Setting ‘Homo sapiens’ and ‘pulmonary nodules’ as keywords, the GSE135304 dataset (15) was downloaded from the NCBI GEO database (https://www.ncbi.nlm.nih.gov/). The dataset consisted of three main types of samples: Whole-blood samples from patients with malignant nodules, patients with benign nodules and nodule-free patients. After removing duplicate and treated samples, 587 samples remained in the following groups: Malignant (n=303), benign (n=196) and no nodules (n=88). Association between RNASE2 expression and nodule size was also analyzed.

The identification of statistically different genes was completed using the ‘Limma’ package (R Core Team) with false discovery rate <0.05 and log(fold-change) >0.05.

Overlapping DEGs from malignant/benign and malignant/(benign + no nodules) nodules were identified using VENNY 2.0 (https://bioinfogp.cnb.csic.es/tools/venny/index2.0.2.html). The KM Plotter (http://kmplot.com/analysis/index.php?p=service) was used to assess the association between the expression of all genes and survival in lung cancer, and all analysis of results has been taken directly from Kaplan-Meier Plotter without reanalysis.

The GSE68465 dataset, which contained expression data for 462 samples [lung adenocarcinoma (n=443) and normal (n=19)] and survival information, was also downloaded from NCBI GEO in order to verify the expression of RNASE2.

The online databases miRDB (https://mirdb.org/) and TargetScanHuman (http://www.targetscan.org/) were used to predict the microRNA (miR/miRNA) targets.

Human lung adenocarcinoma cell lines (A-549 and ABC-1) were purchased from Nanjing Cobioer Gene Technology Co., Ltd. The A-549 cells were cultured in F12K + 10% FBS (Nanjing Cobioer Gene Technology Co., Ltd.) and the ABC-1 cells were cultured in MEM + 1% non-essential amino acids + 10% FBS + 1 mM sodium pyruvate (Nanjing Cobioer Gene Technology Co., Ltd.), both in a 37°C incubator with 5% CO₂.

The ribonuclease A family member 2 (RNASE2) pVITRO2 overexpression vector (RNASE2 OE), small interfering RNA (siRNA) against RNASE2 (RNASE2 siRNA), miR-185-5p mimic and negative controls were designed, synthesized and obtained from GenePharma Co., Ltd. Empty vector and miR-NC were used as negative controls.

A-549 and ABC-1 cells were infected with RNASE2 siRNA, RNASE2 OE, RNASE2 OE, miR-185-5p mimic using Lipofectamine 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 24 h according to the manufacturer's instructions. The cells were collected after 48 h of culture for subsequent experiments.

The sequence of the miR-185-5p mimic was as follows: Sense, 5′-UGGAGAGAAAGGCAGUUCCUGA-3′ and antisense, 5′-AGGAACUGCCUUUCUCUCCAUU-3′. The sequence of the mimic-NC was as follows: Sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 3′-TTAAGAGGCUUGCACAGUGCA-5′.

An EZ Magna RNA immunoprecipitation Kit (cat. no. 17-701; MilliporeSigma) was used according to the manufacturer's guidelines. Briefly, A-549 and ABC-1 cells were lysed in 500 µl RIP lysis buffer. Centrifugation was performed at 21,367 × g at 4°C for 15 min to extract the supernatant. Magnetic beads (100 µl) were preincubated with 5 µg anti-protein argonaute-2 (AGO2) or 5 µg immunoglobulin G (IgG) antibodies for 30 min at room temperature. Cell lysates (100 µl) were immunoprecipitated with beads for 6 h at 4°C. After incubation and brief centrifugation, the EP tube was placed on a magnetic rack, waiting for the solution to clear, and the supernatant discarded. A total of 500 µl RIP Wash Buffer was added, and then the contents of the EP tube was vortexed and placed back on the magnetic rack. The supernatant was discarded again and this step was repeated five times for a total of six washes. qPCR was used to confirm the target protein.

The proliferation of the two cell lines (A-549 and ABC-1) was measured using the CCK-8 assay (MilliporeSigma). The cells were routinely digested using trypsin and seeded onto 96-well plates. After 24 h, 10 µl CCK-8 solution was added per well and incubated for 2 h at 37°C. Finally, absorbance was measured at 450 nm using a microplate reader (Molecular Devices, LLC).

After coating the upper chamber with 50 mg/l Matrigel (BD Biosciences) and leaving to air dry at 4°C, 1×10⁵ A-549 or ABC-1 cells with 100 µl serum-free medium (Thermo Fisher Scientific, Inc.) were seeded into Transwell chambers (8.0-µm pore size; MilliporeSigma). The complete medium was added to the lower chamber. After 48 h of incubation at 37°C, the upper chamber was cleaned using cotton swabs, whereas the cells in the lower chamber were fixed with 70% ethanol for 20 min and stained with 0.1% crystal violet for 15 min, both at 25°C. The number of invasive cells was counted using a light microscope (Olympus Corporation). The steps for the cell migration assay were the same as those for the invasion assay, except that the upper chamber was not pre-coated.

pmirGLO vectors (Promega Corporation) were inserted with binding site-amplified RNASE2 3′UTR, through which RNASE2 3′UTR wild-type reporter (RNASE2-Wt) and a mutated isoform (RNASE2-Mut) with altered binding sites were constructed. Transfection of RNASE2-Wt and RNASE2-Mut with Lipofectamine was conducted along with the aforementioned miR-185-5p mimic. Next, the fluorescence intensity indicating luciferase activity was measured using a Reporter Assay Kit (cat. no. 16186; Thermo Fisher Scientific, Inc.) at 36 h post-transfection, while normalization was performed with reference to Renilla luciferase activity, according to the manufacturer's protocol.

Using GraphPad Prism (version 8.0.1; Dotmatics), data from three independent experiments (with the exception of clinical data) were analyzed and exhibited as the mean ± SD. The statistically significant differences between tumor tissues and adjacent normal tissues were determined using unpaired Student's t-test. The statistically significant differences between other two groups comparisons were determined using Mann-Whitney U-test or unpaired Student's t-test, where appropriate. ANOVA with Tukey's post hoc test was used in datasets containing three or more groups. P<0.05 was considered to indicate a statistically significant difference.

Pulmonary nodules are the main manifestations of early lung cancer (14). The GSE135304 dataset, which contains the sequencing data of whole blood from patients with malignant nodules and benign nodules, and from nodule-free patients, was downloaded from the GEO database. Duplicates and treated samples were removed, leaving 587 samples (303 samples with malignant nodules, 196 with benign nodules and 88 with no nodules). Fig. 1A shows that 14 DEGs were upregulated in the malignant nodule blood samples compared with the benign nodule blood samples. Fig. 1B shows that 5 DEGs were upregulated and one was downregulated in the malignant nodule blood samples compared with the benign nodule and no nodules blood samples. Four overlapping DEGs [S100 calcium binding protein P (S100P), RNASE2, cytochrome c oxidase subunit 7C (COX7C) and mast cell expressed membrane protein 1 (C19orf59)] from malignant/benign and malignant/(benign + no nodules) nodules were obtained using VENNY (Fig. 1C).

The four overlapping DEGs (S100P, RNASE2, COX7C and C19orf59) were upregulated in the malignant nodule blood samples compared with those in the benign nodule samples and no nodules samples, and their RNA expression levels did not differ between the benign and no nodules blood samples (Fig. 2A). Using the KM Plotter, the connection between these DEGs and the overall survival of patients with lung cancer was analyzed. Patients with high S100P and RNASE2 expression exhibited a significantly poorer prognosis compared with those patients with low expression, whereas patients with high C19orf59 expression exhibited a better prognosis compared with the patients with low expression (Fig. 2B). The survival of patients with lung cancer was not affected by COX7C expression.

A literature search revealed that, to the best of our knowledge, no study has yet reported the association between RNASE2 and lung cancer. Therefore, RNASE2 was selected as a novel target to explore its role in lung cancer. The analysis of the GSE135304 dataset revealed an association between RNASE2 expression and nodule size; in general, the larger the nodule, the higher the gene expression level (Fig. 3A). Using the GSE68465 dataset, it was found that the expression of RNASE2 was significantly higher in lung adenocarcinoma tissues than that in normal tissues (Fig. 3B). RNASE2 was knocked down and RNASE2 was overexpressed in A-549 and ABC-1 cells (Fig. 3C), and the proliferation of transfected A-549 and ABC-1 cells was measured using a CCK-8 assay. Proliferation was inhibited by RNASE2 siRNA and promoted by RNASE2 overexpression (Fig. 3D). The ability of the cells to migrate and invade was also tested, which similarly showed inhibition after RNASE2 siRNA treatment and promotion by RNASE2 overexpression (Fig. 3E and F).

The upstream regulatory mechanism of RNASE2 was also explored. As a number of mRNAs are regulated by miRNAs that play a role in the occurrence and development of tumors (16–18), 28 miRNAs were predicted to target RNASE2 in miRDB (https://mirdb.org/) (Table SI). The 18 miRNAs with target scores of >60 were analyzed. Additionally, the miRNA targets were predicted using TargetScanHuman (http://www.targetscan.org/) (Table SII). In TargetScanHuman, the context++ score is an indicator used to assess the reliability and strength of the binding between a miRNA and its target genes. Specifically, the context++ score is calculated based on various features within the target mRNA sequence, including the position of binding sites, sequence specificity, and whether there are evolutionary conservation features such as immunohistochemistry staining. A lower context++ score indicates a stronger predicted binding and a more likely miRNA-target relationship. In total, 17 miRNAs with a context ++ score percentile of 99 were selected. Using VENNY, 10 overlapping miRNAs were identified between the miRDB and TargetScan miRNAs (Fig. 4A). Among these, miR-185-5p is closely associated with lung cancer (19–21). The miR-185-5p mimic was therefore used to transfect A-549 and ABC-1 cells to upregulate miR-185-5p level (Fig. 4B), and RNASE2 was found to be inhibited by the miR-185-5p mimic (Fig. 4C). Next, RNASE2-Wt and RNASE2-Mut were constructed (Fig. 4D). The results of a luciferase reporter assay showed that the luciferase activity of RNASE2-Wt was inhibited by the miR-185-5p mimic, whereas no such effect was shown for RNASE2-Mut (Fig. 4E). The RNA-induced silencing complex (RISC) is a complex protein complex in cells that regulates gene expression. This complex can target a specific mRNA through miRNA guidance, inhibiting its translation or promoting its degradation, thus affecting gene expression levels. RISC contains miRNA, mRNA and Ago2 protein. To confirm whether miR-185-5p and RNASE2 form a RISC, RIP experiments were conducted, which found that both miR-185-5p and RNASE2 were enriched in the Ago2 group (Fig. 4F). This indicates that RNASE2 can bind to miR-185-5p.

Using the miR-185-5p mimic alone or in combination with RNASE2 OE, transfection was conducted in the ABC-1 cells. RNASE2 expression was significantly inhibited by the miR-185-5p mimic in comparison with the control (Fig. 5A). Moreover, significantly suppressed cell proliferation was observed in the ABC-1 cells transfected with miR-185-5p mimic compared with the control, which partially reversed the enhanced cell proliferation induced by RNASE2 OE (Fig. 5B). The migration and invasion of ABC-1 cells were also inhibited by the miR-185-5p mimic, which also partially reversed the enhanced cell migration and invasion induced by RNASE2 OE (Fig. 5C and D).