The prognostic value of miR-323b-5p in non-small cell lung cancer and its mechanism of targeting IL1A in regulating the proliferation and cell cycle of non-small cell lung cancer cells

Abstract

Background

MicroRNA (miRNA) plays a crucial role in initiating and progressing non-small cell lung cancer (NSCLC).

Aim

This study aims to investigate the expression and prognostic value of miR-323b-5p in NSCLC patients, and to clarify the mechanisms of miR-323b-5p and IL1A on the proliferation and cell cycle of NSCLC cells.

Methods

Differentially expressed miRNAs were screened from the GSE171517 dataset. 120 NSCLC patients and 60 healthy controls were collected. RT-qPCR was used to detect miR-323b-5p and interleukin 1α (IL1A). Prognostic value was analyzed via Kaplan-Meier and Cox regression. Target genes of miR-323b-5p were predicted using TargetScan/miRDB and validated by dual-luciferase assay. Cell proliferation was detected by cell counting kit-8 (CCK-8). Cell cycle was detected by flow cytometry. The expression of cyclin-dependent kinase inhibitor 1 A (P21), cyclin D1 (CCND1) and cyclin dependent kinase 4 (CDK4) was detected by western blot and RT-qPCR.

Results

miR-323b-5p was significantly downregulated in NSCLC serum samples and A549, HCC827, and NCI-H1299 cell lines. Low miR-323b-5p correlated with poorer 5-year survival rate and was a prognostic risk factor for NSCLC. IL1A was a direct target of miR-323b-5p. miR-323b-5p mimics can inhibit the proliferation of A549, HCC827, and NCI-H1299 cells, arrest the cell cycle to the S phase, promote P21, and inhibit the expression of CCND1 and CDK4. Overexpression of IL1A can partially reverse the above phenomena.

Conclusions

Low levels of miR-323b-5p predict a poor prognosis for NSCLC. miR-323b-5p regulates the cell cycle of NSCLC cells by targeting IL1A, thereby inhibiting cell proliferation.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12672-026-04409-2.

Introduction

Lung cancer ranks among the most common and deadly forms of malignant tumors globally [1]. Worldwide, lung cancer was responsible for nearly one-fifth of all cancer fatalities in 2020 [2]. Non-small cell lung cancer (NSCLC) is the primary subtype of lung cancer. Compared with small cell lung cancer (SCLC), NSCLC generally exhibits slower growth and delayed metastasis. Owing to the vague and inconspicuous presentation of initial symptoms, most cases are identified only in intermediate or advanced phases, which poses greater therapeutic challenges and poorer prognosis. Although the development of targeted therapy and immunotherapy has shown good efficacy in recent years [3, 4], the high heterogeneity of NSCLC and its tendency to recur lead to drug resistance in a significant number of patients [5]. Consequently, the 5-year survival rate for NSCLC patients remains relatively low, at approximately 30% [6, 7]. Thus, delving deeper into the molecular basis of NSCLC development and discovering new treatment targets hold significant importance in improving patient outcomes.

MicroRNAs (miRNAs) can be involved in regulating multiple processes in organisms. Emerging research increasingly suggests that miRNAs play crucial regulatory roles in various cancers, acting either as oncogenic miRNAs or as tumor suppressor miRNAs. For instance, miR-143 inhibits the proliferation of colorectal cancer by targeting KRAS [8], while miR-183 promotes the invasion and metastasis of breast cancer cells by down-regulating PTEN [9]. In NSCLC, the abnormal expression of multiple miRNAs has been confirmed to be closely related to tumor occurrence, progression, and drug resistance. For example, the miR-21 promotes the proliferation of NSCLC cells [10], while miR-34a exerts a tumor suppressor effect by inhibiting TGFβR2 [11]. Therefore, in-depth research on the regulatory mechanism of miRNAs in NSCLC may provide foundational theories that underpin the development of new diagnostic markers and therapeutic strategies.

miR-323b-5p is a member of the miR-323 family and has been reported to play a significant regulatory role in various diseases and cancers. For instance, miR-323b-5p serves as a serum biomarker for type 2 diabetic patients progressing to severe limb ischemia [12]. Another study indicates that miR-323b-5p can be used as a biomarker to distinguish glaucoma patients from control subjects [13]. In cancer, miR-323b-5p is associated with the occurrence and development of colorectal cancer [14]. However, there has been no research on miR-323b-5p in NSCLC. We hypothesize that miR-323b-5p is involved in the occurrence and development of NSCLC, explore the prognostic value of miR-323b-5p in NSCLC and its mechanism of action on the proliferation of NSCLC cells, provide more effective guidance for the prognosis assessment of patients with NSCLC, and preliminarily explain its mechanism of action.

Materials and methods

Patient enrollment and sample collection

The study enrolled a total of 120 patients with NSCLC. The clinical baseline characteristics included age, gender, tumor type, tumor differentiation, tumor size and Tumor Node Metastasis (TNM) stage. The inclusion criteria were: meeting the diagnostic criteria for NSCLC; all received surgery; and having a good general condition; voluntarily participate in this study and sign the informed consent form. The exclusion criteria were: having other malignant tumors; having pulmonary infectious diseases within the recent 3 months; dying due to severe complications after surgery; and being unable to cooperate with follow-up. Additionally, 60 healthy controls with matched baseline characteristics to the NSCLC patients were included. The mean expression of miR-323b-5p was used as the grouping criterion, and NSCLC patients were accordingly divided into the low-miR-323b-5p group and the high-miR-323b-5p group. The 5-year survival was monitored through follow-up, with death as the survival endpoint. This study has been approved by The First People’s Hospital of Zhengzhou ethics review committee, and all experimental operations involving human subjects strictly adhere to the Declaration of Helsinki.

Fasting venous blood was collected on the day of admission for NSCLC patients and during physical examinations for healthy controls. The collected venous blood was placed in a coagulation tube and left to stand at room temperature for 30 min until the blood coagulated. Then, it was centrifuged at 3000 r/min for 10 min. Carefully draw the upper layer of serum to avoid touching the blood cell precipitate. Aliquot the serum into enzyme-free centrifuge tubes and store at −80℃ for subsequent experiments.

Screening of miRNA and its downstream genes

The GSE171517 dataset was obtained from the GEO database (www.ncbi.nlm.nih.gov), which includes miRNA expression profiles derived from the serum of patients with NSCLC and healthy controls. Using the GEO2R online analysis tool, the complete dataset was downloaded based on the criteria of |log2FoldChange| > 1.5 and p < 0.05 to identify differentially expressed miRNAs. The potential target genes of miR-323b-5p were predicted using the miRDB (https://mirdb.org) and TargetScan (https://www.targetscan.org/). For miRDB, targets with a prediction score of ≥ 85 were selected, while for TargetScan, the filtering criterion was a Cumulative weighted context + + score ≤ −0.5.

Cell culture and cell transfection

Human normal lung epithelial cells (BEAS-2B) were cultured in DMEM/F12 containing 10% fetal calf serum (FBS). Human NSCLC cell lines (A549, HCC827, and NCI-H1299) were cultured in F-12 K, and RPMI-1640 medium containing 10% FBS. All cells were provided by the National Biomedical Laboratory Cell Resource Bank (NSTI-BMCR, China) and were cultured under conditions of 37 °C, 5% CO₂ and a relative humidity of 90% – 95%.

The control mimic (mimic NC), control inhibitor (inhibitor NC), miR-323-5p mimic (mimic MIR) and miR-323-5p inhibitor (inhibitor MIR) were designed by Syngeneiech (Beijing, China). The control plasmid (ovNC), interleukin 1α (IL1A) overexpression plasmid (ovIL1A) and small interfering RNA targeting IL1A (siIL1A) were designed by Beyotime (Shanghai, China). The A549, HCC827, and NCI-H1299 cell lines were grouped as follows. The experimental groups are as follows: Control, mimic NC, mimic MIR, mimic MIR + ovIL1A, inhibitor NC, inhibitor MIR + siIL1A. All transfection experiments were conducted using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer’s instructions. After transfection for 48 h, transfected cells were obtained and the transfection efficiency was verified.

Luciferase reporter assays

Based on the binding site of miR-323b-5p and IL1A, General Biosystems designed and constructed IL1A wild-type (IL1A WT) and IL1A mutant (IL1A MUT) reporter plasmids. After co-culturing A549, HCC827, and NCI-H1299 cells with mimic NC or mimic MIR and IL1A WT or IL1A MUT for 48 h using Lipofectamine 3000, the fluorescence intensity was detected (Beijing Solarbio Science & Technology Co., Ltd.).

Cell proliferation assay

A549, HCC827, and NCI-H1299 cells were seeded at a density of 2000 cells per well. According to the treatment of each group, CCK-8 reagent (Beyotime, Shanghai) was added and incubated for 2 h at 0, 24, 48 and 72 h respectively, and then the absorbance at 450 nm was measured.

RT‑qPCR

Total RNA from the serum of all subjects and A549, HCC827, and NCI-H1299 cells was collected using RNAiso Plus (TAKARA). Reverse transcription was performed according to the kit (TAKARA) instructions. The reaction was heated at 95 °C for 1 min, then heated at 95 °C for 10 s, heated at 60 °C for 30 s, and cycled at 70 °C for 10 s, with 40 cycles. The primer sequences were shown in Supplementary Table 1. Select U6 as the internal reference of miR-323b-5p. The internal reference for IL1A, cyclin-dependent kinase inhibitor 1 A (P21), cyclin D1 (CCND1) and cyclin dependent kinase 4 (CDK4) is GAPDH. Ct values were normalized using the 2^−ΔΔCt method. The amplification curves of all primers presented a clear S-shaped pattern, with no obvious non-specific amplification peaks, and the repeatability of the samples was consistent.

Cell cycle assay

After collecting and washing the transfected A549, HCC827, and NCI-H1299 cells, they were fixed with 70% ethanol at 4 °C for overnight. The cells were centrifuged at 1000 rpm for 5 min, washed with PBS, and re-suspended in the PI staining solution (BD Company, catalog number 550825). The cells were stained in the dark at room temperature for 30 min, and then analyzed using a flow cytometer (BD LSRFortessa) from the United States. The data were analyzed using the ModFit LT 5.0 software (Verity Software House, TopSalem, Maine, USA).

Western blot

A549, HCC827, and NCI-H1299 cells were lysed using RIPA lysis buffer (Solabao), protein concentrations were determined using the BCA protein Kit (Sangon), and then protein samples were isolated using SDS-polyacrylamide gel electrophoresis (10% and 12%). And transferred to PVDF membrane (Merck Millipper, USA). After sealing the membrane with skimmed milk powder for 1 h, incubate the membrane with the primary antibody at 4℃ overnight. The antibodies used include: IL1A (Wuhan Sanying, 1:1000), P21 (Wuhan Sanying, 1:1500), CCND1 (Wuhan Sanying, 1:5000), cyclin dependent kinase 4 (CDK4, Wuhan Sanying, 1: 1000), β-actin (Wuhan Sanying, 1:2000). These membranes were first washed three times with TBST solution for 10 min each time, and then incubated at room temperature with horseradish peroxidase-labeled goat anti-rabbit (CST, 1:2000)/mouse (CST, 1:2000) secondary antibodies for 1 h. Then, these membranes were washed three times with TBST solution, each time for 10 min. Finally, bands were detected using the ECL Western blotting substrate (Pierce) and the Chemidoc XRS gel recording system (BioRad). The bands were quantitatively analyzed by ImageJ software.

Statistical analysis

The experiment was repeated 3 times independently with 3 biological replicates. GraphPad Prism 9.0 was utilized for all statistical analyses. Normally distributed data were analyzed using the student’s t-test or one-way ANOVA, and non-normally distributed data were analyzed using the chi-square test or Mann–Whitney U-test. According to the median expression of miR-323b-5p, the patients were divided into two groups: cases with high expression of miR-323b-5p and cases with low expression of miRNA. The survival differences among different groups (Kaplan–Meier survival analysis) were analyzed by log-rank (Mantel–Cox), and the influencing factors of NSCLC were analyzed by multivariate Cox regression. Significance was denoted as: *p < 0.05, **p < 0.01, ***p < 0.001.

Results

miR-323b-5p was lowly expressed in NSCLC

A total of 41 down-regulated miRNAs and 122 up-regulated miRNAs were obtained in the GSE171517 dataset (Fig. 1A). Among them, the log2FoldChange value of miR-323b-5p was − 1.99382, and the p-value was 0.0010096. The expression values of miR-323b-5p in the NSCLC group and the control group in the GSE171517 dataset are shown in Fig. 1B. Compared with the included healthy controls (n = 60), miR-323b-5p was significantly down-regulated in NSCLC patients (n = 120) (Fig. 1C). Taking the mean expression of miR-323b-5p as the cutoff value, NSCLC patients were divided into the miR-323b-5p high-expression group (n = 55) and the miR-323b-5p low-expression group (n = 65). As listed in Table 1, the abnormal expression of miR-323b-5p was significantly correlated with the tumor differentiation (p = 0.037), tumor size (p = 0.041) and TNM stage (p = 0.031). Compared with stage I (n = 36), miR-323b-5p was significantly lowly expressed in the serum of patients in stages II (n = 32), III (n = 30), and IV (n = 22) (Fig. 1C). The survival analysis results indicated that the survival rate of the low-miR-323b-5p group (n = 65) was significantly lower than that of the high-miR-323b-5p group (n = 55) (Fig. 1D). The multivariate Cox regression model showed that miR-323b-5p was identified as a predictor of 5-year mortality in NSCLC patients (HR = 0.337, 95% CI 0.161 ~ 0.706, p = 0.004, Fig. 1E). Moreover, compared with the BEAS-2B cell group, the expression of miR-323b-5p in A549, HCC827, and NCI-H1299 cell groups showed a downward trend (Fig. 1F).

Fig. 1.

miR-323b-5p is significantly downregulated in NSCLC. A The volcano plot derived from the GSE171517 dataset. B Expression values of miR-323b-5p in each subject of the GSE171517 dataset. C Relative expression levels of miR-323b-5p in the serum of NSCLC patients (n = 120), healthy subjects (n = 60) and patients at clinical stages Ⅰ (n = 36), Ⅱ (n = 32), Ⅲ (n = 30), Ⅳ (n = 22). D 5-year Kaplan-Meier survival curves of patients in the low-miR-323b-5p group (n = 65) and the high-miR-323b-5p group (n = 55). E Results of multivariate Cox regression analysis assessing the impact of miR-323b-5p, age, gender, tumor differentiation, tumor size and TNM stage on NSCLC patients. F RT-qPCR results of miR-323b-5p in human normal lung epithelial BEAS-2B cells and human NSCLC A549, HCC827, and NCI-H1299 cells. The experiment was repeated 3 times independently. (*compared with Control, p < 0.001***. ^#compared with TNM stage I, p < 0.01^##, p < 0.001^###)

Table 1.

The clinical characteristics of miR-323b-5p in the NSCLC patients

Characteristics	low-miR-323b-5p (n = 65)	high-miR-323b-5p (n = 55)	P value
Gender			0.818
Male	45	37
Female	20	18
Age			0.510
< 60	28	27
≥ 60	37	28
Pathological pattern			0.900
LUAD	29	25
LUSC	20	15
Others	16	15
Differentiation			0.037
High and moderate	29	35
Poor	36	20
Tumor size (cm)			0.041
<3	28	34
≥ 3	37	21
TNM stage			0.031
Ⅰ + Ⅱ	31	37
Ⅲ + Ⅳ	34	18

LUAD, Lung Adenocarcinoma. LUSC, Lung Squamous Cell Carcinoma. Others, this case predominantly involves large cell carcinoma. TNM stage, Tumor Node Metastasis stage

IL1A was the target of miR-323b-5p

Two candidate mRNAs, IL1A and mesenchyme homeobox 2 (MEOX2), were obtained from the two databases (Fig. 2A). The RT-qPCR results showed that IL1A was highly expressed in the A549, HCC827, and NCI-H1299 cell lines, while there was no statistically significant difference in the expression of MEOX2 (Fig. 2B). Meanwhile, IL1A was highly expressed in the serum of NSCLC patients (n = 120) (Fig. 2C). The binding site of miR-323b-5p to IL1A is shown in Fig. 2D. The dual-luciferase assay report results indicated that the fluorescence intensity of miR-323b-5p and IL1A-WT group was significantly reduced (Fig. 2E). In the A549, HCC827, and NCI-H1299 cells, the mimic MIR could reduce the expression of IL1A, while the inhibitor MIR could increase the relative expression of IL1A (Fig. 2F). The above results indicate that IL1A is the target gene of miR-323b-5p.

Fig. 2.

miR-323b-5p targets IL1A. A The intersection of predicted targets from TargetScan and miRDB databases. B The expression of IL1A and MEOX2 in A549, HCC827, NCI-H1299 and BEAS-2B cells. C The expression of IL1A in the serum of NSCLC patients (n = 120) and healthy subjects (n = 60). D The binding site of miR-323b-5p and IL1A. E Dual-luciferase reporter assay indicates that miR-323b-5p directly regulates IL1A. F Expression of IL1A in A549, HCC827, and NCI-H1299 cells after transfection with miR-323b-5p mimics. The experiment was repeated 3 times independently. (p < 0.05*, p < 0.001***)

miR-323b-5p affected the proliferation of A549, HCC827, and NCI-H1299 cell lines and blocks the cell cycle through IL1A

The transfection efficiencies of each group in A549 cells were shown in Fig. 3A. The CCK-8 results showed that with the extension of time, mimic MIR significantly inhibited the proliferation of A549 cells, inhibitor MIR promoted cell proliferation, while ovIL1A or siIL1A could reverse the above effects (Fig. 3B). As shown in Fig. 3C, ovIL1A partially promotes the S-phase effect inhibited by mimic MIR, while siIL1A partially inhibits the S-phase effect accelerated by inhibitor MIR, causing the cell cycle to stagger at the G1 phase. The RT-qPCR results (Fig. 3D) indicated that mimic MIR promoted the expression of P21 protein in cells, inhibited the expression of CCND1 and CDK4 proteins, while ovIL1A could reverse the expression trend of the above proteins. Inhibitor MIR inhibited the expression level of P21 and promoted the protein levels of CCND1 and CDK4. siIL1A can reverse the above expression trends. The protein bands in the WB results also showed a trend consistent with that of the RT-qPCR results (Fig. 3E). The result trends of the HCC827 and NCI-H1299 cell lines were the same as those of the A549 cells (Figs. 4 and 5).

Fig. 3.

miR-323b-5p affects A549 cell proliferation and blocks the cell cycle through IL1A. After transfection or non-transfection of A549 cells with the control mimic (mimic NC), miR-323b-5p mimic (mimic MIR), IL1A overexpression plasmid (ovIL1A), control inhibitor (inhibitor NC), miR-323b-5p inhibitor (inhibitor MIR) and IL1A knockdown plasmid (siIL1A). The transfection efficiency of each group of cells was detected by RT-qPCR (A), cell proliferation was detected by CCK-8 (B), the cell cycle status of A549 cells was detected by flow cytometry (C), and the expression of P21, CCND1 and CDK4 mRNA (D) and protein (E) in the cells was detected by RT-qPCR and WB respectively. The experiment was repeated 3 times independently with 3 biological replicates (n = 3). (*compared with Control, p < 0.05*. ^#compared with mimic NC, p < 0.05^#.)

Fig. 4.

miR-323b-5p affects HCC827 cell proliferation and blocks the cell cycle through IL1A. After transfection or non-transfection of A549 cells with the control mimic (mimic NC), miR-323b-5p mimic (mimic MIR), IL1A overexpression plasmid (ovIL1A), control inhibitor (inhibitor NC), miR-323b-5p inhibitor (inhibitor MIR) and IL1A knockdown plasmid (siIL1A). The transfection efficiency of each group of cells was detected by RT-qPCR (A), cell proliferation was detected by CCK-8 (B), the cell cycle status of A549 cells was detected by flow cytometry (C), and the expression of P21, CCND1 and CDK4 mRNA (D) and protein (E) in the cells was detected by RT-qPCR and WB respectively. The experiment was repeated 3 times independently with 3 biological replicates (n = 3). (*compared with Control, p < 0.001***. ^#compared with mimic NC, p < 0.001^###. ^&compared with inhibitor NC, p < 0.05^&)

Fig. 5.

miR-323b-5p affects NCI-H1299 cell proliferation and blocks the cell cycle through IL1A. After transfection or non-transfection of A549 cells with the control mimic (mimic NC), miR-323b-5p mimic (mimic MIR), IL1A overexpression plasmid (ovIL1A), control inhibitor (inhibitor NC), miR-323b-5p inhibitor (inhibitor MIR) and IL1A knockdown plasmid (siIL1A). The transfection efficiency of each group of cells was detected by RT-qPCR (A), cell proliferation was detected by CCK-8 (B), the cell cycle status of A549 cells was detected by flow cytometry (C), and the expression of P21, CCND1 and CDK4 mRNA (D) and protein (E) in the cells was detected by RT-qPCR and WB respectively. The experiment was repeated 3 times independently with 3 biological replicates (n = 3). (^#compared with mimic NC, p < 0.001^###.)

Discussion

More and more studies have shown that miRNA plays an irreplaceable and crucial role in the occurrence and development of NSCLC. The abnormal expression of miRNA is closely related to the malignant biological behavior of NSCLC [15]. In-depth research on the regulation and mechanism of miRNA is helpful for the diagnosis, treatment and prognosis of NSCLC. This study found that miR-323b-5p was significantly lowly expressed in the serum of NSCLC patients. As the clinical stage progressed from stage I to stage IV, the expression level of miR-323b-5p in the patients’ serum gradually decreased, suggesting that the downregulation of this miRNA may be involved in the malignant processes such as tumor invasion and metastasis. Its low expression state can potentially serve as a reference indicator for evaluating tumor burden and malignancy. In our study, patients with a low expression level of miR-323b-5p had a lower 5-year overall survival rate. Meanwhile, an HR value less than 1 also indicated that miR-323b-5p was a protective factor for the prognosis of NSCLC. In addition, TNM stage is commonly used in clinical practice to predict the risk of tumor progression in patients with NSCLC, providing a basis for the formulation of postoperative follow-up frequency and treatment effect detection cycle. An HR value greater than 1 in the TNM stage indicates that it is a risk factor for the prognosis of NSCLC, which is the same as the results of previous studies [16]. The above results all indicate the clinical prognostic value of miR-323b-5p and TNM staging in NSCLC. This suggests that we can conduct closer monitoring of patients with low expression levels of miR-323b-5p, and combine it with the TNM stage assessment model to provide a more precise basis for clinical prognosis stratification and new ideas for the clinical management of NSCLC.

Inflammation has been increasingly recognized as a critical factor in carcinogenesis. In the past decade, the exploration of the relationship between inflammatory responses and malignant tumors has gradually become a hot topic in clinical oncology research [17]. In the field of lung cancer, an increasing number of studies have begun to focus on the connection between inflammatory factors and the survival prognosis of lung cancer patients [18–20]. As a crucial member of the IL-1 family, IL1A serves as a pro-inflammatory cytokine that is vital for immune regulation and inflammatory responses [21–23]. IL1A promotes tumor cell proliferation, survival, and angiogenesis by activating NF-κB, MAPK, and other signaling pathways [24], and contributes to the recruitment of immunosuppressive cells [25]. Existing research had demonstrated an association between IL1A and NSCLC patient prognosis. Peng et al. reported that elevated IL1A expression correlates with poorer clinical outcomes [26]. In a prognostic model for lung adenocarcinoma incorporating IL1A along with apoptosis- and necrosis-related genes (NRGs), the risk score derived from this model was identified as an independent prognostic factor, with higher scores indicating worse prognosis and reduced immune infiltration [27]. Furthermore, Wen et al. identified IL1A as a significant prognostic predictor for early-stage NSCLC patients [28]. Based on this background, we screened for downstream target genes of miR-323b-5p. Two candidate genes, IL1A and MEOX2, were predicted through databases, but only IL1A showed significant overexpression in both NSCLC patient serum and A549, HCC827, and NCI-H1299 cell lines. Therefore, we ultimately selected IL1A for further validation as the target gene of miR-323b-5p. In our study, we hypothesized that IL1A was a target gene of miR-323b-5p and observed its high expression levels in NSCLC cell lines. Moreover, a dual-luciferase reporter assay validated the direct regulatory interaction between miR-323b-5p and IL1A.

Given the core characteristics of NSCLC, such as high heterogeneity and frequent somatic mutations, it is difficult for a single cell line study to fully reflect the universality of the disease’s molecular mechanism. In this study, three representative NSCLC cell lines, A549, HCC827, and NCI-H1299, were specially selected. It covers the key molecular subtypes and pathological types in the clinical practice of NSCLC, providing a reliable model for verifying the extensiveness of the target mechanism. Firstly, the results of the functional experiments showed that miR-323b-5p could significantly inhibit cell proliferation in the above three cell lines with different genetic backgrounds. However, when IL1A is overexpressed, this proliferative inhibitory effect is significantly reversed. The progression of the cell cycle depends on the precise regulation of the cyclin-CDK complex [29]. CCND1, a crucial regulator of G1 phase progression, binds CDK4 to form an active kinase complex that phosphorylates Rb protein. This phosphorylation releases E2F transcription factors, enabling G1-S phase transition and subsequent cell proliferation [30]. As a key CDK inhibitor, the upregulation of P21 expression can directly interact with the CCND1-CDK4 complex, thereby preventing the phosphorylation of Rb protein, inactivating the E2F transcription factor, and ultimately causing the cell to be unable to pass the G1/S checkpoint and remain in the G1 phase [31]. In this study, miR-323b-5p increased the percentage of cells in the S phase. After further detection of the expression of key regulatory proteins in the cell cycle, it was found that miR-323b-5p arrested the cell cycle at the G1/S phase by up-regulating the expression of P21 and down-regulating the expression of CCND1 and CDK4, ultimately inhibiting cell proliferation. Most importantly, the regulatory patterns of the above-mentioned miR-323b-5p/IL1A axis on P21, CCND1 and CDK4 were completely consistent in the three cell lines with different genetic backgrounds. This indicates that this regulatory mechanism is not an accidental phenomenon of a specific molecular subtype, but a universal mechanism that may widely exist in NSCLC. It not only eliminates the interference of tumor heterogeneity on the experimental results, but also significantly enhances the clinical translation potential and practical application value of the research conclusion.

However, this study still has obvious limitations. Firstly, all clinical samples were sourced from a single medical center and only included 120 NSCLC patients and 60 healthy controls, resulting in insufficient population representativeness and making it difficult to rule out the interference of factors such as regional diagnosis and treatment standards and baseline characteristics of the population. Secondly, although three NSCLC cell lines, namely A549, HCC827, and NCI-H1299, were selected for the cell experiments, no in vivo animal experiments were conducted, lacking the functional verification of miR-323b-5p/IL1A axis regulating tumor growth in a living tumor model. The inability to clearly define the applicability and tissue specificity of this mechanism in the in vivo microenvironment also limits the in-depth assessment of its clinical translation value. In the future, the sample size will continue to be expanded, and multi-center research will be conducted to cover NSCLC patients with different pathological subtypes and clinical characteristics. A nude mouse xenograft tumor model will also be constructed to evaluate the inhibitory effect of miR-323b-5p on tumor growth in vivo, further enhancing the scientific nature and translational potential of the research conclusion.

Conclusion

miR-323b-5p acted as a prognostic protective factor for NSCLC and affected the proliferation of A549, HCC827, and NCI-H1299 cells by targeting IL1A. Specifically, it regulates the expression of P21, CCND1 and CDK4 to arrest the cell cycle at the G1/S phase. This brings new ideas to the clinical prognosis and treatment strategies of NSCLC.

Author contributions

D Y and JJ Y conceived and designed the experiments. Y W, ZM D, D Y, JJ Y and CX L performed the experiments. D Y and JJ Y contributed sample collection and statistical analysis. D Y and JJ Y wrote the manuscript. Y W, ZM D, D Y, JJ Y and CX L revised it critically for important intellectual content. All authors read and approved the final manuscript.

Funding

No funding was received to assist with the preparation of this work.

Data availability

The GSE171517 dataset can be obtained from the GEO database (https://www.ncbi.nlm.nih.gov/geo/).

Declarations

Ethics approval consent to participate

The study protocol was approved by The Ethics Committee of The First People’s Hospital of Zhengzhou and followed the principles outlined in the Declaration of Helsinki. In addition, informed consent has been obtained from the participants involved.

Consent for publication

Not Applicable.

Competing interests

The authors declare no competing interests.