Disturbing miR-182 and -381 Inhibits BRD7 Transcription and Glioma Growth by Directly Targeting <i>LRRC4</i>

<div><p>Inactivated <i>LRRC4</i> has been clinically detected in gliomas, and promoter hypermethylation has been implicated as the mechanism of inactivation in some of those tumors. Our previous researches indicated that <i>LRRC4</i> is a target gene of miR-381, the interaction of miR-381 and <i>LRRC4</i> is involved in glioma growth. In this study, we demonstrate that <i>LRRC4</i> is a target gene of the other microRNA, miR-182. We found that the high expression of miR-182 and miR-381 in gliomas are involved in pathological malignant progression. The silencing of miR-182 and miR-381 inhibited the proliferation <i>in vitro</i> and growth of glioma cell with <i>in vivo</i> magnetic resonance imaging by intracranial transplanted tumor model in rats. We also demonstrated that BRD7, a transcriptional cofactor for p53, is highly expressed and negatively correlated with <i>LRRC4</i> expression in gliomas. Disturbing miR-182 and miR-381 affected transcriptional regulation of the <i>BRD7</i> gene. This finding was verified by ectopic overexpression of <i>LRRC4</i> or restoration of endogenous <i>LRRC4</i> expression by treatment with the DNA demethylating agent 5-Aza-dC. Taken together, miR-182 and miR-381 may be a useful therapeutic target for treatment of glioma.</p></div

The Roles of Variants in Human Multidrug Resistance (MDR1) Gene and Their Haplotypes on Antiepileptic Drugs Response: A Meta-Analysis of 57 Studies

<div><p>Objective</p><p>Previous studies reported the associations between the ATP-binding cassette sub-family B member 1 (ABCB1, also known as MDR1) polymorphisms and their haplotypes with risk of response to antiepileptic drugs in epilepsy, however, the results were inconclusive.</p><p>Methods</p><p>The Pubmed, Embase, Web of Science, CNKI and Chinese Biomedicine databases were searched up to July 15, 2014. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using a fixed-effects or random-effects model based on heterogeneity tests. Meta-regression and Galbraith plot analysis were carried out to explore the possible heterogeneity.</p><p>Results</p><p>A total of 57 studies involving 12407 patients (6083 drug-resistant and 6324 drug-responsive patients with epilepsy) were included in the pooled-analysis. For all three polymorphisms (C3435T, G2677T/A, and C1236T), we observed a wide spectrum of minor allele frequencies across different ethnicities. A significantly decreased risk of AEDs resistance was observed in Caucasian patients with T allele of C3435T variant, which was still significant after adjusted by multiple testing corrections (T vs C: OR=0.83, 95%CI=0.71-0.96, p=0.01). However, no significant association was observed between the other two variants and AEDs resistance. Of their haplotypes in ABCB1 gene (all studies were in Indians and Asians), no significant association was observed with AEDs resistance. Moreover, sensitivity and Cumulative analysis showed that the results of this meta-analysis were stable.</p><p>Conclusion</p><p>In summary, this meta-analysis demonstrated that effect of C3435T variant on risk of AEDs resistance was ethnicity-dependent, which was significant in Caucasians. Additionally, further studies in different ethnic groups are warranted to clarify possible roles of haplotypes in ABCB1 gene in AEDs resistance, especially in Caucasians.</p></div

<i>LRRC4</i> is a target gene of miR-182.

<p>(A) Schema of the interaction sites between miR-182 and the 3′-UTRs of <i>LRRC4</i> (B) (B) Luciferase assay of U251 glioma cells co-transfected with pMIR-REPORT–WT/mutant 3′-UTR <i>LRRC4</i> and miR-182 or scrambled control as indicated. * <i>p</i><0.05. (C) Western blot showing the protein expression of <i>LRRC4</i> after miR-182 was transfected into U251/L cells for 48 h. miR-182 mimics inhibited the protein expression of <i>LRRC4</i>. GAPDH was used as a loading control. (D) qRT-PCR showing the mRNA level of <i>LRRC4</i> after miR-182 mimic was transfected into U251/L cells for 48 h. miR-182 mimic down-regulated the mRNA level of <i>LRRC4</i>. * <i>p</i><0.05.</p

LNA-anti-miR-182 and -381 induces <i>LRRC4</i> up-regulation and <i>BRD7</i> down-regulation.

<p>(A) qRT-PCR showing down-regulation of miR-182 and miR-381 in U251 cells after LNA-anti-miRs transfection. * <i>p</i><0.05. (B) LNA-mediated miR-182 and -381 silencing restored endogenous levels of <i>LRRC4</i> protein and decreased BRD7 expression. U251 cells were transfected with either LNA-scrambled, LNA-anti-miR-182 or -381 for 48 h. <i>LRRC4</i> and <i>BRD7</i> expression was assessed by Western blot. GAPDH was used as a loading control. (C) qRT-PCR confirmed re-expression of <i>LRRC4</i> and decreased <i>BRD7</i> expression after LNA-anti-miR-182 and -381 transfection. (D) Ectopic <i>LRRC4</i> expression decreased endogenous levels of <i>BRD7</i> protein in U251 cells. <i>LRRC4</i> and <i>BRD7</i> expression were assessed by Western blot (left) and gray image scanning (right). * <i>p</i><0.05 compared with mock (control). (E) 5-Aza-dC restored endogenous levels of <i>LRRC4</i> protein and decreased that of <i>BRD7</i> expression in U251, SF126, and SF767 cells. <i>LRRC4</i> and <i>BRD7</i> expressions were assessed by Western blot (left) and gray image scanning (right). * <i>p</i><0.05 compared with LNA-scrambled control.</p

Odds ratio (OR) estimates for the association between the ABCB1 C3435T polymorphism and AEDs resistance.

<p>The sizes of the squares reflect theweighting of the included studies. Bars represent 95% CIs. The center of the diamond represents the summary effect; left and right points of the diamond represent the 95% CI. AEDs: antiepileptic drugs; CI: confidence interval; ABCB1: ATP-binding cassette sub-family B member 1.</p

LNA-anti-miR-182 and -381 suppressed the promoter activity of <i>BRD7</i> by down-regulating AP2, SP1, and E2F6, and up-regulating c-Myc.

<p>(A) LNA-mediated miR-182 and -381 silencing down-regulated expression of K-Ras, p-c-Raf, pERK, PI-3K, and pAKT, but the silencing had no effect on N-Ras, total ERK and AKT expression, as shown by Western blot (left) and gray image scanning (right). (B) LNA-mediated miR-182 and -381 silencing down-regulated the expression of AP2, SP1, and E2F6, and up-regulated the expression of c-Myc, as shown by Western blot (top) and gray image scanning (bottom). (C) PD98059 or LY294002 reversed the miR-182 and miR-381 mimics-induced expression of AP2, SP1, E2F6, and c-Myc. AP2, SP1, E2F6, and c-Myc expression were assessed by Western blot (left) and gray image scanning (right). (D) EMSA confirmed that LNA-mediated miR-182 and -381 silencing or ectopic <i>LRRC4</i> expression promoted the <i>BRD7</i> promoter association of c-Myc and disrupted that of AP2, SP1, and E2F6. Mutant, nuclear protein +200×mutant probe + wild biotin-probe; Competitor, nuclear protein +200×competitor cold probe + wild biotin-probe; No extracts, no nuclear protein + wild biotin-probe; Scrambled, nuclear protein of transfected miRNA negative control + wild biotin-probe; LNA-182, nuclear protein of transfected LNA-miR-182 inhibitors + wild biotin-probe; LNA-381, nuclear protein of transfected LNA-miR-381 inhibitors + wild biotin-probe; LRRC4, nuclear protein of transfected LRRC4 gene + wild biotin-probe. (E) Luciferase assays confirmed the inhibition of LNA-mediated miR-182 and -381 silencing, or siRNA of AP2, SP1, and E2F6, and c-Myc overexpression on the promoter activity of <i>BRD7</i> gene. * <i>p</i><0.05 compared with the control (siRNA including SP1, AP2, and E2F6 <i>vs.</i> siRNA scrambled or pCMV-HA-c-Myc <i>vs.</i> pCMV-HA). (F) EMSA indicated that PD98059 and LY294002 reversed the association of AP2, SP1, and E2F6 or c-Myc with the <i>BRD7</i> promoter that was induced by miR-182 and miR-381. Mutant, nuclear protein+200×mutant probe + wild biotin-probe; Competitor, nuclear protein +200×competitor wild probe + wild biotin-probe; No extracts, no nuclear protein + wild biotin-probe; Mock, nuclear protein+ wild biotin-probe; PD98059, nuclear protein with PD98059 + wild biotin-probe; LY294002, nuclear protein with PD98059 + wild biotin-probe; Scrambled, nuclear protein of transfected miRNA negative control + wild biotin-probe; LNA-182, nuclear protein of transfected LNA-miR-182 inhibitors + wild biotin-probe; LNA-381, nuclear protein of transfected LNA-miR-381 inhibitors + wild biotin-probe; 182M, nuclear protein of transfected miR-182 mimics + wild biotin-probe; 381M, nuclear protein of transfected miR-381 mimics + wild biotin-probe. (G) siRNA-AP2, siRNA-SP1, siRNA-E2F6, and c-Myc overexpression affected endogenous expression of <i>BRD7</i> at the protein (left) and mRNA (right) levels. * <i>p</i><0.05 compared with control (LNA-182 and LNA-381 <i>vs.</i> scrambled; si-SP1, si-AP2 and si-E2F6 <i>vs</i>. si-SC; pCMV-HA-c-Myc <i>vs</i>. pCMV-HA).</p

LNA-anti-miR-182 and -381 had anticancer effects on intracranial transplanted tumors by surpassing the blood-brain barrier.

<p>(A) Intraperitoneal injection of LNA-anti-miR-182 and/or -381 oligonucleotides inhibited the growth of intracranial transplanted tumors in Sprague-Dawley rats (top and middle, MRI; bottom, HE staining of coronal section). (B) LNA-anti-miR-182 and/or -381 oligonucleotides reduced expression of miR-182 and -381 (ISH), increased expression of LRRC4, and reduced expression of BRD7 and Ki-67 (IHC).</p

Study selection procedures for a meta-analysis of MDR1 gene polymorphisms (C3435T, G2677T, and C1236T) with AEDs resistance.

<p>AEDs: antiepileptic drugs; ABCB1: ATP-binding cassette sub-family B member 1.</p

LNA-anti-miR-182 and -381 had anticancer effects on glioma cells and subcutaneously transplanted tumors in nude mice.

<p>(A) MTT assays confirmed the effects of the ectopic miR-381 mimic or LNA-mediated miR-381 silencing on glioma cell proliferation. The ectopic miR-381 mimic promoted the proliferation of glioma cells and LNA-mediated miR-381 silencing inhibited it. * <i>p</i><0.05 compared with control (mock or scrambled). (B) LNA-mediated miR-182 and -381 silencing blocked cell cycle progression in the G0/G1 phase, induced pRb expression, and decreased E2F3 expression. (C) <i>BRD7</i> silencing inhibited the proliferation of glioma cells and blocked the cell cycle in the G0/G1 phase. * <i>p</i><0.05 compared with control (siRNA scrambled). (D) LNA-mediated miR-182 and -381 silencing up-regulated GFAP expression in U251 cells (top, Western blot; bottom, indirect immunofluorescence). Expression of GAPDH was used as an internal loading control for Western blotting. DAPI staining was used as an internal control for immunofluorescence.</p

Summary odds ratios and heterogeneity of the C3435T polymorphism in ABCB1 gene on drug response in patients with epilepsy stratified by age, language, ethnicity, sample size and date of publication.

<p>CI: confidence interval; HWE: Hardy-Weinberg equilibrium; No: Number of studies; OR: odds ratio; P_h: P-value for heterogeneity tests.</p><p>Summary odds ratios and heterogeneity of the C3435T polymorphism in ABCB1 gene on drug response in patients with epilepsy stratified by age, language, ethnicity, sample size and date of publication.</p