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

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

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

Image2_Identification and validation of critical genes with prognostic value in gastric cancer.JPEG

Background: Gastric cancer (GC) is a digestive system tumor with high morbidity and mortality rates. Molecular targeted therapies, including those targeting human epidermal factor receptor 2 (HER2), have proven to be effective in clinical treatment. However, better identification and description of tumor-promoting genes in GC is still necessary for antitumor therapy.Methods: Gene expression and clinical data of GC patients were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Last absolute shrinkage and selection operator (LASSO) Cox regression were applied to build a prognostic model, the Prognosis Score. Functional enrichment and single-sample gene set enrichment analysis (ssGSEA) were used to explore potential mechanisms. Western blotting, RNA interference, cell migration, and wound healing assays were used to detect the expression and function of myosin light chain 9 (MYL9) in GC.Results: A four-gene prognostic model was constructed and GC patients from TCGA and meta-GEO cohorts were stratified into high-prognosis score groups or low-prognosis score groups. GC patients in the high-prognosis score group had significantly poorer overall survival (OS) than those in the low-prognosis score groups. The GC prognostic model was formulated as PrognosisScore = (0.06 × expression of BGN) - (0.008 × expression of ATP4A) + (0.12 × expression of MYL9) - (0.01 × expression of ALDH3A1). The prognosis score was identified as an independent predictor of OS. High expression of MYL9, the highest weighted gene in the prognosis score, was correlated with worse clinical outcomes. Functional analysis revealed that MYL9 is mainly associated with the biological function of epithelial-mesenchymal transition (EMT). Knockdown of MYL9 expression inhibits migration of GC cells in vitro.Conclusion: We found that PrognosisScore is potential reliable prognostic marker and verified that MYL9 promotes the migration and metastasis of GC cells.</p

<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

Image3_Identification and validation of critical genes with prognostic value in gastric cancer.JPEG

Background: Gastric cancer (GC) is a digestive system tumor with high morbidity and mortality rates. Molecular targeted therapies, including those targeting human epidermal factor receptor 2 (HER2), have proven to be effective in clinical treatment. However, better identification and description of tumor-promoting genes in GC is still necessary for antitumor therapy.Methods: Gene expression and clinical data of GC patients were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Last absolute shrinkage and selection operator (LASSO) Cox regression were applied to build a prognostic model, the Prognosis Score. Functional enrichment and single-sample gene set enrichment analysis (ssGSEA) were used to explore potential mechanisms. Western blotting, RNA interference, cell migration, and wound healing assays were used to detect the expression and function of myosin light chain 9 (MYL9) in GC.Results: A four-gene prognostic model was constructed and GC patients from TCGA and meta-GEO cohorts were stratified into high-prognosis score groups or low-prognosis score groups. GC patients in the high-prognosis score group had significantly poorer overall survival (OS) than those in the low-prognosis score groups. The GC prognostic model was formulated as PrognosisScore = (0.06 × expression of BGN) - (0.008 × expression of ATP4A) + (0.12 × expression of MYL9) - (0.01 × expression of ALDH3A1). The prognosis score was identified as an independent predictor of OS. High expression of MYL9, the highest weighted gene in the prognosis score, was correlated with worse clinical outcomes. Functional analysis revealed that MYL9 is mainly associated with the biological function of epithelial-mesenchymal transition (EMT). Knockdown of MYL9 expression inhibits migration of GC cells in vitro.Conclusion: We found that PrognosisScore is potential reliable prognostic marker and verified that MYL9 promotes the migration and metastasis of GC cells.</p

Supplementary Table 4 from Integrated Analysis of Multiple Gene Expression Profiling Datasets Revealed Novel Gene Signatures and Molecular Markers in Nasopharyngeal Carcinoma

PDF file - 66K</p

Supplementary Figure 2B from Integrated Analysis of Multiple Gene Expression Profiling Datasets Revealed Novel Gene Signatures and Molecular Markers in Nasopharyngeal Carcinoma

PDF file - 1.5MB</p

Supplementary Figure 2C from Integrated Analysis of Multiple Gene Expression Profiling Datasets Revealed Novel Gene Signatures and Molecular Markers in Nasopharyngeal Carcinoma

PDF file - 1.5MB</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