MiR-291b-3p Induces Apoptosis in Liver Cell Line NCTC1469 by Reducing the Level of RNA-binding Protein HuR

Background: There is increasing evidence that miRNAs are involved in cellular apoptosis. However, the specific role of miR-291b-3p in apoptosis has not been elucidated. In the present study, we investigated the effect of miR-291b-3p on NCTC1469 cell growth and apoptosis. Methods: Cell viability and apoptosis were examined in NCTC1469 cells transfected with miR-291b-3p mimics, inhibitor miRNA or negative control. Using computational miRNA target prediction databases, HuR was predicted as a target of miR-291b-3p. Luciferase assay, immunofluorescence and western blot were used to further explore the effects of miR-291b-3p on HuR expression. In addition, the effect of HuR on cell apoptosis was evaluated using a HuR-specific siRNA. Results: TNF-α-induced hepatocyte apoptosis was accompanied by enhanced expression of miR-291b-3p, suggesting that miR-291b-3p might contribute to the apoptotic process. Follow-up experiments showed that upregulation of miR-291b-3p decreased cell viability and induced NCTC1469 cell apoptosis. Additionally, similar to the activity of miR-519, which is another member of the same miRNA family, miR-291b-3p suppressed HuR translation through binding to the HuR coding region (CR). We further showed that the downregulation of HuR expression by miR-291b-3p was accompanied by reduced Bcl-2 expression. Moreover, knockdown of HuR also impaired Bcl-2 expression and increased the ratio of Bax/Bcl-2. More significantly, downregulation of miR-291b-3p failed to increase Bcl2 expression in NCTC1469 cells that were co-transfected with siRNA-HuR. Finally, inhibition of miR-291b-3p led to reduced apoptosis, while knockdown of HuR by siRNA promoted apoptosis, even in NCTC1469 cells that were co-transfected with the miR-291b-3p inhibitor. Conclusion: The current data suggested that miR-291b-3p contributed to NCTC1469 cell apoptosis by regulating the expression of HuR, which in turn increased Bcl-2 stability

Cell surface glycan alterations in epithelial mesenchymal transition process of Huh7 hepatocellular carcinoma cell.

BACKGROUND AND OBJECTIVE: Due to recurrence and metastasis, the mortality of Hepatocellular carcinoma (HCC) is high. It is well known that the epithelial mesenchymal transition (EMT) and glycan of cell surface glycoproteins play pivotal roles in tumor metastasis. The goal of this study was to identify HCC metastasis related differential glycan pattern and their enzymatic basis using a HGF induced EMT model. METHODOLOGY: HGF was used to induce HCC EMT model. Lectin microarray was used to detect the expression of cell surface glycan and the difference was validated by lectin blot and fluorescence cell lectin-immunochemistry. The mRNA expression levels of glycotransferases were determined by qRT-PCR. RESULTS: After HGF treatment, the Huh7 cell lost epithelial characteristics and obtained mesenchymal markers. These changes demonstrated that HGF could induce a typical cell model of EMT. Lectin microarray analysis identified a decreased affinity in seven lectins ACL, BPL, JAC, MPL, PHA-E, SNA, and SBA to the glycan of cell surface glycoproteins. This implied that glycan containing T/Tn-antigen, NA2 and bisecting GlcNAc, Siaα2-6Gal/GalNAc, terminal α or βGalNAc structures were reduced. The binding ability of thirteen lectins, AAL, LCA, LTL, ConA, NML, NPL, DBA, HAL, PTL II, WFL, ECL, GSL II and PHA-L to glycan were elevated, and a definite indication that glycan containing terminal αFuc and ± Sia-Le, core fucose, α-man, gal-β(α) GalNAc, β1,6 GlcNAc branching and tetraantennary complex oligosaccharides structures were increased. These results were further validated by lectin blot and fluorescence cell lectin-immunochemistry. Furthermore, the mRNA expression level of Mgat3 decreased while that of Mgat5, FucT8 and β3GalT5 increased. Therefore, cell surface glycan alterations in the EMT process may coincide with the expression of glycosyltransferase. CONCLUSIONS: The findings of this study systematically clarify the alterations of cell surface glycan in cancer EMT, and may provide novel insight for HCC metastasis

Meta-Analysis of the Association between COX-2 Polymorphisms and Risk of Colorectal Cancer Based on Case–Control Studies

<div><p>Objective</p><p>Cyclooxygenase-2 (COX-2) is an inducible enzyme converting arachidonic acid to prostaglandins and playing important roles in inflammatory diseases as well as tumor development. Previous studies investigating the association between COX-2 polymorphisms and colorectal cancer (CRC) risk reported conflicting results. We performed a meta-analysis of all available studies to explore this association.</p><p>Methods</p><p>All studies published up to October 2013 on the association between COX-2 polymorphisms and CRC risk were identified by searching electronic databases PubMed, EMBASE, and Cochrane library. The association between COX-2 polymorphisms and CRC risk was assessed by odds ratios (ORs) together with their 95% confidence intervals (CIs).</p><p>Results</p><p>Ten studies with 6,774 cases and 9,772 controls were included for −1195A>G polymorphism, 13 studies including 6,807 cases and 10,052 controls were available for −765G>C polymorphism, and 8 studies containing 5,121 cases and 7,487 controls were included for 8473T>C polymorphism. With respect to −765G>C polymorphism, we did not find a significant association with CRC risk when all eligible studies were pooled into the meta-analysis. However, in subgroup analyses by ethnicity and cancer location, with a Bonferroni corrected alpha of 0.05/2, statistical significant increased CRC risk was found in the Asian populations (dominant model CC+CG vs. GG: OR = 1.399, 95%CI: 1.113–1.760, P = 0.004) and rectum cancer patients (CC vs. GG: OR = 2.270, 95%CI: 1.295–3.980, P = 0.004; Recessive model CC vs. CG+GG: OR = 2.269, 95%CI: 1.297–3.970, P = 0.004). In subgroup analysis according to source of control, no significant association was detected. With respect to −1195A>G and 8473T>C polymorphisms, no significant association with CRC risk was demonstrated in the overall and subgroup analyses.</p><p>Conclusions</p><p>The present meta-analysis suggests that the COX-2 −765G>C polymorphism may be a risk factor for CRC in Asians and rectum cancer patients. Further large and well-designed studies are needed to confirm this association.</p></div

Meta-analysis of COX-2 8473T>C polymorphism and CRC risk.

<p><i>P</i> = P values for Z test. <i>P</i>_h = P values of Q-test for heterogeneity test. OR, odds ratio; CI, confidence intervals; HB, Hospital–based studies; PB, Population-based studies.</p

Forest plot of the COX-2 8473T>C polymorphism and CRC risk using a fixed-effect model (dominant model CC+CT vs. TT).

<p>Forest plot of the COX-2 8473T>C polymorphism and CRC risk using a fixed-effect model (dominant model CC+CT vs. TT).</p

Meta-analysis of COX-2 −765G>C polymorphism and CRC risk.

<p><i>P</i> = P values for Z test. <i>P</i>_h = P values of Q-test for heterogeneity test. OR, odds ratio; CI, confidence intervals; HB, Hospital–based studies; PB, Population-based studies.</p

Meta-analysis of COX-2 −1195A>G polymorphism and CRC risk.

<p><i>P</i> = P values for Z test. <i>P</i>_h = P values of Q-test for heterogeneity test. OR, odds ratio; CI, confidence intervals; HB, Hospital–based studies; PB, Population-based studies; HWE, Hardy–Weinberg equilibrium.</p

Funnel plot analysis to detect publication bias.

<p>Each point represents a separate study for the indicated association. <b>A</b> Funnel plot for dominant model GG+AG vs. AA of COX-2 −1195 G>A polymorphism in overall analysis (P = 0.890); <b>B</b> Funnel plot for dominant model CC+CG vs. GG of COX-2 −765G>C polymorphism in overall analysis (P = 0.703); C Funnel plot for dominant model CC+CT vs. TT of COX-2 8473T>C polymorphism in overall analysis (P = 0.518).</p

Characteristics of eligible studies.

<p>SNP, Single nucleotide polymorphism; HC, Histologically confirmed; PC, Pathologically confirmed; NA, Not available; PB, Population–based; HB, Hospital–based; HWE, Hardy–Weinberg equilibrium in control population; PCR–RFLP, Polymerase chain reaction-restriction fragment length polymorphism; PCR-CTTP, Polymerase chain reaction with confronting two-pair primers.</p