.0 µg/kg per minute for 4 weeks. The mice were exposed to 0.4 W/cm2 intensity ultrasonic radiation for 15 min at 4 h post injection of sinoporphyrin sodium (DVDMS) (4 mg/kg) into the caudal vein was repeated weekly for 4 treatments. The kidney from mice was stained with masson's trichrome staining for collagen fiber expression, while alpha-smooth muscle actin (α-SMA) expression was determined via immunohistochemical staining. The protein levels of fibrosis parameters (α-SMA, collagen I, vimentin), pathway-related proteins (PI3K, AKT, mTORC1) and autophagy-related protein LC3B were determined using western blotting. Intracellular reactive oxygen species (ROS) levels were detected using DCFH-DA probe. Immunofluorescence was also used to observe the expression of α-SMA and E-cadherin in cells. Pathway-related protein inhibitors (the autophagy-related inhibitor 3-methyladenine (3-MA), chloroquine (CQ), ROS inhibitor N-acetyl-L-cysteine (NAC) were applied, and autophagosome changes were observed under transmission electron microscopy. Immunofluorescence was used to observe LC3 spot formation within cells.We obtained the following results via animal and cellular research. In vivo, (1) The collagen area of renal tissue was increased significantly in Ang II group (50.6%). The positive expression of α-SMA was increased significantly (37.8%). (2) The collagen area decreased after NL-SDT treatment (34.8%). The expression of α-SMA was decreased too (48.9%). The expression of LC3B increased in NL-SDT group. (3) The effect of NL-SDT on reducing renal fibrosis can be changed by rapamycin and CQ. In vitro. (1) The expression of α-SMA, collagen I and vimentin were increased significantly in TGF-β1-induced NRK-52E cells. (2) The increase of autophagosomes was observed in TGF-β1-induced NRK-52E cells after NL-SDT. The levels of ROS were increased after NL-SDT (24.8%). The effect of NL-SDT on autophagy was reversed after administration of NAC. The expression of PI3K, P-AKT and P-mTORC1 was decreased in TGF-β1-induced NRK-52E cells after NL-SDT. NL-SDT inhibited the transition of epithelial cells into myofibroblasts by activating PI3K-AKT-mTORC1-autophagy pathway in TGF-β1-induced NRK-52E cells. (3) The administration of the pathway inhibitors showed a reciprocal effect on NL-SDT-inhibited epithelial-mesenchymal transition (EMT).(1) NL-SDT reduced blood pressure temporarily in mice model of hypertensive renal fibrosis induced by Ang II. (2) NL-SDT alleviated renal fibrosis in mice model of hypertensive renal fibrosis induced by Ang II. (3) NL-SDT promoted autophagy by inhibiting PI3K-AKT-mTORC1 signaling pathway and alleviated renal fibrosis in mice model of hypertensive renal fibrosis induced by Ang II. NL-SDT is a non-invasive and efficacious regimen to inhibit renal fibrosis. It may be a new approach for clinical treatment of renal fibrosis, delaying or reducing the occurrence of ESRD.

Liu D ，Wang H ，Li J ，Sheng S ，Wang S ，Tian Y ... -  《Scientific Reports》

Upregulated SAE1 Drives Tumorigenesis and Is Associated with Poor Clinical Outcomes in Breast Cancer.

The purpose of this study was to analyze SUMO activating enzyme subunit 1 (SAE1) expression in breast cancer (BC). Through bioinformatics analysis and in vitro experiments, the biological function and possibly associated signal pathways of SAE1 in BC were further analyzed. Bioinformatics analysis was applied to analyze SAE1 expression in BC and normal breast tissues, its relationship with clinicopathologic characteristics and prognosis in BC patients, and data from the Cancer Genome Atlas database and Gene Expression Omnibus dataset. We performed immunohistochemistry to analyze SAE1 expression in BC tissues and para-cancer tissues in 79 breast cancer patients. BC cell proliferation was detected with the Cell Counting Kit-8 and by the colony formation assay. Cell cycle progression was analyzed by flow cytometry, and the expression of cell cycle-related proteins (E2F1, cyclin D3, and cyclin-dependent kinase 2) was determined by western blots in SAE1 small interfering RNA (siRNA) transfected cells. The GSE1456 dataset was used to analyze possible signal pathways associated with SAE1 by gene set enrichment analysis (GSEA), and the expression of PI3K/AKT/mTOR pathway-related proteins (such as p-PI3K, p-AKT, and mTOR) in SAE1-siRNA cells was detected by western blots. The bioinformatics and immunohistochemical results showed that SAE1 mRNA and protein expression in BC tissues were significantly higher than those in normal tissues. The SAE1 overexpression was significantly associated with the tumor size, tumor-node-metastasis stage, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, and whether or not it was a triple-negative BC. Patients with SAE1 overexpression had a worse overall survival (OS), recurrence-free survival (RFS), and distant metastasis-free survival compared with lower expression patients. Multivariate Cox regression analysis showed that SAE1 may be an independent prognostic factor for OS of BC patients. The proliferation and cell cycle process of BC cells were inhibited by SAE1-siRNA in vitro. The result of GSEA showed that SAE1 was significantly associated with 12 gene sets, including unfolded protein reaction, DNA repair, oxidative phosphorylation, and cell cycle, among others. Additionally, two signal pathways, mTORC1 and PI3K/Akt/mTOR, were significantly correlated with SAE1 overexpression. Western blots confirmed that the expression of PI3K/Akt/mTOR pathway-related proteins (p-PI3K, p-AKT, and mTOR) in BC cells was decreased after knocking down SAE1. SAE1 was highly expressed in BC. Its overexpression was associated with poor BC prognosis. Additionally, it was an independent prognostic factor for BC patients. We demonstrated that in vitro SAE1 knockdown effectively inhibited BC proliferation and its cell cycle process. Furthermore, the biological function of SAE1 may be associated with the PI3K/Akt/mTOR pathway. SAE1 will be a potential target for BC treatment.

Liu H ，Wang J ，Li Y ，Luo F ，Xing L ... -  《-》