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Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2010 May 28;137(4):557–566. doi: 10.1007/s00432-010-0918-4

MiR-21 overexpression in human primary squamous cell lung carcinoma is associated with poor patient prognosis

Wen Gao 1, Hua Shen 1, Lingxiang Liu 1, Jian Xu 2, Jing Xu 1, Yongqian Shu 1,✉
PMCID: PMC11828261  PMID: 20508945

Abstract

Purpose

This study compared miRNA expression patterns in primary squamous cell lung carcinoma specimens with those of matched normal lung tissue in order to determine their potential relevance to clinicopathological factors and patient postoperative survival times.

Methods

Locked nucleic acids miRNA microarray expression profiling was performed on four matched pairs of tissues. After microarray validation by quantitative real-time reverse transcription polymerase chain reaction assays (qRT-PCR) (real-time PCR), miR-21 was selected for further TaqMan real-time PCR study in 30 matched tissue pairs.

Results

Seven miRNAs of hsa-miR-21, hsa-miR-31, hsa-miR-34a, hsa-miR-22*, hsa-miR-504, hsa-miR-18a, and hsa-miR-412 were observed to be upregulated greater than twofold in the squamous cell lung carcinoma tissues compared with normal tissues, whereas 23 miRNAs of hsa-miR-30a, hsa-miR-30d, hsa-miR-126, hsa-miR-652, hsa-miR-100, hsa-miR-143, hsa-miR-130a, hsa-miR-145, hsa-miR-30e, hsa-miR-126*, hsa-miR-181a, hsa-miR-125b, hsa-miR-886-3p, hsa-miR-451, hsa-miR-29c, hsa-miR-26b, hsa-miR-101, hsa-miR-320, hsa-miR-30b, hsa-miR-886-5p, hsa-miR-29a, hsa-miR-26a, and hsa-miR-99a were found to be downregulated greater than twofold. MiR-21 was overexpressed in 73.3% of the squamous cell lung carcinoma specimens examined (P = 0.022). The relationship between the miR-21 expression level and various clinicopathologic factors was also analyzed. High-level expression of miR-21 was significantly correlated with shortened survival time (P = 0.022, log-rank test; Kaplan–Meier). Multivariate Cox proportional hazard regression analysis revealed this significant prognostic impact (P = 0.000; HR 1.293; 95% CI 1.123–1.489) to be independent of clinical disease stage (P = 0.013; HR 2.660; 95% CI 1.229–5.758) and other clinicopathologic factors.

Conclusions

Expression patterns of miRNAs were found to be systematically altered in squamous cell lung carcinoma tissue. High miR-21 expression is associated with shortened survival time, indicating that miR-21 may serve as a molecular diagnostic and prognostic marker for patients with squamous cell lung carcinoma.

Keywords: MiR-21, Microarray, Prognosis, QRT-PCR, Real-time PCR, Squamous cell lung carcinoma

Introduction

Lung cancer is the leading cause of cancer deaths worldwide, with a low, 5-year survival rate of less than 15% after initial diagnosis. Non-small-cell lung cancers (NSCLC), including adenocarcinoma and squamous cell carcinoma, comprise more than 80% of lung cancers; 75% of patients have unresectable, advanced stage tumors at the time of diagnosis (Laskin and Sandler 2005). In recent decades, a variety of tumor markers in NSCLC have, to a large extent, improved early diagnosis and patient care. In addition, cisplatinum-based combination chemotherapy and targeted drugs have greatly improved both survival times and quality of life in patients with NSCLC. However, the efficacy of the new drugs used for the treatment of squamous cell lung carcinoma, such as tyrosine kinase inhibitors, pemetrexed and bevacizumab, is significantly inferior to that of drugs used for the treatment of adenocarcinoma. Thereby, innovative and reliable diagnostic or prognostic biomarkers and therapeutic strategies are urgently needed for the treatment of squamous cell lung carcinoma.

MicroRNAs (MiRNAs) are an abundant class of small non-protein-coding RNAs that regulate the expression of genes by targeting mRNAs and triggering either translation repression or mRNA cleavage. Bioinformatic data indicate that a single miRNA could bind to approximately hundreds of mRNA targets, thereby playing an important role in various biological processes (Bartel 2004). Accumulating evidence has indicated a robust association between miRNAs and carcinogenesis (Calin and Croce 2006a, b). The let-7 family (Takamizawa et al. 2004) and mir-155 (Yanaihara et al. 2006) have been reported to be associated with the clinical outcomes of lung adenocarcinoma. The let-7 family can negatively regulate the oncogene RAS (Johnson et al. 2005), so that it acts biologically as a tumor suppressor. Yu et al. identified a five-miRNA signature that can predict survival in patients with lung cancer, of which hsa-miR-221 and hsa-let-7a were protective; hsa-miR-137, hsa-miR-372, and hsa-miR-182* were non-protective (Yu et al. 2007). In NSCLC, we have identified MicroRNA expression profiles and that its expression is associated with prognosis of patients, strongly suggesting that miRNA is involved in the initiation and progression of this disease (Gao et al. 2010). Since there is no known separate miRNA expression profile study regarding squamous cell lung carcinoma, and in light of the fact that miRNA expression profiling can act as potential biomarkers for diagnosis, prognosis and personalized therapy, an miRNA profiling study was conducted on pairs of primary squamous cell lung carcinoma specimens and matched normal lung tissues using LNA microarray and quantitative real-time reverse transcription polymerase chain reaction assays (qRT-PCR) (real-time PCR). Further analysis of the association of miRNAs with squamous cell lung carcinoma clinicopathologic factors and clinical outcomes were carried out in order to better understand the particular miRNA involvement in multistep carcinogenesis and progression.

Materials and methods

Human cancer tissue specimens

All primary squamous cell lung carcinoma samples with matched normal tissue were derived from patients who had undergone surgical resection at the Department of Cardio-thoracic Surgery, First Affiliated Hospital of Nanjing Medical University. This study was approved by the Institutional Review Boards of the Hospitals. All carcinoma tissues were stained with H&E for histological validation and tumor cell evaluation by pathologist (Fig. 1). Only those cases with >60–70% tumor cell population in the section were used in this study (Yan et al. 2008; Yi et al. 2009). Moreover, we also evaluated the quality and the size of specimens and completeness of the patient’s information, then, 34 pairs of snap-frozen primary squamous cell lung carcinoma specimens and their corresponding normal lung tissue were from 34 individual patients with primary squamous cell lung carcinoma were selected. Among them, four cases obtained in April 2008 were used in miRNA microarray study. An additional 30 cases obtained between February 2004 and January 2005, which could be followed up at least 4–5 years, were used for a qRT-PCR and survival analysis. For each case, carcinoma samples with matched normal tissues were collected during surgical resections and stored in liquid nitrogen immediately, and the clinical characteristics of some of the patients have been defined and described previously (Gao et al. 2010). The clinical stage was determined according to AJCC/UICC guidelines (Sobin and Wittekind 2002; Greene et al. 2002). Information on other clinicopathological factors, including sex, age, histological grade of tumor, tumor diameter, lymph node status, and smoking status, was also collected. Telephone follow-up was conducted every 3 months post surgery in order to evaluate the survival status. The last follow-up date in this study was September 30, 2009.

Fig. 1.

Fig. 1

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A representative histopathologic image of squamous cell lung carcinoma of the resection specimen. Tissues were collected at the indicated time, fixed in l0% neutral buffered formalin, embedded in paraffin and then stained with hematoxylin and eosin (H&E) for histology examination. (magnification ×200)

Extraction of total RNA

Total RNA was obtained using mirVanaTM miRNA isolation kit (Ambion 1560, Austin, TX) in accordance with the manufacturer’s instructions. Frozen tissue was ground to a powder with liquid nitrogen in a pre-chilled mortar and pestle. Specimens were then disrupted in a denaturing lysis buffer. Next, specimens were subjected to acid-phenol:chloroform extraction. RNA species were immobilized on glass-fiber filters, and then washed several times via miRNA wash solution. Finally, RNA was eluted with nuclease-free water provided by the kit and stored at −80°C. RNA concentration was assessed by measuring absorption (A260/A280) on the NanoDrop Spectrophotometer ND-1000 (NanoDrop, Wilmington, DE). The integrity of RNA was assessed by 1.5% agarose gel electrophoresis; RNA specimens that did not show intact 5S, 18S and 28S bands or A260/A280 ratio greater than 1.8–2.1 were excluded from the study.

MiRNA microarray analysis

The microarray used in the present study covered complete human, rat, mouse miRNA in miRBase10.0 database: 730 for human, 350 for rats, 570 for mice. The microarray also contains 144 miRPLUS capture probes, 8 negative control probes, 12 small nuclear RNA-positive control probes and 10 spike-in control probes.

Four pairs of squamous cell lung carcinoma specimens and their matched normal tissue specimens were used for locked nucleic acids (LNA) miRNA microarray analysis. All miRNA microarray experimentation was conducted using an Exiqon A/S platform (Vedbaeck, Denmark). Each sample of total RNA and the common reference were labeled using the miRCURY Hy3/Hy5 labeling kit and hybridized on the miRCURY LNA array (v. 10.0). LNA-modified capture probes corresponding to human, mouse, and rat mature miRNA sequences were observed in quadruplicate on a slide. The slides were scanned using the Genepix 4000B (Axon Instruments, Sunnyvale, CA), and data were analyzed by Genepix Pro 6.0 (Axon Instruments, Sunnyvale, CA). After normalization, the statistical significance of differentially expressed miRNA was analyzed using the t-test. Only miRNAs that were altered at least twofold with P value < 0.05 were considered significant candidates. Eisen CLUSTER and TREEVIEW software (Eisen et al. 1998) were used to perform unsupervised hierarchical cluster analysis.

Validation of miRNA microarray

To confirm the results obtained by microarray analysis, real-time PCR analysis of six representative miRNAs differentially expressed or non-differentially expressed (miR-21, miR-29a, miR-130a, miR-181a, miR-412, let-7b) (Yan et al. 2008) was carried out as previously described (Gao et al. 2010). Total RNA was prepared from one pair of specimens (Tumor 4 and Normal4) (Yan et al. 2008; Murakami et al. 2006). Real-time PCR analysis was performed on a Rotor-Gene 3000 fluorescence quantitative PCR instrument (Corbett Research, Sydney, Australia).

TaqMan real-time PCR analysis of miR-21

To quantify the expression level of the miR-21 on 30 pairs of squamous cell lung carcinoma specimens and their matched normal tissues, TaqMan real-time PCR of miR-21 expression was carried out using TaqMan miR-21 assay kits (ABI P/N 4373090, Applied Biosystems, Foster City, CA) according to manufacturer’s protocol. The kit contains gene-specific stem–loop reverse transcription primers and TaqMan probes to detect mature miRNA in a two-step RT-PCR analysis. CDNAs were synthesized from total RNA using miRNA-specific primers and the TaqMan miRNA reverse transcription (RT) kit (ABI P/N 4366596), according to the manufacturer’s instructions. Reverse transcriptase reactions contained 10 ng of total RNA (5 μl), 3 μl stem–loop RT primer, 0.15 μl 100 mM dNTPs (with dTTP), 1 μl MultiScribe™ reverse transcriptase (50 U/μl), 1.50 μl 10× reverse transcription buffer, 0.19 μl RNase inhibitor (20 U/μl) and 4.16 μl nuclease-free water. The 15-ml reactions were incubated on a GeneAmp PCR System (Bio-Rad, Hercules, CA) for 30 min at 16°C, 30 min at 42°C, 5 min at 85°C, and then held at 4°C. All reverse transcriptions and no-template controls were run at the same time. PCR amplification was carried out using sequence-specific primers on the Applied Biosystems 7500 real-time PCR system. The 20-μl PCRs included 1.33 μl RT product, 10 μl gene Expression Master Mix (ABI P/N 4369016), 7.67 μl nuclease-free water, and 1 μl of primers and probe mix of the TaqMan miRNA assay kit. The assay was carried out in a 96-well optical plate at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. The threshold cycle (CT) data and baselines were determined using auto settings. The threshold cycle was defined as the fractional cycle number at which the fluorescence reached the fixed threshold. U6 small RNA (RNU6B) was also identified using the TaqMan RNU6B assay kit (ABI P/N 4373381) for normalizing the levels of miR-21. Each sample that included no template was analyzed in triplicate. Data were analyzed using SDS software, version 1.4. The quantity of miR-21 in each squamous cell lung carcinoma specimen relative to its matched normal specimen was calculated using the algorithm RQ = 2-DELTA DELTACT, where DELTA DELTACT = (CTmiRNA − CTU6) carcinoma − (CTmiRNA − CTU6) normal (Chen et al. 2005).

Statistical analysis

Microarray and real-time PCR statistical analysis have been described earlier. The Wilcoxon signed-rank test was used to determine differences in miR-21 expression, analyzed by real-time PCR, between squamous cell lung carcinoma specimens and their matched normal tissue specimens. The patients were divided into two groups miR-21 low expressers (n = 15) and miR-21 high expressers (n = 15) according to the median relative expression level of miR-21 (median fold 1.47 was used as the cut-off). Fisher’s exact text was used to analyze the relationship between miR-21 expression level and various clinicopathologic factors. Associations with postoperative survival were estimated by the Kaplan–Meier method, and the resulting curves were compared using the log-rank test. Postoperative survival was calculated as the interval between the date of lung cancer surgery and either the date of the patient’s death or the date of the last patient follow-up. A Cox proportional hazard regression model analysis of clinicopathological factors potentially related to survival was performed to identify which independent parameters might jointly have had a significant influence on postoperative survival. Statistical analyses were performed in SPSS 17.0 for Windows (SPSS Inc.). Any differences were considered to be statistically significant when the P value was <0.05.

Results

Total RNA extraction and quality control

The quantity and quality of the RNA specimens applied to the microarray and real-time PCR were checked by gel electrophoresis and absorbance at A260/280 ratio, respectively. The gel electrophoresis results and A260/280 ratio confirmed the good quality of total RNA isolated.

Microarray results and real-time PCR validation

MiRNAs that were differentially expressed between squamous cell lung carcinoma and their matched normal specimens were then identified. Thirty miRNAs were altered at least twofold: 23 miRNAs were downregulated and seven miRNAs were upregulated in squamous cell lung carcinoma tissues compared with normal tissues. Average fold change was summarized (Table 1). The number of miRNAs that showed downregulation in squamous cell lung carcinoma was higher than the number of upregulated miRNAs, which is consistent with published miRNA expression profiling studies (Yanaihara et al. 2006; Lu et al. 2005). Microarray data are in agreement with the real-time PCR verification results in (tumor sample 4 and normal sample 4) (Yan et al. 2008) fold change of miR-21 is 4.28 and 3.84, respectively; miR-29 is −2.56 and −1.37; miR-130 is −2.08 and −2.63; miR-181a is −1.75 and −2.38; miR-412 is 6.57 and 3.61; let-7b is −2.86 and −2.78. Subsequently, clustering analysis was done to classify the clinical tissue specimens according to their miRNA expression patterns. The clustering analysis accurately and successfully separated the squamous cell lung carcinoma specimens from the normal specimens, as visualized by the hierarchical tree (Fig. 2).

Table 1.

Differentially expressed miRNAs identified in squamous cell lung carcinoma

MiRNAs upregulated MiRNAs downregulated
Name Fold change P value Name Fold change P value
hsa-miR-21 3.31 0.0027* hsa-miR-30a 0.11 1.98E-6*
hsa-miR-31 7.26 0.0067* hsa-miR-30d 0.22 1.59E-5*
hsa-miR-34a 2.83 0.0096* hsa-miR-126 0.11 3.8E-5*
hsa-miR-22* 2.57 0.0150* hsa-miR-652 0.30 7.56E-5*
hsa-miR-504 3.53 0.0315* hsa-miR-100 0.28 0.0001*
hsa-miR-18a 2.24 0.0383* hsa-miR-143 0.24 0.0002*
hsa-miR-412 3.82 0.0479* hsa-miR-130a 0.37 0.0002*
hsa-miR-145 0.48 0.0003*
hsa-miR-30e 0.43 0.0004*
hsa-miR-126* 0.16 0.0005*
hsa-miR-181a 0.40 0.0005*
hsa-miR-125b 0.50 0.0005*
hsa-miR-886-3p 0.36 0.0009*
hsa-miR-451 0.22 0.0010*
hsa-miR-29c 0.32 0.0011*
hsa-miR-26b 0.44 0.0012*
hsa-miR-101 0.39 0.0024*
hsa-miR-320 0.49 0.0028*
hsa-miR-30b 0.48 0.0036*
hsa-miR-886-5p 0.35 0.0063*
hsa-miR-29a 0.27 0.0085*
hsa-miR-26a 0.40 0.0148*
hsa-miR-99a 0.44 0.0382*
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*P < 0.05

Fig. 2.

Fig. 2

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Cluster analysis of the miRNA differentially expressed in squamous cell lung carcinoma. Dendrogram generated by cluster analysis showed the separation of carcinoma from normal lung tissue specimens on the basis of miRNA profiling (fold change at least twofold)

MiR-21 expression in squamous cell lung carcinoma and its relationship with clinicopathological factors

TaqMan real-time PCR was used to evaluate expression levels of miR-21 in 30 paired squamous cell lung carcinoma and matched normal lung specimens. Consistent with the microarray data, most of the squamous cell lung carcinoma specimens (73.3%) showed increased levels of miR-21 compared to the normal tissues, with a media upregulation of 1.47-fold (P = 0.022). However, no statistically significant correlation was found between mir-21 level and any of the various clinicopathologic characteristics (sex, age, smoking status, tumor size, clinical stage, histological grade, and lymph node status). Results are summarized in Table 2.

Table 2.

Comparison of several clinicopathologic factors and expression levels of miR-21 in squamous cell lung carcinoma specimens

Parameter N MiR-21
High expressiona Low expressiona P valueb
Sex
 Male 25 13 12 1
 Female 5 2 3
Age
 ≤63 15 9 6 0.466
 >63 15 6 9
Clinical stage
 I 17 8 9 1
 II–III 13 7 6
Histological grade
 Well/moderately differentiated 13 6 7 1
 Poorly differentiated 17 9 8
Tumor diameter
 ≤5 cm 16 8 8 1
 >5 cm 14 7 7
Lymph node
 Positive 11 7 4 0.45
 Negative 19 8 11
Smoking status
 Never smoker 6 4 2 0.651
 Current or former smoker 24 11 13
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amiR-21 low expressers (n = 15) and miR-21 high expressers (n = 15) according to the median relative expression level of miR-21:1.47

bFisher’s exact text

Survival analysis

MiR-21 expression levels were significantly correlated with postoperative survival of the squamous cell lung carcinoma patients. Kaplan–Meier survival analysis revealed that the patients with high miR-21 expression had significantly poorer survival times compared with those with low miR-21 expression (P = 0.022, log-rank test; Fig. 3.) In addition, patients with advanced clinical stage and lymph node metastases also had poorer survival times than those with less-advanced clinical stage and no metastases (P = 0.012 and P = 0.008, respectively, log-rank test). Univariate Cox hazard regression model analysis showed that high expression level of miR-21 (P = 0.001, hazard ratio [HR] 1.246; 95% CI 1.093–1.419) as well as advanced clinical stage (P = 0.007, HR 2.112; 95% CI 1.231–3.625) and lymph node metastases (P = 0.012, HR 3.064; 95% CI 1.282–7.323) were associated with poor survival time, while other clinicopathologic factors were not. The multivariate Cox regression model revealed that high expression level miR-21 expression (P = 0.000, HR 1.293; 95% CI 1.123–1.489) was also a significantly unfavorable prognostic factor, independent of advanced clinical stage (P = 0.013, HR 2.660; 95% CI 1.229–5.758) (Table 3).

Fig. 3.

Fig. 3

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Kaplan–Meier estimates of postoperative survival times of squamous cell lung carcinoma patients according to the miR-21 relative expression. The expression levels of miR-21 in 30 patients were measured by quantitative reverse transcription polymerase chain reaction. High expression is based on the median fold change (1.47-fold higher than normal). Log-rank P values are from Kaplan–Meier analysis

Table 3.

Cox regression analyses of the relationship between expression levels of miR-21 and clinicopathologic factors related to squamous cell lung carcinoma specimens

Factor Univariate analysis Multivariate analysis
HR (95% CI) P value HR (95% CI) P value
MiR-21 High vs low 1.246 (1.093–1.419) 0.001* 1.293 (1.123–1.489) 0.000*
Clinical stage III vs II vs I 2.112 (1.231–3.625) 0.007* 2.660 (1.229–5.758) 0.013*
Sex Male vs female 0.819 (0.277–2.424) 0.718
Age >63 vs ≤63 years 0.907 (0.394–2.084) 0.817
Smoking status Smoker vs never 0.420 (0.148–1.196) 0.104
Histological grade III vs II vs I 1.098 (0.603–1.997) 0.760
Tumor diameter >5 cm vs ≤5 cm 0.812 (0.357–1.847) 0.620
Lymph node status Positive vs negative 3.064 (1.282–7.323) 0.012* 1.097 (0.336–3.582) 0.878
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*P < 0.05

Discussion

Using the novel locked nucleic acid (LNA)-modified probes miRNA microarray, the most comprehensive miRNA array available, miRNA expression profile signatures in squamous cell lung carcinoma were obtained. Unlike the conventional oligo-array, which requires very different hybridization conditions for accurate detection and which cannot discriminate among closely related miRNA family members, LNA microarray has a normalized 72°C melting temperature and can offer higher specificity and sensitivity for miRNA detection by achieving uniform hybridization conditions. LNA microarray can also measure more accurately the levels of highly homologous miRNAs (Castoldi et al. 2006). Moreover, compared to traditional markers at the mRNA and protein level, microRNA expression patterns are more reliable and sensitive. One important reason is that levels of mRNA and protein are often not proportional. Perhaps, this is due to the regulatory influences of the microRNAs themselves. Another reason is one miRNA can target to about 100 mRNAs. So miRNA profiling does so more accurately and effectively than an mRNA or protein abundance profile (Lu et al. 2005). Thus, as some other studies (Yan et al. 2008), we only use small size samples to analyze the miRNA profiling. As the microarray requires high quality samples, we chose the specimens that had a shorter storage time (Obtained in 2008). Moreover, before RNA extraction, all sections both used in microarray and real-time PCR were stained with H&E for histological diagnosis and tumor cell evaluation. Only those cases with >60–70% tumor cell population in the section were used in this study. Hierarchical cluster analysis, based on the miRNA expression profiling, generated a tree with a clear distinction between squamous cell lung carcinomatous and normal tissues. The cluster tree suggested that miRNA expression profiling was associated with the development and progression of cancer, and thus it can be served as the source of potential biomarkers to help with earlier diagnosis of squamous cell lung carcinoma. In the present study, the number of miRNAs that showed downregulation in squamous cell lung carcinoma specimens (n = 23) was higher than the number of upregulated (n = 7) miRNAs. This trend is in agreement with most previously published miRNA profiling studies, which have reported that downregulation of miRNAs seems to be more common than upregulation in carcinomatous tissues (Yanaihara et al. 2006; Lu et al. 2005). Overall, decreased expression of miRNAs may account for defects in the miRNA-processing mechanisms of cancer (Karube et al. 2005; Thomson et al. 2006); another explanation could be that global miRNA expression levels reflect the stage of cell differentiation (Calin and Croce 2006a, b). Interestingly, some of the miRNAs identified in the profiling are not consistent with those reported in a previous lung cancer study (Yanaihara et al. 2006). This discrepancy could be due to the different fundamental methodology used in the study. First, the array probes used in the earlier study only detected precursors, rather than mature miRNAs. Secondly, the probe databases used in the present microarray study (miRBase 10.0) have been upgraded, so they have more novel probes than earlier versions did (Yanaihara et al. 2006). In addition, the origin of the specimens or patients’ individual variation may also account for this discrepancy. Finally, real-time PCR is a more quantitative and sensitive technique than high-throughput miRNA microarray. Therefore, in order to warrant the accuracy of the microarray, real-time PCR validation was carried out in the present study, and the direction of the change in miRNA expression was concordant between microarray and real-time PCR.

In general, among the clinical biomarker candidates, clinical physicians are more likely to choose the gene that up-regulated in carcinoma samples and functional studies of which are relatively more enriched. In the present microarray study, miR-21 was upregulated (fold change: 3.31; the P value: 0.0027, which was the lowest), and the expression level was high in all 4 patient’s carcinoma specimens. Moreover, the function of miR-21 is well studied among all the up-regulated miRNAs, and the known miR-21-target genes and miR-21 pathways related to cancer is the most comprehensive. In our another ongoing study, we found miR-21 was significantly up-regulated in cisplatin-resistant lung cancer cell line (A549/CDDP). Moreover, cisplatin-sensitive lung cancer cell line (A549) was less sensitive after pre-miR-21 transfection, while cisplatin-resistant lung cancer cell line was less resistant after anti-miR-21 transfected (data not show). Combination chemotherapy, usually platinum based, is commonly served as the first therapy of choice for NSCLC. However, a considerable number of patients are not sensitive to treatment, and cancer cells subsequently developed drug resistance, which usually leads to a relapse and worsening of prognosis. Therefore, miR-21 was selected in this study for further TaqMan real-time PCR investigation and survival analysis on 30 pairs of squamous cell lung carcinoma specimens compared to their matched normal tissues (Ribas et al. 2009; Neely et al. 2010). MiR-21 was found to be significantly upregulated, with a median upregulation of 1.47-fold (P = 0.022) in TaqMan real-time PCR analysis of squamous cell lung carcinoma, suggesting that miR-21 may play an important role in the molecular pathogenesis of squamous cell lung carcinoma and may have clinical diagnostic value. MiR-21 is expressed at high levels in most solid tumors such as breast (Yan et al. 2008), colon (Schetter et al. 2008; Slaby et al. 2007) gastric (Chan et al. 2008), and ovarian cancer (Iorio et al. 2007). Additional studies have indicated that miR-21 is also upregulated in leukemic cancers (Fulci et al. 2007) and aggressive diffuse large B-cell lymphoma (DLBCL) (Lawrie et al. 2007). However, data concerning the relationship between miR-21 expression, clinicopathologic factors and the prognosis of squamous cell lung carcinoma remains unclear.

The potential relationship between the expression level of miR-21 and various clinicopathological factors and postoperative survival times was also analyzed. Expression levels of miR-21 did not correlate with all clinicopathological factors, which could be due to the relatively small sample size. However, in a previously published miRNA profiling study of cancer, no association was found between the clinicopathological factors and any analyzed miRNAs (Yanaihara et al. 2006). It is worth noting that high miR-21 expression in tumors appears to be significantly correlated with poor survival of patients with squamous cell lung carcinoma. Multivariate Cox hazard regression analysis revealed that the correlation was independent of other clinicopathological factors, indicating that high-level expression of miR-21 may be a useful indicator of an unfavorable prognosis (P = 0.000, HR 1.293; 95% CI 1.123–1.489) independent of advanced clinical stage (P = 0.013, HR 2.660; 95% CI 1.229–5.758) and other clinicopathological factors. The potential prognostic value of miR-21 may be able to help physicians identify and select the patients who are most likely to benefit from therapy, in order to improve the treatment outcome of squamous cell lung carcinoma.

The exact molecular mechanism of miR-21 elevation and its role in cancer development and progression is currently unclear. It may be related to multi-level regulatory control such as apoptosis, proliferation, and invasion-related gene. A miR-21 knockdown experiment in multiple glioblastoma cell lines suggested that overexpression of miR-21 has anti-apoptotic function (Chan et al. 2005); anti-miR-21 suppressed both tumor growth in the xenograft mouse model and cell proliferation in vitro, which could be in part due to downregulation of the anti-apoptotic factor, B-cell lymphoma 2(Bcl-2) in anti-miR-21-treated tumor cells (Si et al. 2007). In recent years, studies have indicated that miR-21 can target a number of well-known tumor suppressor genes such as tropomyosin 1 (TPM1) (Zhu et al. 2007) and phosphatase and tensin homolog (PTEN) (Meng et al. 2007). The miR-21 and PTEN mRNA connection location has not yet been completely identified, so whether or not miR-21 could regulate PTEN directly remains unknown. Downregulation of PTEN using miRNA technology can reduce the effects of anti-miR-21 on hepatocellular carcinoma cell growth and invasion (Meng et al. 2007). Regulation of PTEN expression by miR-21 was also identified in colon cancer (Asangani et al. 2008); however, researchers did not find this regulatory role in breast cancer MCF-7 cells (Frankel et al. 2008), lung adenocarcinoma A549 cells (Blower et al., 2008), or glioma cells (Gabriely et al. 2008). Therefore, miR-21 regulation of PTEN seems to be specific to certain cancer types rather than being common to a variety of cancers. Two studies found two direct miR-21 target genes: programmed cell death 4 (PDCD4) (Frankel et al. 2008) and maspin (Zhu et al. 2008), both of which are involved in the inhibition of tumor invasion. PDCD4 is downregulated in lung cancer and was associated with poor prognosis (Chen et al. 2003), suggesting that miR-21-PDCD4 may be able to become a pair of oncogenes; this tumor suppressor gene has clinical significance for cancer development and progression.

Data from multiple studies have already demonstrated that oncomiR miR-21 acts as a practicable clinical tool in cancer diagnosis and prognosis. For example, a robust association of high miR-21 expression in colon cancer with poor survival and therapeutic outcome was observed in two cohorts (Schetter et al. 2008). Overexpression of miR-21, one of the most significantly altered miRNAs in breast cancer, is associated with progression and poor prognosis (Yan et al. 2008). Results of the present study also indicate that miR-21 could serve as both a diagnostic and a prognostic biomarker for squamous cell lung carcinoma patients. However, in gastric cancer, it has been suggested that miR-21 can only be a diagnostic marker rather than a prognostic one (Chan et al. 2008). Interestingly, Lawrie’s studies (Lawrie et al. 2007, 2008), which focus on the role of miR-21 in large B-cell lymphoma (DLBCL), have yielded completely opposite results: DLBCL patients with low expression levels of miR-21 have shorter survival times. These adverse outcomes could be due to a different target gene of miR-21 in different cancer types and to different end points used for analysis. A single miRNA can control many unrelated gene targets, resulting in the control of opposing activities. Perhaps, the ability of the miR-21 to participate in either one pathway or another depends on cell types and specific tissues. However, a single miRNA can involve many pathways; whether these pathways collaborate with each other or contradict each other is still unclear.

Recent studies suggest that in addition to being a diagnostic and prognostic tool for patients with cancer, miR-21 might also be potential drug targets or a biomarker to predict the risk of chemoresistance that could be used in a broad range of cancer therapies. In one in vivo cytologic experiment, inhibition of miR-21 increased the sensitivity of cholangiocarcinoma cell to gemcitabine (Meng et al. 2006). Si et al. transfected breast cancer MCF-7 cells with anti-miR-21 oligonucleotides and found that the growth inhibition of cancer cell induced by topotecan can be increased by 40% (Si et al. 2007). Changing the cellular levels of mir-21 significantly affected the potencies of a number of the anti-cancer agents (Blower et al. 2008). Recently, clinical crowd experiments focused on the cancer treatment–related miRNA have gradually attracted attention. One study (Schetter et al. 2008) analyzed associations with miR-21 expression and therapeutic outcomes in stage II and III colon cancer patients treated with adjuvant chemotherapies that were primarily 5-fluorouracil-based; high miR-21 expression was associated with a poor response to therapy and a more rapid disease recurrence. That research supports a substantial role for miR-21 in chemotherapy response, suggesting novel potential approaches to the improvement of cancer treatment.

Although it is currently uncertain whether miRNA expression profiling is merely concomitantly associated with cancer or directly causal to the development and progression of cancer, data from the present study, together with data from previous published studies, provide evidence for the notion that miRNA expression profiling may generate a unique molecular signature for cancer. However, a single miRNA is thought to be capable of targeting multiple unrelated target genes, while a target gene can be regulated by several miRNAs. This fact emphasizes the complexities of miRNA network as well as the intricacies of miRNA-mediated target genes regulation. Overexpression of miR-21 might either inhibit a series of cell proliferation-related genes and apoptosis-related genes or target multiple tumor suppressor genes and metastasis suppressor gene in tumor growth and metastasis, thereby playing an important role in cancer development and progression.

In conclusion, specific miRNA profiles in squamous cell lung carcinoma were obtained by both microarray and real-time PCR analysis. Cancer-specific miRNAs as biomarkers of cancers could be vital in detecting early stage cancers and may be able to guide a patient’s course of treatment. The principal finding is that a high expression level of miR-21 was associated with poor postoperative survival times in patients with squamous cell lung carcinoma, independent of clinical staging and other clinicopathological factors. Although further testing on larger populations will be necessary, results indicate that miR-21 may be a promising candidate as a molecular diagnostic and prognostic biomarker for squamous cell lung carcinoma.

Conflict of interest statement

None.

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