Diagnostic Valuation of Serum miR-184 and miR-191 in Patients With Non-Small-Cell Lung Cancer.

OBJECTIVE: This study aims to determine the diagnosis and prediction value of serum miR-184 and miR-191 levels in patients with non-small-cell lung cancer (NSCLC).
METHODS: One hundred patients with NSCLC were enrolled (NSCLC group) and treated with gefitinib. In addition, 59 pneumonia cases (pneumonia group) and 51 healthy cases in the corresponding period (normal group) were included. Serum miR-184 and miR-191 expressions were detected by real-time quantitative polymerase chain reaction. Furthermore, the relationships between serum miR-184 and miR-191 expressions and clinicopathological parameters were analyzed. The use of serum miR-184 and miR-191 levels in the diagnosis of NSCLC and the prediction of treatment effectiveness and 3-year overall survival (OS) were assessed by the receiver operating characteristic curve. Hazard factors affecting the efficacy of treatment in patients with NSCLC were determined by logistic regression.
RESULTS: The serum levels of miR-184 in the NSCLC group were significantly lower than those in the pneumonia group and normal group, whereas miR-191 expression was significantly higher in the NSCLC group. Serum miR-184 and miR-191 levels were closely correlated with smoking history, the tumor node metastasis (TNM) stage, and the degree of pathological differentiation. The area under curve (AUC) of serum miR-184 combined with miR-191 in the diagnosis of patients in the NSCLC group and normal group, NSCLC group and pneumonia group, and the efficacy of treatment in patients with NSCLC was 0.925, 0.929, and 0.916, respectively. The AUC of serum miR-184 and miR-191 for the 3-year OS in patients with NSCLC was 0.869 and 0.879, respectively. Smoking history, the degree of pathological differentiation, local treatment, miR-184, and miR-191 were independent risk factors that affected treatment efficacy.
CONCLUSION: Serum miR-184 and miR-191 levels can potentially be used as molecular markers to diagnose and predict the curative effect of treatment in patients with NSCLC.

Introduction

Lung cancer is the third most common cancer in the USA, and it is also the main cause of death in patients with cancer.[1] Statistics showed that there were 1,820,000 new cases and 1,590,000 deaths in 2012, and its 5-year survival rate was below 20%.[2] Non-small-cell lung cancer (NSCLC) is the main type of lung cancer and accounts for 85% of all lung cancer cases. Adenocarcinoma, squamous cell carcinoma, and large cell neuroendocrine carcinoma are its histologic subtypes.[3]

Surgery, radiotherapy, and other local treatments are the main methods used to treat patients with NSCLC, and the type of treatment is decided by the tumor node metastasis (TNM) stage. Patients whose NSCLC was identified at an early stage generally received radical surgery, whereas patients with a later stage mainly received chemotherapy and targeted therapy.[4] NSCLC is currently diagnosed with imaging methods, including histological examination of tumor biopsies. However, because of the lack of typical clinical symptoms and the deficiency of early screening procedures, the majority of patients with NSCLC are not diagnosed until later stages.[5] Therefore, simple and sensitive biomarkers have important clinical significance for the early diagnosis and accurate prediction of NSCLC in patients.

MicroRNAs (miRNAs) are short-stranded noncoding RNAs that exist in almost all eukaryotes. They can regulate several human biological functions. They are involved in the occurrence of human cancers, including NSCLC, by regulating a variety of oncogenes and tumor suppressor genes.[6-8] Both miR-184 and miR-191 belong to the miRNA. MiR-184 can play a role as a tumor suppressor by inhibiting the proliferation and invasion of glioma and breast cancer cells, and it can act as an oncogene by inhibiting apoptosis of renal cancer cells.[9,10] Similarly, miR-191 can act as a tumor promoter in the regulation of cell growth and invasion of prostate cancer and a tumor suppressor to prevent the growth and migration of thyroid follicular cancer cells.[11,12] However, some studies on miR-184 and miR-191 in NSCLC have shown conflicting results. Such as Lin et al.[13] found that miR-184 can be used as a tumor suppressor to target CDC25A and c-Myc during the inhibition of NSCLC cell growth and invasion. Zhao and others[14] found that miR-191 acts as a tumor promoter targeting NFIA to accelerate the proliferation and migration of NSCLC cells.

At present, there are only a few studies that have investigated the serological diagnosis and prediction of NSCLC in patients using miR-184 and miR-191 as molecular markers. We explored this area by detecting the serum expression of miR-184 and miR-191.

Materials and Methods

Baseline Data

One hundred patients with NSCLC admitted from April 2015 to April 2016 were enrolled as the NSCLC group, which included 61 males and 39 females aged 27–69, with an average age of 55.83 ± 5.71. The inclusion criteria were that NSCLC was confirmed by cytology or histology,[15] the TNM staging of NSCLC was based on the eighth edition of the TNM staging for non-small lung cancer published by the American Cancer Association (AJCC) and the International Union Against Cancer (UICC),[16] the patients did not take any drugs that are known to impact the relevant indicators of this study during the past 6 months, and those who had not undergone treatment or surgery before participating in this study. The exclusion criteria included patients whose expected survival time was <3 months, patients with incomplete clinical and follow-up data, patients with a history of allergies to medication used in this study, and patients who had other types of tumors. Another 59 patients with pneumonia during the same period were included in the pneumonia group, and 51 healthy controls were enrolled as the normal group. Thirty-one males and 28 females aged 25–68 were included in the pneumonia group, with a mean age of 54.18 ± 5.53. Twenty-six males and 25 females aged 26–65 were included in the normal group, with a mean age of 56.05 ± 5.81. This study has been approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University, and informed consent forms were signed by subjects and their guardians.

Treatment Methods

Fifty-three patients with NSCLC underwent radical surgery, including 22 patients with stage I, 18 patients with stage II, and 13 patients with stage IIIA; and 47 patients with stages IIIb and IV NSCLC received 250 mg of gefitinib once a day (China Shanghai Source Leaf Biotechnology Co., Ltd., S31076). There were 15 cases of local treatment, 8 cases of thoracic chemotherapy, 3 cases of chemotherapy for brain metastases, and 4 cases of chemotherapy for bone metastases.

Efficacy Evaluation

One month after treatment, the curative effect was evaluated according to the Response Evaluation Criteria in Solid Tumors (version 1.1).[17] On the basis of the range and duration of the lesion, the effect was divided into a complete response (CR), partial response (PR), stable disease), and progressive disease (PD). On the basis of the efficacy of gefitinib, CR and PR were considered as effective cases, whereas stable disease and PD were considered as ineffective cases.

Detection Method

In the NSCLC group, 5 mL of cubital venous blood were collected at 8 am in the morning 1 week before and after treatment, and in the normal group, 5 mL of cubital venous blood were extracted at 8 am in the morning, placed in blood collection tubes containing EDTA-K2, and centrifuged at 850 g for 15 min. The upper serum (2 mL) was pipetted, transferred to EP tubes, and centrifuged at 16,000 g for 10 min. Cell debris was pelleted. The supernatants were kept in new EP tubes and stored at −75°C. Total RNA extraction was performed following the instructions of the mirVanaTM miRNA Isolation Kit (Shanghai Xinle Biotechnology Co., Ltd., China, RMI050). The RNA concentration was determined using an ultraviolet–visible spectrophotometer (Shanghai Lianqiao Biotechnology Co., Ltd., China, LPV 441.99.00002). RNA reverse transcription into cDNA was performed using the TaqMan MicroRNA Reverse Transcription Kit (Beijing Jiehui Bogao Biotechnology Co., Ltd., 23311s), and polymerase chain reaction (PCR) amplification was conducted using cDNA as a template. U6 was used as the internal reference with primer sequences that were designed by Shanghai Kelton Biotechnology Co., Ltd., China. MiR-184 and miR-191 were detected by a real-time polymerase chain reaction system (Shanghai Yanqi Biotechnology Co., Ltd., China 4447930001) using a reference miRNA RT-qPCR Kit (Shanghai Jitai Ecosai Biotechnology Co., Ltd., 110001S). The PCR conditions were as follows: 5 min at 90 °C, 5 s at 90 °C, 30 s at 60 °C, and 5 s at 72 °C for 40 cycles. All samples were repeatedly tested 3 times. The relative expression of miR-184 and miR-191 was determined using the 2−ΔΔCT method.

Follow-Up

The NSCLC group was followed up for a period of 3 years, and the patients were followed up once every 3 months mainly by telephone and interviews to understand the ultimate prognosis. The overall survival (OS) was the duration from the beginning of treatment to death or the last follow-up.

Statistical Analysis

The statistical analysis was performed using SPSS 20.0 (IBM Corp, Armonk, NY, USA), and the figures were constructed in GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). The measured data were expressed as the mean ± standard deviation (SD). The measurement data between groups were compared by independent t-tests. The data before and after treatment were compared by paired t-tests. The count data were expressed by n (%). The count data between groups were compared by the chi-squared test. The diagnostic value of serum miR-184 and miR-191 in patients with NSCLC was evaluated by a receiver operating characteristic (ROC) curve. The predictive value of miR-184 and miR-191 in patients with NSCLC was evaluated by a logistic regression equation and an ROC curve. The risk factors in NSCLC and therapeutic effects were analyzed by multiple logistic regressions. OS was calculated by the Kaplan–Meier method, and the difference in survival time between groups was evaluated by the log-rank test. The factors affecting NSCLC prognosis were assessed by univariate and multivariate Cox regressions. If P < 0.05, the differences were statistically significant.

Results

Clinical Value of Serum miR-184 and miR-191 in Patients With NSCLC

The relative expression of miR-184 and miR-191 in the serum of the NSCLC group, were significantly and successively decreased (P < 0.001), suggesting that miR-184 and miR-191, as serological indicators, may have the potential of specific recognition of pneumonia or NSCLC. The ROC curve indicated that for distinguishing the NSCLC group from the normal group, area under curve (AUC) values of serum miR-184 and miR-191 for NSCLC diagnosis were 0.865 and 0.859, respectively, and the AUC of the combined diagnosis was 0.925, indicating that miR-184 and miR-191, as separate indicators, have higher value in distinguishing the NSCLC group from the normal group, but the combined diagnosis value is higher. For distinguishing the NSCLC group from the pneumonia group, the AUC of serum miR-184 and miR-191 for NSCLC diagnosis was 0.848 and 0.838, respectively, and the AUC of the combined diagnosis was 0.929, suggesting that miR-184 and miR-191, as separate indicators, have good screening value in distinguishing the NSCLC group from the pneumonia group, but the combined screening value is higher. Serum miR-184 and miR-191 levels were closely related to smoking history, the TNM stage, and the degree of pathological differentiation (P < 0.001), suggesting that serum miR-184 and miR-191 may help reflect the above 3 pathological parameters of patients, and have certain predictive value for the above pathological parameters (Figure 1 and Tables 1 and 2).

Figure 1.

Clinical value of serum miR-184 and miR-191 in patients with NSCLC. (A) Serum miR-184 expression in the normal group, pneumonia group, and NSCLC group. (B) Serum miR-191 expression in the normal group, pneumonia group, and NSCLC group. (C) ROC curves of serum miR-184 and miR-191 for the diagnosis of patients in the NSCLC group and normal group. (D) ROC curves of serum miR-184 and miR-191 for the diagnosis of patients in the NSCLC group and pneumonia group. Note: ***P < 0.001.

Table 1.

ROC Parameters of Serum miR-184, miR-191 for Diagnosis of NSCLC Patients.

Group	Index	AUC	95%CI	SE	Cut-off	Sensitivity (%)	Specificity (%)
NSCLC and normal groups	miR-184	0.865	0.792-0.939	0.038	3.11	88.00	82.35
	miR-191	0.859	0.801-0.918	0.030	1.31	78.00	84.31
	miR-184+miR-191	0.925	0.879-0.971	0.023	0.66	87.00	86.27
NSCLC and pneumonia groups	miR-184	0.848	0.782-0.913	0.033	2.92	92.00	71.19
	miR-191	0.838	0.778-0.898	0.031	1.78	71.00	86.44
	miR-184+miR-191	0.929	0.888-0.970	0.021	0.54	89.00	84.75

Table 2.

Relationship Between Serum miR-184, miR-191 and Clinicopathological Characteristics of Patients With NSCLC (mean ± SD).

Group	n	miR-184	t/F	P	miR-191	t/F	P
Gender			1.096	0.276		1.398	0.165
Male	61	2.13 ± 0.56			2.05 ± 0.43
Female	39	2.25 ± 0.49			1.93 ± 0.40
Age (year)			1.032	0.305		1.198	0.234
≤65	66	2.27 ± 0.61			1.97 ± 0.57
>65	34	2.14 ± 0.57			2.10 ± 0.38
Smoking			2.681	0.009		4.148	<0.001
No	35	2.36 ± 0.68			1.83 ± 0.30
Yes	65	2.01 ± 0.59			2.22 ± 0.51
Drinking			1.057	0.293		0.700	0.486
No	43	2.23 ± 0.55			1.96 ± 0.46
Yes	57	2.10 ± 0.65			2.03 ± 0.52
TNM stage			3.531	<0.001		3.647	<0.001
I, II	40	2.46 ± 0.73			1.87 ± 0.31
III, IV	60	1.98 ± 0.62			2.23 ± 0.57
Degree of pathological differentiation			2.661	0.009		3.921	<0.001
Medium / highly differentiated	44	2.36 ± 0.58			1.83 ± 0.32
Poorly differentiated	56	2.04 ± 0.61			2.17 ± 0.50
Tumor diameter (cm)			1.874	0.064		1.916	0.058
≤5	42	2.31 ± 0.57			1.89 ± 0.44
>5	58	2.08 ± 0.63			2.07 ± 0.48
Histological typing			0.219	0.804		0.287	0.751
Adenocarcinoma	82	2.23 ± 0.54			1.92 ± 0.46
Squamous cell carcinoma	13	2.17 ± 0.49			1.98 ± 0.47
Large cell carcinoma	5	2.09 ± 0.43			2.06 ± 0.50

Predictive value of miR-184 and miR-191 in NSCLC serum

The relative expression of miR-184 and miR-191 in the serum of patients with NSCLC after treatment was significantly lower than that before treatment (P < 0.01), indicating that serum miR-184 and miR-191 can respond to the treatment of patients and help predict the efficacy of patients. In 100 patients with NSCLC, 72 cases were CR or PR, and 28 cases were stable disease or PD. The relative expression of miR-184 and miR-191 in the serum of CR and PR patients before treatment was significantly lower than that of stable disease and PD patients (P < 0.001). The AUC of miR-184 in NSCLC diagnosis was 0.849, and the optimal cutoff value was 3.17. The AUC of miR-191 in NSCLC diagnosis was 0.844, and the optimal cutoff value was 1.34. A binomial logistic regression analysis was performed, and miR-184 and miR-191 were used as independent variables to obtain the logistic regression model logit (P) = 2.714 + 1.740. The AUC value of the combined diagnosis was 0.916. The above data indicate that miR-184 and miR-191 have certain predictive value for the efficacy of patients as single indicators, but have greater predictive value as joint indicators (Figure 2 and Table 3).

Figure 2.

Serum miR-184 and miR-191 expressions before and after treatment in patients with NSCLC and their predictive value for efficacy. (A) Expression of serum miR-184 and miR-191 before and after treatment in patients with NSCLC. (B) Expression of serum miR-184 and miR-191 in patients whose treatment was effective and ineffective. (C) ROC curve of serum miR-184 and miR-191 before treatment. Note: **P < 0.01.

Table 3.

Predictive Value of Serum miR-184 and miR-191 in Patients With NSCLC.

Index	AUC	95%CI	SE	Cut-off	Sensitivity (%)	Specificity (%)
miR-184	0.849	0.775-0.923	0.038	3.17	75.00	89.29
miR-191	0.844	0.753-0.936	0.047	1.34	76.39	85.71
miR-184+miR-191	0.916	0.858-0.975	0.030	0.67	88.89	82.14

Multivariate logistic regression analysis of factors influencing NSCLC efficacy

The clinical parameters and indicators of patients with effective and ineffective treatments were compared. The optimal cutoffs of miR-184 and miR-191 in the accurate prediction of NSCLC were 3.17 and 1.34, respectively. Between patients with effective treatment and those with ineffective treatment, the differences in gender, age, history of alcohol abuse, and tumor diameter were not statistically significant (P > 0.05), whereas the differences in smoking history, TNM stage, pathological differentiation, local treatment, miR-184, and miR-191 were statistically significant (P < 0.05). The differential factors were analyzed by multiple logistic regressions. The results showed that smoking history (P = 0.023), the degree of pathological differentiation (P = 0.011), local treatment (P = 0.008), miR-184 (P = 0.002), and miR-191 (P = 0.005) were independent risk factors affecting efficacy. The history of smoking, poor differentiation, local treatment, low miR-184 (≤3.17) expression, and high miR-191 (>1.34) expression increased the risk of ineffective treatment (Tables 4 –6).

Table 4.

Relationship Between Clinical Parameters of NSCLC Patients and Curative Effect [n(%)].

Index	n	Effective (n = 72)	Ineffective (n = 28)	χ2	P
Gender				1.978	0.160
Male	61	47 (65.28)	14 (50.00)
Female	39	25 (34.72)	14 (50.00)
Age (year)				1.360	0.244
≤65	66	50 (69.44)	16 (57.14)
>65	34	22 (30.56)	12 (42.86)
Smoking history				5.024	0.025
No	35	30 (41.67)	5 (17.86)
Yes	65	42 (58.33)	23 (82.14)
Drinking history				1.870	0.171
No	43	39 (54.17)	4 (14.29)
Yes	57	33 (45.83)	24 (85.71)
TNM stage				4.762	0.029
I, II	40	24 (33.33)	16 (57.14)
III, IV	60	48 (66.67)	12 (42.86)
Degree of pathological differentiation				4.409	0.036
Medium / highly differentiated	44	27 (37.50)	17 (60.71)
Poorly differentiated	56	45 (62.50)	11 (39.29)
Tumor diameter (cm)				1.773	0.183
≤5	42	28 (38.89)	15 (53.57)
>5	58	44 (61.11)	13 (46.43)
Local treatment				8.964	0.003
No	85	66 (91.67)	19 (64.29)
Yes	15	6 (8.33)	9 (35.71)
miR-184				12.823	<0.001
≤3.17	43	23 (31.94)	20 (71.43)
>3.17	57	49 (68.06)	8 (28.57)
miR-191				10.674	0.001
≤1.34	58	49 (68.06)	9 (32.14)
>1.34	42	23 (31.94)	19 (67.86)

Table 5.

Logistic Multivariate Regression Analysis Assignments.

Factor	Variables	Assignment
Smoking history	X1	No = 0, Yes = 1
TNM stage	X2	I, II = 0, III, IV = 1
Degree of pathological differentiation	X3	Moderate/high = 0, Low = 1
Local treatment	X4	No = 0, Yes = 1
miR-184	X5	It is continuous variables and analyzed with raw data
miR-191	X6	It is continuous variables and analyzed with raw data

Table 6.

Multivariate Logistic Regression Analysis Affecting the Curative Effect of NSCLC Patients.

Variables	B	SE	Wals	P	OR	95% CI
Smoking history	1.023	0.449	5.109	0.023	2.682	1.138-6.755
TNM stage	0.106	0.437	0.04 3	0.796	1.098	0.436-2.832
Degree of pathological differentiation	0.738	0.302	6.617	0.011	2.115	1.179-5.489
Local treatment	0.582	0.186	7.037	0.008	1.782	1.167-1.341
miR-184	1.901	0.602	9.961	0.002	6.692	2.055-13.790
miR-191	1.348	0.469	8.623	0.005	4.013	1.603-10.253

Relationship Between Serum miR-184 and miR-191 Before Treatment and the 3-year OS in Patients With NSCLC

A 3-year follow-up was successfully performed in 100 patients with NSCLC, and a 3-year OS of 58.00% (58/100) was observed. On the basis of the 3-year OS in patients with NSCLC, ROC curves of miR-184 and miR-191 in the prediction of 3-year OS was plotted. The AUCs of miR-184 and miR-191 in the prediction of 3-year OS were 0.869 and 0.879, respectively. In addition, the optimal cutoff values for the 3-year OS diagnosis were 2.94 and 1.58. The differences in the 3-year OS between patients with serum miR-184 ≤2.94 and miR-191 ≤1.58, as well as serum miR-184 >2.94 and miR-191 >1.58, were statistically significant (P < 0.05). The above data suggest that serum miR-184 and miR-191 have certain predictive value for 3-year OS in patients, and high level of miR-184 (or low level of miR-191) was significantly correlated with higher 3-year OS in NSCLC patients (Figure 3 and Table 7).

Figure 3.

Relationship between serum miR-184, miR-191, and 3-year OS in patients with NSCLC. (A) ROC curves of serum miR-184 and miR-191 in the prediction of 3-year OS in patients with NSCLC. (B) High levels of serum miR-184 were closed to longer 3-year OS. (C) Low levels of serum miR-191 were closed to longer 3-year OS.

Table 7.

Predictive Value of Serum miR-184 and miR-191 on 3-Year OS in NSCLC Patients.

Index	AUC	95%CI	SE	Cut-off	Sensitivity (%)	Specificity (%)
miR-184	0.869	0.794-0.944	0.038	2.94	77.59	92.86
miR-191	0.879	0.810-0.948	0.035	1.58	89.66	76.19

Discussion

NSCLC is a malignant tumor that has the highest incidence and death rates in the world. Its morbidity is continuously increasing, and the 5-year OS is only 15%.[18] The early symptoms of NSCLC in patients usually go unnoticed. Therefore, most patients diagnosed with lymph node metastases and distant metastases have missed the opportunity for operation.[19] Clinical targeting therapy is a new treatment for patients with NSCLC, which can be achieved by the drug-targeted inhibition of the epidermal growth factor receptor (EGFR) mutant gene or anaplastic lymphoma kinase gene.[20] Gefitinib is an EGFR tyrosine kinase inhibitor that can effectively treat patients with late-stage NSCLC with activating EGFR mutations. However, the resistance of patients with NSCLC to gefitinib still causes unsatisfactory OS.[21] Therefore, exploring potential biomarkers related to the diagnosis and accurate prediction of treatment in patients with NSCLC can help improve the OS of patients with NSCLC.

Dysregulated miRNA expression is related to the development and progression of cancer. Many studies have indicated that serum miRNAs can be used as non-invasive biomarkers for cancer diagnosis and the prediction of treatment responses.[22-24] Some research has reported the abnormal expression of serum miRNAs in lung cancer patients. For example, Bica-Pop and others[25] found that the increased expression of miR-184 in serum was related to disease progression in patients with NSCLC. MiR-184 promotes carcinogenesis, such as tumor invasion and metastasis, as well as chemical and radioresistance, and can be used as a potential prognostic marker of NSCLC. Zhou and others[26] found that the miR-184, which is expressed at low levels in serum, acts as a cancer suppressor in small-cell lung cancer (SCLC) and can significantly inhibit the metastasis of SCLC. In this study, the relative expression of serum miR-184 in the NSCLC group was significantly lower than that in the normal and pneumonia groups, whereas miR-191 expression was significantly higher in the NSCLC group. The ROC curve demonstrated that serum miR-184 and miR-191 showed high diagnostic efficacy in the NSCLC and normal groups and NSCLC and pneumonia groups (AUC = 0.865 and 0.859; 0.848 and 0.838, respectively), and the combined diagnosis of miR-184 and miR-191 was more effective (AUC = 0.925 and 0.929, respectively). These results indicate that serum miR-184 and miR-191 have high potential to be used as molecular markers for NSCLC diagnosis. Wang[27] found that the serum levels of miR-125a-5p, miR-145, and miR-146a were increased in patients with NSCLC, and the AUC used in distinguishing NSCLC from healthy controls was 0.71, 0.84, and 0.78, respectively, when used as non-invasive biomarkers of NSCLC.

We also explored the relationship between miR-184, miR-191, and pathological parameters in patients with NSCLC. Both miR-184 and miR-191 were closely related to smoking history, the TNM stage, and the degree of pathological differentiation. Patients with NSCLC with high miR-191 and low miR-184 often have a history of smoking, a high TNM stage, and poor differentiation. The clinical diagnosis of TNM staging and pathological differentiation in patients with NSCLC mainly involve histology and imaging.[28,29] Therefore, the detection of miR-184 and miR-191 is expected to be helpful for the diagnosis of NSCLC pathological features. We also found that miR-184 levels were significantly increased, whereas miR-191 levels were significantly decreased. In addition, the AUC of miR-184 and miR-191 in NSCLC diagnosis was 0.916, suggesting that miR-184 and miR-191 levels can be used to predict the effects of treatment. However, there have been few studies on whether miR-184 and miR-191 will affect the effect of targeted therapy in patients with NSCLC.

In this study, patients with low miR-184 (≤3.17) expression and high miR-191 (>1.34) expression had a higher risk of ineffective treatment. Thus, it is helpful to predict the effect of gefitinib therapy in patients with NSCLC by detecting the levels of miR-184 and miR-191 expressions. Finally, the 3-year OS of patients was 58.00%. The AUC values of miR-184 and miR-191 in the prediction of 3-year OS for patients with NSCLC were 0.869 and 0.879, respectively, indicating that miR-184 and miR-191 can be used as biomarkers to determine the prognosis. Zhang et al.[30] found that serum mi-191 was significantly related to the 3-year OS and could be used to predict the prognosis in patients with advanced NSCLC, which is similar to the results of this study. However, the regulatory mechanism needs to be further explored.

The effective use of serum miR-184 and miR-191 levels in predicting NSCLC diagnosis and prognosis was demonstrated in this study. However, there were a few limitations in this study. First, the relationships between miR-184, miR-191, and toxic side effects were not analyzed. Second, the specific regulatory mechanism of miR-184 and miR-191 in predicting NSCLC targeted therapy was not explored. In addition, the diagnostic value of miR-184 and miR-191 in TNM staging and pathological differentiation of patients with NSCLC was not analyzed. These issues need to be addressed in future research.

In summary, serum miR-184 and miR-191 can be used as potential molecular markers in the diagnosis and prediction of treatment outcomes in patients with NSCLC.