LncRNA MAGI2-AS3 and ZEB2 Plays a Crucial Role in the Lung Squamous Cell Carcinoma:a Bioinformatics Study


 PurposeLung squamous cell carcinoma (LUSC) is a common type of non-small-cell lung cancer. Because of the limitations of targeted therapy and immunotherapy, LUSC treatments are ineffective. To better understand the underlying mechanisms of LUSC carcinogenesis, the present study aimed to identify novel factors and their signaling networks in LUSC, with a primary focus on the construction of a competing endogenous RNA (ceRNA) regulatory network. MethodsWe conducted a transcriptomic analysis of LUSC samples from The Cancer Genome Altas database. Furthermore, we analyzed the data using bioinformatics methods.Results533 samples were selected for analysis, including 485 “PrimaryTumor” samples and 48 “SolidTissueNormal” samples. A total of 1853 DEgenes were identified. Bioinformatics analyses identified the most physiologically relevant and significantly dysregulated lncRNAs and mRNAs. MAGI2-AS3 and ZEB2 were found to play key roles in LUSC. Furthermore, we mapped these signaling pathway based on its role as a miRNA sponge to predict the binding of miRNA to MAGI2-AS3 and ZEB2.ConclusionWe concluded that MAGI2-AS3/ZEB2 loop exhibited tumor-suppressor activity in LUSC by inhibiting miR-374a-5p and miR-374b-5p and modulating the downstream signal transduction through a ceRNA network.


Introduction
Lung cancer has the highest morbidity and mortality rates among all cancer types worldwide. According to GLOBOCAN 2018 statistics(1), lung cancer was the most frequent cause of cancer-related death in most regions globally (14/20 world regions). In males, lung cancer accounted for the highest proportion of all estimated new cases and deaths in 2018. By contrast, lung cancer is the second most common cancer among females, followed by breast cancer. In the past two decades, lung cancer became the most common cancer in China (2). The WHO categorizes lung cancer into the two following major classes based on its biology, therapy, and prognosis: non-small-cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC accounts for more than 80% of all lung cancer cases and includes two subtypes: nonsquamous (including adenocarcinoma, large-cell carcinoma, and other subtypes) and squamous cell carcinoma (3). Lung adenocarcinoma (LUAD) is the most common type of NSCLC, accounting for 40% of lung cancer cases. Lung squamous cell carcinoma (LUSC) represents 25% to 30% of lung cancer cases, and it tends to originate from cells located in the airway epithelium (4).
Lung cancer therapies include surgical resection, chemotherapy (neoadjuvant chemotherapy and adjuvant chemotherapy), immunotherapy, and targeted therapy. Signi cant progress in targeted therapy and immunotherapy for NSCLC has been made. Thus, the death rates for lung cancer patients have been declining (3). Although LUAD and LUSC are both subtypes of NSCLC, there are signi cant differences in the targeted therapies used to treat them. Targeted therapies for speci c LUAD molecular subtypes are well-established, but those for LUSC are still under development. Furthermore, EGFR (27%) and KRAS (32%) are the most commonly mutated genes in LUAD but not in LUSC (<9% and 3%, respectively)(5).
Therefore, targeted therapy is limited to non-LUSC histological subtypes containing actionable driver mutations. To further improve survival outcomes, a better understanding of the molecular mechanisms underlying LUSC carcinogenesis is essential for effective disease prevention or treatment.
In recent decades, the roles of long non-coding RNAs (lncRNAs) in cancer have received increasing attention. LncRNAs exhibit a variety of biological functions in cancer, including the regulation of epigenetics, DNA damage, cell cycle progression, microRNAs, and signal transduction pathways (6,7).
LncRNAs are involved in NSCLC pathogenesis by modulating fundamental cellular processes, such as proliferation, cell growth, apoptosis, migration, stem cell maintenance, and epithelial to mesenchymal transition. Furthermore, lncRNAs represent very promising biomarkers in early-stage NSCLC patients and may become particularly useful in noninvasive screening protocols. For example, lncRNAs may be used as predictive biomarkers of the patient response to chemotherapy and targeted therapies (8). Our research team focuses on the emerging roles of RNA-RNA crosstalk in tumor-related processes. It has become increasingly clear that numerous miRNA-binding sites exist on a wide variety of lncRNAs, leading to the hypothesis that lncRNAs containing miRNA-binding sites can communicate with and regulate messenger RNA (mRNAs) or microRNAs by speci cally competing for shared miRNAs, thereby acting as competing endogenous RNAs (ceRNAs) (9).
To establish if lncRNAs play a role in the progression of LUSC, we constructed a global triple RNA network based on the ceRNA theory using data from The Cancer Genome Altas (TCGA) database (10). Important hub lncRNAs were identi ed in the lncRNA-microRNA-mRNA network, and new subnetworks were formulated that centered on these lncRNAs. These data provide valuable insight into the molecular progression of LUSC and contribute to the identi cation of potential pathogenic mechanisms.
Furthermore, these ndings may contribute to improvements in the diagnosis and prognosis of LUSC patients and help with the identi cation of putative drug targets.

Data collection and preprocessing
The data for analysis were downloaded from the TCGA database website using the GDC tools provided by the TCGA itself (https://portal.gdc.cancer.gov/). The downloaded data consisted of raw nonnormalized quantitative gene expression data (count form data), microRNA isoform expression data, and general clinical data, including the age, gender, survival status, and tumor stage of patients, to further validate our results. The downloaded data were collated using the "GDCRNATools" package (11). Ethical approval was not necessary for our study because the expression pro les were downloaded from the public TCGA database, and no new experiments in patients or animals were conducted.

Screening of differentially expressed genes (DEgenes)
The "edgeR" package(12) of the R program was used to screen differentially expressed mRNAs (DEmRNAs), lncRNAs (DElncRNAs), and miRNAs (DEmiRNAs) between primary tumor and solid normal tissue samples in the count form expressing data. Values of |Log 2 FC| > 2 and FDR < 0.01 were used as the criteria to identify signi cant differences in gene expression.

Functional enrichment analysis and gene set enrichment analysis (GSEA)
To further study the function of hub mRNAs, gene ontology (GO) and KEGG pathway enrichment analyses were conducted using the "enrichDAVID" function of the "clusterPro ler" package (14) in the R program. Values of P < 0.05 were used as the cutoff criteria. The "gseGO" and "gseKEGG" functions of the GSEA "clusterPro ler" package of the R program were used to identify the potential functions of the identi ed lncRNAs and mRNAs. GSEA was conducted to detect whether previously de ned biological processes (BPs) were enriched in the gene rank derived from hub lncRNAs and mRNAs between the two groups. Terms with an FDR < 0.01 were identi ed for all enriched DEGs.

Survival analysis
The "gdcSurvivalAnalysis" function in the "GDCRNATools" package of the R program was used to conduct a survival analysis of the hub lncRNAs and mRNAs. P < 0.05 was considered signi cant.

DEmRNAs, DElncRNAs, and DEmiRNAs in LUSC samples
The RNA expression pro les-labeled TCGA-LUSC and corresponding clinical information were downloaded from the TCGA database website. According to the GDC data dictionary, samples with "01B" at the end of the sample ID are formalin-xed samples with low sequencing quality, which were removed from our study ( ve samples were removed). One duplicate sample (sample ID: TCGA-21-1076-01A) was also removed. To remove signi cant outlier samples, correlation and cluster analyses were conducted.
3.4 Predicated function of hub mRNAs within the ceRNA network GSEA was conducted on the identi ed DEmRNA set, focusing on the BP subclass. The results showed that "regulation of signal transduction," "positive regulation of signal transduction," "regulation of cell migration," "regulation of locomotion," and "regulation of cell motility" were the top ve signi cantly enriched processes ( Figure S6). ZEB2 was simultaneously enriched by multiple functions (Figure 4).
The protein-coding genes contained in the ceRNA network were used for GO and KEGG pathway enrichment analyses. The DAVID database (19,20) was used as the basis for these analyses. The 10 most signi cant BP, CC, and KEGG pathway terms are shown in Figure S7. These genes are widely involved in tumor-related functions and pathways.

Discussion
The molecular mechanisms that contribute to LUSC remain unclear, which hinders early diagnosis, prognosis predictions, and therapeutic target selection in LUSC. Using LUSC samples collected in the TCGA database, we identi ed potential DEgenes and DElncRNAs. We then constructed a ceRNA network by combining these data with microRNA expression pro les. Additionally, the detailed post-transcriptional regulatory mechanisms of genes involved in the pathological process of LUSC were analyzed, and hub lncRNAs and genes were screened. The present study found that MAGI2-AS3 might be a hub lncRNA involved in LUSC progression. It formed ceRNA networks with miR-374 family members (miR-374a-5p and miR-374b-5p), which are known to participate in cell signal transduction, cell metabolism, and cell migration in LUSC. These processes play an important role in the occurrence, progression, and metastasis of LUSC.
According to the ceRNA hypothesis, lncRNAs can act as molecular sponges of miRNAs and effectively occupy their binding sites, thereby releasing target mRNAs and increasing their post-transcriptional levels.
The present study showed that MAGI2-AS3 expression was signi cantly positively correlated with ZEB2 expression; both of which were downregulated in LUSC samples. Through the construction of the ceRNA network and corresponding analysis, it was shown that MAGI2-AS3 or ZEB2 could act as a molecular sponge of miR-374 family members (miR-374a-5p and miR-374b-5p). The downregulation of MAGI2-AS3 might increase the available binding sites of miR-374 and promote the degradation of downstream mRNAs, thereby leading to the downregulation of ZEB2, and vice versa.
Recently, it was con rmed that MAGI2-AS3 functioned as a sponge of miR-374a/b-5p in vitro (21). The present study veri ed that MAGI2-AS3 overexpression could inhibit the colony-forming ability of LUSC cells and signi cantly reduce cell metastasis and invasion. Bioinformatics analysis of prognostic biomarkers for human bladder cancer (22) showed that MAGI2-AS3 was downregulated in bladder cancer samples, and it was identi ed as the hub lncRNA in the ceRNA network. The tumor-suppressive role of MAGI2-AS3 has also been con rmed in multiple studies related to breast cancer (23)(24)(25). In a study on hepatocellular carcinoma (HCC) (26), the tumor-suppressive role of MAGI2-AS3 and targeted regulatory relationship between MAGI2-AS3 and miR-374b-5p were also con rmed. A previous study on epithelial ovarian cancer (27) also con rmed that MAGI2-AS3 played a tumor-suppressive role, which was achieved by targeting miR-374a/b. However, these studies did not further determine the downstream targets of miR-374a/b-5p in the ceRNA network using reliable experiments.  (21). The results of the present study showed that MAGI2-AS3 expression was signi cantly reduced in LUSC samples and that MAGI2-AS3 was involved in the ceRNA network together with miR-374a/b-5p, which is consistent with previous studies. However, no differential expression of SMG1, PTEN, or CADM2 was found in the LUSC samples in our study. Therefore, we consider that the MAGI2-AS3-mediated regulation of cell functions via acting as a molecular sponge of miR-374a/b-5p was achieved through other targets.
The miR-374 family has been shown to be involved in many physiological and pathological processes. This family has been well studied in digestive system tumors but not in other tumors. It is speculated that this family may regulate cell differentiation and proliferation and be associated with the occurrence of tumors (28). In the present study, microRNAs targeting ZEB2 were investigated using the TargetScan online database (http://www.targetscan.org/vert_72/), and the results revealed that the miR-374 family targeted and regulated ZEB2 ( Figure S8). Based on the results of the present study, MAGI2-AS3, the miR-374 family, and ZEB2 constitute a ceRNA regulatory network and participate in the regulation of LUSC progression.
We suggest that ZEB2 plays very diverse roles in LUSC. ZEB2 expression was low in tumor tissues, suggesting that ZEB2 may play a tumor-suppressive role and that a decrease in ZEB2 expression promotes tumor progression. However, the results of survival analyses demonstrated that low ZEB2 expression was a good prognostic factor for patients with LUSC and contributed to improvements in the 5 year survival rate. This seemingly contradictory result stimulated great interest by our research team. It has been proposed that mRNAs and proteins encoded by the same sequence of a gene may have different biological effects in certain situations. ZEB2 is a practical example that supports this hypothesis (29).
The protein encoded by ZEB2 is a member of the Zfh1 family of two-headed zinc nger/homeodomain proteins that function as DNA-binding transcriptional repressors (15,30). It has been con rmed that ZEB2 is widely involved in various tumor-related pathological processes, including proliferation induction, differentiation arrest, apoptosis suppression, and angiogenesis (29). ZEB2 promotes tumor progression and invasion by controlling epithelial-to-mesenchymal transition (EMT) in epithelial cancers (31). ZEB2 has been identi ed as a molecular switch of EMT (29). In prostate cancer (PCa), miR-200c-3p was con rmed to inhibit EMT by targeting ZEB2 and thus reducing the migration and invasion of PCa cells (32). Similarly, miR-145 and ZEB2 form a two-way negative feedback loop that achieves complex functional regulation during PCa invasion and migration and plays a key regulatory role in different pathological processes in the bone metastasis of PCa (31). ZEB2 is also con rmed to be a necessary factor for tumor metastasis in melanoma cells, and the ZEB2-mediated regulation of EMT is one of the most essential mechanisms for the metastasis and ectopic growth of melanoma (33). Furthermore, in a study on triple-negative breast cancers (TNBCs), the researchers con rmed that p53 promoted the transcription of miR-30a-3p and miR-30a-5p, which directly target ZEB2, to inhibit the invasion and distant metastasis of tumor cells. This mechanism is related to the ability of ZEB2 to regulate EMT.
Moreover, the inactivation of p53 could affect the biological behavior of TNBCs (34).
Therefore, ZEB2 plays an important role in regulating the invasion and migration of tumor cells. This conclusion may explain the contribution of low ZEB2 expression to the good prognosis of LUSC patients.
However, compared with paracancerous normal tissues, we found that ZEB2 mRNA was downregulated in the LUSC samples. According to previous studies, the downregulation of ZEB2 expression promotes the apoptosis of tumor cells but inhibits their invasion and migration. Therefore, we believe that the downregulation of ZEB2 mRNA in LUSC samples achieved the post-transcriptional regulation of other molecules at the mRNA level. TP53 and PTEN, two important tumor-related molecules, are commonly inactivated in LUSC (5). Thus, although TP53 and PTEN are important components of the ZEB2/miR-374 regulatory axis in other tumors (24,34), they likely do not contribute to the regulatory mechanism of the ZEB2/miR-374 axis in LUSC. The ceRNA regulatory network composed of ZEB2, MAGI2-AS3, and miR-374 family members might play key roles in extensive post-transcriptional regulation. ZEB2 might target the miR-374 family and competitively bind to miR-374, thereby modulating MAGI2-AS3 expression and further regulating the function of downstream molecules. Downregulated ZEB2 mRNA expression may increase the available competitive binding sites on miR-374, thereby enhancing its targeted regulation of other downstream factors. Moreover, MAGI2-AS3 may be directly targeted and regulated by miR-374, resulting in the subsequent downregulation of MAGI2-AS3 expression and induction of tumor progression in LUSC. However, low MAGI2-AS3 expression was also associated with a good prognosis of LUSC patients, indicating that MAGI2-AS3 may have more unknown functions in LUSC. We speculate that the ceRNA network composed of MAGI2-AS3, miR-374, and ZEB2 might have a complex two-way regulatory effect on different biological functions in various stages of tumor development. Additionally, as a transcription factor, ZEB2 might also directly target miR-374/MAGI2-AS3 or other downstream molecules, such as SMADs, to participate in cell signal transduction and more complex regulatory mechanisms.
We believe that the ceRNA regulatory network composed of MAGI2-AS3, miR-374, and ZEB2 has signi cant research potential. In the future, our team plans to further study the relevant molecular mechanisms involved in the occurrence and progression of LUSC to explore potential therapeutic targets and improve the e cacy of LUSC treatments.

Declarations
Ethics Approval and Consent to Participate: Not applicable.

Consent fot Publication:
Not applicable.
Availability of Data and Materials: The data that support the ndings of this study are available in TCGA database at https:// portal.gdc.cancer.gov/projects/TCGA-LUSC.
Con ict of Interest: The authors declare that they have no con ict of interest.
Funding Statement: No funding was recived fot this study.
Author's Contribution: Doctor CH and Professor JL made contribution to conception and design of the study. Doctor ZW made contribution to collection and analysis the TCGA data and the interpretation of the results. DoctorCH and Doctor QW worked together to draft the manuscript. Doctor JianjunL produced the gures. Professor JL revised the manuscript critically for important intellectual content. And Professor JL agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the nal manuscript.