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Expression of apoptosis-regulating miRNAs and target mRNAs in oral squamous cell carcinoma
Cancer Genetics
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(2015)
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Expression of apoptosis-regulating miRNAs and target mRNAs in oral squamous cell carcinoma udia Malheiros Coutinho-Camillo a,*, Silvia Vanessa Lourenc‚o b, Cla jo Lima c, Luiz Paulo Kowalski d, Fernando Augusto Soares a,b Leandro de Ara u a b
~ o Paulo, Brazil; Department of General Pathology, Department of Anatomic Pathology, AC Camargo Cancer Center, Sa ~ o Paulo, Sa ~ o Paulo, Brazil; c Interinstitutional Graduate Program on Bioinformatics, University Dental School, University of Sa ~ o Paulo, Sa ~ o Paulo, Brazil; d Department of Head and Neck Surgery and Otorhinolaryngology, AC Camargo Cancer of Sa ~ o Paulo, Brazil Center, Sa Aberrations in the apoptotic mechanisms that cause excessive or deficient programmed cell death have been linked to a wide array of pathological conditions. In this study, using real-time reverse transcriptaseePCR, we analyzed the expression of apoptosis-regulating miRNAs (miR-15a, miR-16, miR-17-5p, miR-20a, miR-21, miR-29a, and miR-34a) in 20 oral squamous cell carcinoma and 5 normal oral mucosa tissue samples. Bioinformatic algorithms were used to identify the target genes of these miRNAs (BCL2, CASP2, CASP7, CASP8, DIABLO). The expression transcript levels of the target genes were measured in 50 oral squamous cell carcinoma and 10 normal oral mucosa tissue samples. We observed downregulation of miR-15a, miR-29a, and miR-34a in 50, 75, and 70% of samples, respectively. miR-16, miR-17-5p, miR20a, and miR-21 expression was normal in 80, 75, 90, and 60% of samples, respectively. BCL2 transcripts were downregulated in 60% of samples, and normal-like expression was observed for CASP2, CASP7, CASP8, and DIABLO transcripts in 66, 82, 68, and 60% of samples, respectively. BCL2 expression was negative to weak, and that of proteins CASP2, CASP7, CASP8, and DIABLO was moderate to strong. Our study provides evidence of alterations in the expression of apoptosis-regulating miRNAs and genes in the apoptotic pathway, demonstrating that regulation of apoptosis is a hallmark of oral squamous cell carcinoma pathogenesis. Keywords miRNA, oral squamous cell carcinoma, apoptosis, real-time PCR, gene expression ª 2015 Elsevier Inc. All rights reserved.
MicroRNAs (miRNAs) are noncoding, single-stranded RNAs of approximately 22 nucleotides, and they constitute a novel class of gene regulators. They govern gene expression by binding to the 30 untranslated region (30 UTR) of the target mRNA, leading to translational inhibition or degradation of mRNA, depending on the degree of sequence complementarity (1,2). The significance of miRNAs in cancer has been highlighted by evidence that approximately 50% of miRNA genes lie in cancer-associated genomic regions or fragile sites (3); they are frequently amplified or deleted in tumorigenesis and might be related to altered tumor suppressor gene or oncogene function, causing uncontrolled cell proliferation or Received October 17, 2014; received in revised form March 12, 2015; accepted April 10, 2015. * Corresponding author. E-mail address: [email protected] 2210-7762/$ - see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cancergen.2015.04.004
survival (4). The precise function of miRNAs in cell survival and apoptotic signaling is unknown, but several studies have implicated them in these processes (5). At least 30 individual miRNAs have been reported to control apoptosis. In general, miR-17-5p, miR-20a, miR-21, miR-133, miR-145, miR-146a, miR-146b, miR-155, miR-191, miR-14, bantam, miR-1d, miR-7, miR-148, miR-204, miR210, miR-216, miR-296, and miR-Lat are antiapoptotic. Conversely, the let-7 family, miR-15a, miR-16-1, miR-29, miR-34a, miR-34b, miR-34c, miR-1, miR-101, and miR-214 are considered proapoptotic (3,6e12). Large-scale profiling studies of microRNA expression in human cancers have demonstrated that dysregulation of miRNA is frequently associated with many cancer types, including those that originate from the brain, breast, lung, and prostate (3). Several studies have analyzed the expression of miRNAs in head and neck cancers (1,13e16), but few have examined apoptosis-regulating miRNAs in oral cancer (17,18).
udiaM. Coutinho-Camillo et al. Cla
2 Increasing evidence suggests that oral carcinogenesis correlates with the progressive accumulation of genetic alterations in molecules that mediate apoptosis (19e27). Oral squamous cell carcinoma (OSCC) is the eighth-most common cancer worldwide (28), and its prevalence is high in Brazil, where 15,290 new cases of oral cancer were estimated to develop in 2014 (29). Insights into the general molecular mechanisms of OSCC, for which the function of miRNAs remains undetermined, could guide the development of new therapies. Using quantitative real-time reverse transcriptaseePCR (qPCR), we characterized the expression of apoptosisregulating miRNAs (miR-15a, miR-16, miR-17-5p, miR-20a, miR-21, miR-29a, and miR-34a) in OSSC.
Materials and methods
Table 1 Summary of the demographic and clinicopathologic characteristics of OSCC patients Characteristic
Category
Age, y
60 > 60 n/a Male Female n/a No Yes n/a No Yes n/a No Yes n/a No Yes n/a Poorly/moderately differentiated Well-differentiated n/a No Yes n/a Oral tongue Floor of mouth Others n/a I/II III/IV n/a
Gender
Tobacco smoking status Alcohol consumption status Lymph node metastasis
Tissue samples Tumor tissue samples from 50 OSCC cases and 10 normal oral mucosa samples were provided by the AC Camargo ~o Paulo, Brazil. All cases had Cancer Center Biobank, Sa been treated at the hospital for at least 5 years. Tumor samples were collected before any systemic treatment was administered, and samples were dissected to remove residual normal tissue. Paraffin-embedded tissue samples from 37 of the 50 OSCC cases were obtained from the archives of the Department of Pathology, AC Camargo Cancer ~o Paulo, Brazil. The clinical and histological details Center, Sa of the cases are provided in Table 1. Informed consent was obtained from all patients, and the institutional ethics committee approved this study (protocol number 985/07).
RNA isolation Total RNA was extracted from the OSCC and normal tissue samples using Life Technologies Trizol (Thermo Fisher Scientific, Waltham, MA) and treated with Life Technologies DNase I (Thermo Fisher Scientific) per the manufacturers’ instructions. The RNA was quantified on a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific), and its quality was assessed on a 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA).
qPCR: miRNA expression Ten nanograms of total RNA was reverse-transcribed from 20 OSCC and five normal oral tissue samples using the Applied Biosystems TaqMan microRNA reverse transcription kit (Thermo Fisher Scientific). PCR was performed on an ABI 7900HT sequence detection system (Thermo Fisher Scientific) using Applied Biosystems TaqMan universal master mix (Thermo Fisher Scientific). The program comprised 40 cycles of denaturation for 15 seconds at 95 C and annealing for 1 minute at 60 C after an initial denaturation step (95 C for 10 min). The expression of apoptosis-regulating miRNAs (miR-15a, miR-16, miR-17-5p, miR20a, miR21, miR-29a, and miR-34a) was analyzed by TaqMan miRNA assays (Thermo Fisher Scientific; TM000389, TM000391, TM000393, TM000580, TM000397, TM002112, and TM000426).
Perineural infiltration
Histological grade
Vascular invasion
Tumor site
Clinical stage
Number of patients 18 30 2 36 13 1 30 9 11 7 30 13 16 23 11 26 20 4 21 27 2 38 9 3 11 15 23 1 22 21 7
Abbreviaiton: n/a, information not available.
The relative miRNA expression level was normalized to two reference small nucleolar RNAsdSNORD44 and SNORD48 (TM001094 and TM001006)dand to a calibrator sample. A pool of five normal oral mucosa samples served as the calibrator sample. Experiments were performed in duplicate for each data point. The final results, expressed as n-fold differences in expression relative to the reference small nucleolar RNAs and calibrator sample, were calculated as follows: Relative expression Z 2 ðDCt; sample DCt; calibrator Þ where cycle threshold difference (DCt) values of the sample and calibrator were determined by subtracting the average Ct value of the apoptosis-regulating miRNA from the geometric average Ct value of SNORD44 and SNORD48.
Real-time RT-PCR (qPCR): mRNA expression Two micrograms of total RNA was reverse transcribed from 50 OSCC and 10 normal oral tissue samples using a HighCapacity cDNA reverse transcription kit (Thermo Fisher Scientific). PCR amplification was performed on an ABI
miRNAs and target mRNAs in oral squamous cell carcinoma 7900HT sequence detection system (Thermo Fisher Scientific) using TaqMan universal master mix (Thermo Fisher Scientific); the program consisted of 40 cycles of denaturation for 15 seconds at 95 C and annealing for 1 minute at 60 C after an initial denaturation step at 95 C for 10 minutes. BCL2, CASP2, CASP7, CASP8 and DIABLO expression was examined using TaqMan gene expression assays (Hs00153350_m1, Hs00154242_m1, Hs00169152_m1, Hs00154256_m1, and Hs00219876_m1). Relative expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) and 18S using TaqMan gene expression assays (4326317E and 4319413E) and to a calibrator sample. A pool of 10 normal oral mucosa samples served as the calibrator sample. Experiments were performed in duplicate for each data point. The final results were expressed as n-fold differences in expression relative to the reference genes and calibrator sample, as follows:
3 and the staining intensity: 0, negative/weak staining (10% of cells stained and visible only at 40); 1, moderate staining (10e50% of cells stained and visible at 20); and 2, strong staining (>50% of cells stained and easily detectable at 10). For the statistical analysis, we grouped the cases into two categories: negative (negative/weak expression) and positive (moderate/strong expression).
Statistical analysis
where DCt values of the sample and calibrator were determined by subtracting the average Ct value of the target gene from the geometric average Ct value of GAPDH and 18S.
The association between the clinicopathologic characteristics of the patients and miRNA, mRNA, and protein expression was analyzed by the c2 test. We analyzed differences in expression between the following groups: clinical stage (I/II and III/IV), tumor site (oral tongue, floor of mouth, or other sites), tobacco smoking (yes or no), alcohol consumption (yes or no), gender (male or female), lymph node metastasis (yes or no), vascular invasion (yes or no), perineural infiltration (yes or no), and histological grade (well-differentiated or poorly/moderately differentiated). The significance level was 5% for all statistical tests. All analyses were performed using R, version 2.13 (30).
Immunohistochemistry
Results
The expression of BCL2, CASP2, CASP7, CASP8, and DIABLO was examined in OSCC tissue samples on a tissue microarray. The immunostaining was performed on duplicate tissue slides; duplicate sections were separated by 40 mm. The slides were deparaffinized, rehydrated, and subjected to antigen retrieval. Details on the antigen retrieval methods and primary antibody clones, sources, and titers are listed in Table 2. The sections were incubated in 3% aqueous hydrogen peroxide for 15 minutes to quench endogenous peroxidase activity, and then they were incubated with Dako Protein Block, Serum-Free (Agilent Technologies) for 20 minutes at room temperature to eliminate nonspecific binding of subsequent reagents. Next, the tissue sections were incubated with the primary antibodies for 2 hours at room temperature. The antigenantibody complexes were visualized using the Dako advance detection system (Agilent Technologies) and incubated with Dako 30 3-diaminobenzidine tetrachloride (Agilent Technologies) for 5 minutes. The sections were then counterstained with Mayer’s hematoxylin, dehydrated, and mounted with a glass coverslip and xylene-based mounting media. Positive controls were used per the manufacturer’s recommendation. Semiquantitative analysis of the results was performed using a conventional optical microscope Olympus BX41 (Olympus America Inc., Center Valley, PA) using the following scores, based on the number of cells that stained positively
Apoptosis-regulating miRNAs were analyzed in 20 OSCC and five normal oral mucosa tissue samples. Considering upregulation as two-fold greater expression and downregulation as two-fold lower expression relative to that in normal tissue, miR-15a, miR-29a, and miR-34a were downregulated in 50, 75, and 70% of samples, respectively. miR-16, miR-17-5p, miR-20a, and miR-21 expression was normal in 80, 75, 90, and 60% of samples, respectively. miR-21 was overexpressed in 15% of samples (Figure 1). Magia software (31) was used to predict miRNA target genes. Based on the PITA (http://genie.weizmann.ac.il/pubs/ mir07/index.html), miRanda (http://www.microrna.org/micror na/home.do), and TargetScan (http://www.targetscan.org/) algorithms, a network of predictions was constructed (Figure 2), and the expression of target gene transcripts (BCL2, CASP2, CASP7, CASP8, and DIABLO) was also examined in 50 OSCC and 10 normal oral mucosa tissue samples. BCL2 transcripts were downregulated in 60% of samples, and CASP2, CASP7, CASP8, and DIABLO transcript expression was normal in 82, 68, 50, and 60% of the samples, respectively (Figure 3). The expression of proteins that were encoded by these genes was analyzed in 37 specimens that had paraffinembedded material available, out of the 50 samples. BCL2 expression was negative to weak, and CASP2, CASP7, CASP8, and DIABLO were moderate to strong (Figure 4).
Relative expression Z 2 ðDCt; sample DCt; calibrator Þ
Table 2
Primary serum, clones, source, working titer, and antigen retrieval for immunohistochemical analysis
Primary serum
Clone
Source
Working titer
Antigen retrieval
Bcl-2 Caspase-2 Caspase-7 Caspase-8 SMAC/Diablo
124 Y154 7CSP01 11B6 Y12
Dako (Agilent Technologies, Santa Clara, CA) Epitomics (Epitomics, Burlingame, CA) Chemicon (EMD Millipore, Billerica, MA) Novocastra (Leica Biosystems, Nussloch, Germany) Epitomics
1:200 1:100 1:150 1:250 1:100
Citrate pH 6.0 EDTA/Tris pH 9.0 EDTA/Tris pH 9.0 Citrate pH 6.0 Citrate pH 6.0
4
udiaM. Coutinho-Camillo et al. Cla
Figure 1 Expression analysis of apoptosis-regulating miRNAs in OSCC samples. Relative expression was measured by qPCR, normalized to the SNORD44 and SNORD48 small nucleolar RNA reference genes. The height of the bars represents the number of cases with increased or decreased expression compared with that of the pool of normal oral mucosa tissue (pool N) as a calibrator sample. Up- and downregulation was considered a two-fold higher and lower difference in expression relative to normal tissue, respectively.
Increased expression of miR-20a was associated with alcohol consumption (P Z 0.03). Increased expression of CASP2 mRNA was associated with tumors in the floor of the mouth (P Z 0.04), and upregulation of CASP7 mRNA was associated with well-differentiated tumors (P Z 0.02). Increased expression of the BCL2 protein was associated with tobacco smoking (P Z 0.01), and increased expression of the DIABLO protein was associated with the presence of lymph node metastasis (P Z 0.04). These results are shown in Figure 5. All other associations have shown no statistical significance.
Discussion The expression of the proapoptotic miRNAs miR-15a, miR29a, and miR-34a declined in the OSCC samples that we analyzed.
Figure 2 The interaction network between apoptosisregulating miRNAs and their target mRNAs. Downregulated miRNAs are shown in red, and normal-like miRNAs are shown in yellow. Target mRNAs are shown in gray circles. (Color versions of these illustrations are available on the journal’s website at www.cancergeneticsjournal.org.)
Downregulation of miR-15a and normal expression of miR-16 were observed in our OSCC cases. miR-15 and miR-16 have been identified as regulators of the antiapoptotic factor BCL2; underexpression of these miRNAs has been associated with overexpression of BCL2 in leukemia cells. In normal CD5þ lymphoid cells, the levels of both miRNAs were high and the BCL2 protein was expressed at low levels (32). Dysregulation of BCL2 family members is common in human malignancies and is a significant cause of resistance to therapy. An imbalance between the expression of antiapoptotic and proapoptotic BCL2 family genes promotes the survival of oral cancer cells. Several histopathological and tissue microarray studies have reported abnormal expression of BCL2 family members in oral cancer tissue and have suggested that these factors are prognostic markers (23e25,33,34). Our results strengthen this evidence that BCL2 family members are good prognostic factors in OSCC. The downregulation of miR-34a that we observed is significant. Several recent studies have implicated the highly conserved miR-34 family of miRNAs as an important component of the p53 tumor suppressor pathway (35e39). Ectopic expression of miR-34 induces cell cycle arrest and apoptosis, whereas its downregulation attenuates p53dependent apoptosis (35,37,38). Mutation of the TP53 tumor suppressor gene is the most common and one of the earliest genetic alterations in head and neck squamous cell carcinoma (HNSCC), occurring in more than half of all cases (40,41), underscoring the importance of our finding. p53 is a tumor suppressor that regulates cell cycle progression and apoptosis in response to cellular stress, primarily DNA damage and genomic instability (42). In addition to the direct effects of p53 on the promoters of protein-coding genes, p53 activation has recently been shown to modulate the expression of miRNAs. The miRNAs that are upregulated by p53 constitute a likely mechanism that explains the post-transcriptional inhibition of gene
miRNAs and target mRNAs in oral squamous cell carcinoma
5
Figure 3 Expression analysis of target mRNAs of apoptosis-regulating miRNAs in OSCC samples. Relative expression was measured by qPCR, normalized to GAPDH and 18S (reference genes). The height of the bars represents the number of cases with increased or decreased gene expression relative to that of the pool of normal oral mucosa tissue (pool N) as a calibrator sample. Upand downregulation was considered a two-fold higher and lower difference in expression relative to normal tissue, respectively.
expression on p53 activation. Further, miRNAs regulate p53 through direct or indirect mechanisms, suggesting that miRNAs are key components in the p53 network (43). miR-29 has been identified as a positive regulator of p53 and might upregulate p53 through a PI3K-, AKT-, and MDM2mediated mechanism (44). miR-29 was downregulated in our
OSCC cases. Another study identified MCL1 as a target of miR-29 in cholangiocarcinoma cells (45). MCL1 is an antiapoptotic member of the BCL2 family that binds to the proapoptotic members BCL2L11 (BIM) and BID, preventing TRAIL-induced apoptosis (46). This mechanism has not been examined in OSCC.
Figure 4 Expression of apoptosis-regulating proteins in OSCC samples. (A1) BCL2; (A2) BCL2; (B) CASP2; (C) CASP7; (D) CASP8; (E) DIABLO.
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udiaM. Coutinho-Camillo et al. Cla
Figure 5 Association between the demographic, clinical and pathological characteristics of oral squamous cell carcinoma patients and miRNA, mRNA, and protein expression. (A) miR-20a expression was associated with alcohol consumption; (B) caspase-2 mRNA expression was associated with tumors in the floor of mouth; (C) caspase-7 mRNA expression was associated with well differentiated tumors; (D) Bcl-2 protein expression was associated with tobacco smoking; (E) Diablo protein expression was associated with lymph node metastasis. The relative expression observed for each sample correspond to a dot and the red bars represent the mean value. (Color versions of these illustrations are available on the journal’s website at www.cancergeneticsjournal.org.)
The miR-17-92 cluster is a novel target for p53-mediated transcriptional repression under hypoxia, and its overexpression significantly inhibits hypoxia-induced apoptosis (47). The miR-17-92 cluster modulates E2F1 expression and is positively regulated by MYC (48). The miR-17-92 gene is transcribed as a single primary miRNA and processed into seven mature miRNA molecules: miR-17-5p, miR-17-3p, miR-18, miR-19a, miR-20, miR-19b-1, and miR-92-1 (5,49). The mechanism by which overexpression of the miR-1792 cluster promotes tumorigenesis is unknown, but we did not observe any abnormalities in it in this study (miR-17-5p and miR-20 from miR-17-92 cluster). Although the majority of the samples presented miR-20a expression similar to that observed in the normal tissue, an increase in the expression of this miRNA was associated with alcohol consumption. He et al. (2005) reported a potential function of the miR-17-92 cluster in the evasion of apoptosis by B-cell lymphomas (49). Matsubara et al. demonstrated that inhibition of two miRNAs (miR-17-5p, miR-20a) in the miR-17-92 cluster induces apoptosis in the Calu6 and ACC-LC-172 lung cancer cell
lines, suggesting that overexpression of these miRNAs effects inappropriate proliferation (50). The function of the miR-17-92 cluster evading apoptosis has been strengthened by the validation of proapoptotic BCL2L11 (BIM) as a target of these miRNAs (51). miR-21 is overexpressed in various carcinomas, including glioblastomas (52), hepatocellular cancers (53), and breast cancers (54), and it appears to be a useful prognostic factor for many malignant tumors. In contrast, we did not observe any differences in the expression of miR-21 in OSCC compared with that in the normal samples. Severino et al. also reported no difference in the expression of miR-21 when analyzing an OSCC cell line (SCC25) and normal oral keratinocyte cell line (55). miR-21 binds to the 30 UTR of the tropomyosin 1 (TPM1) and phosphatase tensin homologue (PTEN ) genes (53,56) and inhibits their expression. Restoring TPM1 expression in breast cancer cells suppresses tumor growth by inhibiting anchorage-independent growth and inducing apoptosis through caspase-dependent pathways and cytochrome c release. miR-21 also promotes
miRNAs and target mRNAs in oral squamous cell carcinoma breast tumorigenesis through BCL2 upregulation (54) and correlates with advanced tumor stage, lymph node metastasis, and poor prognosis in breast cancers (57). In addition, miR-21 overexpression in colon carcinomas indicates poor survival and therapeutic outcomes (58). Li et al. reported that increased expression of miR-21 correlates with shorter survival in tongue squamous cell carcinoma patients and is a prognostic factor that is independent of other clinicopathologic factors (18). Avissar et al. also noted that high miR-21 expression correlates with poor prognosis in HNSCC patients, and that correlation has not been demonstrated in OSCC (16). A network comprising microRNA expression profiles and putative target mRNAs, which were identified by target prediction algorithms and demonstrated to be involved in apoptosis, was constructed, and the expression of target genes and proteins was also examined. We found negative or weak expression of BCL2 and downregulation of BCL2 transcripts. Increased expression of BCL2 was associated with tobacco smoking. Expression of BCL2 might be regulated by miR-15a, miR-16, and miR-34, but miR-15a and miR-34 were also downregulated. BCL2 expression might also be governed by miR-21; miR-21 was upregulated in a subset of OSCC samples, suggesting that other mechanisms regulate BCL2. Expression of DIABLO might be controlled by miR-29. DIABLO is a proapoptotic mitochondrial protein that is released into the cytosol, where it interacts and antagonizes inhibitors of apoptosis, activating caspases and apoptosis. In most of our samples, DIABLO expression was moderate/ strong and DIABLO transcript levels were normal. Increased expression of DIABLO was associated with the presence of lymph node metastasis. Downregulation of miR-29 might mediate the overexpression of DIABLO in a subset of OSCC samples. However, other mechanisms might underlie the regulation of DIABLO in OSCC. Expression of CASP2, CASP7, and CASP8 might be regulated by miR-17-5p and miR-20a. mRNA levels of these genes were similar to those in normal tissue. However, CASP2, CASP7, and CASP8 expression was moderate/ strong. A subset of OSCC samples upregulated CASP2, CASP7, and CASP8, but other mechanisms might regulate these genesdCASP2 could be a target of miR-34a, and CASP7 might be a target of miR-29a. Increased CASP2 expression was linked to tumors in the floor of the mouth, and higher CASP7 levels were associated with well-differentiated tumors. In summary, in OSCC, the expression of apoptosisregulating miRNAs is altereddspecifically, proapoptotic miRNAs are downregulateddsuggesting that negative regulation of apoptosis is a significant property of OSCC behavior. This evidence might be able to guide the development of prognostic marker profiles and target-based chemotherapies.
Acknowledgment This work was supported by grants 98/14335-2 and 07/ ~o Paulo Research Foundation 50608-4 from the Sa (FAPESP).
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(2015)
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Expression of apoptosis-regulating miRNAs and target mRNAs in oral squamous cell carcinoma udia Malheiros Coutinho-Camillo a,*, Silvia Vanessa Lourenc‚o b, Cla jo Lima c, Luiz Paulo Kowalski d, Fernando Augusto Soares a,b Leandro de Ara u a b
~ o Paulo, Brazil; Department of General Pathology, Department of Anatomic Pathology, AC Camargo Cancer Center, Sa ~ o Paulo, Sa ~ o Paulo, Brazil; c Interinstitutional Graduate Program on Bioinformatics, University Dental School, University of Sa ~ o Paulo, Sa ~ o Paulo, Brazil; d Department of Head and Neck Surgery and Otorhinolaryngology, AC Camargo Cancer of Sa ~ o Paulo, Brazil Center, Sa Aberrations in the apoptotic mechanisms that cause excessive or deficient programmed cell death have been linked to a wide array of pathological conditions. In this study, using real-time reverse transcriptaseePCR, we analyzed the expression of apoptosis-regulating miRNAs (miR-15a, miR-16, miR-17-5p, miR-20a, miR-21, miR-29a, and miR-34a) in 20 oral squamous cell carcinoma and 5 normal oral mucosa tissue samples. Bioinformatic algorithms were used to identify the target genes of these miRNAs (BCL2, CASP2, CASP7, CASP8, DIABLO). The expression transcript levels of the target genes were measured in 50 oral squamous cell carcinoma and 10 normal oral mucosa tissue samples. We observed downregulation of miR-15a, miR-29a, and miR-34a in 50, 75, and 70% of samples, respectively. miR-16, miR-17-5p, miR20a, and miR-21 expression was normal in 80, 75, 90, and 60% of samples, respectively. BCL2 transcripts were downregulated in 60% of samples, and normal-like expression was observed for CASP2, CASP7, CASP8, and DIABLO transcripts in 66, 82, 68, and 60% of samples, respectively. BCL2 expression was negative to weak, and that of proteins CASP2, CASP7, CASP8, and DIABLO was moderate to strong. Our study provides evidence of alterations in the expression of apoptosis-regulating miRNAs and genes in the apoptotic pathway, demonstrating that regulation of apoptosis is a hallmark of oral squamous cell carcinoma pathogenesis. Keywords miRNA, oral squamous cell carcinoma, apoptosis, real-time PCR, gene expression ª 2015 Elsevier Inc. All rights reserved.
MicroRNAs (miRNAs) are noncoding, single-stranded RNAs of approximately 22 nucleotides, and they constitute a novel class of gene regulators. They govern gene expression by binding to the 30 untranslated region (30 UTR) of the target mRNA, leading to translational inhibition or degradation of mRNA, depending on the degree of sequence complementarity (1,2). The significance of miRNAs in cancer has been highlighted by evidence that approximately 50% of miRNA genes lie in cancer-associated genomic regions or fragile sites (3); they are frequently amplified or deleted in tumorigenesis and might be related to altered tumor suppressor gene or oncogene function, causing uncontrolled cell proliferation or Received October 17, 2014; received in revised form March 12, 2015; accepted April 10, 2015. * Corresponding author. E-mail address: [email protected] 2210-7762/$ - see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cancergen.2015.04.004
survival (4). The precise function of miRNAs in cell survival and apoptotic signaling is unknown, but several studies have implicated them in these processes (5). At least 30 individual miRNAs have been reported to control apoptosis. In general, miR-17-5p, miR-20a, miR-21, miR-133, miR-145, miR-146a, miR-146b, miR-155, miR-191, miR-14, bantam, miR-1d, miR-7, miR-148, miR-204, miR210, miR-216, miR-296, and miR-Lat are antiapoptotic. Conversely, the let-7 family, miR-15a, miR-16-1, miR-29, miR-34a, miR-34b, miR-34c, miR-1, miR-101, and miR-214 are considered proapoptotic (3,6e12). Large-scale profiling studies of microRNA expression in human cancers have demonstrated that dysregulation of miRNA is frequently associated with many cancer types, including those that originate from the brain, breast, lung, and prostate (3). Several studies have analyzed the expression of miRNAs in head and neck cancers (1,13e16), but few have examined apoptosis-regulating miRNAs in oral cancer (17,18).
udiaM. Coutinho-Camillo et al. Cla
2 Increasing evidence suggests that oral carcinogenesis correlates with the progressive accumulation of genetic alterations in molecules that mediate apoptosis (19e27). Oral squamous cell carcinoma (OSCC) is the eighth-most common cancer worldwide (28), and its prevalence is high in Brazil, where 15,290 new cases of oral cancer were estimated to develop in 2014 (29). Insights into the general molecular mechanisms of OSCC, for which the function of miRNAs remains undetermined, could guide the development of new therapies. Using quantitative real-time reverse transcriptaseePCR (qPCR), we characterized the expression of apoptosisregulating miRNAs (miR-15a, miR-16, miR-17-5p, miR-20a, miR-21, miR-29a, and miR-34a) in OSSC.
Materials and methods
Table 1 Summary of the demographic and clinicopathologic characteristics of OSCC patients Characteristic
Category
Age, y
60 > 60 n/a Male Female n/a No Yes n/a No Yes n/a No Yes n/a No Yes n/a Poorly/moderately differentiated Well-differentiated n/a No Yes n/a Oral tongue Floor of mouth Others n/a I/II III/IV n/a
Gender
Tobacco smoking status Alcohol consumption status Lymph node metastasis
Tissue samples Tumor tissue samples from 50 OSCC cases and 10 normal oral mucosa samples were provided by the AC Camargo ~o Paulo, Brazil. All cases had Cancer Center Biobank, Sa been treated at the hospital for at least 5 years. Tumor samples were collected before any systemic treatment was administered, and samples were dissected to remove residual normal tissue. Paraffin-embedded tissue samples from 37 of the 50 OSCC cases were obtained from the archives of the Department of Pathology, AC Camargo Cancer ~o Paulo, Brazil. The clinical and histological details Center, Sa of the cases are provided in Table 1. Informed consent was obtained from all patients, and the institutional ethics committee approved this study (protocol number 985/07).
RNA isolation Total RNA was extracted from the OSCC and normal tissue samples using Life Technologies Trizol (Thermo Fisher Scientific, Waltham, MA) and treated with Life Technologies DNase I (Thermo Fisher Scientific) per the manufacturers’ instructions. The RNA was quantified on a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific), and its quality was assessed on a 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA).
qPCR: miRNA expression Ten nanograms of total RNA was reverse-transcribed from 20 OSCC and five normal oral tissue samples using the Applied Biosystems TaqMan microRNA reverse transcription kit (Thermo Fisher Scientific). PCR was performed on an ABI 7900HT sequence detection system (Thermo Fisher Scientific) using Applied Biosystems TaqMan universal master mix (Thermo Fisher Scientific). The program comprised 40 cycles of denaturation for 15 seconds at 95 C and annealing for 1 minute at 60 C after an initial denaturation step (95 C for 10 min). The expression of apoptosis-regulating miRNAs (miR-15a, miR-16, miR-17-5p, miR20a, miR21, miR-29a, and miR-34a) was analyzed by TaqMan miRNA assays (Thermo Fisher Scientific; TM000389, TM000391, TM000393, TM000580, TM000397, TM002112, and TM000426).
Perineural infiltration
Histological grade
Vascular invasion
Tumor site
Clinical stage
Number of patients 18 30 2 36 13 1 30 9 11 7 30 13 16 23 11 26 20 4 21 27 2 38 9 3 11 15 23 1 22 21 7
Abbreviaiton: n/a, information not available.
The relative miRNA expression level was normalized to two reference small nucleolar RNAsdSNORD44 and SNORD48 (TM001094 and TM001006)dand to a calibrator sample. A pool of five normal oral mucosa samples served as the calibrator sample. Experiments were performed in duplicate for each data point. The final results, expressed as n-fold differences in expression relative to the reference small nucleolar RNAs and calibrator sample, were calculated as follows: Relative expression Z 2 ðDCt; sample DCt; calibrator Þ where cycle threshold difference (DCt) values of the sample and calibrator were determined by subtracting the average Ct value of the apoptosis-regulating miRNA from the geometric average Ct value of SNORD44 and SNORD48.
Real-time RT-PCR (qPCR): mRNA expression Two micrograms of total RNA was reverse transcribed from 50 OSCC and 10 normal oral tissue samples using a HighCapacity cDNA reverse transcription kit (Thermo Fisher Scientific). PCR amplification was performed on an ABI
miRNAs and target mRNAs in oral squamous cell carcinoma 7900HT sequence detection system (Thermo Fisher Scientific) using TaqMan universal master mix (Thermo Fisher Scientific); the program consisted of 40 cycles of denaturation for 15 seconds at 95 C and annealing for 1 minute at 60 C after an initial denaturation step at 95 C for 10 minutes. BCL2, CASP2, CASP7, CASP8 and DIABLO expression was examined using TaqMan gene expression assays (Hs00153350_m1, Hs00154242_m1, Hs00169152_m1, Hs00154256_m1, and Hs00219876_m1). Relative expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) and 18S using TaqMan gene expression assays (4326317E and 4319413E) and to a calibrator sample. A pool of 10 normal oral mucosa samples served as the calibrator sample. Experiments were performed in duplicate for each data point. The final results were expressed as n-fold differences in expression relative to the reference genes and calibrator sample, as follows:
3 and the staining intensity: 0, negative/weak staining (10% of cells stained and visible only at 40); 1, moderate staining (10e50% of cells stained and visible at 20); and 2, strong staining (>50% of cells stained and easily detectable at 10). For the statistical analysis, we grouped the cases into two categories: negative (negative/weak expression) and positive (moderate/strong expression).
Statistical analysis
where DCt values of the sample and calibrator were determined by subtracting the average Ct value of the target gene from the geometric average Ct value of GAPDH and 18S.
The association between the clinicopathologic characteristics of the patients and miRNA, mRNA, and protein expression was analyzed by the c2 test. We analyzed differences in expression between the following groups: clinical stage (I/II and III/IV), tumor site (oral tongue, floor of mouth, or other sites), tobacco smoking (yes or no), alcohol consumption (yes or no), gender (male or female), lymph node metastasis (yes or no), vascular invasion (yes or no), perineural infiltration (yes or no), and histological grade (well-differentiated or poorly/moderately differentiated). The significance level was 5% for all statistical tests. All analyses were performed using R, version 2.13 (30).
Immunohistochemistry
Results
The expression of BCL2, CASP2, CASP7, CASP8, and DIABLO was examined in OSCC tissue samples on a tissue microarray. The immunostaining was performed on duplicate tissue slides; duplicate sections were separated by 40 mm. The slides were deparaffinized, rehydrated, and subjected to antigen retrieval. Details on the antigen retrieval methods and primary antibody clones, sources, and titers are listed in Table 2. The sections were incubated in 3% aqueous hydrogen peroxide for 15 minutes to quench endogenous peroxidase activity, and then they were incubated with Dako Protein Block, Serum-Free (Agilent Technologies) for 20 minutes at room temperature to eliminate nonspecific binding of subsequent reagents. Next, the tissue sections were incubated with the primary antibodies for 2 hours at room temperature. The antigenantibody complexes were visualized using the Dako advance detection system (Agilent Technologies) and incubated with Dako 30 3-diaminobenzidine tetrachloride (Agilent Technologies) for 5 minutes. The sections were then counterstained with Mayer’s hematoxylin, dehydrated, and mounted with a glass coverslip and xylene-based mounting media. Positive controls were used per the manufacturer’s recommendation. Semiquantitative analysis of the results was performed using a conventional optical microscope Olympus BX41 (Olympus America Inc., Center Valley, PA) using the following scores, based on the number of cells that stained positively
Apoptosis-regulating miRNAs were analyzed in 20 OSCC and five normal oral mucosa tissue samples. Considering upregulation as two-fold greater expression and downregulation as two-fold lower expression relative to that in normal tissue, miR-15a, miR-29a, and miR-34a were downregulated in 50, 75, and 70% of samples, respectively. miR-16, miR-17-5p, miR-20a, and miR-21 expression was normal in 80, 75, 90, and 60% of samples, respectively. miR-21 was overexpressed in 15% of samples (Figure 1). Magia software (31) was used to predict miRNA target genes. Based on the PITA (http://genie.weizmann.ac.il/pubs/ mir07/index.html), miRanda (http://www.microrna.org/micror na/home.do), and TargetScan (http://www.targetscan.org/) algorithms, a network of predictions was constructed (Figure 2), and the expression of target gene transcripts (BCL2, CASP2, CASP7, CASP8, and DIABLO) was also examined in 50 OSCC and 10 normal oral mucosa tissue samples. BCL2 transcripts were downregulated in 60% of samples, and CASP2, CASP7, CASP8, and DIABLO transcript expression was normal in 82, 68, 50, and 60% of the samples, respectively (Figure 3). The expression of proteins that were encoded by these genes was analyzed in 37 specimens that had paraffinembedded material available, out of the 50 samples. BCL2 expression was negative to weak, and CASP2, CASP7, CASP8, and DIABLO were moderate to strong (Figure 4).
Relative expression Z 2 ðDCt; sample DCt; calibrator Þ
Table 2
Primary serum, clones, source, working titer, and antigen retrieval for immunohistochemical analysis
Primary serum
Clone
Source
Working titer
Antigen retrieval
Bcl-2 Caspase-2 Caspase-7 Caspase-8 SMAC/Diablo
124 Y154 7CSP01 11B6 Y12
Dako (Agilent Technologies, Santa Clara, CA) Epitomics (Epitomics, Burlingame, CA) Chemicon (EMD Millipore, Billerica, MA) Novocastra (Leica Biosystems, Nussloch, Germany) Epitomics
1:200 1:100 1:150 1:250 1:100
Citrate pH 6.0 EDTA/Tris pH 9.0 EDTA/Tris pH 9.0 Citrate pH 6.0 Citrate pH 6.0
4
udiaM. Coutinho-Camillo et al. Cla
Figure 1 Expression analysis of apoptosis-regulating miRNAs in OSCC samples. Relative expression was measured by qPCR, normalized to the SNORD44 and SNORD48 small nucleolar RNA reference genes. The height of the bars represents the number of cases with increased or decreased expression compared with that of the pool of normal oral mucosa tissue (pool N) as a calibrator sample. Up- and downregulation was considered a two-fold higher and lower difference in expression relative to normal tissue, respectively.
Increased expression of miR-20a was associated with alcohol consumption (P Z 0.03). Increased expression of CASP2 mRNA was associated with tumors in the floor of the mouth (P Z 0.04), and upregulation of CASP7 mRNA was associated with well-differentiated tumors (P Z 0.02). Increased expression of the BCL2 protein was associated with tobacco smoking (P Z 0.01), and increased expression of the DIABLO protein was associated with the presence of lymph node metastasis (P Z 0.04). These results are shown in Figure 5. All other associations have shown no statistical significance.
Discussion The expression of the proapoptotic miRNAs miR-15a, miR29a, and miR-34a declined in the OSCC samples that we analyzed.
Figure 2 The interaction network between apoptosisregulating miRNAs and their target mRNAs. Downregulated miRNAs are shown in red, and normal-like miRNAs are shown in yellow. Target mRNAs are shown in gray circles. (Color versions of these illustrations are available on the journal’s website at www.cancergeneticsjournal.org.)
Downregulation of miR-15a and normal expression of miR-16 were observed in our OSCC cases. miR-15 and miR-16 have been identified as regulators of the antiapoptotic factor BCL2; underexpression of these miRNAs has been associated with overexpression of BCL2 in leukemia cells. In normal CD5þ lymphoid cells, the levels of both miRNAs were high and the BCL2 protein was expressed at low levels (32). Dysregulation of BCL2 family members is common in human malignancies and is a significant cause of resistance to therapy. An imbalance between the expression of antiapoptotic and proapoptotic BCL2 family genes promotes the survival of oral cancer cells. Several histopathological and tissue microarray studies have reported abnormal expression of BCL2 family members in oral cancer tissue and have suggested that these factors are prognostic markers (23e25,33,34). Our results strengthen this evidence that BCL2 family members are good prognostic factors in OSCC. The downregulation of miR-34a that we observed is significant. Several recent studies have implicated the highly conserved miR-34 family of miRNAs as an important component of the p53 tumor suppressor pathway (35e39). Ectopic expression of miR-34 induces cell cycle arrest and apoptosis, whereas its downregulation attenuates p53dependent apoptosis (35,37,38). Mutation of the TP53 tumor suppressor gene is the most common and one of the earliest genetic alterations in head and neck squamous cell carcinoma (HNSCC), occurring in more than half of all cases (40,41), underscoring the importance of our finding. p53 is a tumor suppressor that regulates cell cycle progression and apoptosis in response to cellular stress, primarily DNA damage and genomic instability (42). In addition to the direct effects of p53 on the promoters of protein-coding genes, p53 activation has recently been shown to modulate the expression of miRNAs. The miRNAs that are upregulated by p53 constitute a likely mechanism that explains the post-transcriptional inhibition of gene
miRNAs and target mRNAs in oral squamous cell carcinoma
5
Figure 3 Expression analysis of target mRNAs of apoptosis-regulating miRNAs in OSCC samples. Relative expression was measured by qPCR, normalized to GAPDH and 18S (reference genes). The height of the bars represents the number of cases with increased or decreased gene expression relative to that of the pool of normal oral mucosa tissue (pool N) as a calibrator sample. Upand downregulation was considered a two-fold higher and lower difference in expression relative to normal tissue, respectively.
expression on p53 activation. Further, miRNAs regulate p53 through direct or indirect mechanisms, suggesting that miRNAs are key components in the p53 network (43). miR-29 has been identified as a positive regulator of p53 and might upregulate p53 through a PI3K-, AKT-, and MDM2mediated mechanism (44). miR-29 was downregulated in our
OSCC cases. Another study identified MCL1 as a target of miR-29 in cholangiocarcinoma cells (45). MCL1 is an antiapoptotic member of the BCL2 family that binds to the proapoptotic members BCL2L11 (BIM) and BID, preventing TRAIL-induced apoptosis (46). This mechanism has not been examined in OSCC.
Figure 4 Expression of apoptosis-regulating proteins in OSCC samples. (A1) BCL2; (A2) BCL2; (B) CASP2; (C) CASP7; (D) CASP8; (E) DIABLO.
6
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Figure 5 Association between the demographic, clinical and pathological characteristics of oral squamous cell carcinoma patients and miRNA, mRNA, and protein expression. (A) miR-20a expression was associated with alcohol consumption; (B) caspase-2 mRNA expression was associated with tumors in the floor of mouth; (C) caspase-7 mRNA expression was associated with well differentiated tumors; (D) Bcl-2 protein expression was associated with tobacco smoking; (E) Diablo protein expression was associated with lymph node metastasis. The relative expression observed for each sample correspond to a dot and the red bars represent the mean value. (Color versions of these illustrations are available on the journal’s website at www.cancergeneticsjournal.org.)
The miR-17-92 cluster is a novel target for p53-mediated transcriptional repression under hypoxia, and its overexpression significantly inhibits hypoxia-induced apoptosis (47). The miR-17-92 cluster modulates E2F1 expression and is positively regulated by MYC (48). The miR-17-92 gene is transcribed as a single primary miRNA and processed into seven mature miRNA molecules: miR-17-5p, miR-17-3p, miR-18, miR-19a, miR-20, miR-19b-1, and miR-92-1 (5,49). The mechanism by which overexpression of the miR-1792 cluster promotes tumorigenesis is unknown, but we did not observe any abnormalities in it in this study (miR-17-5p and miR-20 from miR-17-92 cluster). Although the majority of the samples presented miR-20a expression similar to that observed in the normal tissue, an increase in the expression of this miRNA was associated with alcohol consumption. He et al. (2005) reported a potential function of the miR-17-92 cluster in the evasion of apoptosis by B-cell lymphomas (49). Matsubara et al. demonstrated that inhibition of two miRNAs (miR-17-5p, miR-20a) in the miR-17-92 cluster induces apoptosis in the Calu6 and ACC-LC-172 lung cancer cell
lines, suggesting that overexpression of these miRNAs effects inappropriate proliferation (50). The function of the miR-17-92 cluster evading apoptosis has been strengthened by the validation of proapoptotic BCL2L11 (BIM) as a target of these miRNAs (51). miR-21 is overexpressed in various carcinomas, including glioblastomas (52), hepatocellular cancers (53), and breast cancers (54), and it appears to be a useful prognostic factor for many malignant tumors. In contrast, we did not observe any differences in the expression of miR-21 in OSCC compared with that in the normal samples. Severino et al. also reported no difference in the expression of miR-21 when analyzing an OSCC cell line (SCC25) and normal oral keratinocyte cell line (55). miR-21 binds to the 30 UTR of the tropomyosin 1 (TPM1) and phosphatase tensin homologue (PTEN ) genes (53,56) and inhibits their expression. Restoring TPM1 expression in breast cancer cells suppresses tumor growth by inhibiting anchorage-independent growth and inducing apoptosis through caspase-dependent pathways and cytochrome c release. miR-21 also promotes
miRNAs and target mRNAs in oral squamous cell carcinoma breast tumorigenesis through BCL2 upregulation (54) and correlates with advanced tumor stage, lymph node metastasis, and poor prognosis in breast cancers (57). In addition, miR-21 overexpression in colon carcinomas indicates poor survival and therapeutic outcomes (58). Li et al. reported that increased expression of miR-21 correlates with shorter survival in tongue squamous cell carcinoma patients and is a prognostic factor that is independent of other clinicopathologic factors (18). Avissar et al. also noted that high miR-21 expression correlates with poor prognosis in HNSCC patients, and that correlation has not been demonstrated in OSCC (16). A network comprising microRNA expression profiles and putative target mRNAs, which were identified by target prediction algorithms and demonstrated to be involved in apoptosis, was constructed, and the expression of target genes and proteins was also examined. We found negative or weak expression of BCL2 and downregulation of BCL2 transcripts. Increased expression of BCL2 was associated with tobacco smoking. Expression of BCL2 might be regulated by miR-15a, miR-16, and miR-34, but miR-15a and miR-34 were also downregulated. BCL2 expression might also be governed by miR-21; miR-21 was upregulated in a subset of OSCC samples, suggesting that other mechanisms regulate BCL2. Expression of DIABLO might be controlled by miR-29. DIABLO is a proapoptotic mitochondrial protein that is released into the cytosol, where it interacts and antagonizes inhibitors of apoptosis, activating caspases and apoptosis. In most of our samples, DIABLO expression was moderate/ strong and DIABLO transcript levels were normal. Increased expression of DIABLO was associated with the presence of lymph node metastasis. Downregulation of miR-29 might mediate the overexpression of DIABLO in a subset of OSCC samples. However, other mechanisms might underlie the regulation of DIABLO in OSCC. Expression of CASP2, CASP7, and CASP8 might be regulated by miR-17-5p and miR-20a. mRNA levels of these genes were similar to those in normal tissue. However, CASP2, CASP7, and CASP8 expression was moderate/ strong. A subset of OSCC samples upregulated CASP2, CASP7, and CASP8, but other mechanisms might regulate these genesdCASP2 could be a target of miR-34a, and CASP7 might be a target of miR-29a. Increased CASP2 expression was linked to tumors in the floor of the mouth, and higher CASP7 levels were associated with well-differentiated tumors. In summary, in OSCC, the expression of apoptosisregulating miRNAs is altereddspecifically, proapoptotic miRNAs are downregulateddsuggesting that negative regulation of apoptosis is a significant property of OSCC behavior. This evidence might be able to guide the development of prognostic marker profiles and target-based chemotherapies.
Acknowledgment This work was supported by grants 98/14335-2 and 07/ ~o Paulo Research Foundation 50608-4 from the Sa (FAPESP).
7
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