Experimental Lung Research, 35:646–664, 2009 Copyright C Informa Healthcare USA, Inc. ISSN: 0190-2148 print / 1521-0499 online DOI: 10.3109/01902140902822312 DIFFERENTIALLY EXPRESSED MicroRNAs IN SMALL CELL LUNG CANCER Edit Miko and Zsolt Czimmerer ✷ Department of Biochemistry and Molecular Biology, University of Debrecen Medical and Health Science Center, Debrecen, Hungary Eszter Cs ´ anky ✷ Pulmonology Clinic, University of Debrecen Medical and Health Science Center, Debrecen, Hungary G´ abor Boros and J ´ ulia Buslig ✷ Department of Biochemistry and Molecular Biology, University of Debrecen Medical and Health Science Center, Debrecen, Hungary Bal ´ azs Dezs˝ o ✷ Pathology Institute, University of Debrecen Medical and Health Science Center, Debrecen, Hungary Be ´ ata Scholtz ✷ Department of Biochemistry and Molecular Biology, University of Debrecen Medical and Health Science Center, Debrecen, Hungary ✷ Expression of microRNAs (miRNAs) is characteristically altered in cancer, and they may play a role in cancer development and progression. The authors performed microarray and real-time quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analyses to determine the miRNA expression profile of primary small cell lung cancer. Here we show that at least 24 miRNAs are differentially expressed between normal lung and primary small cell lung cancer (SCLC) tu- mors. These include miR-301, miR-183/96/182, miR-126, and miR-223, which are microRNAs deregulated in other tumor types as well; and other miRNAs, such as miR-374 and miR-210, not previously reported in association with lung cancer. The aberrant miRNA profile of SCLC may offer Received 1 February 2009; accepted 13 February 2009. E. Miko and Z. Czimmerer contributed equally to this study. This work was supported by grants from the National Office for Research and Technology (NKFP 2004 OM-00427 and GVOP-3.1.1.-2004-05-0263/3.0). The authors thank Dr. L´ aszl´ o Nagy (Department of Biochemistry and Molecular Biology, University of Debrecen Medical and Health Science Center, Hungary) for critical reading of the manuscript, and M´ aria Balogh and Zsuzsanna Juh´ asz for technical assistance. Address correspondence to Be´ ata Scholtz, Department of Biochemistry and Molecular Biology, University of Debrecen Medical and Health Science Center, Debrecen, Nagyerdei krt. 98. 4012 Hungary. E-mail: [email protected]646 Exp Lung Res Downloaded from informahealthcare.com by University Library Utrecht on 10/17/13 For personal use only.
Edit Miko and Zsolt Czimmerer � Department of Biochemistry andMolecular Biology, University of Debrecen Medical and Health Science Center,Debrecen, Hungary
Eszter Csanky � Pulmonology Clinic, University of Debrecen Medical andHealth Science Center, Debrecen, Hungary
Gabor Boros and Julia Buslig � Department of Biochemistry andMolecular Biology, University of Debrecen Medical and Health Science Center,Debrecen, Hungary
Balazs Dezso � Pathology Institute, University of Debrecen Medical andHealth Science Center, Debrecen, Hungary
Beata Scholtz � Department of Biochemistry and Molecular Biology,University of Debrecen Medical and Health Science Center,Debrecen, Hungary
� Expression of microRNAs (miRNAs) is characteristically altered in cancer, and they may playa role in cancer development and progression. The authors performed microarray and real-timequantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analyses to determine themiRNA expression profile of primary small cell lung cancer. Here we show that at least 24 miRNAsare differentially expressed between normal lung and primary small cell lung cancer (SCLC) tu-mors. These include miR-301, miR-183/96/182, miR-126, and miR-223, which are microRNAsderegulated in other tumor types as well; and other miRNAs, such as miR-374 and miR-210, notpreviously reported in association with lung cancer. The aberrant miRNA profile of SCLC may offer
Received 1 February 2009; accepted 13 February 2009.E. Miko and Z. Czimmerer contributed equally to this study.This work was supported by grants from the National Office for Research and Technology (NKFP
2004 OM-00427 and GVOP-3.1.1.-2004-05-0263/3.0). The authors thank Dr. Laszlo Nagy (Departmentof Biochemistry and Molecular Biology, University of Debrecen Medical and Health Science Center,Hungary) for critical reading of the manuscript, and Maria Balogh and Zsuzsanna Juhasz for technicalassistance.
Address correspondence to Beata Scholtz, Department of Biochemistry and Molecular Biology,University of Debrecen Medical and Health Science Center, Debrecen, Nagyerdei krt. 98. 4012 Hungary.E-mail: [email protected]
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MicroRNAs in SCLC 647
new insights in the biology of this aggressive tumor, and could potentially provide novel diagnosticmarkers.
Keywords microRNA, small cell lung cancer
Neuroendocrine tumors of the lung can be divided into 4 subtypes: typi-cal and atypical carcinoid tumors, large cell neuroendocrine cancers (LC-NECs), and small cell lung cancer (SCLC). The 4 subtypes can be differ-entiated by morphology, immunohistochemistry, and specific patterns ofchromosomal aberrations [1–4]. Global gene expression profiling, on theother hand, appears to identify only 2 prognostically different subtypes: car-cinoid tumors and high-grade neuroendocrine tumors (HGNTs), the latterincluding SCLC and LCNEC [5, 6]. SCLC accounts for 15% to 20% of lungcancers, and has a very dismal prognosis [1, 7]. In spite of recent advancesin understanding the molecular biology of lung cancer, and a number ofclinical trials to develop better therapeutic protocols, most SCLC patientsstill succumb to the disease within a year from diagnosis. Two character-istics contribute to SCLC-related mortality: although initially responsive tochemotherapy, SCLC tumors usually develop chemoresistance, and tend toreappear in the lung; in addition, SCLC is highly metastatic, and almostalways diagnosed as a late-stage disease. More intensive research into theunique molecular biology behind this aggressive phenotype is warranted toimprove the management of SCLC.
MicroRNAs (miRNAs) are special in that they are exclusively negativeregulators of gene expression, mostly at the level of translation in animals.According to the current paradigm, a single miRNA may target a large num-ber of mRNAs, which could theoretically result in the coordinate regulationof proteins participating in the same biological pathway. Not surprisingly,miRNAs are considered to be critical in the initiation, maintanence, andfine-tuning of several cellular programs, including embryonic development,hematopoietic differentiation, organ development, or glucose homeostasis[8–13]. There is a growing body of evidence that expression patterns ofmiRNAs change characteristically in cancer, and in fact, expression profilesmonitoring a few hundred miRNAs can accurately distinguish tumor sub-types, and tumors from normal tissue [14].
The purpose of our work was to characterize the miRNA expressionprofile of primary SCLC tumors. First, we compared the expression of 319miRNAs in 3 SCLC cell lines and in normal lung tissue by microarray.Next, 62 miRNAs expressed differentially were selected for validation withreal-time quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR), and their expression levels were compared between SCLC cell lines,normal lung tissue, and primary SCLC tumors. Lastly, the expression of 15prominently deregulated miRNAs was analyzed in a panel of primary SCLCtumors.
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MATERIALS AND METHODS
Cell Lines and Human Tissue Samples
Established SCLC cell lines were purchased from the American TypeCulture Collection (HTB-172, HTB-184, HTB-119 [= H69]). The cell lineswere maintained in RPMI 1640 supplemented with 10% fetal bovine serum(FBS) and penicillin/streptomycin at 37◦C with 5% CO2.
All primary clinical samples were collected with the approval of theUniversity’s Ethical Committee. Normal human lung tissue samples wereobtained from the 2nd Department of Surgery at the University of Debre-cen (Hungary). Patients were heavy smokers, tumor-free, with an averageage of 45 (range, 40 to 50), and had chronic obstructive pulmonary dis-ease (COPD). Peripheral blood samples were collected at the PulmonologyClinic (University of Debrecen, Hungary) from tumor-free individuals.Twenty-three archived formalin-fixed, paraffin-embedded (FFPE) SCLC tu-mor samples were obtained from the Department of Pathology at theUniversity of Debrecen (Hungary). Clinical data for these patients is shownin Supplementary Data (Supplementary Table 5); like the control patients,they also had COPD. Histologic diagnosis of the selected tumors were val-idated by an experienced pathologist, based on the 1999 World HealthOrganization (WHO) classification. In this regard, all SCLC tumors werepositive for neuron-specific enolase (NSE), chromogranin A, and/or synap-tophysin by standard immunohistochemical methods (data not shown).
Microdissection of FFPE Tumors
Blocks of tumors were cut to yield minimum 12 serial sections. The firstand last sections (4 µm) were used for hematoxylin and eosin (H&E) stain-ing. Five sections, cut at 20 µm to prevent loss of small RNA species duringisolation, were used for microdissection. These sections were microdissectedby an experienced pathologist to separate tumor and normal tissue, underdirect observation with a microscope, guided by the H&E-stained sections,using a fine needle. The remaining 4-µm sections were used for immuno-histochemical staining (data not shown).
Nucleic Acid Extraction
Total RNA was isolated from surgical lung samples and SCLC cell linesusing Trizol reagent (Invitrogen) according to the manufacturer’s instruc-tions. Genomic DNA was extracted from SCLC cell lines and peripheralblood mononuclear cells using the Qiagen Blood and Cell Culture kit, ac-cording to the instructions of the manufacturer. Total RNA and DNA wereisolated from FFPE samples with RecoverAll Total Nucleic Acid Isolation Kit(Ambion) according to the manufacturer’s protocol.
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Microarray Analysis
miRNA profiling of normal lung and SCLC cell lines was per-formed using a service provider (LC Sciences, Houston, TX, http://www.lcsciences.com, miRNA dual-color microarray platform, Human V7.1). Abrief description of the method is given in Supplementary Methods 1. Themicroarray interrogated 319 mature miRNAs, each represented by 4 individ-ual probes on the chip. These chemically modified probes have normalizedTm-s ensuring uniform hybridization on the chip under high-stringency hy-bridization conditions enhancing both the sensitivity and specificity of theprobes [15, 16]. RNA from 3 SCLC cell lines (H69, HTB-172, HTB-184)was used for the initial microarray screen, as a surrogate for primary tumorRNA, and compared to RNA from fresh surgical samples of lung tissue asnormal control. RNA from primary SCLC tumors was available only fromFFPE samples, because SCLC patients are rarely operated for their cancer.However, RNA isolated from FFPE tumor samples could not be used in themicroarray experiment, mainly because the FFPE sections did not provideenough material for the microarray platform. Normal lung RNA was pooledfrom 6 samples isolated from surgical material, and the 3 SCLC cell lineswere assayed individually on 3 chips, each chip comparing the expressionprofile of 1 cell line to the common normal lung reference. miRNAs withraw signals above the averaged microarray background +5 SD were selectedfor further analysis. Data were normalized to hsa-miR-342, and analyzed bythe GeneSpring 7.3 software. Relative miRNA expression values (SCLC celllines relative to normal lung) were derived after normalization from aver-aged values of 3 SCLC cell line microarrays, and averaged values of 3 nor-mal lung microarrays. miRNAs showing statistically significant 5-fold changewere selected for further analysis by qRT-PCR (paired t test, 2-tailed P value,95% confidence interval [CI]). Because the microarray has lower sensitivitycompared with real-time qRT-PCR, some miRNAs that may have a role inSCLC were not detected by the platform.
miRNA Real-Time Quantitative RT-PCR Analysis
miRNAs with above-background expression in SCLC cell lines or nor-mal lung in the microarray experiment were tested by qRT-PCR analy-sis either by using the TaqMan MicroRNA Assays Early Access Kit or byindividual miRNA qRT-PCR assays, according to the manufacturer’s pro-tocol, with minor modifications (Applied Biosystems). In the first screenassaying 62 miRNAs, pooling of RNA was necessary due to the limitingamount of RNA obtained from the FFPE sections. In subsequent experi-ments assaying fewer miRNAs per sample (7 downregulated and 8 over-expressed miRNAs in a panel of 17 SCLC tumors), pooling of the RNAwas not necessary. qPCR was performed on the ABI 7900 HT Sequence
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Detection System. Composition of the RT mixes, the PCR reactions andthe RT-PCR protocols are provided in Supplementary Methods 2. RelativemiRNA expression values (tumor versus normal) were calculated usingthe 2−��Ct method [17]. miRNA expression levels in FFPE SCLC primarytumor samples were compared to their own FFPE normal lung tissue coun-terparts, isolated from the vicinity from the tumor. miRNA expression levelsin SCLC cell lines were compared to fresh surgical normal lung tissue. Thenormalizing gene was hsa-miR-342, which showed the smallest variation be-tween normal lung, SCLC cell lines, and primary tumors (SupplementaryFigure 1).
Copy Number Analysis
Five primer sets were designed to measure the genomic copy number of5 miRNAs (miR-183, miR-182, miR-95, miR-301, and the miR-17-92 cluster)in SCLC cell lines (H69, HTB-172, HTB-173, HTB-184), in 4 FFPE SCLC tu-mors and their normal tissue counterparts isolated by microdissection, andin peripheral blood mononuclear cell (PBMC) genomic DNA mixed fromthe samples of 5 tumor-free individuals. Relative gene copy numbers werecalculated using the 2−��Ct method [17]. Because tumor cell lines and pri-mary tumors harbor many amplified chromosomal regions, we tested severalcandidate normalizing genes. The albumin gene had variable copy numbersin SCLC (data not shown), but the PP1A, TBP, and H19 genes proved tobe suitable candidates to normalize miRNA copy number in our samples.Primer sequences, positions and amplicon lengths are listed in Supplemen-tary Table 4. Copy numbers of the 5 miRNA genomic regions, and the TBPand H19 normalization genes, were measured using SYBR Green I qPCR,and a TaqMan assay was used to quantify PP1A. The detailed protocol isprovided in Supplementary Methods 3. Ct values for miRNAs and the nor-malizer genes were the average of 3 or 5 independent PCR measurements,each sample tested in duplicate in each PCR. Significance of differencesfor the means was assessed with paired t test, using 2-tailed P value, 95%CI. Numerical representation of Figure 2 and data for SCLC cell lines is inSupplementary Table 3A, B.
RESULTS
Comparative miRNA Expression Profiling of SCLC Cell Lines
and Normal Lung
We characterized the miRNA expression profile of normal lung and3 SCLC cell lines, HTB-184, HTB-172, and H69, using the LC SciencesmiRNA microarray platform, interrogating 319 mature miRNAs. The ref-erence normal lung RNA was pooled from 6 RNA samples isolated from
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TABLE 1 miRNA Microarray Profiling of SCLC Cell Lines H69, HTB-184 and HTB-172,Compared to Normal Lung
Note. miRNAs located close to each other, or transcribed as a polycistron are boxed—some ofthese clustered miRNAs are shown, even if the relative expression values did not reach the thresholdvalues (5-fold change). miRNAs quantified by qRT-PCR as well are bold-faced. Chr = chromosomallocalization. Significance was assessed by paired t -test, two-tailed P value, CI 95%.
surgically removed lung tissue. Because the patients providing the sampleswere tumor-free, but middle-aged and heavy smokers, the miRNA expres-sion profile of “normal“ lung probably reflects the effects of age, smoking,and COPD-associated chronic inflammation as well.
The 3 replica arrays for normal lung demonstrated that the individual ar-rays have low variability. Comparison of normalized data allowed the identi-fication of 19 miRNAs that are significantly overexpressed, by at least 10-foldin SCLC cell lines compared to normal lung (Table 1). We also identified36 miRNAs that are down-regulated in SCLC cell lines compared to normallung, and a large region in 19q13.41 containing several downregulated miR-NAs (miR-512 to miR-373), albeit these latter were detected at low levels inboth sample types (Table 1).
A number of the differentially expressed miRNAs is organized inclusters, such as the miR-17-92 cluster, the miR-200b-429 cluster, or the
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miR-145/143 cluster. As observed before by others [18], miRNAs in onecluster are up- or downregulated in concert, indicating that they are mem-bers of the same regulatory unit. It is also evident that miRNAs present inmultiple copies on different chromosomes (miR-29b, miR-199a, miR-133a,and miR-153 [data not shown]), or closely related miRNAs (miR-199a/b,miR-200a/b/c, miR-181-a/b/c/d, miR-106a-363, and miR-17-92 clusters)are often coordinately regulated.
Validation of miRNA Expression Profile With qRT-PCR in SCLC
Cell Lines and Primary Tumor Samples
Based on the microarray experiment, we selected 21 significantly up-or downregulated miRNAs for validation; the remaining 41 miRNAs wereselected because of their potential role in cancer biology, based on litera-ture data. Expression levels of the 62 miRNAs were validated by qRT-PCRin 4 sample types: (a) 3 SCLC cell lines (pooled RNA from H69, HTB-184,and HTB-172); (b) normal lung tissue samples (pooled RNA from 6 sam-ples); (c) formalin-fixed, paraffin-embedded (FFPE) primary SCLC tumorsamples (pooled RNA, isolated from 6 samples); and (d) FFPE normal lungtissue derived from the vicinity of the tumors (pooled RNA, isolated from 6samples). The pooled RNA from 6 normal lung samples was the same thatwas used for the microarray experiment. Sections from FFPE tumor sam-ples were microdissected under the microscope by an experienced pathol-ogist, and RNA was isolated separately from the normal lung tissue and thetumor tissue. Although the microdissected tumor tissue samples still con-tained some normal cells, they can be considered significantly enriched fortumor cells. Successful separation of normal tissue from tumor tissue wasverified by the barely detectable neuron-specific enolase (NSE) mRNA lev-els in the FFPE normal lung samples, versus the high expression of NSE inthe enriched SCLC tumor samples, as determined by qRT-PCR (data notshown).
qRT-PCR analysis confirmed overexpression of 16 miRNAs in primarySCLC tumors, as well as in SCLC cell lines (Table 2, rows 1 to 16), in ac-cordance with the microarray results. qRT-PCR analysis also demonstrateddownregulation of 8 miRNAs in primary SCLC tumors and in SCLC celllines (Table 2, rows 17 to 24).
Expression Patterns of Selected miRNAs in Primary SCLC
Tumors
Seven down regulated and 8 overexpressed miRNAs were selected forfurther analysis in a panel of 17 SCLC tumors, resected from the lung orfrom metastatic sites (Supplementary Table 5). qRT-PCR analysis verified
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TABLE 2 Relative Expression Values of miRNAs Measured by qRT-PCR
Note. RNA isolated from 6 microdissected, primary SCLC tumors (S1-6) were pooled. RNA from H69,HTB-184, and HTB-172 was also pooled before RT-qPCR. miR-342 is the normalizing miRNA. miRNAsin rows 1–24 (left side of figure) show at least two-fold change in both primary tumors and in SCLC celllines when compared to normal lung. miRNAs in bold were also quantified in additional primary SCLCtumor samples (see results in Figure 1A–B).
that miR-126 is uniformly down-regulated in all SCLC samples, and miR-150,miR-222, and miR-223 are down regulated in the majority of the samples(Figure 1A), whereas miR-29a, miR-214, and miR-199a showed a variable ex-pression pattern. Two miRNAs, miR-301 and miR-183, were uniformly over-expressed in SCLC tumors, and miR-106a, miR-25, and miR-95 were upreg-ulated in the majority (77% to 65%) of the samples (Figure 1B). We did notfind any evidence of the tumor location (lung or metastatic site) influencingthe expression of the miRNAs studied in these samples.
Gene Amplification Is Not the General Mechanism Causing
Overexpression of Several miRNAs in SCLC Tumors
Many microRNA genes are found in chromosomal regions with fre-quent genetic aberrations in tumors. Therefore, we used qPCR to deter-mine the copy numbers for 5 genomic regions harboring prominently over-expressed miRNAs: miR-17-92, miR-183/96, miR-182, miR-95, and miR-301in SCLC cell lines and in primary SCLC tumors (Figure 2). Gene amplifi-cation was clearly detected for all 5 miRNA genomic regions in the SCLCcell lines, including for the miR-17-92 cluster, which is in accordance withprevious observations [19] (Supplementary Table 3A). Surprisingly, how-ever, in primary SCLC tumors, only the miR-183/96/182 genomic region
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FIGURE 1 Overexpressed and down-regulated miRNAs in primary SCLC tumors. Relative miRNA ex-pression was determined in FFPE primary SCLC tumors relative to FFPE normal lung tissue, by stem-loopqRT-PCR. The solid horizontal lines at 0.5 (A) or at 2 (B) indicate the threshold value. Significance forthe differences of the mean expression values (tumor vs normal lung) was determined by paired t test,95% CI. miR-214, miR-199a, and miR-29a relative expressions were not significantly different.
was characteristically amplified, in 3 out of 4 samples (Figure 2, and Sup-plementary Table 3B). Although miR-301 was uniformly and highly overex-pressed in SCLC tumors, gene copy number gain was detected in only 1sample (S6), and another sample in fact lost one allele (S5).
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FIGURE 2 Gene copy numbers of 5 overexpressed miRNAs in SCLC tumors. Genomic DNA isolatedfrom 4 microdissected primary SCLC tumors (S1, S4, S5, and S6) was analyzed by qPCR for copy numberchanges, as described in the Materials and Methods. By custom, 2n represents the value for normal,diploid genome, whereas 3n indicates one gained copy, and 1n indicates loss of heterozygosity. miR-182and miR-183 are located nearby, and are represented on the same chart. Data are shown as mean ±SEM.
DISCUSSION
In summary, we combined microarray and qRT-PCR approaches to iden-tify microRNAs aberrantly expressed in small cell lung cancer. We were ableto identify several miRNAs not implicated before in lung cancer, such asmiR-105, miR-429, miR-494, miR-377, miR-374, miR-375, miR-7, and miR-497. As expected, we also found disregulated expression of a number ofmiRNAs in SCLC, which were detected before in other tumor types, orhave known functions in cancer. For example, overexpression of miR-301or miR-183/96 is associated with pancreatic cancer or colorectal cancer, re-spectively [20, 21], the miR-17-92 or miR-106b-363 clusters are known onco-genic clusters [19, 22–24], and miR-214 may regulate apoptosis [25]. Amongthe downregulated miRNAs, miR-145/143, miR-135b, and miR-133b wereshown before to be downregulated in colorectal cancer [20, 26], whereasmiR-34a expression is activated by p53, and induces apoptosis [27, 28]. Onthe other hand, expression of miR-451 is increased during erythroid matu-ration [29, 30], and it is possible that high signals for this miRNA in normal
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TABLE 3 Comparison of miRNA Relative Expression Patterns in SCLC and NSCLC
SCLC NSCLC Ref.
miR-95 Up Down 41miR-9 Up Down 41miR-200c Up Down 42miR-128b Up Down 43let-7 Unchanged Down 44–48miR-34a Down Up 28, 49miR-150 Down Up 41miR-155 Down Up 41miR-223 Down Up in serum 43, 50miR-29 Down Down 41, 51, 52miR-143 Down Down 41miR-145 Down Down 41miR-126 Down Down 41, 53miR-33 Down Down 41miR-224 Down Down 41miR-25 Up Up in serum 43, 50mir-17-92 cluster Up Up 19, 41, 47, 48miR-106a cluster Up Up 41
Note. Up: upregulated; Down: downregulated in tumors relative to normal lung tissue.
lung reflects the presence of blood in the surgical samples. Lastly, we wereable to identify several miRNAs expressed differentially in SCLC versus non–small cell lung cancer (NSCLC), as listed in Table 3. Interestingly, the let-7family of miRNAs are among the highest expressed miRNAs in SCLC, but,unlike in NSCLC tumors, their expression levels are similar to normal lungtissue. miR-9, miR-95, miR-200c, and miR-128b are 4 miRNAs overexpressedin SCLC, but not in NSCLC, which may provide the basis of differential di-agnostics.
Currently there is little known about the role of miRNAs in inflamma-tion, even though it has a central role in several chronic diseases, includ-ing COPD, and is also strongly linked with tumorigenesis. Altered expres-sion of some miRNAs, including miR-223 and miR-146a, was implicated inacute inflammatory responses in the lung [31, 32]; in addition, miR-203and miR-146a may be involved in psoriasis and rheumatoid arthritis [33–35]. Because the lung tissue samples included in our study were derivedfrom patients with COPD, the miRNA expression profile of the nontumorlung samples likely has a COPD-specific component as well. Further studieswill be required to dissect which miRNAs are involved in the inflammatoryprocesses, contributing to the tissue destruction evident in COPD.
Several studies investigating chromosomal aberrations in SCLC iden-tified characteristically lost genomic regions [4, 36–40]. Some of thedown regulated miRNAs are found in regions with frequent loss of heterozy-gosity in SCLC: the 5q32-ter region, containing the miR-143/145 cluster
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(5q33.1), is lost in 79% of SCLC tumors; the 9q22-32 region, containingmiR-32 (9q31.3), is lost in 64% of SCLC tumors; and the region 17p12-ter,containing miR-497, as well as the p53 gene (17p13.1), is deleted in 93% ofSCLCs [36]. Interestingly, the overexpressed miRNAs are not embedded inchromosomal regions frequently amplified in SCLC, with the possible ex-ception of the miR-200b-200a-429 cluster (1p36.33), located in the vicinityof the antiapoptotic gene TNFRSF4, which is amplified in SCLC [44]. There-fore, we analyzed DNA copy number changes in primary SCLC tumors for 5genomic regions with overexpressed miRNAs: miR-17-92, miR-183/96, miR-182, miR-95, and miR-301. Our experiments identified a novel amplifiedregion in SCLC, in the 7q32.2 chromosomal region. 7q32.2 contains themiR-183-96-182 cluster, and copy number gains were clearly detected in 3 of4 SCLC tumors. In contrast, 3 other genomic regions studied showed no orinfrequent amplification (miR-17-92, miR-95, and miR-301).
Taken together, our data and previous studies clearly show that althoughmiRNAs located in the same cluster are up- or downregulated together,the levels of mature miRNAs can be quite different for the cluster mem-bers. In addition, closely related miRNAs, or miRNAs with multiple copieson different chromosomes are often coregulated in SCLC and in other tu-mors, suggesting that aberrant transcriptional regulation is not the only fac-tor contributing to the overexpression or downregulation of many miRNAs.In fact, it is likely that regulation of the post-transcriptional steps, includ-ing Drosha cleavage, export to the cytoplasm, RNA editing, and the cyto-plasmic processing steps, play a very significant, albeit currently less wellcharacterized, role in determining the final microRNA profile for normaland cancer cells.
Declaration of interest: The authors report no conflicts of interest. Theauthors alone are responsible for the content and writing of the paper.
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