Chitinase 3 like-1 activates the Akt pathway, inducing NF-κB-dependent release of pro-inflammatory cytokines and promoting the proliferative ability in nasopharyngeal carcinoma cells.

BACKGROUND: Chitinase 3 like-1 (CHI3L1) has been reported to function as an oncogene in many types of cancer. However, the biological function of CHI3L1 in nasopharyngeal carcinoma (NPC) remains unknown. METHODS: Differentially expressed genes (DEGs) in NPC tissues in GSE64634 and GSE12452 were downloaded from Gene Expression Omnibus (GEO). CHI3L1, interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α) mRNA expression was examined by qRT-PCR. Cell proliferation was evaluated by CCK-8 and EdU incorporation assays. Western blot analysis was used to measure the changes of CHI3L1, nuclear factor-κappaB (NF-κB), and protein kinase B (Akt) pathways. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway analyses were performed using DAVID database. RESULTS: We identified 3 overlapping DEGs using Draw Venn diagram, among which CHI3L1 was chosen for the following analyses. CHI3L1 was upregulated in NPC tissues and cells. CHI3L1 silencing suppressed inflammatory response by inactivating the NF-κB pathway and inhibited cell proliferation in NPC cells. On the contrary, CHI3L1 overexpression induced inflammatory response by activating the NF-κB pathway and promoted cell proliferation in NPC cells. According to GO and KEGG analyses, CHI3L1 positive regulates Akt signaling and is enriched in the PI3K-Akt pathway. CHI3L1 knockdown inhibited the Akt pathway, and CHI3L1 overexpression activated the Akt pathway in NPC cells. Akt overexpression abolished the effects of CHI3L1 knockdown on inflammatory response, NF-κB pathway, and proliferation in NPC cells. On the contrary, Akt knockdown abolished the effects of CHI3L1 overexpression on inflammatory response, NF-κB pathway, and proliferation in NPC cells. CONCLUSION: CHI3L1 knockdown inhibited NF-κB-dependent inflammatory response and promoting proliferation in NPC cells by inactivating the Akt pathway.

Tumor suppressor LHX6 upregulation contributes to the inhibitory effect of miR-346 knockdown on colorectal cancer cell growth.

Colorectal cancer (CRC) is one of the prevalent types of human malignancies and ranks as the second leading cause of cancer-associated death worldwide. Dysregulated miRNAs have been promulgated as oncogenes or tumor-suppressive genes participating in the initiation and progression of CRC. A recent study reported that miR-346 was highly expressed in CRC patients. However, the biological role and underlying mechanism of miR-346 in CRC remain elusive. qRT-PCR and western blot assays were employed to detect miR-346 and LIM homeobox domain 6 (LHX6) expression in CRC cells. Cell proliferation was evaluated by CCK-8 and BrdU assays. Apoptosis was evaluated by TUNEL assay. The interaction between miR-346 and LHX6 was assessed by luciferase reporter assay. Results showed that miR-346 expression was increased and LHX6 expression was reduced in CRC cells. miR-346 knockdown and LHX6 overexpression inhibited proliferation and promoted apoptosis of CRC cells. Additionally, we found that miR-346 negatively regulated LHX6 expression in CRC cells by directly targeting LHX6. LHX6 knockdown partially attenuated anti-miR-346-induced proliferation reduction and apoptosis promotion in CRC cells. Furthermore, miR-346 knockdown inhibited the protein kinase B (Akt)/mechanistic target of rapamycin (mTOR) pathway in CRC cells by targeting LHX6. The present study indicated that miR-346 knockdown repressed cell growth in CRC cells by upregulating LHX6, and this was associated with inactivation of the Akt/mTOR pathway.

CASK regulates Notch pathway and functions as a tumor promoter in pancreatic cancer.

Calcium/calmodulin-dependent serine protein kinase (CASK), a member of membrane-associated guanylate kinase (MAGUK) super-family, is implicated in regulating cell proliferation, cytoskeletal remodeling, and cell metastasis. Our study aimed to investigate the effect of CASK on the malignant behaviors of pancreatic cancer cells and to determine the signaling pathway involved. CASK expression in pancreatic cancer tissues based on the TCGA database was analyzed using GEPIA online tool. The overall survival (OS) and disease-free survival (DFS) in patients with pancreatic cancer based on CASK expression was also analyzed using GEPIA. KEGG pathway enrichment analysis was used to show the association of 1522 CASK-related genes and signaling pathways. The expression of CASK, Notch1 and Hey1 was detected by Western blot. Cell proliferation, colony number, invasion, and apoptosis were detected by CCK-8, colony formation assay, Transwell invasion assay, and flow cytometry analysis, respectively. Results showed that CASK was upregulated in pancreatic cancer tissues and cells. Pancreatic cancer patients with high CASK expression showed shorter OS and DFS than patients with low CASK expression. KEGG pathway enrichment analysis proved that CASK and 1522 CASK-associated genes were primarily associated with the Notch pathway. CASK silencing inhibited cell proliferation, colony formation ability, and invasion and elicited apoptosis in pancreatic cancer cells. Additionally, we confirmed that CASK silencing inhibited the Notch pathway in pancreatic cancer cells. Overexpression of Notch1 resisted the anti-tumor functions of CASK knockdown in pancreatic cancer cells. In conclusion, CASK knockdown suppressed the malignant behaviors of pancreatic cancer cells by inactivating the Notch pathway.

The effects of hsa_circ_0000517/miR-326 axis on the progression of breast cancer cells and the prediction of miR-326 downstream targets in breast cancer.

It has been proposed that circular RNAs (circRNAs) play crucial roles in the initiation and progression of various cancers including breast cancer. Our study aimed to determine the function and regulatory mechanism of hsa_circ_0000517 in breast cancer. qRT-PCR was applied to determine hsa_circ_0000517 expression in breast cancer cells. The circular structure of hsa_circ_0000517 was confirmed using RNase R digestion assay. The subcellular distribution of hsa_circ_0000517 was analyzed using nuclear mass separation assay. Effects of hsa_circ_0000517 on the malignant behaviors of breast cancer cells were determined using CCK-8, colony formation assay, flow cytometry analysis, caspase-3 activity assay, and Transwell invasion assay. Bioinformatics analysis, luciferase reporter assay, and RIP were used to predict and confirm the interaction between hsa_circ_0000517 and miR-326. Bioinformatics analysis was used to search the possible targets of miR-326. Hsa_circ_0000517 was upregulated in breast cancer tissues and cells. Hsa_circ_0000517 was a stable circularized transcript that was preferentially distributed in the cytoplasm. Hsa_circ_0000517 knockdown inhibited cell proliferation, colony formation ability, and invasion and triggered apoptosis in breast cancer cells. Hsa_circ_0000517 acted as a sponge of miR-326 to suppress its expression. miR-326 inhibition abolished the effects of hsa_circ_0000517 knockdown on the malignant behaviors of breast cancer cells. Totally 17 genes were identified as the potential targets of miR-326 in breast cancer. In conclusion, hsa_circ_0000517 silencing repressed breast cancer progression by upregulating miR-326 expression.

Blocking VEGF signaling augments interleukin-8 secretion via MEK/ERK/1/2 axis in human retinal pigment epithelial cells.

AIM: To identify proangiogenic factors engaged in neovascular age-related macular degeneration (AMD) except vascular endothelial growth factor (VEGF) from human retinal pigment epithelial (hRPE) cells and investigate the underlying mechanisms. METHODS: VEGF receptor 2 (VEGFR2) in ARPE-19 cells was depleted by siRNA transfection or overexpressed through adenovirus infection. The mRNA and the protein levels of interleukin-8 (IL-8) in ARPE-19 cells were measured by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. The protein levels of AKT, p-AKT, MEK, p-MEK, ERK1/2, p-ERK1/2, JNK, p-JNK, p38 and p-p38 were detected by Western blotting. A selective chemical inhibitor, LY3214996, was employed to inhibit phosphorylation of ERK1/2. Cell viability was determined by MTT assay. RESULTS: Knockdown of VEGFR2 in ARPE-19 cells robustly augmented IL-8 production at both the mRNA and the protein levels. Silencing VEGFR2 substantially enhanced phosphorylation of MEK and ERK1/2 while exerted no effects on phosphorylation of AKT, JNK and p38. Inhibiting ERK1/2 phosphorylation by LY3214996 reversed changes in VEGFR2 knockdown-induced IL-8 upregulation at the mRNA and the protein levels with no effects on cell viability. VEGFR2 overexpression significantly reduced IL-8 generation at the mRNA and the protein levels. CONCLUSION: Blockade of VEGF signaling augments IL-8 secretion via MEK/ERK1/2 axis and overactivation of VEGF pathway decreases IL-8 production in hRPE cells. Upregulated IL-8 expression after VEGF signaling inhibition in hRPE cells may be responsible for being incompletely responsive to anti-VEGF remedy in neovascular AMD, and IL-8 may serve as an alternative therapeutic target for neovascular AMD.

Celastrol Protects RPE Cells from Oxidative Stress-Induced Cell Death via Activation of Nrf2 Signaling Pathway.

PURPOSE: Oxidative stress to retinal pigment epithelial (RPE) cells and inflammation are closely related to the pathogenesis of age-related macular degeneration (AMD). Celastrol is a natural compound isolated from the root of Tripterygium wilfordii. Celastrol has been shown to have potent anti-inflammatory and anti-tumor effects in multiple disease models. The objective of this study was to test the anti-oxidative effects of celastrol in RPE cells and to investigate the underlying mechanisms. METHODS: ARPE-19 cells were treated with hydrogen peroxide (H2O2) and menadione alone or in combination with celastrol. Cell viability and apoptosis were examined by CCK-8 and TUNEL assay, respectively. The expression of Nrf2 and its target genes, such as GCLM and HO-1 was determined by Western blotting. The knockdown of Nrf2 was done by transfecting ARPE-19 cells with lentivirus encoding shRNA against Nrf2. The knockdown efficiency was determined by real-time quantitative PCR and Western blotting. RESULTS: Treatment of ARPE-19 cells with celastrol significantly attenuated the toxic effects of both H2O2 and menadione. Treatment with celastrol enhanced the expression of transcription factor Nrf2 and its targets, GCLM and HO-1. Knockdown of Nrf2 expression by shRNA partially abolished the protective effects of celastrol. Chemical inhibition of glutathione synthesis by L-buthionine-S,R-sulfoximine (BSO) completely abolished the protective effects of celastrol against H2O2 and menadione-induced damage. However, chemical inhibition of HO-1 activity by ZnPPIX did not reduce the protective effects of celastrol. CONCLUSION: This study provides evidence that treatment of RPE cells with celastrol shows potent protective effects against oxidative insults via activation of Nrf2 signaling pathway and upregulation of GCLM expression. This finding suggests that celastrol might be used as a potential therapeutic agent for oxidative stress-related eyes diseases, such as AMD.

Celastrol Ameliorates Inflammation in Human Retinal Pigment Epithelial Cells by Suppressing NF-κB Signaling.

PURPOSE: Celastrol is a triterpenoid quinine methide that exerts important biological effects on a variety of disease models. In this study, we aim to assess the ability of celastrol to inhibit lipopolysaccharide (LPS)-induced inflammation in retinal pigment epithelial (RPE) cells. METHODS: Primary cultures of human RPE (HRPE) cells and ARPE-19 cell lines were treated with celastrol alone or in combination with LPS. The cytotoxic effect of celastrol on RPE cells was determined by the CCK-8 assay. Protein and mRNA levels of inflammatory cytokines, including IL-6, IL-8, and MCP-1, were detected by flow cytometry or by real-time fluorescent quantitative PCR, respectively. The levels of phosphorylated intermediates in the NF-κB signaling pathway (such as IκBα/ß and p65) and MAPK signaling pathway (p38MAPK, SAPK/JNK, and p42/p44MAPK) were detected by western blotting. RESULTS: Celastrol significantly inhibited LPS-induced expression of protein and mRNA expression levels encoding the proinflammatory cytokines, IL-6, IL-8, and MCP-1, in both HRPE and ARPE-19 cells. Cell viability and apoptosis assays revealed that celastrol had no apparent cytotoxic effect and it inhibited apoptosis of RPE cells at concentrations of less than 1 µM. Mechanistically, RPE cells that were pretreated with celastrol exhibited a substantial decrease in phosphorylation of the NF-κB pathway regulators, IKKα/ß and IκBα, and subsequently inactivated P65, suggesting that celastrol ameliorates LPS-induced inflammation by suppressing the NF-κB signaling pathway. CONCLUSION: Our results provide evidence that celastrol is a potent anti-inflammatory agent in RPE cells and it may have potential applications in prevention and treatment of age-related macular degeneration.