MicroRNAs as Biomarkers for Lung Cancer




Summary of Key Points





  • The biologic roles of microRNAs (miRNAs) in lung cancer indicate their correlation with disease status, prognosis, and therapeutic outcome. The discovery of miRNAs has opened a new avenue for individualized disease diagnosis and treatment.



  • Dysfunctions of miRNAs are frequently found in lung cancer. These noncoding RNAs have been recognized as some of the main regulatory gatekeepers of coding genes in the human genome.



  • Owing to their high stability during storage and handling, miRNAs are optimal biomarkers presenting in blood, urine, and other body fluids.



  • Early detection is a key to improve the survival of patients with lung cancer. Recent studies have suggested that circulating miRNAs may become promising biomarkers for risk assessment and diagnosis of lung cancer in blood and sputum.



  • Some single nucleotide polymorphisms (SNPs) are significantly associated with an increased risk of nonsmall cell lung cancer (NSCLC) and its prognosis.



  • Identification of specific miRNAs may provide accurate subclassification of NSCLC.



  • Recent studies have demonstrated that miRNAs may serve as predictive biomarkers for the chemoresistance of lung cancer among patients treated with systemic chemotherapy and/or targeted therapies.



  • Large prospective cohort studies and cross-validation are needed to consolidate the significant findings demonstrated by studies of miRNA profiling.



  • In conjunction with genetic and proteomic signatures and other screening approaches, miRNA biomarkers may represent a new milestone in lung cancer theranostics.



miRNAs are a class of evolutionarily conserved, endogenous, small noncoding RNAs of about 21 to 23 nucleotides in length that participate in diverse biologic pathways and function as posttranscriptional gene regulators during tumorigenesis. These small molecules mainly bind imperfectly to the 3ʹ untranslated region (UTR) of target messenger RNAs (mRNAs). They are encoded in the genome and are generally transcribed by RNA polymerase II. miRNAs work via RNA-induced silencing complexes to target mRNAs in a sequence-specific manner, resulting in mRNA deadenylation followed by exonucleolytic decay, mRNA endonucleolytic cleavage, or translational inhibition. Deregulation of miRNAs is associated with epigenetic and genetic alterations, such as aberrant DNA methylation, amplification, deletion, and point mutation. More than 1000 miRNAs exist in the human genome, and each one can potentially regulate hundreds of mRNAs. miRNAs therefore play an important role in many cellular processes, including apoptosis, differentiation, proliferation, and the stress response.


Lung cancer is the leading cause of cancer mortality worldwide, yet few molecular markers are available for risk screening, subclassification, early diagnosis, survival prognosis, and prediction of treatment response. Researchers have suggested that aberrant miRNA expression profiles may act as oncogenes or tumor suppressors in many types of cancer, including lung cancer. The biologic roles of miRNAs in lung cancer indicate a correlation with disease status, prognosis, and therapeutic outcome. The discovery of miRNAs has opened a new avenue for individualized disease diagnosis and treatment.




The Importance of Micrornas in Lung Cancer


Implications of miRNAs in the Diagnosis of Lung Cancer


Identifying patients with early-stage lung cancer who will benefit the most from effective therapies may reduce the mortality of this deadly disease. Early detection is thus a key to improving the survival of patients with lung cancer. The results of investigational studies suggest that miRNAs may become promising biomarkers for risk assessment and diagnosis of lung cancer ( Tables 15.1 and 15.2 ).



TABLE 15.1

Single MicroRNAs (miRNAs) as Diagnostic Biomarkers for Lung Cancer






















































































































miRNAs Deregulation
in Cancer
Materials Descriptions References
let-7 Downregulated Tissue/TTNA tissue A SNP in a let-7 complementary site in the KRAS 3ʹ untranslated region increases NSCLC risk
Profiling the let-7 family is a promising method for differentiating adenocarcinoma from squamous cell carcinoma, even in small specimens, such as those obtained with TTNA
let-7a Downregulated Serum Expression levels show 0.74-fold change in cases vs. controls
miR-10b Upregulated Serum High serum miR-10b value is associated with elevated level of tissue polypeptide antigen
miR-17-5p Downregulated Serum Expression levels show 0.82-fold change in cases vs. controls
miR-25 Upregulated Serum The copy numbers of miR-25 are much higher in lung cancer than in healthy control samples
miR-27a Downregulated Serum Expression levels show 0.87-fold change in cases vs. controls
miR-29c Upregulated Serum The increased expression levels may reflect an increased systemic concentration of miR-29c in response to cancer processes
miR-106a Downregulated Serum Expression levels show 0.87-fold change in cases vs. controls
miR-141 Upregulated Serum High serum miR-141 value is associated with elevated level of urokinase plasminogen activator
miR-145∗ Downregulated FFPE tissue miR-145∗ inhibits cell invasion and metastasis
miR-146b Downregulated Serum miR-146b is significantly decreased in lung cancer regardless of stage or histology
miR-155 Downregulated Serum Expression levels show 0.77-fold change in cases vs. controls
miR-196a2 Upregulated Blood Functional SNP rs11614913 in miR-196a2 can contribute to lung cancer susceptibility
The rs11614913 TT genotype is associated with a significantly decreased risk of lung cancer for an Asian subgroup
miR-198 Downregulated PE Cell-free miR-198 from patients with lung adenocarcinoma may have diagnostic potential for differentiating malignant PE from benign PE
miR-205 Downregulated FFPE tissue/TTNA tissue miR-205 provides highly accurate subclassification of squamous cell carcinoma in NSCLC
Profiling miR-205 is a promising method for differentiating adenocarcinoma from squamous cell carcinoma, even in small specimens such as those obtained with TTNA
miR-221 Downregulated Serum miR-221 is significantly decreased in lung cancer regardless of stage or histology
miR-223 Upregulated Serum The copy numbers of miR-223 are much higher in lung cancer than in healthy control samples
miR-328 Upregulated FFPE tissue miR-328 has a role in conferring migratory potential to NSCLC cells working in part through PRKCA

FFPE , formalin-fixed, paraffin-embedded; NSCLC , nonsmall cell lung cancer; PE , pleural effusion; PRKCA , protein kinase C, alpha; SNP , single nucleotide polymorphism; TTNA , transthoracic needle aspiration.


TABLE 15.2

Combinations of MicroRNAs (miRNAs) as Diagnostic Biomarkers for Lung Cancer




















































































































































miRNAs Deregulation
in Cancer
Materials Descriptions References
miR-20a Upregulated Serum This 10-miRNA panel is correlated with the stage of NSCLC, especially in younger patients and patients who are current smokers
miR-24 Upregulated
miR-145 Upregulated
miR-152 Upregulated
miR-199a-5p Upregulated
miR-221 Upregulated
miR-222 Upregulated
miR-223 Upregulated
miR-320 Upregulated
miR-21 Upregulated Sputum The combination of these four markers may improve the early detection of lung adenocarcinoma
miR-200b Upregulated
miR-375 Upregulated
miR-486 Downregulated
miR-28-3p Downregulated Plasma These miRNAs in plasma may have a role as molecular predictors of lung cancer development and aggressiveness
miR-30c Downregulated
miR-92a Upregulated
miR-140-5p Downregulated
miR-451 Upregulated
miR-660 Upregulated
miR-30a Downregulated Tissue This five-miRNA signature may be a new diagnostic classifier for squamous cell carcinoma among Chinese patients
miR-140-3p Downregulated
miR-182 Upregulated
miR-210 Upregulated
miR-486-5p Downregulated
miR-30a-30p Upregulated Plasma This six-miRNA diagnostic test can discriminate between lung adenocarcinoma and granuloma
miR-100 Upregulated
miR-151a-5p Upregulated
miR-154-3p Upregulated
miR-200b-5p Upregulated
miR-629 Upregulated
miR-139-5p Upregulated Plasma This four-miRNA screening test is useful to divide nodule and nonnodule groups
miR-200b-5p Upregulated
miR-378a Upregulated
miR-379 Upregulated
miR-205 Upregulated Sputum The combination of these three markers may improve the early detection of lung squamous cell carcinoma
miR-210 Upregulated
miR-708 Upregulated
miR-574-5p
miR-1254
Upregulated
Upregulated
Serum These two markers may be used as minimally invasive screening and triage tools for early-stage NSCLC

NSCLC , nonsmall cell lung cancer.


The let-7 family is a global genetic regulator important in controlling the expression of lung cancer oncogenes. Chin et al. sequenced the let-7 complementary sites (LCS) in the Kirsten rat sarcoma ( KRAS ) 3ʹ UTR from 74 cases of NSCLC and identified a SNP at LCS6 significantly associated with an increased risk for NSCLC among moderate smokers (odds ratio, 2.3; 95% CI, 1.1–4.6). The LCS6 variant allele showed a 2.3-fold increase in risk for NSCLC among patients who smoked the equivalent of less than 40 pack-years.


A survey has reported that the SNP rs11614913 in miR-196a2 may affect mature miR-196a expression and target mRNA-binding activity and is significantly associated with survival from NSCLC. In a case–control study of 1058 patients with incident lung cancer and 1035 cancer-free controls in a Chinese population, Tian et al. found that miR-196a2 rs11614913 variant homozygote CC was associated with a significant increase of approximately 25% (odds ratio, 1.25; 95% CI, 1.01–1.54) in the risk of lung cancer compared with the wild-type homozygote TT and heterozygote TC. To further determine whether any association exists between four common SNPs (miR-196a2 C>T, rs11614913; miR-146a G>C, rs2910164; miR-499 A>G, rs3746444; and miR-149 C>T, rs2292832) and the risk for lung cancer, He et al. performed a meta-analysis of 40 published case–control studies. Their results demonstrated that the rs11614913 TT genotype was significantly associated with a decreased risk of lung cancer for an Asian population subgroup (TT vs. CC: odds ratio, 0.7; 95% CI, 0.57–0.85; p = 0.284). Squamous cell carcinoma is one major subtype of lung cancer for which biomarkers are urgently needed to aid patient management. Measuring the miRNA expression in cancerous and noncancerous tissue pairs collected from 60 Chinese patients with squamous cell carcinoma (stages I–III), Tan et al. identified a panel of five miRNAs (miR-30a, miR-140-3p, miR-182, miR-210, and miR-486-5p) that distinguished squamous cell carcinoma from normal lung tissues with an accuracy of 94%. They also showed that high expression of miR-31 was associated with poor survival among patients with squamous cell carcinoma.


Recent advances in the treatment of lung cancer require greater accuracy in the subclassification of NSCLC. Using a high-throughput microarray to measure the miRNA expression levels in 122 samples of adenocarcinoma and squamous cell carcinoma, Lebanony et al. identified miR-205 as a highly specific marker (96% sensitivity and 90% specificity) for squamous cell carcinoma. This standardized diagnostic assay may provide accurate subclassification of NSCLC. Fassina et al. investigated the accuracy of miRNAs in differentiating squamous cell carcinoma from adenocarcinoma within scant and distorted specimens obtained by transthoracic needle aspiration. Quantification of the let-7 family and miR-205 expression levels in 18 adenocarcinoma and 13 squamous cell carcinoma specimens by quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed a significant upregulation of the let-7 family and a significant downregulation of miR-205 in adenocarcinoma specimens (all, p < 0.05). Xing et al. profiled miRNA expression signatures in 15 samples of lung squamous cell carcinoma and matched normal lung samples with an miRNA array (GeneChip; Affymetrix, Santa Clara, CA, USA). They identified three miRNAs (miR-205, miR-210, and miR-708) that distinguished the sputum samples of patients with stage I lung squamous cell carcinoma from those of healthy individuals with 73% sensitivity and 96% specificity. Early detection is also the key to improving the survival of patients with lung adenocarcinoma. Using miRNA profiling on 20 paired samples of adenocarcinoma and normal lung tissue, Yu et al. identified four miRNAs (miR-21, miR-200b, miR-375, and miR-486) that distinguished the sputum samples of patients with stage I lung adenocarcinoma from those of healthy individuals with 81% sensitivity and 92% specificity.


Other studies were designed to identify serum-based miRNAs with the ability to diagnose NSCLC at an early stage. Owing to their high stability during storage and handling, miRNAs are optimal biomarkers present in the blood, urine, and other body fluids. Foss et al. performed miRNA profiling on total RNA extracted from serum obtained from 11 patients with early-stage NSCLC and 11 controls. The authors found that the expression of miR-574-5p and miR-1254 was significantly increased in the samples of early-stage NSCLC with respect to the controls ( p = 0.0277). Receiver operating characteristic curves plotting these two miRNAs were able to discriminate early-stage NSCLC from control samples with 82% sensitivity and 77% specificity. Using quantitative RT-PCR to measure the circulating levels of miRNAs in paired serum and plasma samples from 220 patients with early-stage NSCLC and 220 matched controls, Heegaard et al. also demonstrated that the expression levels of let-7a, miR-17-5p, miR-27a, miR-106a, miR-146b, miR-155, and miR-221 were significantly reduced in the serum of patients with NSCLC, whereas miR-29c was significantly increased (all, p < 0.05). Performing risk–score analysis to evaluate the diagnostic values of serum miRNA profiling among 400 patients with NSCLC and 200 controls, Chen et al. showed that a panel of 10 serum miRNAs (miR-20a, miR-24, miR-25, miR-145, miR-152, miR-199a-5p, miR-221, miR-222, miR-223, and miR-320) accurately distinguished patients with NSCLC from controls even up to 33 months before the clinical diagnosis of NSCLC.


The levels of miRNAs in serum are stable, reproducible, and consistent among individuals of the same species. Chen et al. used Solexa sequencing on serum miRNAs of human participants and obtained two miRNAs (miR-25 and miR-223) specific for NSCLC. The study conducted expression profiles of serum miRNAs in 21 healthy human patients and 11 NSCLC patients. Their results were validated in an independent cohort of 152 lung cancer patients and 75 healthy controls. The results of these analyses suggest that the copy numbers of these two serum miRNAs may serve as biomarkers for the detection of NSCLC. Cazzoli et al. used exosome-based techniques to analyze the miRNAs of 30 plasma samples (10 lung adenocarcinomas, 10 lung granulomas, and 10 healthy smokers) and subsequently validated them on an independent group of 105 specimens. The results showed that a screening test of four miRNAs (miR-139-5p, miR-200b-5p, miR-378a, and miR-379) was useful to divide the nodule and nonnodule groups (97.5% sensitivity, 72% specificity, and 90.8% area under the receiver operating characteristic curve [AUC]). The authors also developed a diagnostic test of six miRNAs (miR-30a-3p, miR-100, miR-151a-5p, miR-154-3p, miR-200b-5p, and miR-629) to discriminate between lung adenocarcinoma and granuloma (96% sensitivity, 60% specificity, and 76% AUC).


To examine whether circulating miRNAs have the potential to become suitable blood-based markers for the diagnosis and progression of lung cancer, Roth et al. measured the concentrations of four miRNAs in the serum of 35 patients with lung cancer and seven patients with benign lung tumors. The levels of miR-10b ( p = 0.002) and miR-141 ( p = 0.0001) were significantly higher in patients with lung cancer than in patients with benign disease. High serum concentrations of miR-10b were also found to be associated with lymph node metastasis among patients with lung cancer. Circulating cell-free miRNAs in pleural effusion are also potential biomarkers for cancer. Using microarrays to screen miRNAs in 10 malignant pleural effusions associated with lung adenocarcinoma and 10 benign pleural effusions, Han et al. showed that miR-198 was significantly downregulated in malignant pleural effusion compared with benign pleural effusion ( p = 0.002). The results of miRNA microarray analysis were confirmed by quantitative RT-PCR using a validation set comprising 45 malignant pleural effusions associated with lung adenocarcinoma and 42 benign pleural effusions. The AUC for miR-198 was 0.887.


Although early detection using chest x-ray and spiral computed tomography (CT) has resulted in a marked increase in the number of lung cancer diagnoses, these efforts may also lead to unnecessary treatments, and this possibility indicates the need for biomarkers of aggressive disease. Boeri et al. explored the miRNA expression profiles of 74 plasma samples collected during the 5-year screening plan and identified 12 of 15 samples collected before lung cancer detection by spiral CT, with sensitivity of 80% and specificity of 90%. The 5-year screening plan was a longitudinal analysis of 3246 current or former smokers screened for lung cancer beginning in 1998 either in the United States or in Italy to assess whether CT screening might increase the frequency of lung cancer diagnosis. The median amount of follow-up from the initial CT evaluation to the mortality end point was nearly 5 years. The most frequently deregulated miRNAs were miR-28-3p, miR-30c, miR-92a, miR-140-5p, miR-451, and miR-660.


Brain metastasis affects approximately 25% of patients with NSCLC during their lifetime. Arora et al. performed miRNA microarray profiling on samples from seven patients with NSCLC with brain metastasis and six without brain metastasis, and confirmed that the expression of miR-328 was able to correctly classify patients with and without brain metastasis. This miRNA may be incorporated into clinical treatment decision making to stratify patients with NSCLC who have a higher risk for brain metastasis. Profiling miRNAs extracted from 527 stage I NSCLCs on the human miRNA expression profiling panel, Lu et al. found that miR-145∗ was associated with brain metastasis by virtue of inhibiting cell invasion and metastasis. This miRNA holds potential as a target for preventing and treating brain metastasis among patients with stage I NSCLC.


MicroRNAs as Prognostic Biomarkers for Lung Cancer


Despite the availability of effective treatments, recurrence is common even for early-stage lung cancer. Prognostic biomarkers that can predict tumor progression and survival are required to provide better guidance on postoperative surveillance and therapeutic decisions for patients with lung cancer. Recent evidence suggests that specific miRNAs with altered expression levels have great potential to serve as prognostic biomarkers for lung cancer ( Tables 15.3 and 15.4 ).



TABLE 15.3

Single MicroRNAs (miRNAs) as Prognostic Biomarkers for Lung Cancer








































































































































miRNAs Deregulation
in Cancer
Materials Descriptions References
let-7 Downregulated Tissue Overexpression of let-7 in NSCLC cells inhibits cell growth in vitro
let-7a-2 Downregulated Tissue Low expression of let-7a-2 correlates with poor survival of patients with lung adenocarcinoma
let-7f Upregulated Plasma Expression level of let-7f is associated with overall survival in NSCLC
miR-16 Downregulated Serum High expression of miR-16 is associated with significantly better survival in patients with advanced NSCLC
miR-21 Upregulated Tissue Overexpression of miR-21 is an independent negative prognostic factor for overall survival in NSCLC
Inhibition of miR-21 inhibits cell growth in vitro and inhibits tumor growth in xenograft mouse models of lung adenocarcinoma
High expression of miR-21 has a negative impact on relapse-free survival in lung adenocarcinoma and overall survival in NSCLC
miR-30c-1 Downregulated Tissue The expression of the host nuclear transcription factor Y gene is correlated with pri-miR-30c-1, but not with rs928508 genotypes among patients with NSCLC
miR-30e-3p Downregulated Plasma The expression level of miR-30e-3p is associated with short disease-free survival in NSCLC
miR-31 Upregulated Tissue miR-31 represses DICER1 activity but not PPP2R2A or LATS2 in lung squamous cell carcinoma
In vitro functional assays show that miR-31 increases cell migration, invasion, and proliferation in an ERK1/2 signaling-dependent manner in lung adenocarcinoma
miR-34a Downregulated Tissue A relation has been found between MIRN34A methylation and miR-34a expression in NSCLC
miR-34b Downregulated Tissue Overexpression of miR-34b decreases the expression of c-Met in NSCLC cells
miR-34b/c Downregulated Tissue Ectopic expression of miR-34b/c in lung adenocarcinoma cells decreases cell proliferation, migration, and invasion
miR-145 Downregulated Tissue miR-145 regulates SOX2 and OCT4 translation, and p53 regulates miR-145 expression
miR-146b Upregulated Tissue High expression of miR-146b correlates with poor overall survival for patients with lung squamous cell carcinoma
miR-149 Downregulated Tissue miR-149 may be involved in the pathogenesis of NSCLC
miR-155 Upregulated Tissue High expression of miR-155 correlates with poor survival of patients with lung adenocarcinoma
High expression of miR-155 correlates with poor overall survival for patients with lung squamous cell carcinoma
miR-186 Downregulated Cell line Cyclin D1, CDK2, and CDK6 are each directly inhibited by miR-186, and restoring their expression levels reverses miR-186-mediated inhibition of cell cycle progression
miR-196a Upregulated Tissue miR-196a may be involved in the pathogenesis of NSCLC
miR-196a2 Upregulated Tissue rs11614913 CC is associated with a significant increase in mature miR-196a expression, but not with changes in levels of the precursor for NSCLC
miR-367 Upregulated Tissue SOX2 and OCT4 transcription factors regulate the expression of miR-367
miR-651 Downregulated Blood The FAS:rs2234978 G allele is significantly associated with survival in early-stage NSCLC, and the FAS single nucleotide polymorphism created a miR-651 functional binding site
miR-708 Upregulated Tissue Forced miR-708 expression reduces TMEM88 transcript levels and increases the rate of cell proliferation, invasion, and migration in culture

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Mar 20, 2019 | Posted by in CARDIOLOGY | Comments Off on MicroRNAs as Biomarkers for Lung Cancer

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