Introduction

Lung cancer is one of the most frequent cancers worldwide and is responsible for a significant part of cancer-related deaths in the world. In 2025, an estimated 226,650 new cases and 124,730 deaths from lung and bronchus cancer will occur in the United States ¹. Late-stage diagnosis and subsequent ineffective treatment often contribute to the poor prognosis and increased fatality of lung cancer ². Although recent advancements in treatments have resulted in longer survival rates for lung cancer patients ¹, the high mortality associated with the cancer places a significant burden on healthcare systems and communities ³. Therefore, innovative therapeutic approaches based on molecular mechanisms and tumor biology can potentially enhance lung cancer detection and treatment ⁴.

As one of the emerging molecular targets in cancer, Nuclear Factor-kappa B (NF-κB), a pro-inflammatory transcription factor, plays a significant role in various biological processes, including cell survival, proliferation, immune responses, inflammation, cell growth, and apoptosis ^5-7. Persistent activation of NF-κB has been observed in different types of human tumors, including lung cancer ⁸. NF-κB is regarded as a key factor in lung cancer development and invasion ⁹. In many solid tumors, the constant activation of NF-κB is primarily attributed to the presence of inflammatory cytokines within the tumor microenvironment ^10,11.

The regulation of NF-κB signaling pathways involves proteins and non-coding RNAs, specifically long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) ^12,13. lncRNAs interact with various RNAs, including miRNAs, to regulate gene expression at both transcriptional and post-transcriptional levels. Several studies have demonstrated that the interaction between miRNAs and lncRNAs plays a crucial role in the development and progression of cancer ¹⁴. It has been reported that the NF-κB interacting lncRNA (NKILA) suppresses breast cancer progression by binding to the NF-κB inhibitor (IκB), masking the phosphorylation motifs of IκB and inhibiting IκB kinase-induced phosphorylation of IκB, which consequently prevents NF-κB activation ¹⁵. Additionally, miR-103/107 have been found to negatively regulate NKILA and are essential for breast cancer tumorigenesis. The overexpression of miR-103/107 in cancer cells significantly decreases NKILA expression ^15,16. Further studies have shown that NKILA also inhibits the progression of malignant melanoma ¹⁷, non-small cell lung cancer ⁸, and esophageal squamous cell carcinoma ¹⁸. Authors previous study suggested that NKILA may be a potential downstream target of miR-103/107 in colorectal cancer ¹⁹. 

Existing literature suggests that NKILA can potentially serve as a biomarker or therapeutic target in various types of cancer ²⁰. Nevertheless, the contribution of the NKILA-miR103-miR107 signaling pathway to lung cancer tumorigenesis and invasion has not been investigated. Therefore, the aim of this study was to investigate the effects of miR-103/107 inhibition on NKILA expression and its impact on the proliferation and invasive behavior of A549 lung cancer cells.

Materials and Methods

Cell culture

The Pasteur Institute of Iran (Tehran, Iran) was the source of the human lung cancer cell line A549 used in the research. While STR profiling was not performed in this study, we acknowledge that the absence of formal cell line authentication is a limitation. Purchased cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Invitrogen, London, UK). 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich, London, UK) and 100 U/ml penicillin/streptomycin (Invitrogen, London, UK) were added to the media as supplements. Standard cell culture conditions, which consists of 5% CO₂ at 37°C, were provided for the cells in the medium. During incubation, the adherent cells (approximately 80% confluent) were dissociated by trypsinization using 0.25% trypsin (Sigma-Aldrich, London, UK) and then centrifuged at 400×g for 5 min. The cell pellets were washed with 0.1 M phosphate buffer at pH 7.4. The cell line was free of mycoplasma contamination.

 miR-103/107 silencing by LNA-based antisense oligos

miRCURY® LNA® miRNA Power Family Inhibitor (cat no. 339160, Qiagen) was utilized simultaneously to inhibit the miR-103/107 family, as this inhibitor is designed to target conserved sequences shared by both miR-103 and miR-107 members. Approximately 24 hr before transfection, a six-well plate was filled with A549 cells at a density of 300,000 cells per well. Following this, serum-free DMEM was added to the medium to maximize cell culture viability. The LNA Power Inhibitor (140 pmol) was introduced to cells using 25 μl of HiPerFect Transfection Reagent (cat. no. 301704, Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocol. LNA-transfected cells were extracted after 24 hr of incubation at 37°C to make a reliable assessment of the impacts of antisense oligonucleotides.

 Quantitative real-time PCR assay

Total RNA was isolated from LNA-transfected and control A549 cells utilizing the RNeasy Mini spin column procedure (Qiagen, Hilden, Germany). The quality of the purified RNAs was evaluated by using a NanoDrop™ 2000 Spectrophotometer (Thermo Fisher Scientific, USA). The variation in the A260/A280 and A260/A230 ratios ranged from 1.6 to 2, representing the high standards of the extraction procedures. The miScript II Reverse Transcription Kit (Qiagen, Hilden, Germany) was utilized to produce cDNA according to the kit's instructions. miR-103 and miR-107 concentrations were detected using a StepOnePlus Real-Time PCR thermocycler with the miScript SYBR Green PCR Kit (Qiagen, Hilden, Germany, Cat. No. 218073). The PCR reaction volume was 25 µl, consisting of 12.5 µl of miScript SYBR Green Master mix, 2.5 µl of 10x miScript Universal primer, 2.5 µl of 10x miScript Primer Assay for miR103 (Qiagen, Hilden, Germany, Cat. No: MS00031241) and miR107 (Qiagen, Hilden, Germany, Cat. No: MS00003409), 6 µl of RNase-free water, and lastly 1.5 µl of template cDNA. NORD68 (Qiagen, Hilden, Germany, Cat. No: MS00033712) was selected as the reference gene for miRNA normalization based on its stable expression in A549 cells under our experimental conditions. Cycling conditions for real-time PCR were 95°C for 15 min, followed by 40 cycles of 94°C for 15 s, 55°C for 30 s, and 70°C for 30 s.

Extracted RNAs were used to synthesize cDNA for NKILA by the PrimeScript™ 1st strand cDNA Synthesis Kit (Takara, Dalian, China). PCR was performed in a 20 µl reaction volume, including 4 µl 5x HOT FIREPol EvaGreen qPCR Mix Plus (ROX) (Solis BioDyne, Tartu, Estonia), 1 µl of each primer (final concentration 10 µM), 12 µl RNase-free water, and 2 µl cDNA. The forward and reverse primers for NKILA were 5'- AACCAAACCTACCCACAACG-3' and 5'-ACCACT AAGTCAATCCCAGGTG-3'; and for GAPDH were 5'-ATCACCATCTTCCAGGAGCGA-3' and 5'CCTTC TCCATGGTGGTGAAGAC-3'. The PCR amplification was conducted under the following conditions: 95°C for 15 min, followed by 40 cycles at 94°C for 15 s, 59°C for 20 s, and a final extension at 72°C for 20 s. The specificity of PCR was assessed by subjecting all the PCR products to melting curve analysis (Figure 1). GAPDH gene expression was used as the normalization reference to normalize expression data. The quantity of fold changes in gene expression was measured using the 2−ΔΔCt approach.

 Cell proliferation

The impact of miR-103 and miR-107 knockdown on the viability of A549 cells was evaluated using the MTT assay. Transfected and mock-transfected (vehicle-only) cells were plated in 96-well plates at a density of 1×10³ cells per well and maintained under standard culture conditions. At 24, 48, 72, and 96 hr post-transfection, 20 μl of 5 mg/ml MTT solution (Sigma-Aldrich, London, UK) was added to each well and incubated to allow the formation of formazan crystals. Subsequently, 100 μl of dimethyl sulfoxide (DMSO; Merck, Darmstadt, Germany) was added to dissolve the formazan, and absorbance was measured at 570 nm using a microplate spectrophotometer (Awareness Technology Inc., USA). The absorbance values served as an indicator of cell viability following miR-103/107 inhibition.

Trans well invasion assay

The invasive ability of A549 cells was assessed using 24-well Matrigel-coated invasion chambers (8 µM pores; Corning, NY, USA). Lower chambers were filled with DMEM containing 10% FBS as a chemoattractant, and 5×10⁴ LNA-transfected or mock-transfected (vehicle-only) cells were seeded in the upper chambers in serum-free DMEM. After 48 hr of incubation at 37°C with 5% CO₂, non-invading cells were removed from the upper surface. Invaded cells on the lower surface were fixed with 1% formaldehyde, stained with 0.2% crystal violet, and counted under an inverted microscope at 100× magnification across five random fields per well. Results were expressed as mean ± SD of three independent experiments.

Statistical analyses

All the data were analyzed using SPSS version 16.0 (SPSS, Chicago, IL, USA), and diagrams were created by GraphPad Prism 6.0. All cell culture experiments were conducted at least three independent times to ensure the reproducibility of the results. Differences between groups were analyzed using the Mann–Whitney U test due to the small sample size and the assumption of non-normal data distribution. Results are displayed as mean ± S.D., and p<0.05 was considered statistically significant. 

 Results

LNA inhibition of miR-103/107 increased NKILA in A549 cells

To investigate the functional role of the NKILA regulatory axis modulated by miR-103/107, antisense LNAs were employed to specifically inhibit these microRNAs. Quantitative real-time PCR analysis revealed a significant reduction in miR-103/107 levels in LNA-transfected A549 cells compared to controls (p<0.05) (Figures 2A and 2B). Subsequent evaluation of NKILA expression showed a pronounced upregulation in LNA-treated cells relative to control cells, confirming that suppression of miR-103/107 enhances NKILA levels (p=0.0045) (Figure 2C).

 miR-103/107 silencing reduces lung cancer cell viability

Cell viability of A549 cells was assessed at 24, 48, 72, and 96 hr post-transfection using the MTT assay. Compared to the negative control group, inhibition of miR-103/107 by LNA transfection led to a significant reduction in cell proliferation at all time points (Figure 3) (p=0.0041). These results suggest that silencing miR-103/107 impairs the growth capacity of lung cancer cells over time.

 miR-103/107 inhibition limits A549 cell invasion

The impact of miR-103/107 suppression on the invasive and metastatic behavior of A549 cells was explored. Results from Trans well assays demonstrated that inhibition of miR-103/107 significantly reduced the cells’ invasive capacity compared to control cells (p<0.05) (Figures 4A and 4B).

 Discussion

About 57% of lung cancer patients are diagnosed with metastasis, and their survival rate is as low as 5% ²¹. This staggering data implies the invasive identity of most lung cancer types. Furthermore, conventional therapies, including chemotherapy, radiation, and surgery, have proven limited abilities to address challenges such as the emerging chemoresistance of metastatic lung cancers ²² and come with considerable side effects ²³. Newer approaches, including immune checkpoint inhibitors ²⁴, Anaplastic Lymphoma Kinase (ALK) inhibitors ²⁵, Epidermal Growth Factor Receptor (EGFR) inhibitors ²⁶, and Chimeric Antigen Receptor T Cell (CAR-T) therapy ²⁷, have opened new windows into lung cancer treatment, but are not flawless and often come with limitations and side effects. The absence of a flawless and straightforward therapeutic protocol for lung cancer treatment, in particular, and for cancer in general, has led researchers to explore and consider novel and innovative approaches.

Efforts to develop innovative strategies include novel therapeutics like mRNA ²⁸, miRNA ²⁹, lncRNA ³⁰ modulators and novel targets, such as factors involved in cancer-related inflammation ^31,32. The role of inflammation is firmly established in cancer invasion, metastasis, and overall pathogenesis ^33-35. Tumor-associated inflammation contributes to cancer pathogenesis by promoting immune escape, epithelial-mesenchymal transition, tumor angiogenesis, and apoptosis, among other reasons ³⁶. Tumors utilize various inflammatory signaling pathways, including NF-κB, to impair cytokine balance, and ultimately shift tissue dynamics to their advantage ^37,38. The role of NF-κB signaling pathway has been proven to be necessary for lung cancer development and pathology ³⁹. NF-κB can promote tumor resistance to anti-cancer drugs such as EGFR inhibitors ⁴⁰, promote cell cycle dysregulation and apoptosis suppression in lung cancer cells ⁴¹. Inhibition of NF-κB has proven to be effective in promoting tumor cell apoptosis ⁴², drug sensitivity ⁴³ and inhibiting metastasis and angiogenesis ⁴⁴. NF-κB suppression has been achieved through different mechanisms, such as IκBα suppression,  downregulation of TNFR1 protein expression, and inhibition of NF-κB by aptamers ⁴³, among others 44.

The role of NKILA and its inhibitory effect on NF-κB and lung cancer pathogenesis has also been elucidated. NKILA can inhibit invasion ⁴⁵ and proliferation ⁴⁶ of lung cancer cells and generally promote the immune evasion of tumors ⁴⁷. The pro-oncogenic functions of miR-103/107 have become increasingly evident in recent years. These miRNAs enhance tumor invasiveness in part by suppressing multiple tumor-modulating pathways, including DAPK ⁴⁸, KLF4 ^48,49, NF1 ⁵⁰, Wnt/β-catenin/Axin2 ⁵¹, and OLFM4 ⁵², among other targets.

Our previous study demonstrated an inverse expression pattern between miR-103/107 and NKILA in colorectal cancer, suggesting that miR-103/107 may negatively regulate NKILA expression ¹⁹. The findings of the present study in A549 lung cancer cells are consistent with this inverse association. Although NKILA is known to modulate NF-κB signaling in other cancer models, NF-κB activity was not directly measured in the current study (e.g., p65 nuclear translocation or IκB phosphorylation assays were not performed). Therefore, any link between miR-103/107 suppression, NKILA upregulation, and NF-κB modulation in this model should be interpreted as hypothetical and based on prior literature rather than direct experimental evidence. Our data demonstrate that inhibition of miR-103/107 increases NKILA expression and is associated with reduced proliferation and invasion in A549 cells. However, we cannot conclusively establish a causal NKILA–NF-κB mechanistic pathway within this study. Future investigations using NF-κB reporter assays, Western blot analysis of NF-κB components, or rescue experiments are required to validate this proposed signaling interaction.

 Conclusion

In conclusion, authors findings show that modulation of miR-103/107 levels significantly effects NKILA expression and is associated with changes in proliferation and invasion in A549 lung cancer cells. While these observations are consistent with a potential involvement of NF-κB signaling suggested by previous studies, the present work does not directly demonstrate NF-κB pathway inhibition. This relationship may contribute to lung cancer progression and highlights a possible regulatory interaction between miR-103/107 and NKILA. The use of a single lung cancer cell line and the absence of direct NF-κB activity assays limit mechanistic interpretation. Moreover, the findings—derived from in vitro assays—require validation in additional lung cancer models and in vivo systems. Further studies are needed to determine whether the miR-103/107–NKILA axis functionally modulates NF-κB signaling and whether it may represent a viable therapeutic target in lung cancer.

 Ethics approval

This study was approved by the ethics committee of the Tehran University of Medical Sciences (IR.TUMS.CHMC.REC.1398.072).

 Conflict of Interest

 None to declare.

Funding: This study was supported by a grant from the Tehran University of Medical Sciences (grant no. 01-154-42187).