Circular ZDHHC11 supports Burkitt lymphoma growth independent of its miR-150 binding capacity.

We previously showed that MYC promoted Burkitt lymphoma (BL) growth by inhibiting the tumor suppressor miR-150, resulting in release of miR-150 targets MYB and ZDHHC11. The ZDHHC11 gene encodes three different transcripts including a mRNA (pcZDHHC11), a linear long non-coding RNA (lncZDHHC11) and a circular RNA (circZDHHC11). All transcripts contain the same region with 18 miR-150 binding sites. Here we studied the relevance of circZDHHC11, including this miR-150 binding site region, for growth of BL cells. CircZDHHC11 was mainly present in the cytoplasmic fraction in BL cells and its localization was not altered upon miR-150 overexpression. Knockdown of circZDHHC11 caused a strong inhibition of BL growth without affecting the expression levels of MYC, MYB, miR-150 and other genes. Overexpression of circZDHHC11 neither affected cell growth, nor rescued the phenotype induced by miR-150 overexpression. Genomic deletion of the miR-150 binding site region did not affect growth, nor did it change the effect of circZDHHC11 knockdown. This indicated that the miR-150 binding site region is dispensable for the growth promoting role of circZDHHC11. To conclude, our results show that circZDHHC11 is a crucial factor supporting BL cell growth independent of its ability to sponge miR-150.

Proliferation-promoting roles of linear and circular PVT1 are independent of their ability to bind miRNAs in B-cell lymphoma.

Plasmacytoma Variant Translocation 1 (PVT1) is a long non-coding RNA located at 8q24.21 immediately downstream of MYC. Both the linear and circular PVT1 transcripts contribute to cancer pathogenesis by binding microRNAs. However, little is known about their roles in B-cell lymphoma. Here we studied their expression patterns, role in growth, and ability to bind miRNAs in B-cell lymphoma. Linear PVT1 transcripts were downregulated in B-cell cell lymphoma lines compared to germinal center B cells, while circPVT1 levels were increased. Two Hodgkin lymphoma cell lines had a homozygous deletion including the 5' region of the PVT1 locus, resulting in a complete lack of circPVT1 and 5' linear PVT1 transcripts. Inhibition of both linear and circular PVT1 decreased growth of Burkitt lymphoma, while the effects on Hodgkin lymphoma and diffuse large B cell lymphoma were less pronounced. Overexpression of circPVT1 promoted growth of B-cell lymphoma lacking or having low endogenous circPVT1 levels. Contrary to other types of cancer, linear and circular PVT1 transcripts did not interact with miRNAs in B-cell lymphoma. Overall, we showed an opposite expression pattern of linear and circular PVT1 in B-cell lymphoma. Their effect on growth was independent of their ability to bind miRNAs.

The lncRNA KTN1-AS1 co-regulates a variety of Myc-target genes and enhances proliferation of Burkitt lymphoma cells.

Long non-coding RNAs (lncRNAs) are involved in many normal and oncogenic pathways through a diverse repertoire of transcriptional and posttranscriptional regulatory mechanisms. LncRNAs that are under tight regulation of well-known oncogenic transcription factors such as c-Myc (Myc) are likely to be functionally involved in their disease-promoting mechanisms. Myc is a major driver of many subsets of B cell lymphoma and to date remains an undruggable target. We identified three Myc-induced and four Myc-repressed lncRNAs by use of multiple in vitro models of Myc-driven Burkitt lymphoma and detailed analysis of Myc binding profiles. We show that the top Myc-induced lncRNA KTN1-AS1 is strongly upregulated in different types of B cell lymphoma compared with their normal counterparts. We used CRISPR-mediated genome editing to confirm that the direct induction of KTN1-AS1 by Myc is dependent on the presence of a Myc E-box-binding motif. Knockdown of KTN1-AS1 revealed a strong negative effect on the growth of three BL cell lines. Global gene expression analysis upon KTN1-AS1 depletion shows a strong enrichment of key genes in the cholesterol biosynthesis pathway as well as co-regulation of many Myc-target genes, including a moderate negative effect on the levels of Myc itself. Our study suggests a critical role for KTN1-AS1 in supporting BL cell growth by mediating co-regulation of a variety of Myc-target genes and co-activating key genes involved in cholesterol biosynthesis. Therefore, KTN1-AS1 may represent a putative novel therapeutic target in lymphoma.

The Role of the MYC/miR-150/MYB/ZDHHC11 Network in Hodgkin Lymphoma and Diffuse Large B-Cell Lymphoma.

We previously described involvement of the MYC/miR-150/MYB/ZDHHC11 network in the growth of Burkitt lymphoma (BL) cells. Here we studied the relevance of this network in the two other B-cell lymphomas: Hodgkin lymphoma (HL) and diffuse large B-cell lymphoma (DLBCL). Expression levels of the network components were assessed at the RNA and protein level. The effect of modulating levels of the network components on cell growth was determined through GFP competition assay. AGO2-RNA immunoprecipitation was performed to validate targeting by miR-150. Expression levels of MYC, MYB and ZDHHC11 were increased, while miR-150 levels were decreased similar to the pattern observed in BL. The knockdown of MYC, MYB and ZDHHC11 decreased the growth of HL and DLBCL cells. In contrast, overexpression of miR-150 did not induce clear phenotypes in HL, and limited the effects in DLBCL. This could not be explained by the differences in overexpression levels. Furthermore, we showed that in HL, ZDHHC11 and MYB are efficiently targeted by miR-150. To conclude, MYC, MYB and ZDHHC11 are critical for the growth of HL and DLBCL cells consistent with the role observed in BL cells, while low endogenous miR-150 levels appeared to be less critical for the growth of HL and DLBCL cells despite the effective targeting of ZDHHC11 and MYB.

Synthetic Peptides That Antagonize the Angiotensin-Converting Enzyme-2 (ACE-2) Interaction with SARS-CoV-2 Receptor Binding Spike Protein.

The SARS-CoV-2 viral spike protein S receptor-binding domain (S-RBD) binds ACE2 on host cells to initiate molecular events, resulting in intracellular release of the viral genome. Therefore, antagonists of this interaction could allow a modality for therapeutic intervention. Peptides can inhibit the S-RBD:ACE2 interaction by interacting with the protein-protein interface. In this study, protein contact atlas data and molecular dynamics simulations were used to locate interaction hotspots on the secondary structure elements α1, α2, α3, ß3, and ß4 of ACE2. We designed a library of discontinuous peptides based upon a combination of the hotspot interactions, which were synthesized and screened in a bioluminescence-based assay. The peptides demonstrated high efficacy in antagonizing the SARS-CoV-2 S-RBD:ACE2 interaction and were validated by microscale thermophoresis which demonstrated strong binding affinity (∼10 nM) of these peptides to S-RBD. We anticipate that such discontinuous peptides may hold the potential for an efficient therapeutic treatment for COVID-19.

MiR-378a-3p Is Critical for Burkitt Lymphoma Cell Growth.

MicroRNAs (miRNAs) are small RNA molecules with important gene regulatory roles in normal and pathophysiological cellular processes. Burkitt lymphoma (BL) is an MYC-driven lymphoma of germinal center B (GC-B) cell origin. To gain further knowledge on the role of miRNAs in the pathogenesis of BL, we performed small RNA sequencing in BL cell lines and normal GC-B cells. This revealed 26 miRNAs with significantly different expression levels. For five miRNAs, the differential expression pattern was confirmed in primary BL tissues compared to GC-B cells. MiR-378a-3p was upregulated in BL, and its inhibition reduced the growth of multiple BL cell lines. RNA immunoprecipitation of Argonaute 2 followed by microarray analysis (Ago2-RIP-Chip) upon inhibition and ectopic overexpression of miR-378a-3p revealed 63 and 20 putative miR-378a-3p targets, respectively. Effective targeting by miR-378a-3p was confirmed by luciferase reporter assays for MAX Network Transcriptional Repressor (MNT), Forkhead Box P1 (FOXP1), Interleukin 1 Receptor Associated Kinase 4 (IRAK4), and lncRNA Just Proximal To XIST (JPX), and by Western blot for IRAK4 and MNT. Overexpression of IRAK4 and MNT phenocopied the effect of miR-378a-3p inhibition. In summary, we identified miR-378a-3p as a miRNA with an oncogenic role in BL and identified IRAK4 and MNT as miR-378a-3p target genes that are involved in its growth regulatory role.

The miR-26b-5p/KPNA2 Axis Is an Important Regulator of Burkitt Lymphoma Cell Growth.

The expression of several microRNAs (miRNAs) is known to be changed in Burkitt lymphoma (BL), compared to its normal counterparts. Although for some miRNAs, a role in BL was demonstrated, for most of them, their function is unclear. In this study, we aimed to identify miRNAs that control BL cell growth. Two BL cell lines were infected with lentiviral pools containing either 58 miRNA inhibitors or 44 miRNA overexpression constructs. Eighteen constructs showed significant changes in abundance over time, indicating that they affected BL growth. The screening results were validated by individual green fluorescent protein (GFP) growth competition assays for fifteen of the eighteen constructs. For functional follow-up studies, we focused on miR-26b-5p, whose overexpression inhibited BL cell growth. Argonaute 2 RNA immunoprecipitation (Ago2-IP) in two BL cell lines revealed 47 potential target genes of miR-26b-5p. Overlapping the list of putative targets with genes showing a growth repression phenotype in a genome-wide CRISPR/Cas9 knockout screen, revealed eight genes. The top-5 candidates included EZH2, COPS2, KPNA2, MRPL15, and NOL12. EZH2 is a known target of miR-26b-5p, with oncogenic properties in BL. The relevance of the latter four targets was confirmed using sgRNAs targeting these genes in individual GFP growth competition assays. Luciferase reporter assay confirmed binding of miR-26b-5p to the predicted target site for KPNA2, but not to the other genes. In summary, we identified 18 miRNAs that affected BL cell growth in a loss- or gain-of-function screening. A tumor suppressor role was confirmed for miR-26b-5p, and this effect could at least in part be attributed to KPNA2, a known regulator of OCT4, c-jun, and MYC.

MicroRNA High Throughput Loss-of-Function Screening Reveals an Oncogenic Role for miR-21-5p in Hodgkin Lymphoma.

BACKGROUND/AIMS: Classical Hodgkin lymphoma (cHL) is among the most frequent lymphoma subtypes. The tumor cells originate from crippled germinal center (GC)-B cells that escaped from apoptosis. MicroRNAs (miRNAs) play important roles in B-cell maturation and aberrant expression of miRNAs contributes to the pathogenesis of cHL. Our aim was to identify oncogenic miRNAs relevant for growth of cHL using a high-throughput screening approach. METHODS: A lentiviral pool of 63 miRNA inhibition constructs was used to identify miRNAs essential to cell growth in three cHL cell lines in duplicate. As a negative control we also infected cHL cell lines with a lentiviral barcoded empty vector pool consisting of 222 constructs. The abundance of individual constructs was followed over time by a next generation sequencing approach. The effect on growth was confirmed using individual GFP competition assays and on apoptosis using Annexin-V staining. Our previously published Argonaute 2 (Ago2) immunoprecipitation (IP) data were used to identify target genes relevant for cell growth / apoptosis. Luciferase assays and western blotting were performed to confirm targeting by miRNAs. RESULTS: Four miRNA inhibition constructs, i.e. miR-449a-5p, miR-625-5p, let-7f-2-3p and miR-21-5p, showed a significant decrease in abundance in at least 4 of 6 infections. In contrast, none of the empty vector constructs showed a significant decrease in abundance in 3 or more of the 6 infections. The most abundantly expressed miRNA, i.e. miR-21-5p, showed significantly higher expression levels in cHL compared to GC-B cells. GFP competition assays confirmed the negative effect of miR-21-5p inhibition on HL cell growth. Annexin-V staining of cells infected with miR-21-5p inhibitor indicated a significant increase in apoptosis at day 7 and 9 after viral infection, consistent with the decrease in growth. Four miR-21-5p cell growth- and apoptosis-associated targets were AGO2-IP enriched in cHL cell lines and showed a significant decrease in expression in cHL cell lines in comparison to normal GC-B cells. For the two most abundantly expressed, i.e. BTG2 and PELI1, we confirmed targeting by miR-21-5p using luciferase assays and for PELI1 we also confirmed this at the protein level by western blotting. CONCLUSION: Using a miRNA loss-of-function high-throughput screen we identified four miRNAs with oncogenic effects in cHL and validated the results for the in cHL abundantly expressed miR-21-5p. MiR-21-5p is upregulated in cHL compared to GC-B cells and protects cHL cells from apoptosis possibly via targeting BTG2 and PELI1.

Involvement of MicroRNAs in the Aging-Related Decline of CD28 Expression by Human T Cells.

Loss of CD28 is a characteristic feature of T cell aging, but the underlying mechanisms of this loss are elusive. As differential expression of microRNAs (miRNAs) has been described between CD28+ and CD28- T cells, we hypothesized that altered miRNA expression contributes to the age-associated downregulation of CD28. To avoid the confounding effects of age-associated changes in the proportions of T cells at various differentiation stages in vivo, an experimental model system was used to study changes over time in the expression of miRNA associated with the loss of CD28 expression in monoclonal T cell populations at a lower or higher number of population doublings (PDs). This approach allows identification of age-associated miRNA expression changes in a longitudinal model. Results were validated in ex vivo samples. The cumulative number of PDs but not the age of the donor of the T cell clone was correlated with decreased expression of CD28. Principal component analysis of 252 expressed miRNAs showed clustering based on low and high PDs, irrespective of the age of the clone donor. Increased expression of miR-9-5p and miR-34a-5p was seen in clones at higher PDs, and miR-9-5p expression inversely correlated with CD28 expression in ex vivo sorted T-cells from healthy subjects. We then examined the involvement of miR-9-5p, miR-34a-5p, and the members of the miR-23a~24-2 cluster, in which all are predicted to bind to the 3'UTR of CD28, in the IL-15-induced loss of CD28 in T cells. Culture of fresh naive CD28+ T cells in the presence of IL-15 resulted in a gradual loss of CD28 expression, while the expression of miR-9-5p, miR-34a-5p, and members of the miR-23a~24-2 cluster increased. Binding of miR-9-5p, miR-34a-5p, miR-24-3p, and miR-27- 3p to the 3'UTR of CD28 was studied using luciferase reporter constructs. Functional binding to the 3'UTR was shown for miR-24-3p and miR-27a-3p. Our results indicate involvement of defined miRNAs in T cells in relation to specific characteristics of T cell aging, i.e., PD and CD28 expression.

Argonaute 2 RNA Immunoprecipitation Reveals Distinct miRNA Targetomes of Primary Burkitt Lymphoma Tumors and Normal B Cells.

miRNAs are small noncoding RNAs involved in the posttranscriptional regulation of gene expression. Deregulated miRNA levels have been linked to Burkitt lymphoma (BL) pathogenesis. To date, the number of known pathogenesis-related miRNA-target gene interactions is limited. Here, we determined for the first time the miRNA targetomes of primary BL tumors and normal B cells. AGO2-RNA immunoprecipitation of two frozen diagnostic BL tissue samples and three CD19+ B-cell samples isolated from routinely removed tonsils showed distinct miRNA targetomes of BL and normal B cells. In contrast to normal B cells, miRNA target genes in BL were enriched for targets of the oncogenic miR-17 to 92 cluster, and were involved mainly in cell cycle and cell death. Immunohistochemistry on BL and tonsil tissues confirmed altered protein levels for two of six selected miRNA targets, in line with the differential AGO2-IP enrichment between BL and normal B cells. A comparison of AGO2-IP-enriched genes in primary BL cases with BL cell lines indicated that despite a considerable overlap, the miRNA targetomes of BL cell lines show substantial differences with the targetomes of primary BL tumors. In summary, we identified distinct miRNA targetomes of BL and normal B cells, and showed both the necessity and feasibility of studying miRNA-target gene interactions in primary tumors.

miR-24-3p Is Overexpressed in Hodgkin Lymphoma and Protects Hodgkin and Reed-Sternberg Cells from Apoptosis.

miRNAs play important roles in biological processes, such as proliferation, metabolism, differentiation, and apoptosis, whereas altered expression levels contribute to diseases, such as cancers. We identified miRNAs with aberrant expression in Hodgkin lymphoma (HL) and investigated their role in pathogenesis. Small RNA sequencing revealed 84 significantly differentially expressed miRNAs in HL cell lines as compared to germinal center B cells. Three up-regulated miRNAs-miR-23a-3p, miR-24-3p, and miR-27a-3p-were derived from one primary miRNA transcript. Loss-of-function analyses for these miRNAs and their seed family members resulted in decreased growth on miR-24-3p inhibition in three HL cell lines and of miR-27a/b-3p inhibition in one HL cell line. Apoptosis analysis indicated that the effect of miR-24-3p on cell growth is at least in part caused by an increase of apoptotic cells. Argonaute 2 immunoprecipitation revealed 1142 genes consistently targeted by miRNAs in at least three of four HL cell lines. Furthermore, 52 of the 1142 genes were predicted targets of miR-24-3p. Functional annotation analysis revealed a function related to cell growth, cell death, and/or apoptosis for 15 of the 52 genes. Western blotting of the top five genes showed increased protein levels on miR-24-3p inhibition for CDKN1B/P27kip1 and MYC. In summary, we showed that miR-24-3p is up-regulated in HL and its inhibition impairs cell growth possibly via targeting CDKN1B/P27kip1 and MYC.

Argonaute 2 immunoprecipitation revealed large tumor suppressor kinase 1 as a novel proapoptotic target of miR-21 in T cells.

MicroRNA (miR)-21 is an important suppressor of T-cell apoptosis that is also overexpressed in many types of cancers. The exact mechanisms underlying the antiapoptotic effects of miR-21 are not well understood. In this study, we used the Jurkat T-cell line as a model to identify apoptosis-associated miR-21 target genes. We showed that expression of miR-21 rapidly increases upon αCD3/αCD28 activation of Jurkat cells. Inhibition of miR-21 reduced cell growth which could be explained by an increase in apoptosis. MicroRNA target gene identification by AGO2 RNA-immunoprecipitation followed by gene expression microarray (RIP-Chip) resulted in the identification of 72 predicted miR-21 target genes that were at least twofold enriched in the AGO2-IP fraction of miR-21 overexpressing cells. Of these, 71 were at least twofold more enriched in the AGO2-IP fraction of miR-21 overexpressing cells as compared to AGO2-IP fraction of control cells. The target gene for which the AGO2-IP enrichment was most prominently increased upon miR-21 overexpression was the proapoptotic protein LATS1. Luciferase reporter assays and western blot analysis confirmed targeting of LATS1 by miR-21. qRT-PCR analysis in primary T cells showed an inverse expression pattern between LATS1 transcript levels and miR-21 upon T-cell stimulation. Finally, LATS1 knockdown partially rescued the miR-21 inhibition-induced impaired cell growth. Collectively, these data identify LATS1 as a miR-21 target important for the antiapoptotic function of miR-21 in T cells and likely also in many types of cancer.

Long Noncoding RNA Expression Profiling in Normal B-Cell Subsets and Hodgkin Lymphoma Reveals Hodgkin and Reed-Sternberg Cell-Specific Long Noncoding RNAs.

Hodgkin lymphoma (HL) is a malignancy of germinal center (GC) B-cell origin. To explore the role of long noncoding RNAs (lncRNAs) in HL, we studied lncRNA expression patterns in normal B-cell subsets, HL cell lines, and tissues. Naive and memory B cells showed a highly similar lncRNA expression pattern, distinct from GC-B cells. Significant differential expression between HL and normal GC-B cells was observed for 475 lncRNA loci. For two validated lncRNAs, an enhanced expression was observed in HL, diffuse large B-cell lymphoma, and lymphoblastoid cell lines. For a third lncRNA, increased expression levels were observed in HL and part of Burkitt lymphoma cell lines. RNA fluorescence in situ hybridization on primary HL tissues revealed a tumor cell-specific expression pattern for all three lncRNAs. A potential cis-regulatory role was observed for 107 differentially expressed lncRNA-mRNA pairs localizing within a 60-kb region. Consistent with a cis-acting role, we showed a preferential nuclear localization for two selected candidates. Thus, we showed dynamic lncRNA expression changes during the transit of normal B cells through the GC reaction and widely deregulated lncRNA expression patterns in HL. Three lncRNAs showed a tumor cell-specific expression pattern in HL tissues and might therefore be of value as a biomarker.

Inhibition of the miR-155 target NIAM phenocopies the growth promoting effect of miR-155 in B-cell lymphoma.

Several studies have indicated an important role for miR-155 in the pathogenesis of B-cell lymphoma. Highly elevated levels of miR-155 were indeed observed in most B-cell lymphomas with the exception of Burkitt lymphoma (BL). However, the molecular mechanisms that underlie the oncogenic role of miR-155 in B-cell lymphoma are not well understood. To identify the miR-155 targets relevant for B-cell lymphoma, we performed RNA immunoprecipitation of Argonaute 2 in Hodgkin lymphoma (HL) cells upon miR-155 inhibition and in BL cells upon ectopic expression of miR-155. We identified 54 miR-155-specific target genes in BL cells and confirmed miR-155 targeting of DET1, NIAM, TRIM32, HOMEZ, PSIP1 and JARID2. Five of these targets are also regulated by endogenous miR-155 in HL cells. Both overexpression of miR-155 and inhibition of expression of the novel miR-155 target gene NIAM increased proliferation of BL cells. In primary B-cell lymphoma NIAM-positive cases have significant lower levels of miR-155 as compared to NIAM-negative cases, suggesting that NIAM is also regulated by miR-155 in primary B-cell lymphoma. Thus, our data indicate an oncogenic role for miR-155 in B-cell lymphoma which involves targeting the tumor suppressor NIAM.

Long noncoding RNAs as a novel component of the Myc transcriptional network.

Myc is a well-known transcription factor with important roles in cell cycle, apoptosis, and cellular transformation. Long noncoding RNAs (lncRNAs) have recently emerged as an important class of regulatory RNAs. Here, we show that lncRNAs are a main component of the Myc-regulated transcriptional program using the P493-6 tetracycline-repressible myc model. We demonstrate that both Myc-induced mRNAs and lncRNAs are significantly enriched for Myc binding sites. In contrast to Myc-repressed mRNAs, Myc-repressed lncRNAs are significantly enriched for Myc binding sites. Subcellular localization analysis revealed that compared to mRNAs, lncRNAs more often have a specific subcellular localization with a markedly higher percentage of nuclear enrichment within the Myc-repressed lncRNA set. Parallel analysis of differentially expressed lncRNAs and mRNAs identified 105 juxtaposed lncRNA-mRNA pairs, indicative for regulation in cis. To support the potential relevance of the Myc-regulated lncRNAs in cellular transformation, we analyzed their expression in primary Myc-high and Myc-low B-cell lymphomas. In total, 54% of the lncRNAs differentially expressed between the lymphoma subsets were identified as Myc-regulated in P493-6 cells. This study is the first to show that lncRNAs are an important factor within the Myc-regulated transcriptional program and indicates a marked difference between Myc-repressed lncRNAs and mRNAs.

Cellular localization and processing of primary transcripts of exonic microRNAs.

Processing of miRNAs occurs simultaneous with the transcription and splicing of their primary transcripts. For the small subset of exonic miRNAs it is unclear if the unspliced and/or spliced transcripts are used for miRNA biogenesis. We assessed endogenous levels and cellular location of primary transcripts of three exonic miRNAs. The ratio between unspliced and spliced transcripts varied markedly, i.e. >1 for BIC, <1 for pri-miR-146a and variable for pri-miR-22. Endogenous unspliced transcripts were located almost exclusively in the nucleus and thus available for miRNA processing for all three miRNAs. Endogenous spliced pri-miRNA transcripts were present both in the nucleus and in the cytoplasm and thus only partly available for miRNA processing. Overexpression of constructs containing the 5' upstream exonic or intronic sequence flanking pre-miR-155 resulted in strongly enhanced miR-155 levels, indicating that the flanking sequence does not affect processing efficiency. Exogenously overexpressed full-length spliced BIC transcripts were present both in the nucleus and in the cytoplasm, were bound by the Microprocessor complex and resulted in enhanced miR-155 levels. We conclude that both unspliced and spliced transcripts of exonic miRNAs can be used for pre-miRNA cleavage. Splicing and cytoplasmic transport of spliced transcripts may present a mechanism to regulate levels of exonic microRNAs.

Characterization of protocadherin-1 expression in primary bronchial epithelial cells: association with epithelial cell differentiation.

Protocadherin-1 (PCDH1) is a novel susceptibility gene for asthma that is expressed in airway epithelium. We aimed to characterize PCDH1 mRNA transcripts and protein expression in primary bronchial epithelial cells and to determine regulation of PCDH1 during mucociliary differentiation. Total RNA and protein were isolated from human primary bronchial epithelial cells. PCDH1 transcripts were characterized by rapid amplification of cDNA ends in bronchial epithelial cells of 4 subjects. PCDH1 expression was quantified by quantitative RT-PCR and Western blotting in bronchial epithelial cells directly ex vivo and after air liquid interface (ALI) or submerged culture. We identified 5 novel exons on the 5' end and 1 exon on the 3' end of PCDH1. Novel transcripts showed major variation in expression of intracellular conserved motifs. Expression levels of PCDH1 transcripts encoding exon 1-2 were 4-fold higher, and transcripts encoding exon 3-4 were 15-fold higher in freshly isolated bronchial epithelial cells than in submerged cultures. PCDH1 mRNA (3- to 8-fold) and protein levels (2- to 3-fold) were strongly up-regulated during mucociliary differentiation of primary bronchial epithelial cells in ALI cultures. In summary, PCDH1 transcripts display remarkable variability in expression of conserved intracellular signaling domains. Enhanced PCDH1 expression levels strongly correlate with differentiation of bronchial epithelial cells.

Expression of the c-Met oncogene by tumor cells predicts a favorable outcome in classical Hodgkin's lymphoma.

BACKGROUND: The c-Met signaling pathway regulates a variety of biological processes, including proliferation, survival and migration. Deregulated c-Met activation has been implicated in the pathogenesis and prognosis of many human malignancies. We studied the function and prognostic significance of c-Met and hepatocyte growth factor protein expression in patients with classical Hodgkin's lymphoma. DESIGN AND METHODS: Expression of c-Met and its ligand, hepatocyte growth factor, were determined by immunohistochemistry. Prognostic values were defined in cohorts of German and Dutch patients with classical Hodgkin's lymphoma. Functional studies were performed on Hodgkin's lymphoma cell lines. RESULTS: Expression of c-Met was detected in the tumor cells of 52% (80/153) of the patients and expression of its ligand, hepatocyte growth factor, in 8% (10/121) of the patients. c-Met expression correlated with a 5-year freedom from tumor progression of 94%, whereas lack of expression correlated with a 5-year freedom from tumor progression of 73% (P<0.001) in the combined cohort. In multivariate analysis both c-Met (hazard ratio 5.0, 95% confidence interval 1.9-13.3, P<0.001) and stage (hazard ratio 2.8, 95% confidence interval 1.2-6.4, P=0.014) were independent predictors for freedom from tumor progression. In functional studies activation with hepatocyte growth factor did not affect cell growth, while the c-Met inhibitor SU11274 suppressed cell growth by inducing G2/M cell cycle arrest. CONCLUSIONS: Although functional studies showed an oncogenic role of the hepatocyte growth factor/c-Met signaling pathway in cell cycle progression, expression of c-Met in tumor cells from patients with classical Hodgkin's lymphoma strongly correlated with a favorable prognosis in two independent cohorts.

MiR-17/106b seed family regulates p21 in Hodgkin's lymphoma.

Hodgkin's lymphoma (HL) is a B cell-derived lymphoma characterized by a minority of malignant Hodgkin Reed-Sternberg (HRS) cells that have lost their normal B cell phenotype. Alterations in the cell cycle and apoptosis pathways might contribute to their resistance to apoptosis and sustained cell cycle progression. A key player in both cell cycle arrest and apoptosis is CDKN1A, encoding p21$^{{\rm{waf/cip1}}}$ (p21). P21 is regulated by p53 and can function as a cell cycle inhibitor when in the nucleus or as an apoptosis inhibitor when localized in the cytoplasm. We observed expression of p53, p21 and p-p21 in a variable number of HRS cells in 24 of 40 cases. Expression of miR-17 and miR-106a was detected in HRS cells of 10 HL cases. MiR-17/106b seed family members, CDKN1A RNA and p21 protein levels were variable in HL cell lines. We showed effective targeting of the CDKN1A 3' UTR by miR-17/106b in HL cell lines in a luciferase reporter assay and up-regulation of p21 protein levels upon anti-miR-17 treatment of KM-H2 cells. Functional studies indicated a p21-mediated G(1) arrest after miR-17/106b down-regulation in KM-H2, whereas no G(1) arrest was observed for U-HO1 and L428. This difference could not be explained by differences in the 3' UTR, the cellular location of p21 or expression variation during cell cycle progression. A strong correlation was observed for the miR-17/106b:CDKN1A ratio and the responsiveness to miR-17 inhibition, ie a low ratio in KM-H2 and an extremely high ratio in the two unresponsive HL cell lines. In conclusion, we show that miR-17/106b regulates p21 protein levels in HL and that the effect of miR-17/106b-mediated inhibition depends on the miRNA : target gene ratio. Thus, in HL high miR-17/106b expression contributes to a dysfunctional p53 pathway and thereby also to the malignant phenotype.

A high throughput experimental approach to identify miRNA targets in human cells.

The study of human microRNAs is seriously hampered by the lack of proper tools allowing genome-wide identification of miRNA targets. We performed Ribonucleoprotein ImmunoPrecipitation-gene Chip (RIP-Chip) using antibodies against wild-type human Ago2 in untreated Hodgkin lymphoma (HL) cell lines. Ten to thirty percent of the gene transcripts from the genome were enriched in the Ago2-IP fraction of untreated cells, representing the HL miRNA-targetome. In silico analysis indicated that approximately 40% of these gene transcripts represent targets of the abundantly co-expressed miRNAs. To identify targets of miR-17/20/93/106, RIP-Chip with anti-miR-17/20/93/106 treated cells was performed and 1189 gene transcripts were identified. These genes were analyzed for miR-17/20/93/106 target sites in the 5'-UTRs, coding regions and 3'-UTRs. Fifty-one percent of them had miR-17/20/93/106 target sites in the 3'-UTR while 19% of them were predicted miR-17/20/93/106 targets by TargetScan. Luciferase reporter assay confirmed targeting of miR-17/20/93/106 to the 3'-UTRs of 8 out of 10 genes. In conclusion, we report a method which can establish the miRNA-targetome in untreated human cells and identify miRNA specific targets in a high throughput manner. This approach is applicable to identify miRNA targets in any human tissue sample or purified cell population in an unbiased and physiologically relevant manner.