circ-BIRC6, a circular RNA, promotes hepatocellular carcinoma progression by targeting the miR-3918/Bcl2 axis

Circular (circ)RNA is a special type of endogenous RNA consisting of a covalently closed loop structure without 5‘ to 3‘ polarity and a polyadenylated tail. Accumulating evidence suggests that circRNAs play important roles in the development and progression of human cancers. However, the role of circRNAs in the progression of hepatocellular carcinoma (HCC) is largely unknown. This was addressed in the present study using high-throughput sequencing to identify aberrantly expressed circRNAs in HCC patient tissue and cell lines. We found that circ-baculoviral IAP repeat-containing (BIRC)6 was upregulated in HCC tissue samples and cells; this was associated with the overall survival of HCC patients. circ-BIRC6 knockdown reduced HCC cell proliferation, migration, and invasion and enhanced their apoptosis. Additionally, circ-BIRC6 overexpression negatively regulated the expression of microRNA miR-3918, which was identified as an inhibitor of B cell lymphoma (Bcl)2. The tumor-suppressive effect of circ-BIRC6 deletion was abrogated by inhibiting miR-3918. These results indicate that circ-BIRC6 functions as a competing endogenous RNA that regulates Bcl2 expression by sponging miR-3918, and may serve as a prognostic biomarker and therapeutic target for the treatment of HCC.</p

<i>circFAM134B</i> is a key factor regulating reticulophagy-mediated ferroptosis in hepatocellular carcinoma

Ferroptosis is an important mode of regulated cell death (RCD). Its inhibition is closely related to therapeutic resistance and poor prognosis in hepatocellular carcinoma (HCC). Previous reports have demonstrated ferroptosis as a biological process highly dependent on selective autophagy, such as ferritinophagy, lipophagy, and clockophagy. Our study also revealed a role for ER-phagy-mediated ferroptosis in HCC cells treated with multi-targeted tyrosine kinase inhibitors (TKIs). In the current study, we found that the homologous circular RNA (circRNA) of the family with sequence similarity 134, member B (FAM134B), hsa_circ_0128505 (was abbreviated as circFAM134B in the present study), was identified to specifically target ER-phagy to promote lenvatinib (LV)-induced ferroptosis using reactive oxygen species (ROS), Fe2+, malondialdehyde (MDA), and western blot (WB) assays in HCC cells. RNA pull-down and mass spectrometry analyses suggested that circFAM134B and FAM134B mRNA were enriched with several common interacting proteins. Among them, poly (A) binding protein cytoplasmic 4 (PABPC4) was identified as the most enriched binding partner. It was proven to be a novel antagonist against the nonsense-mediated mRNA decay (NMD) mechanism. We then applied RNA immunoprecipitation (RIP), RNA pull-down, luciferase reporter, and NMD reporter gene assays to further explore the exact role and underlying mechanism of circFAM134B-PABPC4-FAM134B axis in HCC cells. circFAM134B was confirmed as a sponge that competitively interacted with PABPC4, thereby influencing FAM134B mRNA nonsense decay. Our results provide novel evidences and strategies for the comprehensive treatment of HCC.</p

Meloxicam Executes Its Antitumor Effects against Hepatocellular Carcinoma in COX-2- Dependent and -Independent Pathways

<div><p>Background</p><p>Cyclooxygenase (COX)-2 is overexpressed in many types of cancers including hepatocellular carcinoma (HCC). Meloxicam, a selective COX-2 inhibitor, has shown potential therapeutic effects against HCC, but the mechanisms accounting for its anti-cancer activities remain unclear.</p><p>Methods and Findings</p><p>Meloxicam inhibited the ability of human HCC cells expressing higher levels of COX-2 to migrate, invade, adhere and form colonies through upregulating the expression of E-cadherin and downregulating the expression of matrix metalloproteinase (MMP) -2. Meloxicam induced cell apoptosis by upregulating pro-apoptotic proteins including Bax and Fas-L, and downregulating anti-apoptotic proteins including survivin and myeloid cell leukemia-1 (Mcl-1), through inhibiting phosphorylation of AKT. Addition of prostaglandin E2 (PGE2), the major product of COX-2, could abrogate the effects of meloxicam on the expression of survivin and myeloid cell leukemia-1 (Mcl-1), but not Bax and Fas-L, indicating that meloxicam induces cell apoptosis via both COX-2-dependent and -independent pathways. Meloxicam also induced cell autophagy by upregulating Beclin 1 and light chain 3-II. Specific inhibition of autophagy by 3-methyladenine and chloroquine had little effect on cell apoptosis but could enhance the pro-apoptotic effects of meloxicam by further upregulating the expression of Bax.</p><p>Conclusions</p><p>Meloxicam executes its antitumor effects by targeting the COX-2/MMP-2/E-cadherin, AKT, apoptotic and autophagic pathways in COX-2-dependent and -independent pathways, and inhibition of cell autophagy could help to overcome the resistance to meloxicam-induced apoptosis in HCC.</p></div

The expression of COX-2 and EP2 in HCC cells and meloxicam reduces cell viability <i>in vitro</i>.

<p>(A) The expression of COX-2 in HCC cell lines SMMC-7402, Bel-7402, HepG2, SMMC-7721 and Huh-7 was detected by Western Blotting. GAPDH served as an internal control. (B) Bel-7402, HepG2 and SMMC-7721 cells that express higher levels of COX-2 were incubated with increasing concentrations of meloxicam, and the rates of viability inhibition were measured. (C) The expression of EP2 in the above five HCC cell lines was detected by Western Blotting. GAPDH served as an internal control.</p

Meloxicam inhibits cell migration, invasion, adhesion and colony formation.

<p>(A) Representative photographs were taken from HepG2 cells incubated for 48 h with meloxicam (80 μM) or vehicle (control) and subjected to cell migration, invasion, adhesion and colony formation assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092864#s2" target="_blank">Materials and Method</a>s. (B) The above assays were quantified. Data represent three independent experiments. “**” indicates a highly significant (<i>P</i>&lt;0.001) difference from controls.</p

Meloxicam inhibits phosphorylation of AKT in a COX-2-depdendent way.

<p>(A) HepG2 cells were incubated with meloxicam (80 μM), and harvested at the indicated time points. (B, C) HepG2 cells were incubated for 72 h with meloxicam (80 μM) in the presence and absence of PGE2 (3 μM) (B) or rh-MMP-2 (25 ng/mL) (C). The above harvested cells were subjected to Western blot analysis. The band density in each assay was measured and normalized to that of GAPDH, respectively. Data represent three independent experiments. “**”, indicates a highly significant (<i>P</i>&lt;0.001) difference.</p

Meloxicam induces cell apoptosis via COX-2-dependent and -independent mechanisms.

<p>(A, B) HepG2 cells were incubated for 72 h with meloxicam (80 μM) in the presence or absence of PGE2 (3 μM) or rh-MMP-2 (25 ng/mL). Untreated cells served as controls. (A) Representative dot plots were taken from cytometrically analyzed cells. (B) The apoptosis rate was calculated. (C) HepG2 cells were incubated with meloxicam (80 μM), and harvested at the indicated time points. (D) HepG2 cells were incubated for 72 h with meloxicam (80 μM) in the presence or absence of PGE2 (3 μM) or MK-2206 (5 μM). (E) HepG2 cells were incubated with meloxicam (80 μM), and harvested at the indicated time points. (F) HepG2 cells were incubated with meloxicam (80 μM) in the presence or absence of PGE2 (3 μM) for 72 h, and harvested. The above harvested cells were subjected to Western blot analysis. The band density in each assay was measured and normalized to that of GAPDH, respectively. Data represent three independent experiments. “*” indicates a significant (<i>P</i>&lt;0.05) difference, and “**”, a highly significant (<i>P</i>&lt;0.001) difference.</p

Meloxicam upregulates E-cadherin and downregulates MMP-2 in a COX-2-dependent way.

<p>HepG2 cells were cultured for 72(80 μM), PGE2 (3 μM) or rh-MMP-2 (25 ng/mL), or the combination. Cell lysates were analyzed by Western blot analysis to detect expression of E-cadherin (A, B) and MMP-1/MMP-2 (C) proteins. The band density in each assay was measured and normalized to that of GAPDH, respectively. (D, E) The concentrations of soluble E-cadherin (sE-cad) in supernatants from the above cell culture were measured by ELISA. (F–H) The above cells were lysed and subjected to quantitative real-time RT-PCR for measuring the levels of E-Cadherin (F) and MMP-2 (G) mRNAs, and to a standard RT-PCR assay, in which PCR products of E-Cadherin (I) and MMP-2 (H) were electrophoresed. GAPDH served as an internal control. Data represent three independent experiments. “*” indicates a significant (<i>P</i>&lt;0.05) difference, and “**”, a highly significant (<i>P</i>&lt;0.001) difference.</p

Proposed mechanisms by which meloxicam executes its antitumor effects in COX-2-depdendent and -independent ways.

<p>Meloxicam inhibits the production of PGE2 by inhibiting COX-2 activity. PGE2 binds to EP2 to upregulate the expression of survivin and Mcl-1 via activation of AKT. Meloxicam induces cell apoptosis by upregulating Bax and Fas-L in a COX-2-independent way. 3-MA inhibits the engulfing of Bax by autophagosome, thus blocks the inhibitory effect of autophagy on apoptosis. PGE2 binds to EP2 to upregulate MMP-2, which in turn promotes the cleavage of E-cadherin. Soluble E-cadherin binds to HER/IGF-1R to activate AKT, while full-length of E-cadherin inhibits activation of AKT. “→” indicates positive regulation or activation; “⊥”, negative regulation or blockade; COX-2, cyclooxygenase-2; E-cad, E-cadherin; EP2, prostaglandin E2 receptor; HER, human epidermal growth factor receptor; IGF-1R, insulin-like growth factor-1 receptor; Mcl-1, myeloid cell leukemia-1; MMP-2, matrix metalloproteinase-2; PGE2, prostaglandin E2; 3-MA, 3-methyladenine.</p

Meloxicam induces autophagy and inhibition of autophagy promotes the apoptosis of HepG2 cells.

<p>(A) Representative images were from HepG2 cells that were incubated for 72 h with meloxicam (Mel) (80 μM) in the presence or absence of 3-MA (2 mM), and then stained by acridine orange. Untreated cells served as control (CTL). (B) The above cells from (A) were further subjected to flow cytometry to measure the degree of autophagic lysosomes as expressed by fold change of acridine orange fluorescence intensity (FL3) in treated cells versus control cells. (C) HepG2 cells were incubated with meloxicam (80 μM), and harvested at the indicated time points. (D–F) HepG2 cells were incubated for 72 h with meloxicam (80 μM) in the presence or absence of 3-MA (2 mM). (E) Representative dot plots were taken from cytometrically analyzed cells. (F) The apoptosis rate in (E) was calculated. (G) The above harvested cells were subjected to Western blot analysis. The band density in each assay was measured and normalized to that of GAPDH, respectively. Data represent three independent experiments. “*” indicates a significant (<i>P</i>&lt;0.05) difference, and “**”, a highly significant (<i>P</i>&lt;0.001) difference.</p