Circ-CCT2 Activates Wnt/ß-catenin Signaling to Facilitate Hepatoblastoma Development by Stabilizing PTBP1 mRNA.

BACKGROUND & AIMS: Circ-CCT2 (hsa_circ_0000418) is a novel circular RNA that stems from the CCT2 gene. However, the expression of circ-CCT2 and its roles in hepatoblastoma are unknown. Our study aims to study the circ-CCT2 roles in hepatoblastoma development. METHODS: Hepatoblastoma specimens were collected for examining the expression of circ-CCT2, TAF15, and PTBP1. CCK-8 and colony formation assays were applied for cell proliferation analysis. Migratory and invasive capacities were evaluated through wound healing and Transwell assays. The interaction between circ-CCT2, TAF15, and PTBP1 was validated by fluorescence in situ hybridization, RNA pull-down, and RNA immunoprecipitation. SKL2001 was used as an agonist of the Wnt/ß-catenin pathway. A subcutaneous mouse model of hepatoblastoma was established for examining the function of circ-CCT2 in hepatoblastoma in vivo. RESULTS: Circ-CCT2 was significantly up-regulated in hepatoblastoma. Overexpression of circ-CCT2 activated Wnt/ß-catenin signaling and promoted hepatoblastoma progression, whereas knockdown of circ-CCT2 exerted opposite effects. Moreover, both TAF15 and PTBP1 were up-regulated in hepatoblastoma tissues and cells. TAF15 was positively correlated with the expression of circ-CCT2 and PTBP1 in hepatoblastoma. Furthermore, circ-CCT2 recruited and up-regulated TAF15 protein to stabilize PTBP1 mRNA and trigger Wnt/ß-catenin signaling in hepatoblastoma. Overexpression of TAF15 or PTBP1 reversed knockdown of circ-CCT2-mediated suppression of hepatoblastoma progression. SKL2001-mediated activation of Wnt/ß-catenin signaling reversed the anti-tumor effects of silencing of circ-CCT2, TAF15, or PTBP1. CONCLUSIONS: Circ-CCT2 stabilizes PTBP1 mRNA and activates Wnt/ß-catenin signaling through recruiting and up-regulating TAF15 protein, thus promoting hepatoblastoma progression. Our findings deepen the understanding of hepatoblastoma pathogenesis and suggest potential therapeutic targets.

LncRNA OIP5-AS1 aggravates the stemness of hepatoblastoma through recruiting PTBP1 to increase the stability of ß-catenin.

BACKGROUND: Hepatoblastoma is a malignancy that occurs in the liver, most of which occur in children younger than 3 years old. It was reported that lncRNA OIP5-AS1 was up-regulated in hepatoblastoma, but the detailed mechanism by which OIP5-AS1 regulates hepatoblastoma development is unclear. METHODS: qRT-PCR, Western blotting, and immunofluorescence were used to examine levels of OIP5-AS1, PTBP1, ß-catenin or proliferation/stemness-related molecules. Colony formation, sphere formation, wound healing assay and transwell were applied to detect cell proliferation, stemness and invasion, respectively. RIP assay was used to investigate the interaction of OIP5-AS1/PTBP1 and PTBP1/CTNNB1. Finally, in vivo model was constructed to detect the function of OIP5-AS1 in hepatoblastoma. RESULTS: OIP5-AS1 was significantly up-regulated in hepatoblastoma cells. OIP5-AS1 silencing notably attenuated the stemness and invasion of hepatoblastoma cells. OIP5-AS1 bound with PTBP1, and silencing of OIP5-AS1 inhibited ß-catenin. Meanwhile, overexpression of PTBP1 or ß-catenin activation significantly reversed OIP5-AS1 silencing-inhibited hepatoblastoma cell proliferation and stemness. Moreover, ß-catenin was found to be the downstream target of PTBP1, and OIP5-AS1 activated ß-catenin signaling via promoting the binding between PTBP1 and ß-catenin to increase the mRNA stability of ß-catenin. Finally, OIP5-AS1 knockdown significantly alleviated the tumor growth of hepatoblastoma by repressing ß-catenin. CONCLUSION: OIP5-AS1 silencing inhibits the growth and stemness of hepatoblastoma through binding with PTBP1 to inhibit ß-catenin signaling pathway. OIP5-AS1 may be the potential target against hepatoblastoma.

Carnitine promotes recovery from oxidative stress and extends lifespan in C. elegans.

Carnitine is required for transporting fatty acids into the mitochondria for ß-oxidation. Carnitine has been used as an energy supplement but the roles in improving health and delaying aging remain unclear. Here we show in C. elegans that L-carnitine improves recovery from oxidative stress and extends lifespan. L-carnitine promotes recovery from oxidative stress induced by paraquat or juglone and improves mobility and survival in response to H2O2 and human amyloid (Aß) toxicity. L-carnitine also alleviates the oxidative stress during aging, resulting in moderate but significant lifespan extension, which was dependent on SKN-1 and DAF-16. Long-lived worms with germline loss (glp-1) or reduced insulin receptor activity (daf-2) recover from aging-associated oxidative stress faster than wild-type controls and their long lifespans were not further increased by L-carnitine. A new gene, T08B1.1, aligned to a known carnitine transporter OCTN1 in humans, is required for L-carnitine uptake in C. elegans. T08B1.1 expression is elevated in daf-2 and glp-1 mutants and its knockdown prevents L-carnitine from improving oxidative stress recovery and prolonging lifespan. Together, our study suggests an important role of L-carnitine in oxidative stress recovery that might be important for healthy aging in humans.

Effects of licorice extracts on the pharmacokinetics of brucine in rats and its possible mechanism.

The detoxification effects of licorice are believed to be related to its pharmacokinetic (PK) interference. This paper aimed to evaluate the effects of licorice water extracts (LWE) on the pharmacokinetics of brucine. Rats were administered brucine and/or LWE. The pharmacokinetic behavior of brucine and bioactive components of licorice were quantified by HPLC-MS/MS. P-glycoprotein (P-gp) inhibitor verapamil, real time PCR, vesicular transport assay and everted gut sacs were employed to investigate its possible mechanism. We found LWE reduced the Cmax and AUC of oral brucine in a dose-dependent way. In contrast, the AUC values of intraperitoneal brucine showed no significant difference between LWE treated and untreated rats, which indicating the intestinal absorption of brucine was influenced by LWE. We found that high dose of LWE activated the transport activity of P-gp in vesicular transport assay, while the mRNA level of P-gp in the intestinal was not affected by licorice. Moreover, high dose of LWE decreased the intestinal absorption of brucine in the everted gut sacs model, which could over turned by verapamil. These results suggested that a single high dose of LWE could impair the intestine absorption of brucine, and its potential mechanism may be mediated by P-gp in intestine.

Long non-coding RNA HULC as a diagnostic and prognostic marker of pancreatic cancer.

BACKGROUND: Long non-coding RNA (lncRNA) is abnormally expressed in various malignant tumors. In recent years, it has been found that IncRNA HULC is increasingly expressed in pancreatic cancer tissues and is involved in the development and progression of pancreatic cancer. However, the clinical value of serum HULC in pancreatic cancer remains unclear, and there are few studies on how HULC regulates the biological function of pancreatic cancer cells. AIM: To determine the value of lncRNA HULC in the diagnosis and prognosis of pancreatic cancer, and its possible biological potential. METHODS: Sixty patients with pancreatic cancer and sixty patients with benign pancreatic diseases admitted to Xiangya Hospital, Central South University were assigned to the pancreatic cancer group and the benign disease group, respectively, and another 60 healthy subjects were enrolled as the normal group during the same period. HULC-siRNA and NC-siRNA were transfected into pancreatic cancer cells. Quantitative real-time polymerase chain reaction was performed to determine the expression of HULC in tissues, serum, and cells. Western Blot was carried out to determine the expression of ß-catenin, c-myc, and cyclin D1 in cells, and the cell counting kit-8, flow cytometry, and Transwell assay were conducted to determine the proliferation, apoptosis and invasion of cells. RESULTS: Highly expressed in the tissues and serum of pancreatic cancer patients, HULC showed good clinical value in distinguishing between patients with pancreatic cancer, patients with benign pancreatic diseases and healthy subjects. HULC was related to pathological parameters including tumor size, T staging, M staging and vascular invasion, and the area-under-the-curve for evaluating these four parameters was 0.844, 0.834, 0.928 and 0.818, respectively. Patients with low expression of HULC had a significantly higher 3-year overall survival (OS) and 5-year OS than those with high expression. T staging, M staging, vascular invasion, and HULC were independent prognostic factors affecting the 3-year OS of patients with pancreatic cancer. Inhibition of HULC expression prevented the proliferation and invasion of pancreatic cancer cells, promoted apoptosis, and inhibited the expression of Wnt/ß-catenin signaling pathway-related proteins, ß-catenin, c-myc, and cyclin D1. The Wnt/ß-catenin signaling pathway agonist (LiCl) restored proliferation, apoptosis, and invasion of pancreatic cancer cells with inhibited expression of HULC. CONCLUSION: HULC is an effective marker for the diagnosis and prognosis of pancreatic cancer, which may affect the biological function of pancreatic cancer cells through the Wnt/ß-catenin signaling pathway.

Hypoxia-induced shedding of MICA and HIF1A-mediated immune escape of pancreatic cancer cells from NK cells: role of circ_0000977/miR-153 axis.

One key to malignant progression of pancreatic cancer (PC) is the acquired ability of tumour cells to escape immune-mediated lysis. Hypoxic microenvironment plays a causal role in PC metastasis. According to previous studies, hypoxia could induce the upregulation of HIF1A, ADAM10 and sMICA, leading to decreased NKG2D in NK cells and tumour cells escape from immune surveillance and NK cell-mediated lysis. In the present study, in NK cells derived from high-HIF1A expression patients, the levels of internalization of MICA/B and NKG2D were obviously higher than those in low-HIF1A expression group; hypoxia dramatically upregulated the levels of sMICA culture supernatant of Panc-1 cells. Regarding the molecular mechanism, dysregulated circRNAs and miRNAs that might modulate HIF1A-mediated immune escape were selected and examined for detailed functions. The expression of circ_0000977 could be induced by hypoxia, and circ_0000977 knockdown enhanced the killing effect of NK cells on PC cells under hypoxia through HIF1A and ADAM10. HIF1 and ADAM10 were direct downstream targets of miR-153; circ_0000977 served as a sponge for miR-153 to counteract miR-153-mediated repression of HIF1 and ADAM10 mRNA through direct targeting in both 293T cells and Panc-1 cells. miR-153 inhibition exerted an opposing effect on HIF1A-mediated immune escape of PC cells to circ_0000977 knockdown; the effect of circ_0000977 knockdown were partially attenuated by miR-153 inhibition. In summary, circ_0000977/miR-153 axis modulates HIF1A-mediated immune escape of PC cells through miR-153 downstream targets HIF1A and ADAM10. We provided a novel mechanism of HIF1A-mediated immune escape of PC cells from the perspective of circRNAs-miRNA-mRNA axis. Abbreviations: Pancreatic cancer (PC); peripheral blood lymphocytes (PBLs); A Disintegrin and Metalloproteinase Domain 10 (ADAM10); MHC class I-related molecule A (MICA); soluble MICA (sMICA); membrane MICA (mMICA); Hypoxia-inducible factor 1-alpha (HI1FA); long non-coding RNAs (lncRNAs); non-coding RNAs (ncRNAs); natural killer (NK); Haematoxylin and eosin (H&E); Immunohistochemistry (IHC); natural killer group 2 member D (NKG2D).

Long noncoding RNA FEZF1-AS1 predicts poor prognosis and modulates pancreatic cancer cell proliferation and invasion through miR-142/HIF-1α and miR-133a/EGFR upon hypoxia/normoxia.

Nowadays, pancreatic cancer (PC) remains the most lethal tumor, partially due to the invasive and treatment-resistant phenotype induced by the extent of hypoxic stress within the tumor tissue. According to previous studies, miR-142/HIF-1α and miR-133a/EGFR could modulate PC cell proliferation under hypoxic and normoxic conditions, respectively. In the present study, FEZF1-AS1, a recently described oncogenic long noncoding RNA, was predicted to target both miR-142 and miR-133a; thus, we hypothesized that FEZF1-AS1 might affect PC cell proliferation through these two axes under hypoxic or normoxic conditions. In PC cell lines, FEZF1-AS1 acted as an oncogene via promoting PC cell proliferation and invasion through miR-142/HIF-1α axis under hypoxic condition; however, FEZF1-AS1 failed to affect the protein levels of HIF-1α and VEGF under the normoxic condition, suggesting the existence of another signaling pathway under normoxic condition. As predicted by an online tool, FEZF1-AS1 could target miR-133a to inhibit its expression; under the normoxic condition, FEZF1-AS1 exerted its effect on PC cell lines through miR-133a/EGFR axis. Taken together, FEZF1-AS1 might be a promising target in controlling the aberrant proliferation and invasion of PC cell lines.

RELA/NEAT1/miR-302a-3p/RELA feedback loop modulates pancreatic ductal adenocarcinoma cell proliferation and migration.

Pancreatic ductal adenocarcinoma (PDAC) remains a challenging malignancy due to distant metastasis. RELA, a major component of the NF-κB pathway, could serve as an oncogene through activating proliferation or migration-related gene expression, including NEAT1, a well-known oncogenic long noncoding RNA. In the current study, the expression and function of RELA and NEAT1 in PDAC were examined. The potential upstream regulatory microRNAs of RELA were screened and verified for their correlation with RELA and NEAT1. The expression and function of the selected miR-302a-3p were evaluated. RELA and NEAT1 expression were upregulated in PDAC tissues, particularly in PDAC tissues with lymph node metastasis, and their expression correlated with clinical parameters. RELA overexpression promoted PDAC cell proliferation and migration, which could be partially attenuated by the NEAT1 knockdown. By binding to RELA, miR-302a-3p inhibited RELA expression, as well as PDAC cell proliferation and migration. RELA downstream NEAT1 expression was negatively regulated by miR-302a-3p; the suppressive effect of NEAT1 knockdown on PDAC cell proliferation and migration was partially attenuated by miR-302a-3p inhibition. Moreover, through direct binding, the expression of miR-302a-3p was also negatively regulated by NEAT1. The expression of miR-302a-3p was downregulated and negatively correlated with RELA or NEAT1 in tissue samples, indicating that rescuing miR-302a-3p expression may inhibit PDAC cell proliferation and migration through RELA/NEAT1. In summary, RELA, NEAT1, and miR-302a-3p form a feedback loop in PDAC to modulate PDAC cell proliferation and migration.