Prognosis and Biological Function of miR-3195 in Non-Small Cell Lung Cancer.

PURPOSE: Lung cancer has the highest mortality and morbidity rates worldwide. Among the subtypes of lung cancer, non-small cell lung cancer (NSCLC) accounts for approximately 85% of cases. The present study evaluated the potential prognostic value and biological function of miR-3195 in NSCLC. PATIENTS AND METHODS: In total, 129 patients with NSCLC were enrolled in this study. The expression of miR-3195 expression in NSCLC tissues and cell lines was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Kaplan-Meier survival curve analysis and multivariate Cox regression analysis were used to elucidate the prognostic value of miR-3195. The Cell Counting Kit-8 (CCK-8) assay and Transwell cell migration experiments were carried out to explore the effective effect of miR-3195 on the biological behavior of NSCLC cells.
RESULTS: The expression of miR-3195 was downregulated in NSCLC tissues and cell lines. Moreover, the decreased expression of miR-3195 was correlated with positive lymph node metastasis and high TNM stage. The overall survival of patients with low expression of miR-3195 was worse than those with high expression of miR-3195. Furthermore, miR-3195 was an independent prognostic indicator for overall survival in patients with NSCLC. Enhanced expression of miR-3195 restrained cell growth, migration, and invasion of NSCLC tumor cells, while attenuation of miR-3195 expression augmented cell proliferation activities, migration, and invasion potential.
CONCLUSION: Our findings suggest that miR-3195 may be used as a prognostic biomarker for NSCLC and is likely to act as a tumor suppressor for NSCLC.

Introduction

Cancer is a major cause of death globally. Among all types of cancers, lung cancer is one of the most common and serious health concern. It is the major contributor to morbidity and mortality due to cancer in the world.1 Smoking is the main risk factor for lung cancer. In economically developed and developing countries, breast cancer in women and lung cancer in men are the most common cancers, and they are also the main causes of death in male and female cancer patients, respectively.1,2 Based on 2012 data, there were about 1.8 million newly diagnosed cases of lung cancer, which accounted for 13% of all diagnosed cancers.3 By 2020, it is estimated that there will be 2.2 million new cases of lung cancer cases and 1.8 million lung cancer related-deaths among all cancer types.4 Lung cancer mainly has two subtypes: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), among which NSCLC is an aggressive disease, accounting for about 80–85% of all lung cancer cases. NSCLC can be treated by surgical resection at the initial stage of discovery, but patients with resectable NSCLC have a poor prognosis. A recurrence in 30–50% of cases has been reported within 5 years of initial resection.5 Since patients with NSCLC have no obvious clinical manifestations at an early stage, most patients are diagnosed at the advanced stage.3,6

MicroRNA (miRNA) is a small single-stranded non-coding RNA molecules that can regulate biological processes at the translational and/or post-translational level.7 Studies have indicated that miRNA has an important role in a variety of cancers including NSCLC, and is a potential prognostic indicator.8–10 miRNAs have been found to be aberrantly expressed in the serum, tissues, and even cells of NSCLC, and have an impact on the cell growth, proliferation, and metastasis of NSCLC tumor cells.11–13 Studies have shown that metastasis is an important cause of death from lung cancer, and microRNAs play an important role in almost all aspects of cancer biology.14,15 Therefore, it is important to understand the role of miRNAs in NSCLC.

Several studies have elucidated the expression of miR-3195 in tumor cells, and it has been found that miR-3195 can be up-regulated or down-regulated to varying degrees, thereby affecting some tumor tissues or cells.16–19 However, there are relatively few studies on miR-3195 expression in NSCLC. Kumar et al demonstrated that the expression of miR-3195 in the serum of patients with NSCLC was significantly down-regulated than the healthy subjects.20 However, the aforementioned study did not evaluate the abnormal expression of miR-3195 in NSCLC tissues and cells.

Considering the role of miR-3195 in NSCLC is still elusive, we investigated the expression pattern of miR-3195 in NSCLC tissues and cells and elucidated its potential clinical and functional role in NSCLC. This study found that miR-3195 was downregulated in human NSCLC tissues and cell lines, and its overexpression restrained cell proliferation, migration, and invasion. These findings may provide a new insight for treatment of NSCLC.

Materials and Methods

Patients and Tissue Samples Collection

The tissue samples used in this study were collected from 129 patients with NSCLC who underwent surgery at the Huadong Hospital Affiliated to Fudan University from 2012 to 2015 and were verified by the pathologists. None of the patients received any anti-tumor therapy before sampling. After the tissue samples were collected, the NSCLC tissue and the adjacent normal tissues were quickly frozen in liquid nitrogen until RNA extraction. The written informed consent form was signed by each patient, and this study was approved by the Ethics Committee of Huadong Hospital Affiliated to Fudan University (No.20120047) and was conducted in accordance with the Declaration of Helsinki. Besides, the data for clinical and pathological parameters, including age, gender, tumor size, lymph node metastasis, TNM staging, and smoking history, were collected and recorded from the electronic medical records of all patients. These data are presented in Table 1. All patients received standard treatment and participated in a 5-year follow-up survey after operation, and the overall survival information was collected and recorded for subsequent clinical survival analysis.

Table 1

Correlation Between miR-3195 Expression and Clinicopathological Characteristics in NSCLC Patients

Clinical Parameter	No. of Cases	miR-3195 Expression Levels		P values
	n = 129	Low (n = 65)	High (n = 64)
Age				0.542
< 60	53	25	28
≥ 60	76	40	36
Diameter of tumor				0.530
< 3 cm	71	34	37
≥ 3 cm	58	31	27
Gender				0.767
Female	48	25	23
Male	81	40	41
Lymph node metastasis				0.017
Negative	69	28	41
Positive	60	37	23
TNM stage				0.002
I–II	77	30	47
III–IV	52	35	17
Smoking history				0.540
No	49	23	26
Yes	80	42	38
Histology type				0.080
Adenocarcinoma	56	27	29
Squamous	58	34	24
Others	15	4	11

Cell Lines Culture and Transfection

Four NSCLC cell lines (A549, H1299, PC9, and NCIH460) and a human bronchial epithelial cell line (16HBE) and were acquired from the Chinese Academy of Science Cell Bank (Shanghai, China). All the cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Gibco, Carlsbad, CA, USA) medium supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) in a humidified incubator set at 5% CO2 and 37°C.

The miR-3195 mimic, miR-3195 inhibitor, and miRNA negative control (miR-NC) sequences were synthesized at GenePharma (Shanghai, China) for in vitro regulation. Cell transfection was performed with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.21 The follow-up cell experiments were performed after 48 h of transfection.

Total RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)

Total RNA from clinical tissues and cells was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) as recommended by the manufacturer, then cDNA was synthesized from 1 μg of the total RNA using an equivalent of by a PrimeScript RT reagent kit (Takara Bio, Shiga, Japan). The qRT-PCR for miRNA was carried out using an SYBR Green I Master Mix kit (Invitrogen, Carlsbad, CA, USA) and 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The relative expression of miR-3195 was calculated by the 2−ΔΔCt method22 and expression data was normalized using U6 as an internal control.

Cell Proliferation Assay

The effects of miR-3195 on cell proliferation of NSCLC cells were investigated using Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) assay.23 The transfected cells (density: 3000 cells/well) were seeded into 96-well plates and incubated for 0, 24, 48 and 72 h, followed by treatment with 10 μL CCK-8 solution for 2 h. After the incubation, the absorbance at 450 nm was measured using a microplate reader (Bio-Tek, Winooski, VT, USA).

Cell Migration and Invasion Assay

The effects of miR-3195 on cell migratory and invasive abilities were evaluated using the Transwell chambers (Corning, NY, USA).24 The upper chambers that pre-coated with Matrigel were used for invasion assay, and uncoat Matrigel chambers were used for migration assay. Transfected cells at a density of 2×105 cells/well in serum-free medium were seeded into the upper chambers and cultured at 37°C. The lower chambers were filled with culture medium supplemented with 10% FBS as a chemoattractant. After 24 h of incubation, the cells from the upper chamber had moved and the migrated or invaded cells in the lower chambers were fixed with paraformaldehyde and stained with crystal violet at room temperature. The cells in random five fields were counted under a light microscope.

Statistical Analysis

The data were statistically and analyzed using Statistical Product and Service Solutions (SPSS) 23.0 software (SPSS Inc., Chicago, IL, USA). The data were all presented as mean ± SD. The differences between the two groups were analyzed using paired Student’s t-test and those among three or more groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test among three or more groups. The chi-square test was used to evaluate the correlation between miR-3195 expression and clinicopathological characteristics. Survival analysis was done using the Kaplan–Meier method and the Log rank test. Multivariate Cox regression analysis was used to identify prognostic risk factors of NSCLC. Among the results of all analyses, the values with P < 0.05 was considered statistically significant.

Results

miR-3195 Expression in Tissue Samples and Cells

In this study, qRT-PCR was used to evaluate the relative expression of miR-3195 in NSCLC tissues and cell lines. The results manifest that the relative expression of miR-3195 in NSCLC tissues was significantly lower than that of matched normal tissues (P < 0.001, Figure 1A). Consistent with the findings in tissue samples, the relative expression of miR-3195 in NSCLC cell lines (A549, H1299, NCIH460, and PC9) was also significantly downregulated than that in the normal cell line 16HBE (all P < 0.001, Figure 1B).

Figure 1

The expression levels of miR-3195 were measured by qRT-PCR analysis. (A) miR-3195 expression in NSCLC tissues and paired normal tissues. Compared to the normal tissue controls, the expression of miR-3195 in tumor tissues was relatively low (***P < 0.001). (B) miR-3195 expression in cell lines. Compared with the normal cells 16HBE, the expression of miR-3195 was downregulated in NSCLC cell lines compared with that in 16HBE (***P < 0.001).

Correlation of miR-3195 with Clinical Characteristics of Patients with NSCLC

To analyze the relationship between the expression of miR-3195 and clinicopathological characteristics, the selected patients were divided into two groups including low expression (n = 65) and high expression (n = 64) based on the median expression level of miR-3195 (0.432) in tumor tissues as a cut-off value, and the chi-square test was used for evaluating differences. The evaluation results are summarized in Table 1. We found that there are differences in indicators between the low expression group and the high expression group. The results indicate that the low expression of miR-3195 was associated with positive lymph node metastasis (P = 0.017) and advanced TNM staging (P = 0.002), but it was not related to age, the diameter of the tumor, gender, smoking history, histology type (all P > 0.05).

Prognostic Significance of miR-3195 for NSCLC

The patient’s survival information was recorded during 5 years of follow-up study, and used to drew the survival curve, which is shown in Figure 2. Compared to the patients with high expression of miR-3195, patients with low expression of miR-3195 had a poorer overall survival (log-rank P = 0.002). In addition, the clinical characteristics of Cox regression analysis also revealed that miR-3195 expression (hazard ratio [HR] = 2.849, 95% confidence interval [CI] = 1.109–7.322, P = 0.030) and TNM stage (HR = 2.975, 95% CI = 1.087–8.145, P = 0.034) are independent prognostic factors for NSCLC patients (Table 2).

Figure 2

Kaplan-Meier survival curve with the Log rank test was conducted to analyze the overall survival of patients with NSCLC. Patients with low miR-3195 expression have a shorter survival time than those with high miR-3195 expression (log-rank P = 0.002).

Table 2

Multivariate Analysis of Risk Factors and Patients’ Survival Outcomes

Variables	Multivariate Cox Regression Analysis
	HR	95% CI	P values
miR-3195 expression	2.849	1.109–7.322	0.030
Age	1.734	0.807–3.727	0.159
Diameter of tumor	2.521	0.922–6.891	0.072
Gender	1.885	0.814–4.364	0.139
Lymph node metastasis	2.289	0.865–6.057	0.095
TNM stage	2.975	1.087–8.145	0.034
Smoking history	1.983	0.899–4.372	0.090
Histology type	1.004	0.312–3.233	0.840

Reduction in miR-3195 Promotes Proliferation, Migration, and Invasion of NSCLC Cells

To elucidate the functional role of miR-3195 in NSCLC, its biological function of miR-3195 in NSCLC was further explored through function gain and loss experiments, and its effect on tumor cell proliferation, migration, and invasion was evaluated. Through cell transfection, it was found that miR-3195 mimic in A549 and H1299 tumor cells promoted the expression of miR-3195, while miR-3195 inhibitor inhibited the expression of miR-3195 (all P < 0.05, Figure 3A and B). The results of CCK-8 analysis showed that the upregulation of miR-3195 in NSCLC cells inhibited cell proliferation, while reduction of miR-3195 promoted cell proliferation (all P < 0.05, Figure 3C and D). Furthermore, the count of migratory and invasive cells results in Figure 4A and B indicated that miR-3195 overexpression inhibited tumor cell migration and invasion while downregulation of miR-3195 enhanced tumor cell migration and invasion (all P < 0.05).

Figure 3

The effect of miR-3195 on the proliferation of NSCLC cells in A549 and H1299 cell lines. (A and (B) miR-3195 mimics can enhance the expression of miR-3195, but miR-3195 inhibitors can reduce its expression (***P < 0.001). (C and D) Inhibition of miR-3195 promote cell proliferation, while its overexpression inhibits cell proliferation (*P < 0.05).

Figure 4

The effect of the miR-3195 expression on the migration and invasion of A549 and H1299 cell lines (magnification 200 ×). (A) The reduction of miR-3195 promoted tumor cell migration, while miR-3195 overexpression inhibited tumor cell migration (*P < 0.05, **P < 0.01, ***P < 0.001). (B) The reduction of miR-3195 promoted tumor cell invasion, while its overexpression inhibited tumor cell invasion (*P < 0.05, ***P < 0.001).

Discussion

miRNA is a key regulator for tumor progression of cancers, including NSCLC.25,26 Abnormal expression levels of a number of miRNAs, such as miR-126-5p,27 miR-198-5p,28 and miR-338-3p,29 have been reported to play a key role in the occurrence and progression of tumors. Consistent with the previous studies, we also found the downregulation in expression of miR-3195 in NSCLC tissues and cells compared to the respective normal control groups. Pan et al found that the expression of miR-3195 was downregulated in lung cancer caused due to air pollution,30 which is consistent with our findings in the tissues and cells of patients with NSCLC. Additionally, the downregulated expression of miR-3195 is related to lymph node metastasis and the TNM stage in patients with NSCLC. These findings suggest that miR-3195 may be involved in the development of NSCLC tumors. Besides NSCLC, the downregulated expression of miR-3195 has been reported in patients with prostate cancer,31 which suggests that indicating that miR-3195 may have a tumor-inhibiting effect.

Most patients with NSCLC are at an advanced stage when they are diagnosed. Therefore, it is urgent to evaluate the utility of miRNAs as biomarkers to predict the prognosis of patients with tumors. Based on survival analysis using survival curve and Cox regression analysis data, we found that miR-3195 can be used as an independent prognostic biomarker for NSCLC patients. Interestingly, the serum expression levels of miR-3195 have been reported in 75 patients with NSCLC and it might act as an independent prognostic factor for overall survival.20 Overall, these findings suggest that serum and tissue expression of miR-3195 may have prognostic significance in NSCLC. Our findings indicate that the reduced expression of miR-3195 may have the potential to predict the poor prognosis of patients with NSCLC.

Previous studies have elucidated the effects of miR-3195 on tumor cell proliferation, migration, and invasion. Jiang et al found that the moderately abnormal expression of miR-3195 in laryngeal cancer is related to the characteristics of clinical cases, which can inhibit the proliferation and promote the apoptosis of Hep-2 by regulating T-box 1 (TBX1) expression.32 In this study, overexpression of miR-3195 inhibited the proliferation, migration, and invasion of NSCLC cells, whereas knockdown of miR-3195 promoted the proliferation, migration, and invasion of NSCLC cells, thus indicating that miR-3195 may have a tumor-inhibitory role in the progression of NSCLC. This confirms that miR-3195 participates in the occurrence and development of NSCLC tumors, and inhibits tumor progression in NSCLC, and can be used as an independent prognostic factor for NSCLC. Previous studies have reported that miR-3195 can target several genes in NSCLC, such as paired-like homeodomain 1 (PITX1), guanine nucleotide-binding protein (G protein), gamma 7 (GNG7), T-cell leukemia homeobox 3 (TLX3), TBX1, Prokineticin 2 (PROK2), and VEGF.32–34 Among these putative genes, PITX1 was upregulated in NSCLC and associated with worse outcomes in patients with lung adenocarcinoma.35 The online database GEDS35 and starBase36 based on TCGA project data also showed that PITX1 was upregulated in lung adenocarcinoma and lung squamous cell carcinoma. These findings imply that PITX1 may be a direct target of miR-3195 in NSCLC, wherein miR-3195 may restrain NSCLC cell cellular activities by targeting PITX1.

However, the precise molecular mechanisms underlying the role of miR-3195 in NSCLC are not yet clear and need elucidation and confirmation in further studies. The results of the present study may be biased due to limited sample size and, therefore, further studies with a larger research cohort are warranted.

Conclusion

These findings of the present study suggest that the expression level of miR-3195 expression is related to the progression of NSCLC, wherein the overexpression of miR-3195 inhibits the proliferation, migration, and invasion of tumor cells and may be used as an independent prognostic factor for NSCLC. The present study suggests a potential prognostic biomarker for NSCLC and a theoretical basis for designing the therapy of NSCLC.