Identification of plasma microRNA-21 as a biomarker for early detection and chemosensitivity of non–small cell lung cancer

Studies have shown cell-free microRNA (miRNA) circulating in the serum and plasma with specific expression in cancer, indicating the potential of using miRNAs as biomarkers for cancer diagnosis and therapy. This study was to investigate whether plasma miRNA-21 (miR-21) can be used as a biomarker for the early detection of non–small cell lung cancer (NSCLC) and to explore its association with clinicopathologic features and sensitivity to platinum-based chemotherapy. We used real-time RT-PCR to investigate the expression of miR-21 in the plasma of 63 NSCLC patients and 30 healthy controls and correlated the findings with early diagnosis, pathologic parameters, and treatment. Thirty-five patients (stages NIB and IV) were evaluable for chemotherapeutic responses: 11 had partial response (PR); 24 had stable and progressive disease (SD+ PD). Plasma miR-21 was significantly higher in NSCLC patients than in age- and sex-matched controls (P < 0.001). miR-21 was related to TNM stage (P < 0.001), but not related to age, sex, smoking status, histological classification, lymph node status, and metastasis (all P > 0.05). This marker yielded a receiver operating characteristic (ROC) curve area of 0.775 (95% CI: 0.681 – 0.868) with 76.2% sensitivity and 70.0% specificity. Importantly, miR-21 plasma levels in PR samples were several folds lower than that in SD plus PD samples (P = 0.049), and were close to that in healthy controls (P = 0.130). Plasma miR-21 can serve as a circulating tumor biomarker for the early diagnosis of NSCLC and is related to the sensitivity to platinum-base chemotherapy.

Lung cancer, one of the most common malignant tumors, is a serious disease threatening human life and health. Of the pathologic types of lung cancer, nonsmall cell lung cancer (NSCLC) is the most prevalent (80%). The lack of effective early diagnostic and treatment methods are the main causes of high mortality in patients with lung cancer, with the 5year survival rate at 15% to 20%. MicroRNA (miRNA) is a class of 20 to 25 nt long noncoding RNA found . miRNA genes are transcribed in the nucleus to primary transcripts (primiRNA) [1] . After digestion by Drosha, precursor miRNA (premiRNA) is then formed and is transported to the cytoplasm and digested by Dicer to produce mature miRNA [2,3] , which regulates gene expression mainly through its partially complementary to 3'untranslated region (3爷UTR) of target mRNA [4] .
Among their functional roles, miRNAs regulate development, organogenesis, hematopoiesis, cell proliferation and apoptosis, and tumorigenesis and progression. It has been confirmed that miRNAs are involved in the development of a variety of tumors, such as leukemia [5] , neuroblastoma [6] , pituitary adenoma [7] , breast cancer [8] , thyroid cancer [9] , hepatocarcinoma [10] , colorectal cancer [11] , and lung cancer [12] . The up or downregulation of different miRNAs in these tumor tissues were found with most of the miRNA targets located in regions of tumorrelated genome, fragile sites, loss of heterozygosity, and amplified regions [5] , thereby exhibiting the same effect as oncogenes or tumor suppressor genes.
An increasing number of studies have shown that analysis of miRNA expression has a potential application value in tumor diagnosis, treatment, and prognosis prediction. It has been observed that miRNAs are closely related to tumor development and stably exist in serum/plasma [1315] . The origin of miRNAs is unclear and lacks experimental evidence, but current assumptions of the origin include cancer cells in peripheral blood [16] or tumorassociated bodies [17,18] . The first miRNA marker found in serum is miRNA21 (miR21). Lawrie . [19] proved that high expression of miR15, miR21, and miR21 0 existed in the serum of patients with diffuse large Bcell lymphoma (DLBCL) using realtime fluorescence quantitative PCR, and miR21 with largest differential expression is related to lymphoma recurrence and patient survival. Subsequently, Mitchell . [16] found that miR141 was highly expressed in patients with metastatic prostate cancer. The level of miR141 expression is highly specific and sensitive for the diagnosis of prostate cancer and thus, can serve as a marker. In addition, the level of miR184 expression in the plasma of patients with tongue squamous cell carcinoma was significantly higher than that in healthy people and was decreased significantly after tumor removal and corresponding treatment [20] . Ng . [21] found that the expression levels of miR173p and miR92 in the plasma and tissue of patients with rectal cancer were significantly increased and that the expression of miR92 was significantly reduced after tumor resection, suggesting that miR92 can function as a serum molecular marker for the diagnosis of colorectal cancer at stage IIV, whereas early diagnosis can improve survival and prognosis. The expression of miR155 in patients with progesterone receptorpositive breast cancer was higher than that in progesterone receptornegative patients, suggesting that miR155 can serve as a molecular marker differentiating hormone sensitive breast cancer from nonsensitive ones [22] . These studies indicate that plasma/serum miRNAs may function as potential biomarkers for the noninvasive diagnosis, treatment, and prognosis of cancers.
In the present study, we measured the plasma level of miR21 and determined its relationship to the early diagnosis of NSCLC, efficacy of chemotherapy, and clinicopathologic characteristics of patients with NSCLC.

Study subjects
Peripheral blood specimens from 63 patients with pathologically confirmed NSCLC were collected between October 2009 and August 2010 from the Departments of Thoracic Surgery and Oncology in the First Affiliated Hospital of Nanjing Medical University. Of the 63 patients, 45 were men and 18 were women, aged of 36 to 83 years with a median of 61 years. The patients did not undergo chemotherapy or radiotherapy prior to admission. Venous blood was drawn before surgery for patients with early stage NSCLC. For those with advanced NSCLC, CT was used to confirm measurable tumor lesions. In addition, functional scores before chemotherapy were greater than 60, with blood and kidney function in the normal range and a normal electrocardiogram. Venous blood was drawn before chemotherapy for all patients. According to the 7th edition of TNM staging of the Union for International Cancer Control (UICC) and American Joint Committee on Cancer (AJCC) in 2009, 18 cases were classified as stage I, 5 as stage II, 5 as stage III, and 35 as stage IV. Thirtyeight patients had adenocarcinoma, 15 had squamous cell carcinoma, 1 had large cell carcinoma, and 9 had unclassified tumors. In addition, 30 had lymph node metastasis and 33 had no lymph node metastasis. The control group was composed of 30 healthy people: 20 men and 10 women aged 41 to 75 years with a median of 57 years. There was no statistical difference in age ( = 0.086, MannWhitney test) and sex ( = 0.803, 字 2 test) between NSCLC patients and the control group.

Chemotherapy regimen and the evaluation of therapeutic efficacy
Of the 63 patients , 40 underwent cisplatin or carboplatinbased chemotherapy; 35 had measurable lesions. The chemotherapy regimen was repeated every 3 to 4 weeks. After 2 to 3 cycles of treatment, an objective evaluation was made according to the standards of Evaluation Criteria in Solid Tumors recommended by the WHO, defined as complete remission (CR), partial remission (PR), stable disease (SD), and progressive disease (PD).

Real鄄 time quantitative RT鄄 PCR detection of miR鄄 21 expression in plasma
Peripheral blood specimen (5 mL) was conventionally drawn from each patient before surgery or chemotherapy. EDTA was used as an anticoagulant. Plasma and blood cells were separated by centrifugation at 1 200 伊 for 10 min at 4益 , and stored in 1.5 mL RNasefree tubes at 80益 . Peripheral blood was processed within 4 h.
Plasma (400 滋 L) from each sample was centrifuged at 1 200 伊 for 10 min at 4益 before RNA extraction. Total RNA was extracted using mirVana PARIS kit according to manufacturer爷s instruction (Ambion, USA). To eliminate differences among samples, synthetic Caenorhabditis elegans celmiR39, which has no homologous gene in human, was added into each sample. RNA was dissolved in 100 滋 L of RNasefree water. Absorbance at 260 and 280 nm was measured using a spectrophotometer to determine the concentration and purity of RNA.
The expression of miR21 in NSCLC patients and healthy controls was detected by using miScript SYBR Green PCR kit (Qiagen, Germany). The reaction was performed in a realtime quantitative PCR machine ABI7300HT. Reverse transcription of miRNA was performed according to manufacturer爷s instruction using the miScript Reverse Transcription kit (Qiagen). The first chain of cDNA was reverse transcribed from 2 滋 L of total RNA using specific stemloop structure RT primers of miR21. The amount of RNA was determined by the volume of plasma: (2.0/80) 伊 400 = 10 mL, 2 滋 L of RNA corresponding to 10 滋 L plasma [16] . The reverse transcription reaction was carried out at 37益 for 60 min and at 95益 for 5 min. Subsequently, PCR was performed using the first chain of cDNA as the template. The reaction system contained 2 滋 L of cDNA, 12.5 滋 L of 1伊 SYBR Green I Master mix, 2.5 滋 L of miRNA specific primers, and 2.5 滋 L of upstream universal primer. The reaction conditions were as follows: denatured at 95益 for 15 min, 40 cycles of 95益 for 15 s, 57益 for 30 s, and 70益 for 34 s. miR16 was used as an internal reference for normalization. An average of triplicate findings was considered as the result for each sample. The sequence of celmiR39 was 5'UCACCGGGUGUAAAUCAGCU UG3'; the sequence of miR16 primer was 5'UAGCAG CACGUAAAUAUUGGCG3'; the sequence of miR21 primer was 5'UAGCUUAUCAGACUGAUGUUGA3' (synthesized by Qiagen). Finally, Ct values were measured using melting curve analysis to represent the expression of target gene mRNA. Relative changes of gene expression were represented by 2 -吟吟Ct , the difference between the original copy number of miR21 in the NSCLC group and that in the normal group. △ △ Ct= (Ct miR21 Ct miR16 ) of the NSCLC group (Ct miR21 Ct miR16 ) mean of the control group.

Statistical analysis
GraphPad Prism V4.03 and SPSS16.0 statistical softwares were used for statistical analysis. Log10 normality conversion was used to analyze the relative expression of miR21. The MannWhitney test was used for data comparison between two groups. The KruskallWallis test was used for comparison among multiple groups. The receiver operating characteristic (ROC) curve was drawn to evaluate the diagnosis value of plasma miR21 for NSCLC. < 0.05 indicates a statistical significance.

Results
The expression levels of plasma miR鄄 21 in NSCLC group and healthy control group detected by real鄄 time quantitative RT鄄 PCR Using miR16 as an internal reference, the expression level of plasma miR21 was detected by realtime fluorescence quantitative PCR. The quantitative PCR amplification and melting curves reveal the expression levels of miR21 and miR16 (Figure 1).

Relationship between plasma miR鄄 21 expression and clinicopathologic features of NSCLC
PCR results showed that miR21 expression was significantly higher in the NSCLC group than in the control group ( < 0.001) (Figure 2). miR21 expression was not related to age, sex, smoking status, pathologic type, lymph node metastasis, and distant metastasis (all > 0.05), but related to TNM stage ( < 0.001). Plasma miR21 expression was higher in stage T34 patients than in stage T12 patients ( = 0.036) and was higher in stage IIIIV patients than in stage III patients ( = 0.043) (Figure 2), indicating that plasma miR21 is important for the diagnosis of stage IIV NSCLC. The area under the ROC curve (AUC) for plasma miR21 was 0.775 (95% CI: 0.6810.868), the sensitivity and specificity were 76.2% and 70.0% , respectively, and the value of the cutoff point was 1.308 ( Figure 3).

Relationship between plasma miR鄄 21 expression and chemotherapeutic efficacy
Thirtyfive patients with advanced NSCLC who underwent 2 to 3 cycles of platinumbase chemotherapy were followed up: 11 had PR, 13 had PD, 11 had SD, and none achieved CR after chemotherapy. The expression of plasma miR21 was significantly higher in the PD plus SD group than in the PR group ( = 0.049), whereas no statistical significance was found between the PR group and the control group ( = 0.130) ( Figure  4), suggesting that plasma miR21 expression may correlate with sensitivity to chemotherapy in NSCLC.
Discussion miR21 is overexpressed in lung cancer, breast cancer, glioblastoma, gastric cancer, pancreatic cancer, pancreatic neuroendocrine tumor, liver cancer, bile duct cancer, colon cancer, and prost ate cancer [23] . miR21 is encoded by a single gene, which locates at the fragile site FRA17B on 17p23.2 and is overlapped with the gene encoding transmembrane protein 49 (TMEM49, also known as VMP1) [24] . miR21 contains 22 nucleotides and is separated from a 3400 nt pretranscript [25] . Abnormal expression of miR21 may be related to transcription and posttranscriptional regulation [24] . miR21 regulates cell differentiation, proliferation, and apoptosis and is involved in tumor cell invasion, vascular infiltration, and metastasis by regulating target genes, including tropomyosin 1 ( ), programmed cell death gene 4 ( ), methyladenosine phosphatase ( ), , genes in the p53mediated pathway and transforming growth factor (TGF茁 ) pathway, , phosphatase and tensin homolog ( ). In addition, the high expression of miR21 in patients with colorectal cancer is correlated to clinical stage, lymph node metastasis, and distant metastasis [26] ; stages of breast cancer and liver metastases accompany with the increasing expression level of miR21 [8] . Several studies have also confirmed that miR21 plays an important clinical role in tumor diagnosis and evaluation. Schetter . [27] found that high expression of miR21 in colon cancer is related to poor efficacy of treatment and prognosis. Yan . [28] found that overexpression of miR21 in breast cancer is associated to lymph node . The expression of miR鄄 21 is significantly higher in NSCLC than in normal control (P = 0.001), higher in stage T3-4 NSCLC than in stage T1-2 NSCLC (P = 0.036), and higher in stage III-IV NSCLC than in stage I-II NSCLC (P = 0.043).  Gao . [29] revealed that high expression of miR21 in lung squamous cell carcinoma indicates poor prognosis. However, it has been reported that miR21 only serves as a diagnostic marker in gastric cancer with no correlation to prognosis [23] .
In this study, we used realtime quantitative PCR to determine the expression of plasma miR21 in patients with NSCLC. Plasma miR21 expression was significantly higher in NSCLC patients than in the control group ( < 0.0001). ROC curve analysis showed the AUC was 0.775; the sensitivity and specificity were 76.19% and 70.0% . miR21 expression was significantly related to TNM stage. The expression of plasma miR21 was higher in patients with advanced NSCLC than in those with early stage NSCLC, suggesting that miR21 expression may be related to factors associated with tumor progression. In particular, the expression of plasma miR21 was significantly higher in patients with stage T12, N0 or III NSCLC than in the control group, suggesting that plasma miR21 can be used as an effective, noninvasive marker for early NSCLC diagnosis.
Recently, studies have shown that miRNAbased treatment has a potential effect. Meng . [30] found that the application of antimiR21 AMOs (antimiRNA oligonucleotides) increase sensitivity of cholangiocarcinoma cells to gemcitabine. Si .
[31] treated miR21/AMOstransfected cancer cells with topotecan and found that antisense inhibition of miR21mediated cell growth was significantly inhibited. Li . [32] found that overexpression of miR21 in malignant glioma cells leads to tumor cell resistance to chemotherapeutic drugs and that inhibiting the expression of miR21 in malignant glioma U373MG cells by specific AMOs increases the sensitivity of U373MG cells Vm26, indicating that miR21 can be used to increase efficacy of drugs on malignant glioma. Hwang . [33] retrospectively studied the effect of miR21 on the chemotherapy efficacy and prognosis of pancreatic cancer: patients with low expression of miR21 have significantly prolonged overall and diseasefree survival after chemotherapy; antisense inhibition of miR21 can enhance chemotherapeutic sensitivity. We followed up 35 patients with advanced NSCLC treated with platinum chemotherapy and found that the expression of plasma miR21 in SD and PD patients was higher than that in PR patients. The expression of plasma miR21 was low in 11 PR patients, which showed no significant difference when compared to that in the control group. Thus, miR21 may be related to platinum sensitivity. The high expression of plasma miR21 may also be involved in cell resistance to platinum chemotherapy. Inhibiting miR21 expression is likely to restore cell sensitivity to platinum chemotherapy. Therefore, our study provides a favorable basis to use miR21 as a predictor of sensitivity to platinum chemotherapy. Due to a short followup duration, we cannot calculate the overall and diseasefree survival, but we will continue to explore the relationship between plasma miR21 expression and . Expression levels of the miR鄄 21 (Log10 scale at Y鄄 axis) are normalized to miR鄄 16. Statistically significant differences were determined using the Mann鄄 Whitney U test. Plasma level of miR鄄 21 is significantly higher in PD plus SD samples than in PR samples (P < 0.05) and healthy control samples (P < 0.001), and is similar between PR samples and control samples. The quantity of miR鄄 21 in plasma may be a potential biomarker for predicting platinum鄄 based chemo鄄 sensitivity of NSCLC. NSCLC prognosis.
In summary, our study demonstrated that the expression of plasma miR21 is sensitive and specific for the early diagnosis of NSCLC and thus can be used as a potential biomarker for NSCLC diagnosis. Plasma miR21 expression reflects sensitivity to platinum chemotherapy for NSCLC, suggesting that plasma miR21 is a predictive indicator for sensitivity to platinum chemotherapy and may be a therapeutic target that reverse resistance to platinum chemotherapy.