MicroRNA-299-3p as a potential marker of tumor size in non-small cell lung cancer

Abstract

Background

Lung cancer is one of the world’s leading causes of cancer-related death. A tumor biopsy is the gold standard for diagnosis but may not be representative of the whole tumor. We therefore need non-invasive biomarkers to guide us in structure, mass, prognosis and treatment. Therefore, alternative non-invasive and dynamic biomarkers are needed to assist physicians in evaluating tumor structure, histology, burden, prognosis, and treatment response. Telomerase dysregulation is key to cancer development. MicroRNAs can be both tumor suppressors and oncogenes. Bioinformatic analyses suggest that microRNA-299-3p interacts with telomerase. This study evaluated serum levels of microRNA-299-3p before and after surgery in patients with non-small cell lung cancer (NSCLC) and explored their potential association with tumor size.

Methods

This observational pilot study included 15 NSCLC patients who underwent anatomical lung resection at a specialist hospital, following a power analysis. Blood samples were collected before and three weeks after surgery. MicroRNA was isolated using kits according to the manufacturer’s protocols. Patients who had received chemotherapy or radiotherapy, had multiple primary cancers, or did not give informed consent were excluded.

Results

There was no significant difference in miRNA-299-3p levels between preoperative and three-week postoperative serum samples. However, a significant negative correlation was identified between preoperative miRNA-299-3p levels and tumor size (r=-0.59; p=0.02).

Conclusion

Preliminary findings suggest that levels of the molecule RNA-299-3p are linked to tumour size. It is thought that lower levels of RNA-299-3p may counteract the effect of telomerase, which can cause uncontrolled cell growth. More research is needed to confirm these results.

Lung cancer is the leading cause of cancer-related death worldwide.^[1] Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases.^[2] At the time of diagnosis, more than 60% of patients present with advanced-stage disease, resulting in a 5-year survival rate of 16.8%.^[3]

Early diagnosis of lung cancer is crucial for improving survival outcomes. However, sensitive methods for early detection remain limited, posing a major clinical challenge.^{[4, 5]} We therefore need non-invasive biomarkers to guide us in structure, mass, prognosis and treatment.^{[6, 7, 8, 9]}

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that bind to target messenger RNAs (mRNAs).^[10] These molecules have been shown to function as both tumor suppressors and oncogenes.^[11] Their circulating levels in serum or plasma can reflect disease stage and are considered potential biomarkers for cancer detection and monitoring.^{[12, 13]} Unlike normal cells, cancer cells exhibit limitless proliferative capacity, largely driven by telomerase and associated enzymes, particularly human telomerase reverse transcriptase (hTERT).^[14] Bioinformatic analysis using the TargetScan miRNA database predicts that microRNA-299-3p (miR-299-3p) interacts strongly with hTERT, with a confidence level of 96%.^[15] Previous studies have also demonstrated that miR-299-3p directly targets hTERT.^{[16, 17, 18]}

This study aimed to evaluate the expression levels of miR-299-3p in blood samples obtained from patients with NSCLC before and after surgical resection. Given its reported association with telomerase activity and its inverse relationship with tumor size, miR-299-3p may serve as a potential non-invasive biomarker for lung cancer diagnosis and prognosis.

Methods

This prospective observational pilot study evaluated circulating miR-299-3p levels in venous blood samples obtained from 15 patients diagnosed with NSCLC who underwent anatomical resection (lobectomy or pneumonectomy) at a tertiary care hospital. Blood samples were collected preoperatively and again three weeks postoperatively, coinciding with the second routine follow-up visit prior to the initiation of oncologic therapy. The primary objective was to assess whether miR-299-3p levels changed following the reduction in tumor burden after surgery and to examine their correlation with tumor size.

Inclusion criteria were age >18 years, suitability for general anesthesia, and eligibility for R0 resection. Exclusion criteria included prior chemotherapy or radiotherapy, inoperable or unresectable disease, multiple primary malignancies, heart failure, age >75 years, or absence of written informed consent. Although the study was initially planned to run for 6 months, it was concluded earlier after a power analysis indicated that a minimum of 13 patients would be sufficient (α=0.05, power =80%). To account for potential data loss, the sample size was increased to 15 patients. Patients were enrolled consecutively to minimize selection bias. The study was approved by the Clinical Research Ethics Committee of Gaziantep University (decision number: 2020/249, date: 19.09.2020) and was conducted in accordance with the Declaration of Helsinki.

Surgical Interventions

All patients provided written informed consent for both the surgical procedure and genetic analyses after undergoing standard anesthetic evaluation. Preoperative staging included positron emission tomography-computed tomography imaging. Lymph nodes with an SUV_max≥2.5 were considered suspicious and were further evaluated using endobronchial ultrasound or mediastinoscopy, depending on anatomical accessibility.^[19] After review by the multidisciplinary tumor board, rigid bronchoscopy was performed to determine bronchial resection margins. Anatomical lung resections were subsequently carried out via posterolateral thoracotomy in accordance with standard surgical protocols. Chest drains were removed when the drainage output was <100 mL within 24 hours and no air leak was detected. Patients were followed up at postoperative weeks 1 and 3. Blood samples for miRNA analysis were collected preoperatively and again at the third postoperative week using ethylenediaminetetraacetic acid (EDTA) tubes.

A graphical abstract illustrating the study design is presented below (Graphical Abstract).

Graphical Abstract.

Illustrating the study design is presented above.

Sample collection and miRNA isolation

Blood samples were collected from patients immediately prior to surgery and again during the third postoperative week, corresponding to the second routine follow-up visit before the initiation of adjuvant therapy. The samples were drawn into 3-mL BD EDTA tubes (purple cap) and processed within 30 minutes of collection. Plasma was separated by centrifugation at 3,000 rpm for 10 minutes at 4 °C and subsequently transferred into sterile microtubes. The extracted plasma samples were stored at -80 °C until miRNA isolation. Total RNA was isolated from plasma using the miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany; cat. no. 217184) according to the manufacturer’s instructions. The purified RNA was stored at -80 °C until subsequent complementary DNA (cDNA) synthesis.^[20]

RNA quality and quantity assessment

The concentration and purity of the extracted RNA were assessed spectrophotometrically using a MicroDrop device (Implen p360, Germany). Measurements were obtained at 260 nm (OD260) to quantify nucleic acid content and at 280 nm (OD280) to assess protein contamination. The OD260/OD280 ratio was calculated for each sample, with values between 1.7 and 2.0 considered indicative of high RNA purity suitable for downstream quantitative polymerase chain reaction (PCR) analyses. Each sample was measured in triplicate to ensure consistency and reliability of the spectrophotometric readings, and the mean concentrations and optical density ratios were recorded. RNA integrity was additionally evaluated by confirming the absence of significant absorbance at 230 nm, which may indicate contamination with phenolic compounds or chaotropic salts. All spectrophotometric measurements were performed at room temperature using RNase-free tips and tubes to minimize the risk of RNA degradation.

cDNA synthesis

cDNA synthesis was performed using the miScript II RT Kit (Qiagen, Hilden, Germany; cat. no. 218161) according to the manufacturer’s protocol. Relative quantification of miRNA expression was carried out using the miScript SYBR Green PCR Kit (Qiagen) on a Corbett Rotor-Gene real-time PCR system (Qiagen Corbett Life Science, Australia). Each 20-μL reaction contained 10 μL of 2× QuantiTect SYBR Green PCR Master Mix, 2 μL of miScript Universal Primer, 2 μL of the specific miScript Primer Assay (targeting either miR-299-3p) or procedures were conducted under RNase-free conditions to minimize RNA degradation. Each sample was analyzed in triplicate to ensure reproducibility. Reverse transcription was performed using a thermal cycler (Eppendorf Mastercycler Nexus, Germany) with the following program: 37 °C for 60 minutes for reverse transcription, followed by 95 °C for 5 minutes for enzyme inactivation. The resulting cDNA samples were stored at -20 °C until qRT-PCR analysis.^[21]

Gene expression analysis

Relative expression of miR-299-3p was determined by quantitative real-time PCR (qRT-PCR) using the miScript SYBR Green PCR Kit (Qiagen, Hilden, Germany) on a Corbett Rotor-Gene real-time PCR system (Qiagen Corbett Life Science, Australia). Each reaction included the miScript Universal Primer and miScript Primer Assays specific for miR-299-3p and the endogenous control U6 (Qiagen). All samples were analyzed in triplicate, and no-template controls were included to monitor assay specificity. Relative expression levels were calculated using the comparative Ct (2^ΔΔCt) method, with miR-299-3p expression normalized to U6.^[22]

Statistical Analysis

The normality of miR-299-3p expression data was assessed using the Shapiro-Wilk test. As the data were not normally distributed, pre- and postoperative values were compared using the Wilcoxon signed-rank test. Correlations between continuous variables were evaluated using Spearman’s rank correlation coefficient. Receiver operating characteristic analysis was performed to assess the discriminative statistical analyses were conducted using SPSS v22, with a p-value <0.05 considered significant. A prior power analysis (MedCalc v18.11.3) indicated that 13 samples were required (α=0.05, power =80%); therefore, 15 samples were included to account for potential data loss.

Results

Patient Characteristics

A total of 15 patients were included in this study, of whom 13 (86.7%) were male and 2 (13.3%) were female. The mean age was 63.6±8.89 years, with a median age of 62 years (range, 41-75). Detailed demographic characteristics and comorbidities are presented in Tables 1 and 2.

Table 1. General descriptive statistics.

(n=15)		n	%
Sex	Male	13	86.7
	Female	2	13.3
Operation	Right upper lobectomy	3	20.0
	Right middle lobectomy	1	6.7
	Right lower lobectomy	1	6.7
	Right lower bilobectomy	1	6.7
	Right pneumonectomy	1	6.7
	Left upper lobectomy	2	13.3
	Left lower lobectomy	2	13.3
	Left pneumonectomy	3	20.0
	Extended left lower lobectomy	1	6.7
Diagnosis	Squamous epithelial carcinoma	9	60.0
	Adeno carcinoma	3	20.0
	Other	3	20.00
pTNM	pT1cN2M0	1	6.7
	pT2aN1M0	1	6.7
	pT4NoM0	1	6.7
	pT2aN0M0	2	13.3
	pT2bN0M0	1	6.7
	pT2bN1M0	2	13.3
	pT2bN2M0	1	6.7
	pT3N0M0	2	13.3
	pT3N1M0	2	13.3
	pT4N0M0	3	20

The eighth edition pTNM stage classification for lung cancer.

Table 2. Mean and median values of descriptive statistics.

-	(n=15)
Descriptive characteristics	Mean ± SD	Median (min-max)
Age (years)	63.6±8.89	62 (41-75)
Length of hospital stay (days)	7.07±4.03	6 (3-20)
PET-CT fasting blood sugar (mg/dL)	107.53±20.18	98 (91-167)
Tumor SUVmax	14.19±7.69	11.6 (5.3-28.5)
Tumor diameter (cm)	5.29±1.7	5 (3-9)

SD: Standard deviation; PET-CT: Positron emission tomography-computed tomography; SUV_max: Maximum standardized uptake value.

Surgical Procedures and Histopathological Findings

Various types of anatomical lung resections were performed, including right upper lobectomy in three patients (20%), right middle lobectomy in 1 (6.7%), right lower lobectomy in 1 (6.7%), right lower bilobectomy in 1 (6.7%), right pneumonectomy in 1 (6.7%), left upper lobectomy in 2 (13.3%), left lower lobectomy in 2 (13.3%), left pneumonectomy in 3 (20%), and extended left lower lobectomy in one patient (6.7%). These surgical details are summarized in Table 1.

Histopathological examination revealed squamous cell carcinoma in nine patients (60%), adenocarcinoma in three patients (20%), and other NSCLC subtypes in three patients (20%). No distant metastases were observed; all cases were classified as M0.

Two patients were unexpectedly found to have N2 lymph node involvement after resection. One patient underwent right upper lobectomy with metastasis to right lymph node station 4, while the other underwent left pneumonectomy with metastasis to lymph node station 5.

miRNA Expression Analysis

Comparison of preoperative and postoperative plasma miR-299-3p levels is shown in Table 3. The median (interquartile range) preoperative level was 30.95 (27.00-31.88), whereas the median level at postoperative week 3 was 30.84 (30.39-31.16). Statistical analysis using the Wilcoxon signed-rank test indicated no significant difference (p=0.532). These findings are illustrated in the box plot shown in Figure 1. Individual patient cycle threshold values for both U6 and miR-299-3p are presented in Table 4.

Table 3. Comparison of miR-299-3p preoperative and postoperative values.

-	Pre-op (n=15)	Post-op (n=15)	-
	Median (25-75%)	Median (25-75%)	p
miR-299-3p Ct	30.95 (27-31.88)	30.84 (30.39-31.16)	0.532

p<0.05; Ct: Cycle threshold; Median (25-75%) threshold.

Figure 1.

Box-plot comparison of miR-299-3p preoperative and postoperative values.

Table 4. Pre-operative and post-operative control U6 and miR-299-3p levels of the case.

Case no	Pre-op U6-Ct	Pre-op miR-299-3p Ct	Post-op U6-Ct	Post-op miR-299-3p Ct
1	27.78	32.1	31.62	29.63
2	31.8	30.95	28.05	30.83
3	33.08	31.89	28.4	31.44
4	31.94	25.31	30.86	30.39
5	31.85	26.51	28.69	30.76
6	30.6	31.74	30.59	31
7	32.72	31.88	29.67	24.07
8	32.79	32.42	27.29	31.09
9	33.7	31.27	26.49	32.75
10	30.27	27.93	32.36	32.5
11	31.96	27	28.87	30.84
12	31.52	30.9	28.25	26.76
13	25.49	30.19	29.91	31.16
14	31.41	31.15	30.86	30.83
15	28.45	25.46	31.59	30.89

Ct: Cycle threshold.

Correlation with Tumor Size

Spearman correlation analysis revealed a significant negative correlation between preoperative plasma miR-299-3p levels and tumor diameter (r=-0.592, p=0.020), as shown in Figure 2. A scatter plot illustrating this relationship across histopathological subgroups is provided in Figure 3.

Figure 2.

Scatter plot for preoperative miR-299-3p plasma levels and tumor diameter.

Figure 3.

Scatter plot of plasma levels of miR-299-3p at preoperative week 3 and tumor diameter in sub diagnostic groups.

Discussion

In this study, no significant change was observed in circulating miR-299-3p levels before and after surgery. However, a strong negative correlation was identified between preoperative plasma miR-299-3p levels and tumor size. Similar observations have been reported for other miRNAs in lung cancer. For instance, miR-148a, miR-148b, and miR-152 were significantly downregulated in patients with larger tumors and lymph node metastases.^{[23, 24]} Conversely, analyses using the the University of ALabama at Birmingham CANcer data analysis Portal database indicate that miR-299-3p is upregulated in lung tumor tissues compared to normal tissues.^{[25, 26]} This apparent discrepancy may reflect differences between circulating and tissue miRNA levels, a phenomenon previously highlighted in multiple studies.

miR-299 is known to directly target hTERT mRNA, thereby reducing telomerase activity and limiting uncontrolled cellular proliferation.^{[17, 18]} Therefore, decreased circulating miR-299-3p levels in patients with larger tumors may reduce inhibition of hTERT, facilitating tumor growth.

This study has several limitations, including a relatively small sample size, short follow-up duration, and heterogeneity of NSCLC subtypes. These factors limited our ability to perform detailed subgroup analyses and may have influenced the statistical outcomes.

In conclusion, our findings suggest a potential role for circulating miR-299-3p levels in reflecting tumor burden in NSCLC. However, larger, well-designed prospective studies are needed to validate these preliminary observations and further explore the clinical utility of miR-299-3p as a prognostic biomarker.

This study demonstrates that circulating miR-299-3p levels are inversely correlated with tumor size in patients with NSCLC. A significant negative correlation was observed between preoperative miR-299-3p levels and tumor diameter (r=-0.592, p=0.020). These findings support the hypothesis that reduced miR-299-3p expression may weaken its inhibitory effect on telomerase, particularly hTERT, thereby facilitating uncontrolled cellular proliferation and tumor growth. No significant change was observed between preoperative and third-week postoperative plasma miR-299-3p levels, which may be attributed to the biological half-life of the miRNA or the relatively short postoperative follow-up period.

Several limitations should be considered. The small sample size, short duration of follow-up, and heterogeneity of tumor histology limit the generalizability of these findings. Additionally, the cohort was insufficient to perform multivariate analyses adjusting for potential confounders such as age, sex, tumor subtype, and metabolic activity.

Larger-scale, prospective studies with extended follow-up are warranted to validate these preliminary observations and to determine whether circulating miR-299-3p can serve as a reliable, non-invasive biomarker for the diagnosis, prognosis, and monitoring of NSCLC.

Ethics

Ethics Committee Approval: The study was approved by the Clinical Research Ethics Committee of Gaziantep University (decision number: 2020/249, date: 19.09.2020) and was conducted in accordance with the Declaration of Helsinki.

Informed Consent: All patients provided written informed consent for both the surgical procedure and genetic analyses after undergoing standard anesthetic evaluation.