MicroRNA Expression Profiling of Lung Cancer with Differential Expression of the RON Receptor Tyrosine Kinase

Background The Ron receptor tyrosine kinase (RON) can act as a protooncogene and may play a prominent role in the initiation and development of lung cancer. microRNAs (miRNA) are master regulators of gene expression through direct or indirect regulation, and impact all aspects of cell biology. Methods Nonsmall-cell lung cancer (NSCLC) samples and small-cell lung cancer (SCLC) were stratified based on RON expression to identify miRNA profiles associated with RON expression levels, differentially expressed miRNA regulated by RON were screened out, and their biological behavior was analyzed. Results miRNA expression was most significantly affected by cancer type, and we found 85 miRNAs that were significantly differentially expressed between NSCLC and SCLC. There were 46 miRNAs differentially expressed between high RON expressing NSCLC compared to low RON expressing NSCLC. Biological processes and pathways found to be significantly influenced by RON expression included epithelial-mesenchymal transition (EMT) and activation of the PI3K-Akt and MAPK signaling pathways. Conclusions These data may provide the basis for a novel strategy to characterize lung cancer by RON expression and microRNA genotyping.


Introduction
Lung cancer is the leading cause of cancer-related deaths in the world, and mutations and deregulation of many proteins and pathways, including EGFR, ALK, ROS1, and MET, play a significant role in lung cancer occurrence and development [1,2]. Better identification and utilization of these disease-related biomarkers could lead to improvement in precision treatment of lung cancer, ultimately reducing disease incidence, lowering disease recurrence, and improving survival and the quality of life for lung cancer patients.
RON is located at the chromosome 3p21.3 region, a region that has tumor suppressor activity and undergoes frequent loss of heterozygosity in human lung and breast cancers. e mature RON is a 180 kDa heterodimer composed of a 40 kDa extracellular chain and a 150 kDa transmembrane β-chain. Stimulation of RON by its ligand, MSP, promotes invasive growth of epithelial cells, resulting from the integration of a number of input pathways, including epithelial-mesenchymal transition (EMT), cellcell dissociation ("scattering"), and extracellular matrix growth and invasion [3,4].
RON is primarily expressed in cells of epithelial origin including lung cells. Several lung cancer cell lines overexpresses Ron and variant isomers, and normal lung tissue exhibits minimal expression of RON compared to adjacent tumor tissue. ese data suggest that RON expression may be related to the occurrence and development of both NSCLC and SCLC and could be used as a prognostic indicator for lung cancer patients [5]. Regulation of how RON expression and splicing is deregulated in NSCLC and SCLC requires further characterization. e frequency and intensity of RON and phospho-RON expression in NSCLC is lower than in SCLC, and the β-RON isoform (around 150 kDa) was found to be the dominant isoform in NSCLC, compared to the 120 kDa isoform in SCLC. Furthermore, northern blot analysis verified that the RON gene is normally transcribed in the lungs. ese data suggest that characterizing mechanisms regulating RON gene expression are of great significance to improve understanding of lung tumor occurrence and development [6]. microRNAs (miRNAs) are small noncoding RNAs, ∼18-25 nucleotides in length, that regulate gene expression at the posttranscriptional level by hybridizing to target mRNAs, and either inhibiting gene translation or promoting degradation of messenger RNAs (mRNA) by miRNAs are thought to regulate at least 30% of human gene expression and are involved in essential biological processes, including cell-cycle control, cell lineage fate decision, cell survival, tissue patterning, vascular development, immune control, and metabolism [7]. miRNAs act as oncogenes or tumor suppressors in various tumors, including lung cancer [8].
RON and miRNAs are closely related to the occurrence and development of lung cancer; however, it is unclear how miRNAs interact with RON to facilitate lung cancer progression. In vitro, RON mutations that alter miRNA expression induce oncogenic and metastatic potential. miRNA profiles are significantly affected by RON expression in pancreatic cancer, indicating the potential role of miRNAs in tumor invasion and metastasis [9]. In the present study, we compare miRNA profiles of lung cancer samples with differential expression of RON in order to investigate how RON regulates miRNA expression in both NSCLC and SCLC.

MicroRNA Expression Analysis.
is research has been carried out in accordance with the World Medical Association Declaration of Helsinki and was approved by the Institutional Review Board (CWO) of Medical School of Ningbo University, Ningbo, China (2020-YXY-0035). All subjects provided written informed consent. Nine NSCLC samples and nine SCLC samples were analyzed by miRNA microarray. Each group was classified based on RON expression levels, no expression, low expression, and high expression, with three samples representing each level. miRNA expression patterns were compared between NSCLC and SCLC samples with different RON expression levels. e expression of RON was determined by Western blot and immunohistochemistry.

Immunohistochemistry of Lung Tissue.
Fragments of lung tissue were incubated with anti-RON monoclonal antibodies Zt/f2 overnight at 4°C, followed by the DAKO Envision DAB System visualization reagents (DAKO, Denmark). All slides were counterstained with hematoxylin. RON expression was assessed using a semiquantitative scoring system.

miRNA Expression by Microarray.
Total RNA was harvested using TRIzol (Invitrogen) and the miRNeasy mini kit (QIAGEN), according to manufacturer's instructions. RNA quantity was determined using a NanoDrop 1000 instrument. RNA was labeled using the miRCURY ™ Hy3 ™ / Hy5 ™ Power labeling kit and hybridized onto the miRCURY ™ LNA Array (v.16.0). e slides were washed and scanned using an Axon GenePix 4000B microarray scanner.
Scanned images were imported into GenePix Pro 6.0 software (axon) for grid alignment and data extraction. miRNAs with intensity ≥50 in all samples were selected for calculating a normalization factor after averaging the replicated miRNAs. After normalization using median normalization, volcano plot filtering was used to identify significantly differentially expressed miRNAs. Hierarchical clustering was performed to visualize distinct miRNA expression profiling among samples.

Statistical Methods
One-way analysis of variance was applied to assess differential expression of miRNAs between groups, and Benjamini-Hochberg FDR correction was applied, in addition to Tukey's honestly significant difference (HSD) post hoc test. A p value of <0.05 was considered to indicate a statistically significant difference.

Results
miRNA expression profiling was performed for NSCLC and SCLC samples with different RON expression levels. miRNA expression levels were more greatly influenced by tumor type than by RON expression levels.
Comparison of miRNA expression between NSCLC and SCLC identified 85 significantly differentially expressed miRNA genes. Among these, 55 (65%) were overexpressed in SCLC vs. NSCLC, and 30 (35%) were underexpressed. miRNAs with fold-change with ≥1.5-fold overexpression or ≤0.5-fold underexpression are given in Table 1 and Figure 1. In addition, RON overexpression was associated with discrete miRNA expression patterns (Table 2), particularly in NSCLC patients (Table 3), compared to RON nonexpressing samples; hierarchical clustering was performed to show the distinct miRNA expression profiling among samples ( Figure 2).
In order to discern the major miRNA genes associated with RON overexpression in NSCLC, differentially expressed miRNA genes were obtained after comparing the RON high-expressing and RON nonexpressing NSCLC samples, and a gene coexpression network was constructed. Using the miRWalk, miRDB, and TargetScan miRNA target prediction databases, 1827 overlapping target genes were identified for the 10 most significantly differentially expressed miRNAs. A regulatory network between the overlapping target genes and their upstream miRNAs was constructed using Cytoscape ( Figure 3). In order to identify the potential mechanism by which RON may promote NSCLC through miRNA-mediated regulation, the 1827 predicted target genes were then subjected to DAVID pathway enrichment analysis.
is pathway enrichment revealed that RON-related miRNAs are predicted to target genes that significantly affect pathways involved in   e top 4 significantly enriched signaling pathways were pathways in cancer, the PI3K-Akt signaling pathway, the MAPK signaling pathway, endocytosis, and proteoglycans in cancer ( Figure 4). In addition, the top 10 overexpressed and top 10 underexpressed miRNAs and their major targets which might have key function were predicted from published data ( Figure 5). Overexpression or underexpression of miRNAs detected by the microarray chip is represented by an upward (red) or a downward (blue) arrow, respectively. Changes of target genes expression levels, which were inversely correlated with the expression of the miRNAs that target them, are also represented by a down (blue) or up (red) arrow.

Discussion
Lung cancer is one of the most common cancers and a leading cause of cancer-related deaths. e identification of critical genetic factors leading to cellular transformation, including EGFR, ALK, and Ros1, has resulted in significant improvements in therapy for advanced lung cancer based on radiotherapy and chemotherapy and has improved the prognosis and quality of life for patients with lung cancer [1,2]. e RON receptor tyrosine kinase is an oncogene that plays an important role in the development of lung cancer. RON overexpression can induce a complex genetic program that results in cell dissociation, migration, and extracellular matrix invasion, which may be important in several tumor types, including lung cancer. e extent of RON overexpression varies widely among different lung cancer cells and between different subtypes of lung cancers. e frequency and intensity of RON expression in different lung cancer subtypes is not well characterized, and the specific mechanism by which RON overexpression contributes to the pathogenesis of different lung cancer subtypes remains unknown. R. Kanteti et al. reported that RON expression in NSCLC was lower than in primary and secondary SCLC tumors [6]. Reports from M. Wang confirm that RON overexpression is common in lung adenocarcinoma, suggesting that RON may initiate oncogenic programs and plays an important role in the pathogenesis of lung adenocarcinoma [10,11].
miRNAs play important roles in lung tumor progression. Using high-throughput RNA sequencing and bioinformatics, many tumor-related miRNAs and their predicted targets that are oncogenes or tumor suppressor genes have been reported. In this study, miRNA expression patterns in RON high-expressing and RON nonexpressing lung cancer samples were profiled, and we predicted potential gene-miRNA interactions with a systematic bioinformatics approach. Our study demonstrates that the type of tumors, such as SCLC or NSCLC, has the greatest impact on differential miRNA expression patterns. Within subgroups, RON expression had a greater impact on overexpression or underexpression of miRNAs in NSLCL samples than in SLCL samples.
Increased expression of hsa-miR-31-5p inhibits cell proliferation, migration, and invasion by regulating the Sp1 transcription factor in hepatocellular carcinoma. However, the function of hsa-miR-31-5p was shown to differ, as hsa-miR-31-5p was also demonstrated to positively influence cell motility in correlation with metastatic status by regulating PGE2, which was mediated by EP1-ERK-MMP9 signaling. Moreover, comprehensive miRNA expression profiling analysis found that hsa-miR-31-5p is a diagnostic biomarker for pancreatic cancer [28,29].  Journal of Oncology Hsa-let-7a expression plays an important role in tumorigenesis through repressing c-Myc and is significantly downregulated in a number of cancers, including hepatocellular cancer, breast cancer, and ovarian cancer [30]. e hsa-miR-320 family, particularly hsa-miR-320e, is downregulated in colorectal adenoma and affects colorectal tumor proliferation by targeting CDK6. hsa-miR-320e plays an important role in the growth of colorectal tumors and is considered as a biomarker for the early detection of colorectal tumors [31].
Upregulated hsa-miR-27b has a protective role in cell proliferation and migration by targeting Smad7 and  hsa-miR-590-5p hsa-miR-151a-5p Figure 5: A network of RON-related miRNA target genes and potential mechanism of action. Upregulation or downregulation of a specific miRNA or target gene is represented by an upward (red) or a downward (blue) arrow, respectively. affecting the TGF-β pathway. hsa-miR-27b-3p directly targets HSP90AA1 and Fzd7 in NSCLC, ROR1 in gastric cancer, and CBLB/GRB2 in breast cancer to suppresses cell proliferation, migration, invasion, and expression of MET [32,33].
miR-590-5p inhibits breast cancer cell stemness and metastasis by targeting SOX2, inhibits colorectal cancer angiogenesis and metastasis by regulating the NF90/VEGFA axis, inhibits gastric cancer cell growth and chemosensitivity through the RECK and AKT/ERK pathway, inhibits growth of HepG2 cells via decrease of S100A10 expression and inhibition of Wnt pathway, and suppresses the proliferation and invasion of NSCLC by regulating GAB1 [36,37].
RON high-expression activates or represses some transcription factors, which likely impacts the expression of a number of miRNAs. e 46 miRNAs that were differentially expressed miRNAs between RON high-expressing and RON nonexpressing NSCLC samples might be important factors that mediate the pathogenesis of NSCLC. ese miRNAs promote tumor development via regulation of numerous targets, multiple signaling pathways, and biological functions and behaviors. Among these, EMT and invasion were identified as important functions enriched in these miRNAs, which is consistent with previous findings. Constitutively, high RON expression leads to morphological scattering or stabilized EMT and TGF-β1, and activation of the MAPK and Ras pathways was closely related to RONmediated EMT. JAK/STAT, MAPK, and Ras signaling are possible common mediators of the functional behavior of these miRNAs, as the JAK/STATsignaling pathway mediates the biological effects of various external stimuli and controls survival, proliferation, and differentiation of several cell types. RON overexpression may be involved in the pathological process of tumorigenesis through these pathways, although interrogation of the specific mechanisms involved is beyond the scope of this study. e miRNA expression between NSCLC and SCLC samples was clustered, and a heat map was constructed to visualize miRNA expression patterns. Our findings suggest that while RON expression levels are associated with distinct miRNA expression patterns in NSCLC, the specific lung cancer subtype (either NSCLC or SCLC) has a greater impact on miRNA expression profiles. Furthermore, we analyzed the interactions between miRNAs and their potential target genes that were differentially expressed based on differential RON expression in NSCLC, and we constructed a network of miRNA interactions using Cytoscape software. Our data suggest that the RON-associated miRNAs may impact a number of pathways, including PI3K-Akt, MAPK, endocytosis, proteoglycans in cancer, focal adhesion, and Ras signaling pathway-related activation state; these pathways may be differentially activated between NSCLC samples that exhibit distinct patterns of RON expression and are consistent with previous research which suggested that RON promotes oral squamous cell carcinoma progression by regulating EMT and the MAPK signaling pathway [38].

Conclusion
In summary, here, miRNA expression patterns of lung cancer associated with differential expression of the human receptor tyrosine kinase RON were profiled. We identified differentially expressed miRNA between NSCLC and SCLC, as well as between NSCLC samples with high RON expression and lacking RON expression. Our results suggest that miRNAs regulating PI3K-Akt, MAPK, endocytosis, proteoglycans in cancer, focal adhesion, and Ras signaling may exhibit significantly different expression patterns associated with differential RON expression in NSCLC. EMT and PI3K-Akt and MAPK signaling pathways are significantly regulated by RON expression levels. e approach and findings of this study may provide new perspectives on the tumor-promoting effect of RON and may lead to the development of improved diagnostic and therapeutic strategies for lung cancer. Further research will verify these differentially expressed miRNAs on larger tissue samples and then evaluate the miRNAs-regulated gene networks predicted by bioinformation analysis. Functional verification of gene-gene interactions can be carried out at both cellular and animal levels.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.