Bioinformatics analysis of the expression and role of microRNA-221-3p in the head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer subtype globally, associated with a high rate of morbidity and mortality. However, the target genes of miR-221-3p and the underlying mechanism involved in HNSCC were not known. Therefore, in the current study, we studied the role of miR-221-3p in the HNSCC. Tissues collected from 48 control and 21 HNSCC patients were processed to check the differential expression of miR-221-3p by Real-time RT-Polymerase Chain Reaction (RT-qPCR). Overexpression of microRNA-221-3p (miR-221-3p) is signicantly correlated to the onset and progression of HNSCC. We also conducted the meta-analysis of the cancer literature from the cancer genome atlas (TCGA) and the Gene Expression Omnibus (GEO) database to estimate the expression of miR-221-3p in HNSCC. The miR-221-3p target genes in the HNSCC were predicted with the miRWalk and TCGA databases, and functionally annotated via the Gene Ontology Finally, Spearman’s analysis was used to determine the role of the related target genes in important pathways involved in the development of HNSCC. HNSCC:Head and neck squamous cell carcinoma; miR:microRNA; RT-qPCR:Real-time RT-Polymerase Chain Reaction; PPI:protein-protein interaction; GO:Gene Ontology; KEGG,Kyoto Encyclopedia of Genes and Genomes; GEO:Gene Expression Omnibus; TCGA: The cancer genome atlas; DAVID:Database for Annotation, Visualization andIntegrated Discovery; BiNGO: Biological Networks Gene Oncology tool; BP:biological process; CC: cellular component; MF: molecular function;


Background
Head and neck squamous cell carcinoma (HNSCC)encompassing the oral squamous cell carcinoma, oropharyngeal squamous cell carcinoma, hypopharyngeal squamous cell carcinoma and laryngeal squamous cell carcinoma is the sixth most common cancer worldwide, accounting for approximately 1-2% of all cancer deaths, with about 600,000 detection of new cases each year globally [1,2,3].The treatment of patients at the early stage is relatively successful, but the overall survival rate of recurrent or metastatic HNSCC remains low and has barely seen any improvements in their survival for decades [4,5].
Although the past three decades have seen several improvements in the diagnostic tools and treatment regimens, the overall survival rate for the advanced (stage -) head and neck squamous cell carcinoma (HNSCC) is only 65% [6,7]. The current standard of care for such patients are surgery along with radiation and chemotherapy, but these have not signi cantly improved the 5-year survival of the HNSCC patients.
There is an urgent need for the e main reason for the decline in patient survival is the lack of a powerful therapeutic target for the development of HNSCC [8,9].
Recent years have seen an increasing concentration of research in, microRNA (miRNA) [10,11], which are a type of highly conserved single-stranded non-coding RNA, containing 17~22 nucleotides and are involved in the process of tumorigenesis, cell survival, and chemosensitivity [12,13,14]. They bind the 3'non-region (3'UTR) of different target mRNA (messenger RNA) genes to degrade or inhibit the mRNA translation of target genes associated with a tumor suppressor function [13,15,16]. In normal healthy individuals, miR-221-3p is observed to play a role in the process of vascular proliferation [17], while the tumor promoter, microRNA-221 is involved in the process of regulation of apoptosis of the tumor cell [18,19,20] and is associated with a variety of cancer types, including hepatocellular cancer [21], cutaneous melanoma [19], prostate cancer [20],non-small cell lung cancer [22].
Studies have shown that speci c miRNA pro les can be identi ed between tumor tissue and adjacent healthy tissue in the HNSCC patients [2,23]. For instance, studies have reported the link between miR-211 and vascular invasion during the incidence of HNSCC [14,15,24,25,26]. However, the de nite target gene of the miR-211-3p and its biological mechanism of action are still not clear. Thus, in the current study, we investigated the expression of miR-221-3p in the HNSCC and attempted to explore the correlation between the two.

RT -qPCR
Tissue specimens were collected from 48 control and 21 HNSCC patients from the Department of Pathology, First A liated Hospital of Guangxi Medical University (Nanning, Guangxi Medical University), and processed for RT-qPCR with the SYBR-Green Master mix (Takara, Tokyo, Japan) in a ABI 7500 cycler (Applied Biosystems) with the following conditions: initial denaturation at 95˚C for 30 sec, followed by 40 cycles of denaturation at 95˚C for 5 sec and annealing at 60˚C for 34 sec. The differential expression of miR-21 in the HNSCC tissues relative to the negative control (NC) tissues was calculated using the 2-ΔΔ Ct method, with U6 as the internal control. The primers of miR-221-3p and U6 were synthesized by TaKaRa (Dalian, Liaoning, China) and the sequences are as follows: miR-221-3p: Forward 5' -AGCUACAUUGUCUGCUGGGUUUC -3' and Reverse 5' mRQ 3'; U6: Forward 5' -GGAACGATACAGAGAAGATTAGC-3' and Reverse 5' -TGGAACGCTTCACGAATTTGCG -3'. All the experiments were repeated three times. Database for this study. We searched the database from the earliest available data beginning to October 1, 2019.The following keywords were used: (HNSCC OR SCC OR "squamous cell cancer" OR "squamous cell carcinoma") AND (oropharynx OR oropharyngeal OR "head and neck" OR nose OR nasopharynx OR "nasal sinus" OR "nasal cavity" OR "oral cavity" OR hypopharynx OR oral OR laryngopharynx OR larynx OR laryngopharyngeal OR laryngeal OR pharyngeal OR tongue OR tonsil OR tonsillar OR cheek OR palatal OR "paranasal sinuses" OR buccal OR lip) AND (microRNA-221-3p OR miRNA-221-3p OR "miR 221-3p" OR "miRNA 221-3p" OR miRNA221-3p OR miR221-3p) 2.3. Selection criteria and data extraction.
The databases were searched independently by two researchers who selected the studies based on the following inclusion criteria: (1) comparison of HNSCC and non-cancerous tissues, (2) validation of miR-221-3p expression levels via reverse transcription quantitative PCR (RT-qPCR), (3) evaluation of the association between miR-221-3p expression and clinical outcomes, and (4) availability of su cient data to calculate the mean, standard deviation (SD) and 95% con dence intervals (95% CI). The articles were eliminated if they met one of the following exclusion criteria: (1) irrelevant to the research focus, (2) inclusion of unquali ed data, (3) publication language other than English or Chinese, (4) overlapping or duplicate publications, and (5) letters, reviews, comments, editorials, conference articles, laboratory studies or case reports. The reviewers independently appraised the quality of data in each eligible study and extracted the rst author name, year of publication, country of origin, sample type, sample size and analysis method. For articles with incomplete information, the authors were contacted to obtain relevant information.

Microarray data collection from GEO.
Microarray data of HNSCC samples uploaded till October 1, 2019 was obtained from GEO database (https://www.ncbi.nlm.nih.gov/gds/) using the following keywords: (HNSCC OR SCC OR "squamous cell cancer" OR "squamous cell carcinoma") AND (oropharynx OR oropharyngeal OR "head and neck" OR nose OR nasopharynx OR "nasal sinus" OR "nasal cavity" OR "oral cavity" OR hypopharynx OR oral OR laryngopharynx OR larynx OR laryngopharyngeal OR laryngeal OR pharyngeal OR tonsil OR tonsillar OR tongue OR cheek OR palatal OR "paranasal sinuses" OR buccal OR lip) AND ( microRNA OR miRNA OR "miR" OR "miRNA"). The inclusion criteria for the microarray datasets were: (1) comprising of data from HNSCC and non-cancerous tissues, (2) evaluation of the association between miR-221-3p expression and clinical outcomes, (3) availability of su ciently data to calculate mean, SD and 95% CI. The exclusion criteria were: (1) unrelated to this study, (2) unquali ed data, (3) publication language other than English.

Statistical analysis
Continuous variables and meta-analysis of the data of the available cancer literature, The Cancer Genome Atlas (TCGA) and GEO Gene Expression Omnibus). The meta-analysis data was veri ed by PCR. Further, we used diagnostic meta-analysis to estimate the potential diagnostic value of miR-221-3p. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were used to comprehensively analyze the possible molecular mechanisms of miR-221-3p in HNSCC.

Comprehensive statistical analysis of the data
Data which were removed from our meta-analysis as well as TCGA data (Nanning, Guangxi Zhuang Autonomous Region, China) were analyzed using the STATA 15.0 software. Standard mean difference (SMD) and 95% con dence interval were used to estimate the expression value of miR-221-3p. In normal and HNSCC, scatter plots prepared with the GraphPad Prism 6 (Nanning, Guangxi Zhuang Autonomous Region, China) of the data of both the normal and HNSCC tissues were used to assess the expression level of miR-221-3p. Evidence of bias was assessed by the visual funnel plots and Egger's regression asymmetry test. In addition, the I 2 index was used to evaluate the potential heterogeneity of the selected data. When I 2 >50% or P<0.05, we used the xed effect model; otherwise, the random effect model was used. SPSS 24.0 software (IBM, Somers, NY) was used for the statistical analysis. Mean ± SD (X ± s) and t test for comparisons between groups were applied all to the measurement data. The count rate was expressed as a percentage (%), and we used the X2 test for comparison. Based on true positive (TP), false positive (FP), false negative (FN) and true negative (TN) to determine the diagnostic value of miR-221-3p, we used the meta-disc software to calculate the speci city, sensitivity, likelihood ratio (negative and positive) receiver operating characteristic curve (sROC). We also did the sensitivity analysis to assess the differences between the sample sizes. Moreover, all the miR-221-3p expression data which included the TCGA sequence was normalized to log2 to improve the normality of the measurements. P<0.05 is considered a statistically signi cant value.

Predicting target genes of mir-221 in HNSCC
The Gene Ontology (GO) [27]analysis was used to de ne biological processes (BP), molecular function (MF), and cellular components (CC), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) [28] pathway enrichment analyses were conducted using R (version 3.6.1) to visualize the ClusterPro ler of R. The Search Tool for the Retrieval of Interacting Genes (STRING) (https://string-db.org/) was then used to establish a protein-protein interaction (PPI) network of the miR-221-3p target genes associated with the signi cantly enriched pathways. The expression levels of the miR-221-3p target genes in the HNSCC and non-tumor tissues were determined with the UALCOULD [29] (http://ualcan.path.uab.edu/index.html).
Finally, the Linkedomics [30] (http://www.linkedomics.org/) Spearman's analysis tool was applied to determine the correlation between the expression levels of the miR-221-3p and the potential target genes involved in notable signaling pathways.

miR-221-3p is highly expressed in the tumor tissues
The RT-qPCR data clearly showed that miR-221-3p is highly expressed in the tissues of the nasopharyngeal carcinoma patients as compared to the control group(p<0.05) (Figure 1).

Bias risk assessment
The results of the visual funnel plots show a symmetrical shape, which indicate absence of any signi cant publication bias as a whole. Sensitivity analysis performed on the heterogeneity samples showed that there did not exist any signi cant difference between the studies (Figure 3).

Expression and meta-analysis of miR-221-3p in the HNSCC
From the total of 911 HNSCC samples and 289 normal samples of the Pubmed, GEO and TGCA datasets, we observed that miR-221-3p is overexpressed in the HNSCC compared to the normal tissues (P<0.05).
Which is consistent with the results obtained by RT-qPCR. Meta-analysis to explore the level of expression of mrR-221-3p in HNSCC indicate a high degree of heterogeneity between these studies. Therefore, the random-effect mode was selected and the combined standard deviation (SMD) was observed to be 0.717(95% CI:0.283, 1.152) (Figure 4).. The meta-analysis data indicate that miR-221-3p expression is signi cantly upregulated in the HNSCC. In addition, the expression levels of mir-221-3p in HNSCC and normal tissues from each included dataset is shown in Figure 5 (A-R).

Clinical -pathological features of HNSCC based on the TCGA
Based on the sample data of the HNSCC extracted from the TCGA database, we analyzed 527 samples, which indicate the absence of any statistically signi cant difference between miR-221-3p expression and age, lymphovascular invasion Neoplasm histologic grade or any other clinical-pathological features. However, it indicated a statistically signi cant difference in the expression of miR-221-3p that mainly refer to the different tissue, gender and clinical stage, even alcohol. (Table 2)..

The prospective target genes of miR-221 in HNSCC
By using MiRWalk2.0 [32] (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/), and GEPIA(http://gepia.cancer-pku.cn/index.html) analysis, we retrieved the number of the differentially expressed genes to be 5311 and 467respectively. Bioinformatics analysis showed a total of 117 overlap genes which were involved, as shown in Figure 9 and Table 3. The enriched GO and KEGG pathway category overlapping genes with p<0.05 are shown in Figure 10 , table 4 and table5. For the cellular component the identi ed target genes are mostly enriched in the actin cytoskeleton, sarcomere, contractile ber part and the molecular function is mainly concerned with the aromatase activity, oxidoreductase activity, and cofactor binding. KEGG enrichment analysis showed that miR-221-3p plays a signi cant role in HNSCC through a variety of pathways, including the drug metabolism pathways of the cytochrome P450 signaling pathway. The protein-protein interaction (PPI) network of drug metabolism -cytochrome P450-related genes is shown in the gure 11.
3.8. Validation of the miR-221-3p target genes in the drug metabolism -cytochrome P450 signaling pathway Seven target genes associated with miR-221-3p are also involved in the drug metabolism -cytochrome P450 pathway and are down-regulated. The expression value of each of the target genes are shown in Figure 12. The Spearman's correlation analysis showed that the correlation of the certi ed target genes with miR-221-3p in HNSCC (Figure 13).

Discussion
The Drug metabolism -cytochrome P450 signaling pathway is an essential signal transduction pathway in cells. It is an important biological function of cell survival, proliferation and apoptosis. For example, Liping Wang and his colleagues suggest [33] that inhibition of CYP3A5 in the cytochrome P450 Drug metabolism can drive the migration, proliferation, and invasion of HNSCC cells. Dongfang Wang.et al show that the cytochrome P450 is correlated with the overall survival and vascular invasion of Hepatocellular carcinoma (HCC) patients. Additionally, inhibiting the P450 pathway on drug metabolismcytochrome can increase the e cacy of the HNSCC [34]. In the target genes of mir-221-3p, MAOB and UGT1A7 is associated with the mir-221-3p and drug-cytochrome P450 signaling pathways, are downregulated in the HNSCC(P<0.05). MAOB is regarded as novel biomarkers for accurate Prostate cancer diagnosis and treatment [35]. In addition, a large number of valuable epidemiological studies have shown that UGT1A7 affects individuals' susceptibility to various cancers, such as pancreatic cancer [36],gastrointestinal carcinomas [37].Now our study suggests that miR-221-3p may play an important role as a gene that promotes the development of HNSCC, by reducing the expression of MAOB and UGT1A7 pathways in HNSCC. Of course, our study has some limitations. The target gene of mir-221-3p have not been experimentally con rmed and we believe that our conclusions will need to be con rmed by clinical or molecular biological methods in the nearly future.
Understanding the pathogenesis of HNSCC and identifying new gene therapy programs are the focus of recent studies. Many recent studies have reported that the miRNAs with different expression patterns in different tumors [38,39,40,41,42], a control the progression of the tumors. For example, miR-221-3p has be regarded as a tumor biomarker that can be used to assess the clinical prognosis of breast cancer [15]. In healthy humans, it has been shown that miR-221-3p plays a role in the process of vascular proliferation, while the tumor promoter, miR-221-3p regulates the apoptosis of tumor cells. Now, the current study focused on the expression pattern of miR-221-3p in HNSCC, identifying the exact target gene of this miRNA and its biological mechanism of action.

Conclusions
In our study, we found that miR-221-3p levels were apparently up-regulated in HNSCC compared to normal tissues and had certain diagnostic value in HNSCC, also provided insights into its potential molecular mechanisms in driving cancer progression. MAOB and UGT1A7 are potentially important targets of miR-221-3p, which plays an important role in HNSCC through the drug metabolismcytochrome P450 signaling pathway. In conclusion, miR-221-3p may can be used as a non-invasive biomarker in the diagnosis and is an extremely important gene locus involved in the process of the Ethics approval and consent to participate All procedures were approved by the Ethics Committee of Guangxi Medical University (Nanning, China). Written informed consent was obtained from all patients or their families prior to enrolment in the present study.

Consent for publication
Not applicable.

Competing interests
The authors declared that they have no competing interests. MK designed the study and accessed the relevant information, and ZZ and LWW collected and analyzed the data. XJL and CL were involved in statistical analysis. XZX, QQL, JMS and XRQ critically revised the manuscript. All authors read and approved the nal manuscript.

Researcher
Year    Figure 1 MiR-221-3p levels in HNSCC and NC tissue specimens. RT-qPCR was used to detect miR-221-3p was signi cantly up-regulated in the tumor tissues of an HNSCC cohort (n = 48) relative to control tissues (n = 21) P 0.01).

Figure 2
The ow chart of literature search and selection of relevant studies. sixteen out of 424 GEO microarray datasets met our inclusion criteria. At the same time, a total of 17 articles were retrieved from the initial search, and one papers (Zhou Cheng. et al) were selected after the full-text review based on the inclusion criteria.