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Reconstruction and analysis of the aberrant lncRNA-miRNA-mRNA network based on competitive endogenous RNA in adenoid cystic carcinoma of the salivary gland

Background The aim of this work was to investigate the competing endogenous RNA (ceRNA) network in adenoid cystic carcinoma of the salivary gland (SACC). Methods Differentially expressed lncRNAs (DElncRNAs), miRNAs (DEmiRNAs), and mRNAs (DEmRNAs) between cancer tissues and normal salivary gland (NSG) in ACC were identified using data from the Gene Expression Omnibus (GEO) database. Functional annotation and pathway enrichment analysis of DEmRNAs were performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The miRNAs that are targeted by lncRNAs were predicted using miRanda and PITA, while the target mRNAs of miRNAs were retrieved from miRanda, miRWalk, and TargetScan. A protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, and then we constructed the lncRNA-miRNA-mRNA networks of ACC. Results Differentially expressed RNAs were identified in SACC. Upon comparing cancer tissues and NSG tissues, 103 upregulated and 52 downregulated lncRNAs and 745 upregulated and 866 downregulated mRNAs were identified in GSE88804; in addition, 39 upregulated and 43 downregulated miRNAs were identified in GSE117275. GO enrichment analyses revealed that the most relevant GO terms were regulation of transcription DNA-templated, transcription DNA-templated, and cell division. KEGG pathway enrichment analysis showed that differentially expressed genes (DEGs) were mainly enriched in the cell cycle, pathways in cancer, PI3K-Akt signaling pathway, breast cancer, and microRNAs in cancer. The PPI network consisted of 27 upregulated and 54 downregulated mRNAs. By constructing ceRNA network, NONHSAT251752.1-hsa-miR-6817-5p-NOTCH1, NONHSAT251752.1-hsa-miR-204-5p/hsa-miR-138-5p-CDK6 regulatory axises were identified and all genes in the network were verified by qRT-PCR. Conclusions The present study constructed ceRNA networks in SACC and provided a novel perspective of the molecular mechanisms for SACC.


Introduction
Adenoid cystic carcinoma (ACC) is a relatively rare cancer, accounting for 1% of head and neck tumors and 10% of salivary gland tumors (1)(2)(3). SACC is characterized by high rates of recurrence, perineural invasion, and distant metastasis (2,3). Current treatments are still mainly surgery and adjuvant radiation, and no systemic chemotherapy has been proven to be fully effective (4). However, over time, most patients develop recurrence and distant metastasis (5). One study had suggested that the overall 5-, 10-, and 15year survival estimates for all stages among ACC were 68%, 52%, and 28%, respectively (6). Despite some remarkable achievements have been made recently, the etiology and pathogenesis of SACC remain largely unclear (7). Therefore, it is important to have a deeper understanding of the molecular mechanism underlying SACC tumorigenesis and to seek new therapeutic strategies for SACC.
Long noncoding RNAs (lncRNAs), a class of noncoding RNAs (ncRNAs) with no known protein-coding functions and a length of more than 200 nucleotides (8), are involved in numerous important cell biological processes, including cellular homeostasis and transcriptional and posttranscriptional regulation (8,9). MicroRNAs (miRNAs), a type of short ncRNA with approximately 22 nucleotides (10), can partially or entirely bind to the 3' untranslated regions (3' UTRs) of target genes to promote the degradation of targeted mRNAs and translation suppression, along with the negative regulation of gene expression at posttranscriptional levels (11,12). The competing endogenous RNA (ceRNA) hypothesis was proposed by Salmena et al. (13) as early as 2011, according to the ceRNA hypothesis, endogenous RNA molecules have miRNA action sites and can competitively bind to miRNAs, thus indirectly regulating the use of miRNA target factors. A series of studies have shown that miRNAs can silence genes by binding to mRNA (14), and lncRNA can be a "molecular sponge" of miRNA, binding miRNA to attenuate its silencing effects on target genes and to regulate them (15). Therefore, the interaction between three types of RNAs results in the formation of lncRNA-miRNA-mRNA networks, which gives rise to ceRNA.
To date, numerous experimental studies have supported the theory of ceRNA network regulation and it is reported that ceRNA network has been constructed in gastric cancer (16), hepatocellular carcinoma (17), cervical cancer (18), ovarian clear cell carcinoma (19) and other cancers. However, ceRNA networks have rarely been reported in the field of SACC. The rapid development of gene sequencing technology and the establishment of online public database provide a resource platform for data mining and research. In our work, the disciplinary advantages of molecular biology and bioinformatics were integrated to construct a molecular regulatory network in ACC, and we screened RNAs that may be associated with SACC. Through this analysis, we aim to better understand the pathogenesis of SACC and reveal some potential lncRNA biomarkers.

Data downloading and preprocessing
We obtained RNA sequencing data from the Gene Expression Omnibus (GEO) database. The lncRNA/ mRNA expression profile data were downloaded from GSE88804 (https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE88804) RNA sequencing datasets, which contained 13 SACC tissues and 7 normal salivary gland (NSG) tissues. The miRNA expression profile data were extracted from the GSE117275 (https://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc=GSE117275) database, which contained 3 SACC tissues and 3 NSG tissues. The downloaded raw lncRNA and mRNA data were identified and annotated by BLAST comparison according to gene annotation files in the NCBI, Ensembl, and NONCODEv5 databases. All data from GEO are publicly available and they do not require the approval of a local ethics committee. The analysis process of this experiment is shown in Figure 1.

Tissue collection
Samples came from SACC patients who had finished the operation at our center from August 2020 to March 2021. These patients met the following inclusion and exclusion criteria: the tumor was primary, the pathological type was ACC, patients with preoperative radiotherapy or chemotherapy were excluded, patients with other malignant tumors were excluded. A total of 3 samples were used in our study. Immediately after tumor resection, tumors and normal salivary gland tissues were frozen in liquid nitrogen, and stored at −80 ℃. Both preoperative biopsy and postoperative histopathology were diagnosed as ACC of the salivary gland. Three matched SACC tissue samples and corresponding normal salivary gland tissues were investigated by quantitative real-time polymerase chain reaction (qRT-PCR). The study was approved by the Ethics Committee and was conducted under the guidance of international ethical standards (IRB number: PKUSSIRB-202056098). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Written informed consent was obtained from all patients.

Functional enrichment analysis of DE-mRNAs
Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) functional enrichment analyses were conducted on DE-mRNAs using the R clusterProfiler package (22). Fisher's test was used to identify the significant GO terms and KEGG pathways, and P value <0.05 was considered statistically significant.

Construction of the PPI network and screening for the hub genes
Protein-protein interaction (PPI) network prediction for target genes was carried out using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (https://string-db.org/). Based on the PPI network, we chose the top several genes with the highest degree of connections to other genes as hub genes. In addition, mRNAs in the modules were selected as core RNAs to constitute a subnetwork.  Table 1. The primer sequences of U6 were not available from the provider. The relative expression of lncRNAs, mRNAs, and miRNAs was calculated by the 2 −△△Ct method. The qPCRs were all repeated three times for the three matched samples.

Statistical analysis
The Student's t-test was used to evaluate the differences of mRNAs, miRNAs and lncRNAs expression between cancer and normal samples. Pathway enrichment analysis was estimated by Fisher's test, and false discovery rate (FDR) was calculated to correct the P values. The results of RT-qPCR were repeated three times for the three matched samples. Data were presented as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8.0.2 (GraphPad Software, USA). Differences between the two groups were determined by t-test. P<0.05 was considered statistically significant.

Identification of DE-mRNAs, DE-lncRNAs, and DE-miRNAs
In total, 13 SACC tissues and 7 NSG tissues were available in the GSE88804 dataset, and 3 SACC tissues and 3 NSG tissues were available in the GSE117275 dataset. Through representational difference analysis, we identified 1,  Table 2, and clustergrams of DE-lncRNAs, DE-miRNAs, and DE-mRNAs were generated (shown in Figure 2).

GO and KEGG analyses of significant DE-mRNAs
The significantly DE-mRNAs were utilized for GO and KEGG analyses. The results revealed that these mRNAs were mainly involved in 853 GO terms and 77 KEGG pathways. The up and down-regulated genes were mainly enriched for the following GO terms: regulation of transcription DNA-templated, transcription, cell division, negative regulation of transcription from the RNA polymerase II promoter, cell proliferation, oxidationreduction process, signal transduction, positive regulation of transcription from RNA polymerase II promoter, neutrophil degranulation, negative regulation of cell proliferation. The top 25 GO functions according to the P value are shown in Figure 3. The most significant KEGG pathway terms for the DE-mRNAs are in cell cycle, pathways in cancer, PI3K-Akt signaling pathway, breast cancer, microRNAs in cancer, metabolic pathways, salivary secretion, PPAR signaling pathway, pathways in cancer, AMPK signaling pathway. By combining GO and KEGG pathway analyses, 539 significant mRNAs were obtained. The top 25 KEGG pathways according to the P value are shown in Figure 4.

Construction of the lncRNA-miRNA-mRNA network
By predicting 155 DE-lncRNAs, 82 DE-miRNAs, and 539 mRNAs, we constructed 535 lncRNA-miRNA interaction pairs and 419 miRNA-mRNA interaction pairs. A total of 76 lncRNAs and 80 miRNAs were included in the lncRNA-miRNA pairs, and 73 miRNAs and 192 mRNAs were included in the miRNA-mRNA pairs. Depending on the interactions between lncRNA-miRNA and miRNA-mRNA, we screened the negative correlation of miRNA-mRNA and miRNA-lncRNA pairs and constructed two lncRNA- GCGGCGGAAAATAGCCTTTG GATCACACGTTCCACCTCATC miRNA-mRNA ceRNA networks. The lncRNA (downregulated)-miRNA (up-regulated)-mRNA (down-regulated) network consisted of 22 lncRNAs, 31 miRNAs, and 71 mRNAs with a total of 480 interactions. The lncRNA (upregulated)-miRNA (down-regulated)-mRNA (up-regulated) network consisted of 38 lncRNAs, 18 miRNAs, and 32 mRNAs with a total of 344 interactions. Subsequently, we combined the files of these pairs and visualized them using Cytoscape, yielding a ceRNA topology network ( Figure 5).

PPI network construction
Based on the STRING database, we used the ceRNArelated DE-mRNAs to build a PPI network for investigating      the function of DE-mRNAs at the protein level and filtering for functional genes. The PPI network is composed of 59 up-regulated mRNAs and 55 down-regulated mRNAs. The PPI network is shown in Figure 6.

Validation of part of the DE-RNAs using qRT-PCR
To verify the results of bioinformation analysis, a total of 33 DE-RNA transcripts were validated, including 4 lncRNAs, 14 mRNAs, and 15 miRNAs, the selected DE-RNAs are shown in Table 3. The results of 29 (87.9%, 29/33) RNA transcriptions were consistent with the sequencing data (P<0.05) (Figure 7) by comparing the expression level in SACC tissues and NSG tissues, except for has-mi-34c-5p, has-mi-135a-3p, has-mi-34b-5p, has-mi-20b-3p.

Discussion
In our study, we present the different expression profiles of lncRNA, miRNA, mRNA in SACC by bioinformation analysis, attempting to further understand the molecular mechanism of SACC.  (41), which indicate that metabolism plays an important role. In addition, we tested the reliability of bioinformatics analysis by qRT-PCR, 29 (87.9%, 29/33) DE-RNAs were consistent with bioinformatics analysis, only 4 DE-miRNAs had the opposite results by comparing with NSG tissues. In the process of verifying the reliability of bioinformatics analysis, we also found a significantly high-expressed gene SOX4, and knockdown of SOX4 expression induces apoptosis in ACC3 cells in the literature (42). In the end, according to the node degree, the regulation and control abilities in the PPI network, P values, and combined with ceRNA network, NONHSAT251752.1-hsa-miR-6817-5p-NOTCH1, NONHSAT251752.1-hsa-miR-204-5p/hsa-miR-138-5p-CDK6 regulatory axises were identified.

NSG SACC
Additionally, a study showed that NOTCH1 mutation has been defined as a distinct group characterized by solid histology, with a propensity to bone and liver metastasis, and poor prognosis (53). Notch-1 knockdown suppresses the growth and migration of SACC cells in vitro and the metastasis of SACC cells in vivo (54). In conclusion, Notch-1 plays an important role in SACC and it is a vital candidate target in SACC. In our study, NOTCH1 has the most complex interaction with other mRNAs in PPI interactions and it was confirmed to be elevated in SACC tissues, which was consistent with the result of the previous study (55). The family of cyclin-dependent kinases (CDKs) covers 13 different serine/threonine kinases. CDK6 is known as classic cell cycle kinases that facilitate the progression of cells through the early G1 phase of the cell cycle by forming complexes with D-type cyclins (D1, D2, and D3). CDK6 plays an important role in promoting cancer initiation, especially in hematologic malignancies, breast cancer, melanoma. At the same time, CDK6 is also inextricably linked to immune functions (56). Overexpression of CDK6 has been reported in T-cell lymphoblastic lymphoma (57) and leukemia (T-LBL or T-ALL) and in B-lymphoid malignancies (58), which is consistent with our result. According to studies, the regulatory axis of ceRNA targeting CDK6 has been reported many times. LncRNA AWPPH accelerates the progression of non-small cell lung cancer by sponging miRNA-204 to upregulate CDK6 (59). RP11-480I12.5-004 Promotes Growth and Tumorigenesis of Breast Cancer by Relieving miR-29c-3p-Mediated AKT3 and CDK6 degradation (60).

Conclusions
Through bioinformatics analyses, the significantly upregulated and downregulated RNA and pathways in SACC were found, constructing the lncRNA-miRNA-mRNA interaction network and PPI network in SACC. Moreover, we build NONHSAT251752.1-hsa-miR-6817-5p-NOTCH1, NONHSAT251752.1-hsa-miR-204-5p/ hsa-miR-138-5p-CDK6 regulatory networks. However, the detailed regulation effect and underlying mechanism of the regulatory axis still need further investigation and it would be our focus in future work. This study provides some key clues for molecular mechanistic investigations of SACC in the future.  Ethical Statement: The authors are 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. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee and was conducted under the guidance of international ethical standards (IRB number: PKUSSIRB-202056098). Written informed consent was obtained from all patients.
Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons