Jianni Qi

The miR-545/374a cluster encoded in the Ftx lncRNA is overexpressed in HBV-related hepatocellular carcinoma and promotes tumorigenesis and tumor progression.

Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC). Previous studies have shown several long noncoding RNAs (lncRNAs) play various roles in HCC progression, but no research has focused on the expression pattern of microRNA clusters encoded in lncRNAs. The Ftx gene encodes a lncRNA which harbors 2 clusters of microRNAs in its introns, the miR-374b/421 cluster and the miR-545/374a cluster. To date, no research has focused on the role of the miR-545/374a and miR-374b/421 clusters in HBV-related HCC. In this study, 66 pairs of HBV-related HCC tissue and matched non-cancerous liver tissue specimens were analyzed for the expression of the Ftx microRNA clusters. Our results showed that the miR-545/374a cluster was upregulated in HBV-HCC tissue and significantly correlated with prognosis-related clinical features, including histological grade, metastasis and tumor capsule. Transfection studies with microRNA mimics and inhibitors revealed that miR-545/374a expression promoted in vitro cell proliferation, cell migration and invasion. The wild-type HBV-genome-containing plasmid or full-length HBx protein encoding plasmid was transfected into the Bel-7402 cell line and observed for their influence on miR-545/374a expression. We found that transfection of the HBV genome or HBx alone resulted in an increase in miR-545/374a expression. Next, by monitoring the expression of sera miR-545/374a before and after surgical tumor excision, we found serum miR-545/374a was tumor-derived and exhibited a sharp decrease 25 days after tumor excision. We also examined the gender-based difference in miR-545/374a expression among HCC patients and utilized microRNA target prediction software to find the targets of miR-545/374a. One of these targets, namely estrogen-related receptor gamma (ESRRG) was inversely correlated with miR-545 expression. In conclusion, the overexpression of miR-545/374a cluster located in the Ftx lncRNA is partially responsible for a poor prognosis, and monitoring sera levels of miR-545/374a may be a useful diagnostic marker for HCC.

SNAI1 promotes the development of HCC through the enhancement of proliferation and inhibition of apoptosis

SNAI1, a zinc‐finger transcription factor, plays an important role in the induction of epithelial–mesenchymal transition (EMT) in various cancers. However, the possible functions of SNAI1 in the proliferation and apoptosis of hepatocellular carcinoma have not been clearly identified. In this study, we investigated the effects and mechanisms of SNAI1 in the proliferation and apoptosis of hepatocellular carcinoma using clinical samples and cell lines. We found that SNAI1 is highly expressed in the tissues of liver cancer compared with adjacent nontumor tissues. SNAI1 is also highly expressed in the hepatoma cell lines HepG2, SMMC‐7721, and BEL‐7402 compared with the human normal liver cell line L02. We also observed that SNAI1 expression was correlated with distal metastasis, incomplete tumor capsule formation, and histological differentiation in hepatocellular carcinoma (HCC). Moreover, we demonstrated that knockdown of SNAI1 via lentiviral vectors of RNAi against SNAI inhibited cell proliferation by inducing G1 arrest, which was accompanied by the downregulation of cyclin D1 but not that of cyclin A. In addition, knockdown of SNAI1 promoted apoptosis by decreasing the expression of Bcl‐2. In conclusion, our findings revealed that SNAI1 is involved in the development of hepatocellular carcinoma via regulating the growth and apoptosis of tumor cells.

MAPK/p38 regulation of cytoskeleton rearrangement accelerates induction of macrophage activation by TLR4, but not TLR3

Toll-like receptor 3 (TLR3) and TLR4 utilize adaptor proteins to activate mitogen-activated protein kinase (MAPK), resulting in the acute but transient inflammatory response aimed at the clearance of pathogens. In the present study, it was demonstrated that macrophage activation by lipopolysaccharide (LPS) or poly(I:C), leading to changes in cell morphology, differed significantly between the mouse macrophage cell line RAW264.7 and mouse primary peritoneal macrophages. Moreover, the expression of α- and β-tubulin was markedly decreased following LPS stimulation. By contrast, α- and β-tubulin expression were only mildly increased following poly(I:C) treatment. However, the expression of β-actin and GAPDH was not significantly affected. Furthermore, it was verified that vincristine pretreatment abrogated the cytoskeleton rearrangement and decreased the synthesis and secretion of proinflammatory cytokines and migration of macrophages caused by LPS. Finally, it was observed that the MAPK/p38 signaling pathway regulating cytoskeleton rearrangement may participate in LPS-induced macrophage cytokine production and migration. Overall, the findings of the present study indicated that MAPK/p38 regulation of the cytoskeleton, particularly tubulin proteins, plays an important role in LPS-induced inflammatory responses via alleviating the synthesis and secretion of proinflammatory cytokines and inhibiting the migration of macrophages.

HBeAg induces the expression of macrophage miR-155 to accelerate liver injury via promoting production of inflammatory cytokines

Activation of Kupffer cells (KCs) induced that inflammatory cytokine production plays a central role in the pathogenesis of HBV infection. The previous studies from our and other laboratory demonstrated miRNAs can regulate TLR-inducing inflammatory responses to macrophage. However, the involvement of miRNAs in HBV-associated antigen-induced macrophage activation is still not thoroughly understood. Here, we evaluated the effects and mechanisms of miR-155 in HBV-associated antigen-induced macrophage activation. First, co-culture assay of HepG2 or HepG2.2.15 cells and RAW264.7 macrophages showed that HepG2.2.15 cells could significantly promote macrophages to produce inflammatory cytokines. Furthermore, we, respectively, stimulated RAW264.7 macrophages, mouse primary peritoneal macrophages, or healthy human peripheral blood monocytes with HBV-associated antigens, including HBcAg, HBeAg, and HBsAg, and found that only HBeAg could steadily enhance the production of inflammatory cytokines in these cells. Subsequently, miRNAs sequencing presented the up- or down-regulated expression of multiple miRNAs in HBeAg-stimulated RAW264.7 cells. In addition, we verified the expression of miR-155 and its precursors BIC gene with q-PCR in the system of co-culture or HBeAg-stimulated macrophages. Meanwhile, the increased miR-155 expression was positively correlation with serum ALT, AST, and HBeAg levels in AHB patients. Although MAPK, PI3K, and NF-κB signal pathways were all activated during HBeAg treatment, only PI3K and NF-κB pathways were involved in miR-155 expression induced by HBeAg stimulation. Consistently, miR-155 over-expression inhibited production of inflammatory cytokines, which could be reversed by knocking down miR-155. Moreover, we demonstrated that miR-155 regulated HBeAg-induced cytokine production by targeting BCL-6, SHIP-1, and SOCS-1. In conclusion, our data revealed that HBeAg augments the expression of miR-155 in macrophages via PI3K and NF-κB signal pathway and the increased miR-155 promotes HBeAg-induced inflammatory cytokine production by inhibiting the expression of BCL-6, SHIP-1, and SOCS-1.

Taurolidine promotes cell apoptosis by enhancing GRIM‑19 expression in liver cancer

Taurolidine (TRD) is a substance derived from the amino sulfonic acid taurine, which was originally used to treat peritonitis and catheter‑associated bloodstream infections, due to its antimicrobial and anti‑inflammatory properties. A recent study reported the anticancer function of TRD in malignant tumors; however, the effects and mechanisms of TRD in liver cancer remain unclear. The present study aimed to investigate the effects and mechanism of TRD treatment on human liver cancer cells. The viability and apoptosis of liver cancer cells were evaluated using the MTT assay and flow cytometry. Subsequently, small interfering RNA (siRNA) was used to knock down the expression of gene associated with retinoid‑interferon‑induced mortality‑19 (GRIM‑19), after which, reverse transcription‑quantitative polymerase chain reaction was used to detect the mRNA expression levels of GRIM‑19, whereas immunofluorescence was used to analyze the location of GRIM‑19. Furthermore, western blotting was performed to detect the protein expression levels of GRIM‑19, cyclinxa0D1, signal transducer and activator of transcriptionxa03 (STAT3), phosphorylated (p)‑STAT3, B‑cell lymphomaxa02 (Bcl‑2) and Bcl‑2‑associated Xxa0protein (Bax). The STAT3 pathway was inhibited using niclosamide. The results revealed that TRD reduced the viability of liver cancer cells and induced apoptosis at higher frequencies. In addition, the expression levels of GRIM‑19 were increased in a time‑ and dose‑dependent manner following TRD treatment. Alongside GRIM‑19 upregulation, the expression levels of Bax were increased, whereas those of cyclinxa0D1, Bcl‑2 and p‑STAT3 were decreased. Furthermore, following GRIM‑19 knockdown, the effects of TRD on the viability and apoptosis of HepG2 cells, and the expression of downstream target genes (including cyclinxa0D1, STAT3, p‑STAT3, Bcl‑2 and Bax) were reversed. Conversely, treatment with a p‑STAT3 inhibitor had an inverse effect on the expression of these genes but did not affect GRIM‑19 expression compared with the TRD group. These results indicated that TRD may contribute to cell apoptosis by inducing GRIM‑19 expression and deactivating the STAT3 signaling pathway in liver cancer cells.

[Corrigendum] RLIP76 decreases apoptosis through Akt/mTOR signaling pathway in gastric cancer

Following the publication of this article, we realize that the title appeared incorrectly: This appeared in print as RLIP76 increases apoptosis through Akt/mTOR signaling pathway in gastric cancer, and the corrected title is now featured above (increases should have read as decreases). Note that this error did not have any bearing on the results reported in the paper (which were reported accurately in terms of the influence of RLIP76 on apoptosis of human gastric cancer SGC‑7901 and MGC‑803xa0cells), or the conclusions therein. We regret any inconvenience that this mistake has caused. [the original article was published in the Oncology Reports 36: 2216-2224, 2016; DOI: 0.3892/or.2016.5043].

Impact of tumor deposits on the prognosis and chemotherapy efficacy in stage III colorectal cancer patients with different lymph node status: a retrospective cohort study in China

BACKGROUNDnThe goal was to determine whether tumor deposits (TDs) had effects on the overall survival (OS), cancer-specific survival (CSS), disease-free survival (DFS) and responses to chemotherapy in advanced colorectal cancer (CRC) patients with different lymph node (N) stages.nnnMATERIALS AND METHODSnThe retrospective cohort study recruited 1455 stage III CRC patients diagnosed at a single institution between January 2010 and July 2016. Patients were divided into TDs negative and positive groups. Based on whether they accepted chemotherapy, patients were further divided into chemotherapy and non-chemotherapy groups. Kaplan-Meier methods, univariate and multivariate analyses, and subset analyses based on the N stage were performed to compare the OS, CSS and DFS between different groups.nnnRESULTSnMultivariate Cox analyses showed that TDs were independent prognostic markers for the OS (adjusted HRu202f=u202f1.929, 95% CI: 1.339-2.777), CSS (adjusted HRu202f=u202f1.789, 95% CI: 1.165-2.748) and DFS (adjusted HRu202f=u202f2.179, 95% CI: 1.612-2.944) in all N stages combined. In addition, subset analyses based on the N stage further demonstrated that TDs were independent risk factors for the OS (Pu202f=u202f0.012), CSS (Pu202f=u202f0.010) and DFS (Pu202f<u202f0.001) in patients with the N1a, 1u202fb stages, and for the OS (Pu202f=u202f0.023) and DFS (Pu202f<u202f0.001) in patients with the N2a, 2u202fb stages. Furthermore, the OS, CSS and DFS in the TDs negative group could be extended significantly after the administration of chemotherapy, whereas patients with positive TDs lost the DFS benefit from chemotherapy.nnnCONCLUSIONSnStage III CRC patients with positive TDs had a poor prognosis, and they did not display a DFS benefit from chemotherapy. TDs had adverse effects on the OS and DFS in patients with the N1a, 1u202fb and N2a, 2u202fb stages, providing evidence for the feasibility of the new TNM category method.

lncRNA Ftx promotes aerobic glycolysis and tumor progression through the PPARγ pathway in hepatocellular carcinoma

Aerobic glycolysis is a phenomenon by which malignant cells preferentially metabolize glucose through the glycolytic pathway, rather than oxidative phosphorylation to proliferate efficiently. The present study aimed to investigate the expression and functional implications of long non-coding (lnc)RNA Ftx in the aerobic glycolysis and tumorigenesis of hepatocellular carcinoma (HCC). It was identified that lncRNA Ftx was upregulated in human HCC tissues and cell lines and, notably, was associated with aggressive clinicopathological features. lncRNA Ftx overexpression promoted the proliferation, invasion and migration of HCC cells, whereas lncRNA Ftx knockdown resulted in the opposite effects. Furthermore, lncRNA Ftx affected the activity and expression of key enzymes in carbohydrate metabolism, suggesting that lncRNA Ftx may be involved in aerobic glycolysis in HCC. The measurement of glucose consumption, lactate production and glucose transporter expression further supported this assumption. Mechanistically, peroxisome proliferator-activated receptor γ (PPARγ) expression in human HCC tissues and cell lines was positively correlated with lncRNA Ftx. Inhibiting PPARγ in Huh7 cells partially abrogated the alterations in glucose uptake, lactate production and relative glycolytic enzyme expression induced by lncRNA Ftx; similarly, PPARγ activation in Bel-7402 cells partially rescued the lncRNA Ftx-mediated alterations. In conclusion, lncRNA Ftx is a promoter of the Warburg effect and tumor progression, partly via the PPARγ pathway, and may serve as a promising therapeutic target for HCC treatment.

The E3 ubiquitin ligase MuRF2 attenuates LPS‐induced macrophage activation by inhibiting production of inflammatory cytokines and migration

Muscle RING‐finger (MuRF) proteins are E3 ubiquitin ligases that are expressed in striated muscle. MuRF2 is an important member of this family, but whether it is expressed in tissues other than striated muscle has not been thoroughly elucidated to date. In this study, we determined that MuRF2 is also expressed in other vital organs, including liver, lung, brain, spleen and kidney. Moreover, we show that the level of MuRF2 expression is significantly decreased in hepatic mononuclear cells of mice with lipopolysaccharide (LPS)/d‐galactosamine‐induced hepatitis and negatively correlated with the serum levels of alanine aminotransferase and aspartate aminotransferase in these mice. Furthermore, the expression of MuRF2 was down‐regulated in RAW264.7 cells activated with LPS but not in cells treated with polyinosinic‐polycytidylic acid (Poly(I:C)) or with lipidosome plus Poly(I:C). We also found that MuRF2 was able to translocate from the cytoplasm to the nucleus in RAW264.7 cells activated with LPS but not in cells treated with Poly(I:C). In addition, we demonstrated that interleukin 6 and tumour necrosis factor α production and macrophage migration were inhibited after MuRF2 was overexpressed in RAW264.7 cells. We further verified that nuclear factor‐κB p65 subunit level was greatly reduced in RAW264.7 macrophage nuclei by gain of function. Taken together, these findings indicate that MuRF2 may rescue LPS‐induced macrophage activation by suppressing the production of proinflammatory cytokines and cell migration. We also identify a novel function of MuRF2 in non‐muscle tissues and cells.

Prognostic and predictive value of the macroscopic growth pattern in patients undergoing curative resection of colorectal cancer: a single-institution retrospective cohort study of 4,080 Chinese patients

Purpose The purpose of this study was to determine whether macroscopic growth patterns had an impact on the prognosis of colorectal cancer (CRC) patients with different tumor–node–metastasis (TNM) stages and responses to chemotherapy in stage III patients. Patients and methods We retrospectively recruited 4,080 stage I–III CRC patients who underwent curative resection at Shandong Provincial Hospital affiliated to Shandong University. All patients were grouped by macroscopic growth patterns (expansive, infiltrative and ulcerative subtypes), and stage III patients were further divided into chemotherapy and nonchemotherapy groups. Kaplan–Meier methods, univariate and multivariate analyses and subset analyses were performed to assess the overall survival (OS), cancer-specific survival (CSS) and disease-free survival (DFS). Results Kaplan–Meier survival curves and univariate analyses revealed better OS (HR=0.731; 95% CI=0.584–0.916), CSS (HR=0.714; 95% CI=0.548–0.932) and DFS (HR=0.722; 95% CI=0.602–0.864) in the expansive subtype and worse OS (HR=2.121; 95% CI=1.457–3.088), CSS (HR=2.499; 95% CI=1.664–3.753) and DFS (HR=2.360; 95% CI=1.756–3.170) in the infiltrative subtype. Subset analyses based on the tumor–node–metastasis stage showed that the infiltrative subtype was associated with inferior DFS in stage II (HR=2.357; 95% CI=1.210–4.595) and stage III patients (HR=1.941; 95% CI=1.394–2.702) and inferior OS and CSS in stage III patients (HR=1.805; 95% CI=1.210–2.693 and HR=1.981, 95% CI=1.280–3.065, respectively). In addition, multivariate Cox proportional hazard regression models revealed similar results. Furthermore, in stage III patients, the OS, CSS and DFS in both the expansive and ulcerative subtypes were significantly extended after the administration of chemotherapy (all, P<0.001). However, the OS, CSS and DFS in the infiltrative subtype did not change significantly after the administration of chemotherapy (P=0.486, 0.290 and 0.731, respectively). Conclusion The macroscopic growth pattern was an independent prognostic factor among stage I–III CRC patients. The infiltrative subtype had the worst prognosis in stage III patients and did not display survival benefits from chemotherapy.