Supplemental Material - Prognostic Factors for Clinical Outcomes in Patients with Newly Diagnosed Advanced-stage Hodgkin Lymphoma: A Nationwide Retrospective Study

Supplemental Material Prognostic Factors for Clinical Outcomes in Patients with Newly Diagnosed Advanced-stage Hodgkin Lymphoma: A Nationwide Retrospective Study by Chieh-Lin Jerry Teng, Tran-Der Tan, Yun-Yi Pan, Yu-Wen Lin, Pei-Wen Lien, Hsin-Chun Chou, Peng-Hsu Chen, and Fang-Ju Lin in Cancer Control</p

The Inhibition of microRNA-128 on IGF-1-Activating mTOR Signaling Involves in Temozolomide-Induced Glioma Cell Apoptotic Death

<div><p>Temozolomide (TMZ), an alkylating agent of the imidazotetrazine series, is a first-line chemotherapeutic drug used in the clinical therapy of glioblastoma multiforme, the most common and high-grade primary glioma in adults. Micro (mi)RNAs, which are small noncoding RNAs, post-transcriptionally regulate gene expressions and are involved in gliomagenesis. However, no studies have reported relationships between TMZ and miRNA gene regulation. We investigated TMZ-mediated miRNA profiles and its molecular mechanisms underlying the induction of glioma cell death. By performing miRNA microarray and bioinformatics analyses, we observed that expression of 248 miRNAs was altered, including five significantly upregulated and 17 significantly downregulated miRNAs, in TMZ-treated U87MG cells. miR-128 expression levels were lower in different glioma cells and strongly associated with poor survival. TMZ treatment significantly upregulated miR-128 expression. TMZ significantly enhanced miR-128-1 promoter activity and transcriptionally regulated miR-128 levels through c-Jun N-terminal kinase 2/c-Jun pathways. The overexpression and knockdown of miR-128 expression significantly affected TMZ-mediated cell viability and apoptosis-related protein expression. Furthermore, the overexpression of miR-128 alone enhanced apoptotic death of glioma cells through caspase-3/9 activation, poly(ADP ribose) polymerase degradation, reactive oxygen species generation, mitochondrial membrane potential loss, and non-protective autophagy formation. Finally, we identified that key members in mammalian target of rapamycin (mTOR) signaling including mTOR, rapamycin-insensitive companion of mTOR, insulin-like growth factor 1, and PIK3R1, but not PDK1, were direct target genes of miR-128. TMZ inhibited mTOR signaling through miR-128 regulation. These results indicate that miR-128-inhibited mTOR signaling is involved in TMZ-mediated cytotoxicity. Our findings may provide a better understanding of cytotoxic mechanisms of TMZ involved in glioblastoma development.</p></div

The microRNA-302b-inhibited insulin-like growth factor-binding protein 2 signaling pathway induces glioma cell apoptosis by targeting nuclear factor IA

<div><p>MicroRNAs are small noncoding RNAs that post-transcriptionally control the expression of genes involved in glioblastoma multiforme (GBM) development. Although miR-302b functions as a tumor suppressor, its role in GBM is still unclear. Therefore, this study comprehensively explored the roles of miR-302b-mediated gene networks in GBM cell death. We found that miR-302b levels were significantly higher in primary astrocytes than in GBM cell lines. miR-302b overexpression dose dependently reduced U87-MG cell viability and induced apoptosis through caspase-3 activation and poly(ADP ribose) polymerase degradation. A transcriptome microarray revealed 150 downregulated genes and 380 upregulated genes in miR-302b-overexpressing cells. Nuclear factor IA (NFIA), higher levels of which were significantly related to poor survival, was identified as a direct target gene of miR-302b and was involved in miR-302b-induced glioma cell death. Higher NFIA levels were observed in GBM cell lines and human tumor sections compared with astrocytes and non-tumor tissues, respectively. NFIA knockdown significantly enhanced apoptosis. We found high levels of insulin-like growth factor-binding protein 2 (IGFBP2), another miR-302b-downregulated gene, in patients with poor survival. We verified that NFIA binds to the IGFBP2 promoter and transcriptionally enhances IGFBP2 expression levels. We identified that NFIA-mediated IGFBP2 signaling pathways are involved in miR-302b-induced glioma cell death. The identification of a regulatory loop whereby miR-302b inhibits NFIA, leading to a decrease in expression of IGFBP-2, may provide novel directions for developing therapies to target glioblastoma tumorigenesis.</p></div

Effects of the miR-302b target gene of NFIA on regulating glioma cell death.

<p>(A) Relative NFIA expression levels according to GBM patient risk group based on GSE16011 profiling. Higher risk patients had higher NFIA expression, denoted by red symbols. Conversely, lower NFIA expression was associated with higher survival, denoted in green. (B) Kaplan—Meier analysis of patient survival data from GSE16011 profiling. The <i>x</i>-axis values indicate the number of patients per group. The red curves indicate high-risk patients. The green curves indicate low-risk patients. The plus sign indicates censored observations. The <i>p</i> values were calculated using the log-rank test. (C) Schematic diagram of potential miR-302b-targeted sites in the NFIA 3′UTR. Three putative sites were predicted and named 3U-1, -2, and -3. (D and E) Effects of miR-302b on NFIA 3′UTR luciferase activity. To test the effect of miR-302b, overnight culture cells were cotransfected with different doses of the miR-302b mimic or 50 nM scrambled miRNA mimic (control) and 500 ng of the pmiRGlo-NFIA 3′ UTR or mutant 3′UTR. Luciferase activity was measured in these cells 24 h after transfection. Effects of miR-302b overexpression on NFIA mRNA (F) and protein (G) expression. After overnight culture cells were transfected with the indicated dose of the miR-302b mimic and 50 nM scrambled miRNA mimic (Ctrl) for 24 h, the relative mRNA and protein levels of NFIA were analyzed using real-time PCR and immunoblotting assays. The lower panel in (G) shows quantitative results of three independent experiments of immunoblotting assays. Overexpression and knockdown of NFIA influenced miR-302b-mediated cell viability (H) and apoptotic markers (I). After overnight culture cells were cotransfected with 50 nM miR-302b mimic and 1 μg of the pcDNA3-NFIA OE plasmid or shRNA for 24 h, the cell viability and apoptotic markers were measured using the MTT assay and immunoblotting assays with anti-poly(ADP ribose) polymerase and caspase-3 antibodies. The scrambled miRNA mimic (50 nM), empty pcDNA3 vectors (1 μg), and scrambled shRNA (1 μg) were used as controls. Data are expressed as the mean ± SD of three independent experiments. After analyzing by one way ANOVA with Tukey—Kramer multiple comparison test, different letters above bars denote samples that were significantly different (<i>p</i> < 0.05) compared with control. *<i>p</i> < 0.05 by unpaired Student’s <i>t</i> test.</p

Effects of NFIA on mediating glioma cell growth.

<p>(A) Detection of endogenous NFIA protein levels in normal human astrocytes and three glioma cell lines. Cells were grown overnight to 80% confluency in serum-containing media. After spent media, total lysates were used to extract the total proteins for measuring NFIA and β-actin levels by immunoblotting assays. (B) IHC detection in formalin-fixed, paraffin-embedded surgical samples showed variations in NFIA expression in tumor tissues (T) compared with normal tissues (N). (C) NFIA overexpression enhanced U87-MG cell growth. Knockdown of NFIA reduced cell viability (D) and enhanced caspase-3 activation and PARP degradation (E). After overnight culture cells were transfected with the indicated dose of the NFIA OE plasmid or NFIA shRNA for 24 h, cell viability and caspase-3 and PARP levels were measured using the MTT assay and immunoblotting assays. The empty pcDNA3 vectors and scrambled shRNA (1 μg) were used as controls. Data are expressed as the mean ± SD of three independent experiments. After analyzing by one way ANOVA with Tukey—Kramer multiple comparison test, different letters above bars denote samples that were significantly different (<i>p</i> < 0.05) compared with control.</p

miR-302b inhibited the IGFBP2-mediated signaling pathway through NFIA regulation.

<p>Overexpression of the miR-302b mimic reduced IGFBP2 mRNA (A) and protein levels (B). After overnight culture U87-MG cells were transfected with the indicated dose of the miR-302b mimic or 50 nM scrambled miRNA mimic (Ctrl) for 24 h, relative mRNA and protein levels of IGFBP2 were measured using real-time PCR and immunoblotting assays. Data are expressed as the mean ± SD of three independent experiments. After analyzing by one way ANOVA with Tukey—Kramer multiple comparison test, different letters above bars denote samples that were significantly different (<i>p</i> < 0.05) compared with control. (C) miR-302b dose dependently reduced IGFBP2 promoter activity. After overnight culture U87-MG cells were cotransfected with the indicated dose of the miR-302b mimic, 50 nM scrambled miRNA mimic, and 500 ng of pGL3-IGFBP2 prom for 24 h, luciferase activity was measured. Cells were also cotransfected with pNL1.1.TK[<i>Nluc</i>/TK] plasmids (5 ng), and the NanoLucR luciferase value was used as an internal control. (D) ChIP revealed that miR-302b could inhibit the binding of NFIA to the IGFBP2 promoter. Data are expressed as the mean ± SD of three independent experiments. After analyzing by one way ANOVA with Tukey—Kramer multiple comparison test, different letters above bars denote samples that were significantly different (<i>p</i> < 0.05) compared with control. (E) Effects of NFIA on the miR-302b-inhibited IGFBP2 signaling pathway. After overnight culture U87-MG cells were cotransfected with 50 nM miR-302b mimic, 50 nM scrambled miRNA mimic, 1 μg of NFIA OE plasmids, empty pcDNA3 vectors, NFIA shRNA, or scrambled shRNA for 24 h, relative protein levels of IGFBP2 signaling downstream regulators were measured using immunoblotting assays.</p

List of temozolomide (TMZ)-mediated microRNAs.

<p>List of temozolomide (TMZ)-mediated microRNAs.</p

The JNK2/c-Jun signaling pathway is involved in temozolomide (TMZ)-upregulated miR-128 expression.

<p>(A) Effects of mitogen-activated protein kinase (MAPK) inhibitors including U0126 (an ERK inhibitor), SB203580 (a p38 inhibitor), and SP600125 (a JNK inhibitor) on endogenous miR-128 levels. (B) Inhibition of JNK2 and c-Jun expressions inhibited TMZ-upregulated miR-128 levels. After cells were respectively pretreated with 10 μM U0126, 5 μM SB203580, and 10 μM SP600125 for 1 h or respectively transfected with 1 μg of shRNAs for 24 h, 400 μM TMZ was added for another 24 h. miR-128 levels were measured through real-time PCR. Data are the mean ± SD of three experiments. * <i>p</i> < 0.05. (C) Time-dependent effects of TMZ on JNK and c-Jun activation. After cells were treated with 400 μM TMZ for the indicated time, phosphorylated and total forms of JNK and c-Jun were detected using immunoblotting assays. (D) SP600125 treatment, mutation of the activator protein (AP)-1-binding site, and inhibition of JNK2 or c-JUN attenuated TMZ-activated miR-128 promoter activity. After cells were transfected with 500 ng pGL3-miR-128-1-prom2500 or a pGL3-miR-128-1-prom2500 mutant for 24 h, luciferase activity was measured with SP600125 pretreatment for 1 h or cotransfected with 1 μg of shRNAs for 24 h followed by TMZ treatment for another 24 h. pNL1.1.TK[<i>Nluc</i>/TK] plasmids at 5 ng were also cotransfected into cells, and the NanoLucR luciferase value was used as an internal control. (E) The ChIP analysis showed that TMZ enhanced c-Jun binding to the miR-128 promoter. Procedures of the ChIP assay are described in “Materials and methods”. As positive or negative controls, protein–DNA complexes were incubated with anti-RNA polymerase or control mouse IgG antibodies. The input DNA represented one-fifth of the starting material. The right panel figure shows quantitative results from the left panel by densitometry. Overexpression and knockdown effects of miR-128 on TMZ-inhibited cell viability (F) and apoptosis-related protein expressions (G). After cells were respectively transfected with 750 ng of miR-128-overexpressing plasmids and 100 nM miR-128 inhibitor followed by TMZ treatment for another 24 h, cell viability and apoptosis-related protein expressions were measured using MTT and immunoblot assays. Data are the mean ± SD of three experiments. * <i>p</i> < 0.05.</p

TMZ upregulates <i>miR-128-1</i> but not <i>miR-128-2</i> activation.

<p>(A) Dose-dependent effects of TMZ on mature (A) and primary (B) miR-128 expression levels. After cells were treated with the indicated doses of TMZ for 24 h, endogenous miR-128, primary (pri)-miR-128-1, and pri-miR-128-2 levels were respectively measured through real-time PCR. U6B and 18S rRNA levels were used as internal controls for the mature and primary miR-128 genes, respectively. (C) TMZ dose-dependent activated <i>miR-128-1</i> but not <i>miR-128-2</i> promoter activity. (D) Identification of the core region in the <i>miR-128-1</i> promoter. After cells were transfected with 500 ng pGL3-Basic, pGL3-miR-128-1-prom2500, or pGL3-128-1 variants for 24 h, luciferase activity was measured with TMZ treatment for another 24 h. pNL1.1.TK[<i>Nluc</i>/TK] plasmids at 5 ng were also cotransfected into cells, and the NanoLucR luciferase value was used as an internal control. Data are the mean ± SD of three experiments. * <i>p</i> < 0.05.</p

TMZ inhibited mammalian target of rapamycin (mTOR) pathways through miR-128 targeting.

<p>(A) Schematic diagram of potential miR-128-targeted sites in the 3’-untranslated region (UTR) of <i>mTOR</i>, <i>RICTOR</i>, <i>IGF1</i>, and <i>PIK3R1</i> genes. (B) Effects of miR-128 on 3’-UTR luciferase activity of <i>mTOR</i>, <i>RICTOR</i>, <i>IGF1</i>, and <i>PIK3R1</i> genes. To examine the effect of miR-128, different doses of miR-128-expressing plasmids were cotransfected with 500 ng pmiRGlo-3’-UTR or mutant 3’-UTR of <i>mTOR</i>, <i>RICTOR</i>, <i>IGF1</i>, and <i>PIK3R1</i> genes. Luciferase activity was measured in these cells 24 h after transfection. Effects of miR-128 overexpression on mRNA (C) and protein (D) expressions of <i>mTOR</i>, <i>RICTOR</i>, <i>IGF1</i>, and <i>PIK3R1</i> genes. After cells were respectively transfected with the indicated dose of miR-128-expressing plasmids for 24 h, relative mRNA and protein levels of the <i>mTOR</i>, <i>RICTOR</i>, <i>IGF1</i>, and <i>PIK3R1</i> genes were analyzed through real-time PCR and immunoblotting assays. Data are the mean ± SD of three experiments. * <i>p</i> < 0.05. (E) miR-128 is involved in TMZ-reduced mTOR signaling. (F) The effects of IGF-1 stimulation on miR-128-targeted genes expression levels. (G) IGF-1 stimulation attenuated miR-128-induced cytotoxicity. After cells were transfected with 750 ng of miR-128-expressing plasmids or 100 nM of a miR-128 inhibitor, 400 μM of TMZ was added for another 24 h. For IGF-1 stimulation assays, cells were treated with 200ng/mL of IGF-1 recombinant proteins for 24 h or transfected with 500 ng of miR-128-expressing plasmids combined with 200ng/mL of IGF-1 treatment for 24 h. Protein levels of mTOR, RICTOR, IGF1, and PIK3R1 were analyzed using immunoblotting assays. Cell viability was measured using the MTT assay. Data are the mean ± SD of three experiments. * <i>p</i> < 0.05.</p