Material and Method: miRNAs obtained from serum samples were converted into cDNA. miRNA-339-3p and miRNA-155-5p expression analyses were performed using SYBR GREEN kit.

Results: In the patient group, serum levels of miRNA 339-3p were found to be reduced compared to those in the control group, whereas miRNA 155-5p levels were significantly higher in the patient group than in the controls.

Conclusion: The serum levels of miRNA-155-5p and miRNA-339-3p, which showed significant statistical differences between the patient group and control groups, suggest that these miRNAs may serve as potential biomarkers for the diagnosis of NSCLC.

MicroRNAs (miRNAs), which are the focus of our study in relation to various molecular mechanisms associated with lung cancer, are small noncoding RNAs ranging from 19 to 25 nucleotides in length and play pivotal roles in the regulation of gene expression^5,6. miRNAs exert their regulatory effects by attac-hing to the 3' untranslated regions (UTRs) of messen-ger RNAs (mRNAs), thus critically influencing post-transcriptional gene regulation⁷. This regulation can manifest itself as either upregulation or downregula-tion, influencing molecular mechanisms that affect lung cancer prognosis. This condition may contribute to favorable outcomes by inhibiting proliferation or inducing apoptosis, or conversely, may negatively impact prognosis by promoting proliferation and metastasis^8,9.

Numerous investigations have demonstrated that miR-NAs are implicated in cancer metastasis by a) modula-ting angiogenesis in tumor cells; b) modulating the transcriptional activity of oncogenes, tumor suppressor genes or genes related to tumor metastasis; c) inhibi-ting enzymes involved in DNA methylation of tumor cells or d) regulating epithelial-mesenchymal transition¹⁰.

miRNA-339 has been identified as a microRNA capab-le of inhibiting cell proliferation and metastasis in several cancer types^11,12. In lung cancer patients, elevated miRNA-339 expression is linked to improved NSCLC prognosis by inhibiting proliferation and supp-ressing invasion and migration in adenocarcinoma¹³. Research on over 600 miRNAs in adenocarcinoma highlighted their potential as diagnostic biomarkers in NSCLC, and miRNA-339-5p has been reported to inhibit migration and invasion, suggesting its potential as a biomarker for this cancer type^14,15.

miRNA-155-5p is a key oncogenic miRNA that is upregulated in various cancer types and contributes to cell growth, migration, invasion, angiogenesis, geno-mic instability, and drug resistance^16,17. miRNA-155-5p has been reported to be upregulated in NSCLC, with disease progression linked to the suppression of key tumor suppressor genes mediated by this miRNA⁵. The overexpression of miRNA-155-5p has emer-ged as a standalone prognostic indicator in adenocarci-noma and serves as a potential diagnostic biomarker in lung cancer^18-20. Inhibition of miRNA-155-5p may promote apoptosis and hinder cancer progression, with its targeted inhibition and modulation of autophagy proposed as a potential approach for future NSCLC therapies^17,21.

In this research, we intended to assess the concurrent serum expression patterns of miRNA-339-3p and miRNA-155-5p, which have been reported in the litera-ture to exhibit variable expression patterns in lung cancer cases. Although some evidence suggests that these miRNAs could function as potential biomarkers in NSCLC development, there is a paucity of studies exploring their expression levels together. We foresee that our findings will provide significant insights into the potential applications of miRNA-339-3p and miR-NA-155-5p as biomarkers for the diagnosis and thera-peutic management of NSCLC. Additionally, we hope that the findings will pave the way for novel diagnostic and therapeutic approaches for lung cancer.

miRNA Isolation
Serum samples from both patients and the control group were stored at -80°C in sterile tubes until miR-NA experiments were performed. The isolation of miRNAs was carried out using Qiagen's miRNeasy Serum Plasma Kit (Qiagen, Hilden, Germany, Cat. No. / ID: 217184) according to the manufacturer’s instruc-tions. The purity and concentration of the isolated miRNAs were measured using a NanoDrop2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA).

cDNA Synthesis
cDNA synthesis was performed using the miRCURY LNA RT Kit (Cat. No. / ID: 339340-Qiagen) to the manufacturer's instructions. The amount of cDNA was quantified using the Qubit miRNA Assay Kit on a Qubit 3.0 Fluorometer (Thermo Scientific).

miRNA339-3p and miRNA155-5p Expression Analysis
cDNA serum samples were used for expression analy-sis. After quantifying the sample concentrations, Real-time PCR analysis was carried out using ROTOR-GENE device for miRNA339-3p (lot:201705190083- Qiagen-Germany) and miRNA155-5p (lot:21002552-1-Qiagen-Germany) gene and U6 control gene (houseke-eping assay, RNU6-lot:20800469-1-Qiagen-Germany). miRNA-339-3p, miRNA-155-5p, U6 were dissolved in appropriate quantity of RNAse-free water before use.

Expression analysis was performed as described in the instructions of the miRCURY LNA SYBR Green PCR Kit (Cat. No. / ID: 339346-Qiagen).

miRNA Calculation
The internal control (housekeeping assay=miR-U6) was used to normalize the ΔCT values and calculate the fold change of miRNA expression levels. To de-termine miRNA levels, the Livak formula (2^(-ΔΔCT)) was applied²². The ΔΔCT value was calculated by subtracting the ΔCT of the target gene from the avera-ge ΔCT of the internal controls. The fold change was then calculated as 2^(-ΔΔCT).

Statistical Analysis
Statistical analysis of the data obtained in the study was performed using the SAS (Statistical Analysis System,) v9.4 program. The normality of the dependent and independent variables used in the study was first examined using the Kolmogorov-Smirnov test. For this purpose, skewness values were reviewed, and paramet-ric tests were applied as the data followed a normal distribution. To evaluate the differences between the two groups, an independent sample t-test was perfor-med. A paired sample t-test was performed to compare the miRNA-339-3p and miRNA-155-5p values. To determine the sensitivity and specificity, ROC analysis was performed. Throughout the study, a significant level of <0.05 was considered statistically significant.

Click Here to Zoom

Table 1: Demographic data of the patients.

The 26 patients (84.62% male, 15.38% female) had a mean age of 61.6±11.4 years. Of these, 3% had never smoked, 69.2% had quit smoking at various times, 48% were suffering from Chronic Obstructive Pulmonary Disease (COPD), and 24% had a family history of cancer. Complaints about cough and shortness of bre-ath were reported at 26.9% and 19.2%, respectively.

In the comparative analysis of miRNA expression levels between patient and control groups, miRNA-339-3p expression was significantly downregulated in the patient group relative to the control group. Conver-sely, the expression of miRNA-155-5p was markedly upregulated in the patient group compared to the cont-rol group (Table 2).

Click Here to Zoom

Table 2: Expression levels of miRNA-339-3p and miRNA-155-5p in patient and control groups.

The independent samples t-test yielded a p-value of <0.05, indicating that the observed differences in miR-NA expression levels between the groups are statisti-cally significant and unlikely to have occurred by chance.

Sensitivity and specificity calculations were performed for various 2’ΔΔCt threshold values to predict disease status. The optimal 2’ΔΔCt threshold and the corresponding sensitivity and specificity values are presented in table 3 and 4.

Click Here to Zoom

Table 3: ROC analysis; miRNA-339-3p

Click Here to Zoom

Table 4: ROC analysis; miRNA-155-5p.

Additionally, based on these findings, an empirical ROC curve was generated using a non-parametric method in the SAS software, which is depicted in figures 1 and 2.

Click Here to Zoom

Figure 1: ROC curve; miRNA-339-3p.

Click Here to Zoom

Figure 2: ROC curve; miRNA-155-5p.

These ROC curves and AUC values indicate that 2’ΔΔCt values possess strong predictive power in distinguis-hing patients from healthy individuals.

To compare the miRNA-339-3p and miRNA-155-5p levels between individuals in patient and control groups, a paired sample t-test was performed. The results are presented in table 5 and figure 3.

Click Here to Zoom

Table 5: Analysis of dependent variables.

Click Here to Zoom

Figure 3: Expression Levels of miRNA-339-3p and miRNA-155-5p in patient and control groups.

A notable statistical difference was observed between the mean values of miRNA-339-3p and miRNA-155-5p in the patient group (p =0.0001). Additionally, the analysis conducted without distinguishing between the patient and control groups also revealed a statistically significant difference between the mean values of miRNA-339-3p and miRNA-155-5p.

The results suggest that the expression levels of these two miRNAs may play a role in disease status and differ accordingly. Statistical significance indicates that this difference is not due to chance.

Numerous studies have emphasized the significance of miRNA in the molecular mechanisms influencing can-cer prognosis^13,14,19. Hence, this study specifically investigates the expression levels of miRNA-339-3p and miRNA-155-5p in lung cancer, both of which have been previously identified as exhibiting differential expression across various cancer types. There is substantial evidence indicating that miRNA-339 plays a pivotal role in suppressing cancer cell proliferation, migration, and invasion, as well as regulating genes that induce apoptosis. Moreover, miRNA-339-5p has been shown to prevent metastasis in lung, ovarian, and pancreatic cancers by regulating epithelial-mesenchymal transition^10,12,25. In the literature, miRNA-339-5p expression was significantly lower in the primary tissues of NSCLC patients compared to the surrounding normal tissues¹⁵.

Furthermore, miRNA-339-3p's inhibitory effect on tumor cell invasion has been validated in various can-cers, including its tumor-suppressive role in melanoma. Also, in gastric cancer (GC), miRNA-339-3p is down-regulated, and its upregulation inhibits proliferation, migration, invasion, and enhances apoptosis, sugges-ting its role in suppressing GC development^26,27. However, contrary to these findings, there is also a study that reports miRNA-339-3p to be upregulated in patients with NSCLC²⁸.

In our study, we found that miRNA-339-3p was signi-ficantly downregulated in patients diagnosed with lung cancer compared to the control group. The literature has shown that miRNA-339-3p suppresses cell proliferation in lung cancer by targeting the 3'-UTR of Skp2 mRNA¹². When considered alongside the existing literature, this suggests that miRNA-339-3p plays a role in inhibiting cancer cell growth.

miRNA 155 has been shown to be involved in the regulation of inflammatory processes²⁹. A meta-analysis highlighted that it may serve as a potential biomarker for the detection of lung cancer³⁰. Increased expression of miRNA-155 has been recognized as an independent prognostic factor associated with ad-verse outcomes in patients with adenocarcinoma. Addi-tionally, it functions as a negative prognostic indicator in adenocarcinomas and a positive prognostic marker in SCC cases with lymph node metastasis^18,31.

miRNA-155-5p is highly expressed in NSCLC, negatively affecting disease progression, patient prognosis, and survival rates. The inhibition of this miRNA has been proposed as a potential strategy to prevent NSCLC progression⁵. Moreover, the transfection of AntimiR-155-5p significantly decreases miRNA-155-5p expression while simultaneously increasing the rate of apoptosis¹⁷. These data underscore the prognostic role of miRNA-155 in various types of lung cancer and suggest that it may represent a promising approach for future NSCLC treatment strategies.

In our study, consistent with findings in the literature, we found that miRNA-155-5p expression levels were significantly higher in individuals diagnosed with lung cancer compared to healthy individuals. The elevated levels of miR-155-5p, particularly in NSCLC and other various cancer types, suggest that this miRNA plays a crucial role in cancer progression and tumor cell proli-feration.

We believe that the potential of miRNA-339-3p and miRNA-155-5p as molecular biomarkers in NSCLC is supported by the findings of this study. However, a limitation of our current research is the relatively small sample size, which restricts the generalizability of the results. Therefore, it is crucial to emphasize that our findings should be validated through further studies involving larger lung cancer cohorts. Such large-scale investigations will provide more robust evidence for considering both miRNA-339-3p and miRNA-155-5p as significant biomarkers and potential therapeutic targets in the pathogenesis of lung cancer.

In conclusion, our findings regarding the serum levels of both miRNAs in patients diagnosed with NSCLC align with the prevailing consensus in the literature. Accordingly, we believe that miRNA-339-3p and miRNA-155-5p have potential as biomarkers in lung cancer and could serve as therapeutic targets, warran-ting further investigation in future studies.

1) Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics. CA Cancer J Clin 2022; 72:7-33.

2) Zeng J, Ding X, Ding J, Wang X. Histological transformation into SCLC: An important resistance mechanism of NSCLC upon immunotherapy. Front Immunol 2023; 14: 1275957.

3) Alduais Y, Zhang H, Fan F, Chen J, Chen B. Non-small cell lung cancer (NSCLC): A review of risk factors, diagnosis, and treatment. Medicine 2023; 102: e32899.

4) Dela Cruz CS, Tanoue LT, Matthay RA. Lung cancer: epidemiology, etiology, and prevention. Clin Chest Med 2011; 32: 605-44.

5) Xue X, Liu Y, Wang Y, et al. MiR-21 and MiR-155 promote non-small cell lung cancer progres-sion by downregulating SOCS1, SOCS6, and PTEN. Oncotarget 2016; 20: 84508-19.

6) Leitão AL, Enguita F J. A Structural View of miRNA Biogenesis and Function. Noncoding RNA 2022; 8: 10.

7) Ratti M, Lampis A, Ghidini M et al. MicroRNAs (miRNAs) and Long Non-Coding RNAs (lncRNAs) as New Tools for Cancer Therapy: First Steps from Bench to Bedside. Target Oncol 2020; 15: 261-78.

8) Gao X, Yang X, He F, Xue LX, Liu D, Yuan X. Downregulation of microRNA 494 inhibits cell proliferation in lung squamous cell carcinoma via the induction of PUMA α mediated apoptosis. Exp Ther Med 2023; 25: 242.

9) Zhou R, Zhou X, Yin Z, et al. MicroRNA-574-5p promotes metastasis of non-small cell lung cancer by targeting PTPRU. Sci Rep 2016; 20: 35714.

10) Li Y, Zhang X, Yang Z, Li Y, Han B, Chen LA. miR-339-5p inhibits metastasis of non-small cell lung cancer by regulating the epithelial-to-mesenchymal transition. Oncol Lett 2018; 15: 2508-14.

11) Wu Z, Wu Q, Wang C, et al. MiR-339-5p inhibits cancer cell migration and invasion in vitro and may be a potential biomarker for breast cancer prognosis. BMC Cancer 2010; 10: 542.

12) Ren H, Zhang Y, Zhu H. MiR-339 depresses cell proliferation via directly targeting S-phase kinase-associated protein 2 mRNA in lung cancer. phase kinase-associated protein 2 mRNA in lung cancer. Thorac Cancer 2018; 9: 408-14.

13) Li P, Liu H, Li Y, Wang Y, Zhao L, Wang H. miR 339 5p inhibits lung adenocarcinoma invasion and migration by directly targeting BCL6. Oncol Lett 2018; V16: 5785-90.

14) Rani S, Gately K, Crown J, O'Byrne K, O'Driscoll L. Global analysis of serum microRNAs as poten-tial biomarkers for lung adenocarcinoma. Cancer Biol Ther 2013; 14: 1104-12.

15) Li Y, Zhao W, Bao P, et al. miR-339-5p inhibits cell migration and invasion in vitro and may be as-sociated with the tumor-node-metastasis staging and lymph node metastasis of non-small cell lung cancer. Oncol Lett 2014; 8: 719-25.

16) Zanoaga O, Braicu C, Chiroi P et al. The Role of miR-155 in Nutrition: Modulating Cancer-Associated Inflammation. Nutrients 2021; 13: 2245.

17) Moayedi F, Shojaei-Ghahrizjani F, Yaghoobi H. Inhibition of miR-155-5p in non-small cell lung cancer, a potential target for induction of autop-hagy, Meta Gene 2021; 28: 100855.

18) Yanaihara N, Caplen N, Bowman E et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell 2006; 9: 189-98.

19) Zhu HZ, Fang CJ, Guo Y et al. Detection of miR-155-5p and imaging lung cancer for early diagno-sis: in vitro and in vivo study. J Cancer Res Clin Oncol 2020; 146: 1941-51.

20) Liu K, Zhao K, Wang L, Sun E. Prognostic value of microRNA-155 in human carcinomas: An upda-ted meta-analysis. Clin Chim Acta 2018; 479: 171-180.

21) Wang Y, Li J, Tong L et al. The prognostic value of miR-21 and miR-155 in non-small-cell lung cancer: a meta-analysis. Jpn J Clin Oncol 2013; 43: 813-20.

22) Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real- time quantitative PCR and the 2(-Delta Delta C(T)) Method. Met-hods 2001; 25: 402-8.

23) Yan H, Tang S, Tang S et al. miRNAs in anti-cancer drug resistance of non-small cell lung can-cer: Recent advances and future potential. Front Famacol 2022; 13: 949566.

24) Wani JA, Majid S, Imtiyaz Z et al. MiRNAs in Lung Cancer: Diagnostic, Prognostic, and Thera-peutic Potential. Diagnostics 2022; 12: 1610.

25) Yu Z, Zhao S, Wang L, Wang J, Zhou J. miRNA-339-5p Plays an Important Role in Invasion and Migration of Pancreatic Cancer Cells. Med Sci Monit 2019; 25: 7509-17.

26) Weber CE, Luo C, Hotz-Wagenblatt A et al. miR-339-3p is a tumor suppressor in melanoma. Cancer Res 2016; 76: 3562-71.

27) Wan H, Wang L, Huo B, Qiao Z, Zhang, Y. CIZ1 aggravates gastric cancer progression via media-ting FBXL19-AS1 and miR-339-3p. Heliyon 2023; 9: e21061.

28) Trakunram K, Chaniad P, Geater SL et al. Serum miR-339-3p as a potential diagnostic marker for non-small cell lung cancer. Cancer Biol Med 2020; 17: 652-63.

29) Mahesh G, Biswas R. MicroRNA-155: A Master Regulator of Inflammation. J Interferon Cytokine Res 2019; 39: 321-30.

30) Shao C, Yang F, Quin Z, Jing X, Shu Y, Shen H. The value of miR-155 as a biomarker for the diag-nosis and prognosis of lung cancer: a systematic review with meta-analysis. BMC Cancer 2019; 19: 1103.

31) Donnem T, Eklo K, Berg T et al. Prognostic im-pact of MiR-155 in non-small cell lung cancer eva-luated by in situ hybridization. J Transl Med 2011; 9: 6.