Matrix metalloproteinase‑1 and microRNA‑486‑5p in urinary exosomes can be used to detect early lung cancer: A preliminary report
- Authors:
- Published online on: January 29, 2024 https://doi.org/10.3892/ol.2024.14261
- Article Number: 127
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Copyright: © Ando et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Lung cancer (LC) was the leading cause of mortality in 2018 in males and the third in females worldwide (1). In Japan, recent mortality statistics (2021) and incidence rate (2019) revealed that LC accounts for the highest mortality and fourth highest incidence rate in males, and the second highest mortality and third highest incidence rate in females (2). However, LC treatment has improved, which is mainly attributed to the increased detection of early-stage LC, especially in cases where tumors are ≤2 cm in size (3). Low-exposure CT and MRI have majorly contributed to the diagnosis of early LC (4). However, the prognosis of LC remains poor. In Japan, the Act for Health and Safety in Labors strictly requires all workers to undergo a chest X-ray examination to prevent tuberculosis and pulmonary diseases due to occupational hazards. However, chest X-ray examination is not suitable for LC detection because it is difficult to diagnose early-stage LC using plain radiography; the specificity and sensitivity of chest X-ray for LC is also insufficient (5). Hence, there remains a need for develop a novel LC screening test with easy access, high quality, and low price, for the early detection of early-stage patients with LC having high sensitivity and specificity at the work-place and community health centers.
We have previously reported a novel breast-cancer screening method using a combination of microRNA-21 (miRNA-21) and matrix metalloproteinase-1 (MMP-1) in urinary exosomes capable of detecting breast cancer in 95% cases before metastasis (6). All cells secrete exosomes that are small vesicles (30–150 nm in size) containing DNA, mRNA, miRNAs, oncogenic genes, proteins, and lipids among others (7–9). These exosomes relay important cell information between cells, migrate through the blood vessels or other routes, and adhere to the surface of distant cells (7–10). Therefore, they are crucial for maintaining harmony with other organs, not only for physiological growth, development, and cell death, but also for pathogenesis. Exosomes from the cancer cells can invade the distant cells of other organs, eventually resulting in metastasis (7–10). Therefore, urinary exosomes contain sufficient information on the oncogenic genes and their related proteins. However, LC screening using urinary exosomes has not been reported to date. Furthermore, MMP-1 is one of the key enzymes of collagen metabolism (11) involved in the carcinogenesis in LC (12–15). An increased expression of MMP-1 evidenced in early stage of LC (15). However, MMP-1 has not been investigated to serve as a biomarker for LC screening.
We selected miRNA-21 and miRNA-486-5p as candidate miRNAs to serve as biomarkers for LC screening in the present study (16–21). Several studies have reported the usefulness of miRNA-21 and miRNA-486-5p in diagnosing LC using the circulating plasma exosomes (16,20). Both miRNA-21 and miRNA-486-5p have multi-faced functions in cancer diagnosis, tumorigenesis, and prognosis (16,17,20). The function of miRNA-21 inhibits several tumor suppressor genes such as PTEN and HOXD10 (17–19). MiRNA-486-5p targeting the PIK3R1 gene is involved in the suppression of tumor cell growth (21).
Herein, we analyze the expression of MMP-1/CD63 as well as miRNA-21 and miRNA-486-5p in urinary exosomes of patients with LC and determine their application in early screening and detection of LC in patients.
Materials and methods
Study population
This study was conducted at the International University of Health and Welfare (IUHW) Hospital (Nasu-Shiobara City, Tochigi Japan) and the Kitasato University Medical Center (Kitamoto City, Saitama, Japan) between October 1, 2018 and October 31, 2022. The study was approved by the ethics committee of the two abovementioned institutions. The details were described in Declarations.
Thirty-five patients with LC (24 males and 11 females; mean age, 71.0±8.4 and 64.8±9.5 years, respectively) included in this study were admitted to the Center for Respiratory Diseases, Department of Chest Surgery, IUHW Hospital. The patients were diagnosed with LC after performing chest X-ray, chest CT, chest MRI, and subsequent needle biopsy before surgery, if necessary. The final pathological diagnosis was performed on the basis of surgical resection at the IUHW Hospital. The clinical stage was classified according to the eighth edition of the TNM classification of LC (22).
Urine samples were collected before surgical treatment in the morning before breakfast and stored at −80°C until the exosomes were separated. All samples were collected before patients received surgery, chemotherapy, or radiation.
The control urine samples were obtained from 40 healthy controls (20 males and 20 females) selected among 533 persons (351 males and 182 females) who visited the Department of Preventive Medicine, IUHW Hospital for a health check from January 20, 2020 to February 8, 2020, because only a few visitors were over 70 years of age. The number of selected individuals in the 40-year-old, 50-year-old, 60-year-old, and >70-year-old groups in each sex group was more than five. All selected controls had no complaints or abnormal physical findings such as obesity, hypertension, abnormal peripheral blood examination and blood chemistry, abnormal ECG findings, or abnormal findings on chest radiography, upper GI endoscopy, or abdominal echography. There was no history of cancer, signs of dysplasia, inflammatory disease, autoimmune disease, or chronic diseases such as cardiac, liver, or kidney diseases. All participants provided written informed consent.
Classification of LC clinical stage
The clinical stage was classified according to the eighth edition of the TNM classification of LC (22). The TNM classification of the 35 patients with LC is listed in Table I.
Isolation of the urinary exosomes
All urine samples were transferred to Kitasato University Medical Center at −80°C. A uniform volume of 2 ml urine was collected from all participants for exosome isolation. Before exosome isolation, the thawed urine sample was centrifuged at 3,000 × g for 15 min at 4°C and passed through a 0.22-µm nylon filter. The urinary exosomes isolated using the Exosome Isolation Kit (Cat. 130-110-912, Miltenyi Biotec, Bergisch Gladbach, Germany) were subjected to western blotting using SDS-PAGE and anti-CD63 biotin-conjugated antibody (BioLegend Inc., San Diego, CA, USA) and identified using transmission electron microscopy (Fig. S1) as reported previously (6). The pellet of isolated exosomes was placed over the Transmission Electron Microscopy (TEM) grid coated with carbon/formvar (Oken-Shoji Co., Tokyo, Japan), stained by the 2% uranyl acetate for 2 min at room temperature, and finally exosomes were observed by transmission electron microscopy (Hitachi H-7600, Hitachi Ltd., Tochigi, Japan).
MMP-1/CD63 expression ratio in urinary exosomes
The levels of MMP-1 and CD63 were determined with western blotting using an anti-MMP-1 antibody (Cat. ab134184, Abcam plc., Cambridge, UK) and biotin-conjugated anti-CD 63 antibodies (Cat. 353017, BioLegend Inc., CA, USA). One-sixth of the urinary exosomes extracted from 2 ml was applied to the wells. Anti-MMP-1 antibody (1:1,000) and anti-CD63 antibody (1:1,000) were used. The ratio of MMP-1/CD63 was pixelated in patients with LC and compared to that in healthy controls (Fig. S2). The analysis was performed using the ImageJ 1.52a software (NIH, Bethesda, MD, USA) (23). To validate reproducibility, all experiments were performed twice, and the average or median values were calculated, as described previously (6).
Analysis of miRNA-21 and miRNA-486-5p extracted from the urinary exosomes
RNA for analyzing miRNAs (miRNA-21 and miRNA-486-5p) was extracted from the isolated exosomes using a Total Exosome RNA and Protein Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA); the isolated miRNA was reverse-transcribed to cDNA using the TaqMan MicroRNA Reverse Transcription Kit (Cat. 4366596, Thermo Fisher Scientific), according to the manufacturer's protocol. RT-qPCR was performed on a Step One Plus® thermal cycler (Applied Biosystems, Foster City, CA, USA) for miRNAs extracted from the urinary exosomes. For determining the miRNA-21 and miRNA-486-5p expression respectively, assays were performed using a commercial miRNA-21 assay kit (Cat. 4427975, Assay ID: 000397, Applied Biosystems) and miRNA-486-5p assay kit (Cat. 4427975, Assay ID 001278, Applied Biosystems) respectively according to the manufacturer's instructions. The assays were performed in duplicate for each sample, as described previously (6). In this study, instead of internal controls, miRNAs were extracted from urinary exosomes and reverse transcription reactions were performed using 5 ng of the recovered miRNAs to correct for miRNA levels between samples, and expression analysis was performed. This method has been proven in previous reports (6).
Relative expression levels of miRNA-21 and miRNA-486-5p in the urine exosomes
Based on the mean number of miRNA-21 and miRNA-486-5p copies in the healthy controls, the copy number of miRNA-21 and miRNA-486-5p in patients with LC was calculated as the relative expression grade, as described previously (6). The ΔΔCq value was determined by subtracting the mean CT value of miRNA-21 and miRNA-486-5p in the healthy controls from the individual CT value of miRNA-21 and miRNA-486-5p in patients with LC. In the present study, the number of RT-PCR cycles in miRNA-486-5p varied between healthy male and healthy female controls, hence miRNA-486-5p was analyzed by gender. Finally, the copy number of miRNA-21 and miRNA-486-5p were compared as the value of 2ΔΔCq.
Statistical analysis
Data are expressed as the mean ± standard deviation (SD) or median [95% confidence interval (CI)]. All statistical analyses were performed using the PRISM 9 software for Mac OS (GraphPad, Inc., La Jolla, CA, USA). Data were tested for normality and equal variance to confirm the appropriateness of the parametric tests, and those that followed a normal distribution were analyzed using the unpaired Student's t-test. Mann-Whitney U-tests were used to compare differences between groups, and Kruskal-Wallis test followed by Dunn's post hoc test were used to compare differences among three or more groups. P<0.05 was considered to indicate a statistically significant difference.
Logistic regression analysis was performed with the presence of lung cancer as dependent variable, and the expression ratio for MMP-1/CD63, and relative expression of miRNA-486-5p as independent variable adjusted with age, sex, and smoking status as covariates. ROC analysis was applied for the prediction of logistic regression model, and cut-off value was calculated by the Youden index. Logistic regression analysis and its ROC analysis were performed by IBM SPSS ver 26.0.
Results
Clinical characteristics and histopathological findings in patients with LC
The included patients with LC were relatively early-detected cases since most have visited the Department of Preventive Medicine of IUHW Hospital for health checkups, including LC screening including chest X-ray and cytological examination on sputum, and their suspicious lesions were examined using lung CT and MRI followed by the further examinations (Tables I and SI).
The pathological findings in the surgically resected tissues were as follows: non-small cell lung cancer was evident in 30 patients, adenocarcinoma (AC) in 24 (15 males and 9 females), squamous cell carcinoma (SC) in 5 (4 males and 1 female), and large cell carcinoma (LCC) in 1 (1 male and 0 female). Small cell lung cancer (SCLC) was evident in 4 (3 males and 1 female); pleomorphic carcinoma was evident in one male.
Among the 35 patients with LC, stage 0 was seen in one man, stage I in 19 (10 males and 9 females), stage II in 5 men, stage III in 3 (2 males and 1 female), and stage IV in 7 (6 males and 1 female).
Comparison of expression ratio of MMP-1/CD63 in patients with LC and those in healthy controls
The individual expression ratios of MMP-1/CD63 in patients with LC and healthy controls are listed in Tables I and SII, respectively. The MMP-1/CD63 expression ratio in both male and female healthy controls showed the same distribution trend, and those in both male and female patients with LC was increased (Fig. 1A-C). The median expression ratio in patients with LC was 1.342 (95% CI: 0.890–1.974), which was significantly higher than that in the healthy controls, 0.600 (95% CI: 0.490–0.900) (P<0.0001) (Tables I and SII; Fig. 1A).
The MMP-1/CD63 expression ratio in female healthy controls who are under 60 years of age was not significant different from those who are older (Table IIA). Male patients with LC showed higher ratio tendency in both under 60 years and over 60 years of age, especially in those under 60 years patients, than in both healthy males under and over 60 years of age (Table IIA). A significantly higher ratio in both female patients under and over 60 years was observed compared with the females healthy controls (P<0.01 and P<0.05, respectively; Table IIA). The MMP-1/CD63 expression ratio of SCLC and those of NSCLC was not statistically different in the limited population.
 | Table II.Expression ratio of MMP-1/CD63 and relative expression of miRNA-21 and miRNA-486-5p in 40 healthy controls and 35 lung cancer patients. |
Expression ratio of MMP-1/CD63 in patients with LC based on the cancer stage
The expression ratio of MMP-1/CD63 in 20 patients with stage 0 and I was 1.342 (95% CI: 0.888–2.319), which was significantly higher than that in 40 healthy controls (P=0.0074). The expression ratio of MMP-1/CD63 in 5 patients with stage II was 1.795 (95% CI: 1.105–4.414), which was higher than that in 40 healthy controls (P=0.0192), as depicted in Fig. 1D (all), 1E (males), and 1F (females). These data suggest that the expression ratio of MMP-1/CD63 is a useful biomarker for early stage patients with LC including both sex.
Expression ratio of MMP-1/CD63 in 35 patients with LC based on the cancer metastasis
Two cases (No. 13 and 32) revealed M1a, their expression ratio of MMP-1/CD63 were 0.41 and 0.88. Three M1b cases (No. 14, 18 and 29) showed 0.40, 5.03 and 0.39, respectively. The rest two M1c cases (No. 17 and 21) revealed 4.01 and 0.27. Among 7 metastasis cases, two cases showed the marked increased value.
Expression ratio of MMP-1/CD63 based on pathology and smoking habits
The expression ratio of MMP-1/CD63 in 11 patients who were never-smokers was higher (2.32; 95% CI: 1.80–4.29) than that in 14 current smokers (1.01; 95% CI: 0.43–4.03) and in 10 former smokers (0.90; 95% CI: 0.39–1.92), as presented in Table IIIA, Fig. 1G (all), Fig. 1H (males), and Fig. 1I (females), although our preliminary study did not show the statistical significance.
| Table III.Comparison of MMP-1/CD63 and miRNA-486-5p expression based on smoking status and cancer type in lung cancer patients. |
Eleven never-smokers (4 males and 7 females), among whom the expression ratio of MMP-1/CD63 was higher than 1.25 in 9 patients (3 males and 6 females). Interestingly, all had AC and 67% (6/9) were female patients with AC. The ratio was slightly higher in the AC group than that in the SC group (Table IIIA).
Relative expression levels of miRNA-21 and miRNA-486-5p in patients with LC and healthy controls
We selected miRNA-21 and miRNA-486-5p as a candidate screening biomarker as they have been reported to be effective in diagnosis/screening for LC using the circulating plasma exosomes (16–21).
Relative expression levels of miRNA-21 in patients with LC and healthy controls
The relative expression level of miRNA-21 in healthy controls was slightly higher in both of male and female over 60 years healthy controls compared with those in both sex under 60 years healthy controls, although not significantly; the expression level was higher in healthy males than females, although not significantly (Table IIB).
The expression level of miRNA-21 in male patients with LC was not significantly different from those in male healthy controls (Table IIB). The same tendency was seen in female patients with LC compared with those in female healthy controls (Fig. 2A-C; Table IIB). The expression levels of miRNA-21 were not significantly different from those of healthy controls for pathology and smoking habits (Fig. 2D-I).
Relative expression levels miRNA-486-5p in patients with LC and healthy controls
The relative expression level of miRNA-486-5p in under 60 years healthy males was significantly lower than that in under 60 years healthy females (P<0.01), while significantly higher in over 60 years healthy males than their healthy females counterparts (P<0.05; Table IIC).
The distribution pattern of the relative expression levels of miRNA-486-5p in male patients with LC compared with those in healthy males was up-regulated, while those in females patients with LC compared with those in healthy controls was down-regulated (Fig. 3A-C). These data show the sex difference in the relative expression levels of miRNA-486-5p.
Then we measured the relative expression levels of miRNA-486-5p in healthy controls and patients with LC by sex separately; the individual relative expression levels of miRNA-486-5p are listed in Tables I and SII.
The median expression levels of miRNA-486-5p in male patients with LC was 2.565 (95% CI: 1.800–4.420), which was significantly higher than that in male healthy controls (1.254, 95% CI: 0.790–1.530, P=0.0004). The median relative expression levels in female patients with LC (0.460, 95% CI: 0.060–2.130) tending to be lower than that in female healthy controls (1.250, 95% CI: 0.470–2.510, P=0.1566), as shown in Fig. 3A-C.
The results of miRNA-21 and miRNA-486-5p significantly varied; we concluded that the relative expression of miRNA-486-5p is a useful screening method for LC at work-place and community health centers.
Signature of miRNA-486-5p in patients with LC based on cancer stages
The expression levels of miRNA-486-5p in stage I and IV all patients with LC were shown in Fig. 3D, and those of male patients slightly higher than those in the healthy male controls (Fig. 3E), whereas the relative expression of miRNA-486-5p tended to be lower in stage I female patients with LC than in healthy female controls (Fig. 3F). That is, miRNA-486-5p showed sex-difference, up-regulation in males and down-regulation in female patients with LC.
Relative expression of miR-486-5p in 35 Patients with LC based on the cancer metastasis
Two cases (No. 13 and 32) revealed M1a, their expression ratio of MMP-1/CD63 were 0.30 and 5.44. Three M1b cases (No. 14, 18 and 29) showed 1.21, 5.52 and 4.42, respectively. The rest two M1c cases (No. 17 and 21) revealed 7.59 and 11.7. Among 7 metastasis cases, six cases showed the extremely very high increased value.
Expression levels of miRNA-486-5p based on smoking habits
Never-smoker female patients with LC showed low expression levels of miRNA-486-5p, except one case. In contrast, among the 4 never-smoker male patients with LC, two had high and two had low miRNA-486-5p expression levels. The relative expression of miRNA-486-5p was not related to smoking habits (Fig. 3G-I).
Receiver operating characteristics analysis
The sensitivity and specificity of MMP-1/CD63 as a primary screening approach for LCThe AUC of MMP-1/CD63 in the patients with LC was 0.755 (95% CI 0.640–0.871), and the cut-off value was 1.25 (Fig. 4A). The sensitivity and specificity of MMP-1/CD63 expression were 0.925 and 0.543, respectively.
The sensitivity and specificity of miRNA-486-5p as a primary screening approach for LC based on sex
The AUC of miRNA-486-5p in male patients with LC was 0.801 (95% CI 0.669–0.939), that in female patients with LC was 0.627 (95% CI 0.396–0.858); the cutoff values were 2.14 and 0.91, respectively (Fig. 4B and C). The sensitivity and specificity of miRNA-486-5p in the male patients with LC were 0.850 and 0.708, respectively, and those in the female patients with LC were 0.700 and 0.727, respectively.
ROC analysis of combined biomarkers of MMP-1/CD63 and miRNA-486-5p
Both the biomarkers of the expression ratio of MMP-1/CD63 and the relative expression of miRNA-486-5p in the urinary exosomes improved ROC analysis: 86% sensitivity and 85% specificity calculated using the cut-off value of 1.25 for MMP-1/CD63, 2.14 in males and 0.91 for females for miRNA-486-5p against 35 patients with LC and 40 healthy controls in the present study as one cohort.
Logistic regression analysis for the usefulness of both biomarkers in LC screening: The association between the presence of lung cancer and the expression ratio for MMP-1/CD63, and relative expression of miRNA-486-5p
The association between the expression ratio for MMP-1/CD63 and the presence of lung cancer showed an odds ratio (OR)=31.70 (95% CI: 4.12–243.67), while that between the relative expression of miRNA-486-5p and the presence of lung cancer had an OR=1.56 (95% CI:1.01–2.42) after adjusting with age, sex, and smoking status (Table IV). AUROC of the logistic regression model including MMP-1/CD63 and miRNA-486-5p was 0.954 (95% CI: 0.908–1.000), with 89% sensitivity and 88% specificity using the cut-off value of 0.48 (Fig. 4D).
| Table IV.Logistic regression analysis between lung cancer, and smoking status, MMP-1/CD63 and microRNA-486-5p. |
The relative expression of miRNA-486-5p is a supportive biomarker for the expression ratio of MMP-1/CD63, which may contribute to the better specificity.
Discussion
To the best of our knowledge, this is the first study that used urinary exosome-derived MMP-1 and miRNA for early LC screening. We demonstrated that the combined screening for both biomarkers of MMP-1/CD63 and miRNA-486-5p targeting urinary exosomes is useful in detecting early-stage LC. Our purpose was to develop a universal and practical screening method for early-stage LC for job workers and community residents to enable a simple, easy to access, cost-effective screening of LC and requires only a small volume of urine.
The expression ratio of MMP-1/CD63 did not show a sex-based difference in the present study; the ratio increased in both male and female patients with LC, as observed in breast cancer patients (6). In 1998, Rutter et al reported a single nucleotide polymorphism (SNP) at −1607 bp in the MMP-1 promoter region, where an additional guanine (G) created an erythroblast transformation specific (Ets) binding site, 5′-GGA-3′ (12). This SNP was associated with transcriptional and protein/DNA binding activities; hence its frequency was investigated in tumor cells. Subsequent studies reported a close relationship between the MMP-1 promoter SNP and the risk of LC (13,14). Sauter et al found that MMP-1 SNPs are related to an increased risk of early onset of LC; this risk is further worsened by smoking in patients who are younger than 51 years of age (15). Moreover, increased MMP-1 levels in serum or plasma (24–26) and increased expression of MMP-1 in the tumor tissues of patients with LC was observed by immunohistochemical staining (26).
Therefore, we consider that the expression ratio of MMP-1/CD 63 in the urinary exosomes of the patients with LC is a useful biomarker for LC screening, and the present study reports the upregulation of MMP-1 in the urinary exosomes in early-stage patients with LC. MMP-1 upregulation in LC can be affected by environmental and/or genetic factors, such as signal transducer and activator of transcription 3 (27), extracellular signal-regulated kinases/mitogen-activated protein kinases pathways in the epidermal growth factor receptor (EGFR) ligands, and their signaling pathways (28). The Capicua (CIC) repressor (29–34) is a similar factor. The missense, nonsense, insertion, deletion, and splice sites for the CIC repressor have been reported (29–34), but the direct relationship between CIC and upregulation of MMP-1 expression in LC has not yet been determined. CIC is a high mobility group-box transcriptional repressor, and its target genes include the polyoma enhancer activator 3, whose group Ets translocation variants (ETVs) have been well characterized (29–32). A recent report revealed the progression of hepatocellular carcinoma related to the CIC-ETV4-MMP-1 axis (33). We have previously reported the increased expression ratio of MMP-1/CD63 in breast cancer patients including early-stage of cancer (6), while the data after operation were low, although high value was observed in the case with metastasis after operation (6). The CIC repressor involvement in Ewing sarcoma, melanoma, prostate-, breast-, lung-, gastric-, or colon cancer has not been fully verified. Since CIC in Drosophila plays an important role in terminal and dorsoventral patterning (30), similar genes are supposed in human (33,34).
Saito et al demonstrated that the EGFR-tyrosine kinase inhibitor (TKI)-resistant AC showed strong MMP-1-positive staining with a significantly independent poor prognosis (35). However, once MMP-1 expression is upregulated, the cell phenotype may change in many organs associated with the hedgehog protein and/or epithelial-mesenchymal transition, thereby leading to carcinogenesis (35,36). This suggests that MMP-1-positive cancer patients have a poor prognosis, as reported in the case of colorectal cancer (37). Our screening tests for early-stage LC illustrated the usefulness of urinary exosomes and can clarify the relationship between CIC and LC occurrence and metastases in future studies. Furthermore, combined screening of MMP-1/CD 63 and miRNA-486-5p using urinary exosomes as a target could detect LC more accurately.
The miRNA-21 was useful for breast cancer screening and can modulate the expression of cancer suppressor genes such as PTEN, HOXD10, TPM1 and mapin (a tumor-suppressing serpin protein) (6,17–19). However, several patients with LC are over the age of 70 years; therefore; hence, relative expression of miRNA-21 in such patients could not be discriminated from those of the healthy controls among the aged individuals. Therefore, we focused on miRNA-486-5p, which targets the PIK3R1 gene, a suppressor of tumor growth (20,21). Surprisingly, miRNA-486-5p showed sex differences here but not in previous reported studies. The obtained signature of miRNA-486-5p in this study is not consistent with the literature (20,21). ElKhouly et al (20) reported that miRNA-486-5p is multifaceted, thereby reflecting the disease conditions on the date of examination. These high-age limitation have not been previously reported in studies on miRNAs in patients with LC (38–44). However, recent reports on miRNA-486 suggest that dysregulation of miRNAs may affect sex hormone signaling via modulation of estrogen receptors and through miRNAs (45,46). Our results suggest that the relative expression of miRNA-486-5p was a useful biomarker for LC screening at work-place and community health centers, with a sensitivity and specificity of nearly 90%. Moreover, the relative expression of miRNA-486-5p increased at the early stage of LC. Although miRNA-486-5p was increased in AC and SCC, the association with smoking habits was not expected. Saito et al reported that the number of cases with high MMP-1 immunoreactivity was significantly higher in smokers (26/54 cases) than non-smokers (8/27 cases) (35), which is inconsistent with our findings, showing higher expression of MMP-1 in never-smoker patients with AC. However, Pao et al reported frequently observed point mutations in codon 858 (exon 21) in EGFR-TKI-sensitive never-smokers patients (47). The results of the ROC analysis for logistic regression model suggests that MMP-1/CD63 and miNA-486-5p could predict lung cancer, although further studies are warranted.
Isolation of exosomes from urine and miRNA analysis may be less methodologically established than isolation from serum or plasma. Hence, it may be difficult to normalize for urine-derived exosomes. One reason for this is the difficulty in selecting appropriate miRNAs as controls for body fluid samples, including urine (48,49). In this study, we did not use internal controls such as small RNAs such as U6 and 5S rRNA, which are commonly used in miRNA analysis (50,51). Since these small RNAs are structurally different from miRNAs in urinary exosome-derived miRNA expression analysis, it is unclear whether they can be used as generic internal controls like GAPDH and β-actin in mRNA expression analysis. In our preliminary study, the measurement of internal standard miRNAs in exosomes did not work well. We next investigated the use of externally added miRNAs as internal standards. However, since RNA added externally as an internal control was not derived from exosomes extracted from patients but from RNA synthesized during RNA extraction (50,51), we considered the external addition of RNA to be insufficient as a control in this study. For example, U6 RNA is not appropriate in patient with liver fibrosis, suggesting that it may not function as an intrinsic control depending on the type of disease complicating the patient (52). Furthermore, we believe that externally added miRNAs cannot eliminate the effects of contamination and the risk of degradation. Furthermore, simple analysis using an electron microscope revealed no differences in the shape, size, or number of urine-derived exosomes per urine volume. Therefore, we believe that there are no major differences in the number of exosomes or the variety of miRNA pools between each sample. Therefore, in this study, exosomes were extracted from 2 ml of urine and all RNA was recovered; 5 ng of RNA was used for reverse transcription, after which the CT values of the target miRNAs were measured; individual samples from LC and healthy controls were normalized using the mean CT values of healthy controls. In other words, they were compared to patients based on the healthy control mean expression levels of the target miRNAs. Although this method has been reported in previous reports (6), this study, which did not use internal controls for miRNAs, is more controversial in the normalization of urine-derived exosomes. It is hoped that more appropriate methods for internal control of urinary exosomes will be established in the future.
This study has several limitations. The study is preliminary, the population is not large, and it has not been conducted on exosome characteristics other than EM. Future studies should identify more precise characteristics on exosomes.
Global statistics estimate that approximately 25% of patients with LC are never smokers (53). Therefore, our screening method can help identify numbers of the early-staged patients with LC that are never-smokers but exposed to passive smoking. In Asian countries, there are never-smoker female patients with LC, which is a major challenge that our novel screening method can address.
Our study proposed a new screening method useful for workers who cannot visit a health-check clinic for cancer screening due to busy work schedules and/or economic limitations.
We are presently investigating the collection of information on the false-positive cases in at least 500 visitors for the annual health checks. Further studies with a larger cohort of patients with LC are needed to validate our findings.
Supplementary Material
Supporting Data
Supporting Data
Acknowledgements
Not applicable.
Funding
This study was supported by the Japanese Society for the Promotion of Science (Kakenhi grant no. 18K08797) given to SI and YK.
Availability of data and materials
The data generated in the present study may be requested from the corresponding author.
Authors' contributions
WA, MS, TU, SI and IO designed and conducted the study. WA and MS analyzed the data. WA, MS and IO confirm the authenticity of all the raw data. HY confirmed morphological analysis. WA and MS performed statistical analysis. Logistic regression analysis was done by HF. WA, MS, SI, HY, YK, KO, TN, SE, HT, and IO interpreted and discussed the data. WA, MS, HF and IO wrote the draft of this manuscript. SI, HY, YK, KO, TN, SE and HT edited the paper for English grammar and writing. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The study was approved by ‘The Ethics Committee of IUHW Hospital’ (#13-B-299 IUHW on May 24, 2018; last updated August 7, 2020), and ‘The ethical committee of Kitasato University Medical Center’ (#28–51 Kitasato University Medical Center on March 27, 2017) and conformed to the principles of the 1975 Declaration of Helsinki, as reflected by its prior approval by the institution's human research committee. All the study participants provided written informed consent. All participants signed informed consent for participation in this study.
Patient consent for publication
All participants signed informed consent to publish the results in peer review journal.
Authors' information
IO published a research paper in Nature (1974) entitled ‘Collagenase activity in experimental hepatic fibrosis’, and continued matrix metalloproteinase study. The year before last year he published original paper as a first author entitled ‘Sequential matrix metalloproteinase-1 expression triggered by infiltrating monocytic lineage cells modulates pathophysiological aspects of human nonalcoholic steatohepatitis, Metalloproteinases in Medicine 2020:7 1–13’.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
|
AC |
adenocarcinoma |
|
AUC |
area under the receiver operating characteristic curve |
|
CIC |
Capicua |
|
EGFR |
epidermal growth factor receptor |
|
Ets |
erythroblast transformation specific |
|
ETVs |
Ets translocation variants |
|
LC |
lung cancer |
|
LCC |
large cell carcinoma |
|
miRNA |
microRNA |
|
MMP-1 |
matrix metalloproteinase-1 |
|
OR |
odds ratio |
|
ROC |
receiver operating characteristic |
|
RT-PCR |
reverse transcription polymerase chain reaction |
|
SCLC |
small cell lung cancer |
|
SC |
squamous cell carcinoma |
|
TKI |
tyrosine kinase inhibitor |
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