Analysis Of CircRNA Expression Profile And Function Of MiR-548c-3p In Hypopharyngeal Squamous Cell Carcinoma

Objective:Hypopharyngeal squamous cell carcinoma(HSCC)is a malignant tumor originating from hypopharyngeal tissue.It has the characteristics of occult onset,early metastasis and easy recurrence.Finding out the differentially expressed genes and their possible regulatory molecular mechanisms at the molecular level is of great significance for the early diagnosis and survival of hypopharyngeal cancer.Previous studies have shown that miRNAs play an important regulatory role in tumorigenesis and development,and circRNA has the function of sponge adsorbing miRNAs.In this study,the expression profiles of circRNA in HSCC tissues and adj acent non-cancerous normal tissues were detected by second-generation high-throughput sequencing technology,and the differentially expressed circRNAs were screened out.The sponge-adsorbed miR-548c-3p was analyzed by bioinformatics.The effects of miR-548c-3p and its target gene TP53BP2 on cell proliferation,cloning,migration,invasion,cycle and apoptosis in hypopharyngeal cancer cells were analyzed at cell level.The purpose of this study was to investigate the expression of circRNA in hypopharyngeal cancer and the effect of sponge-adsorbed miR-548c-3p on the biological function of hypopharyngeal cancer cells(FaDu).These findings play an important role in further elucidating the mechanism of hypopharyngeal cancer,and provide a reference for the early diagnosis of hypopharyngeal cancer and the discovery of tumor biomarkers.Methods:1.Sixty-three patients with HSCC who were treated for the first time were selected as the study subjects.HSCC tissue samples and corresponding non-neoplastic mucosa samples were collected during the operation.The expression profiles of circRNA in HSCC tissues and adjacent non-cancerous mucosa tissues were analyzed after high-throughput detection of all circRNA in 3 specimens.2.Scatter plots were drawn with GGPROT2 package in R language,gene lengths of six samples were measured by principal component analysis(PCA),and expression levels were measured by millionth rate reading map reading(RPM).Statistical results showed that there were significant differences in circRNAs among samples.3.Realtime PCR was used to detect 19 strip circRNAs differentially expressed(significantly increased or decreased)in 3 normal tissues of tumors and adjacent non-tumors to verify the sequencing results of circRNAs.4.The distribution of circRNA in different DNA fragments and chromosomes was analyzed,and the characteristics of circRNA ring formation and distribution in chromosomes were clarified.5.Gene Ontology(GO),Kyoto Encyclopedia of Genesand Genomes(KEGG)were used to analyze the possible biological functions of differentially expressed circRNA.6.According to the circbase database,the first 20 up-regulated circRNAs and the last 20 down-regulated circRNAs were numbered and coded in the database.Targetscan algorithm was used to screen differentially targeted miRNAs in the server CircInteractome.KEGG and miRPath databases analyzed the relationship between the top 40 differential circRNAs and miRNAs.7.The role of circRNAs in ErbB and Hippo signaling pathways was elucidated by constructing a miRNA-ceRNA network using Cytoscape software.8.The clinical correlation between the expression of miR-548c-3p and the survival rate of patients and Kaplan-Meier survival rate were analyzed by using the data of miRNA and mRNA in the esophageal cancer cohort of the American Cancer and Tumor Gene Map(TCGA).9.Quantitative RT-PCR was used to detect the expression of miR-548c-3p in 60 HSCC patients'cancer tissues and adjacent normal tissues.Kaplan-Meier method was used to detect the 5-year survival rate.10.miR-548c-3p mimic was transfected into FaDu,HSC2,HSC3 and D562 cells,respectively.The transfection efficiency was detected by qRT-PCR,and cell proliferation was detected by CCK-8 and clone formation assay.11.miR-548c-3p mimic was transfected into FaDu cells,and Transwell was used to detect cell migration and invasion.Annexin V/FITC was used to detect apoptosis.12.Through Targetscan software analysis,TP53BP2 was predicted to be a candidate target gene of miR-548c-3p.Double luciferase reporter gene was used to detect the luciferase activity of FaDu cells co-transfected with miR-548c-3p mimic and TP53BP23'UTR wt vectors.Further,the expression of TP53BP2 was detected by qRT-PCR after co-transfection of miR-548c-3p mic.13.The effect of miR-548c-3p on the growth of HSCC tumors was examined in nude mice.miR-548c-3p mimic was transfected into FaDu cells,and the cells were subcutaneously injected into the right back of the mice.The size of the tumors was measured every 5 days.The tumors were removed and detected by qRT-PCR.14.The effects of miR-548c-3p mimic on cell proliferation and cloning were compared with those of FaDu cells co-transfected with miR-548c-3p mimic and pcDNA-TP53BP2.15.Tanswell assay was used to detect cell migration and invasion.Migration and invasion of miR-548c-3p mimic transfected cells were compared with that of FaDu cells co-transfected with miR-548c-3p mimic and pcDNA-TP53BP2 vectors.Migration and invasion of FaDu cells were reversed by miR-548c-3p mimic.16.Annexin V/FITC assay was used to detect apoptosis.miR-548c-3p mimic was transfected into FaDu cells.Annexin V/FITC assay showed that it promoted cell apoptosis.miR-548c-3p mimic and pcDNA-TP53BP2 vectors co-transfected FaDu cells reversed the effect of miR-548c-3p mimic on cell apoptosis.RESULTS:1.Compared with circRNAs in non-cancerous tissues adjacent to cancer,71 circRNAs in HSCC tissues were significantly increased and 102 circRNAs were significantly decreased.2.Nineteen circRNAs differentially expressed were identified by qRT-PCR in three groups(HSCC and non-cancerous normal tissues)and 12 of them were identical with the sequencing results.3.The precursor RNA produces circRNA by reverse splicing,which includes exonic circRNA(ecircRNA),intron circRNA(ciRNA)and exon-intron circRNA(EIciRNA).Among them,exon source accounts for the majority,followed by intron source.4.The down-regulated expression of circRNAs in KEGG pathway is high,and plays a role in the cellular drinks,ubiquitin-mediated protein hydrolysis,Janus kinase(JAK)/signal transducer and transcriptional activator(STAT)signaling pathway;the up-regulated expression of circRNAs in KEGG pathway is low.GO analysis found that a large number of circRNAs were accumulated in the process of antimicrobial drug response,transcription regulation and cell division,while down-regulation of circRNA was associated with autophagy,mitochondrial actin cytoskeleton,membrane division and cell adhesion.5.CircRNAs regulate ERBB and HIPPO pathways through the miRNA-ceRNA network.CircRNAs act as sponge adsorption of microRNAs,which is the main mechanism of circRNA.For the first 20 up-regulated and the last 20 down-regulated circRNAs,they were numbered by circRNA database,and the possible adsorbed microRNAs by circRNAs were analyzed by Target Scan database.191 increased and 182 decreased circRNAs corresponded to corresponding microRNAs.RNA regulated by microRNAs was significantly enriched in several signaling pathways,such as ERGB,HIPPO,RAS,TGF-beta,phosphatidylinositol serine/threonine kinase Akt/3 kinase and Wnt signaling pathways.6.We found a synergistic transcriptional relationship between circ-0008287 and circ-0005027 and miR-548c-3p in HIPPO and ERGB pathways.The down-regulation of CircRNAs may lead to the release of miR-548c-3p,and the over-expression of miR-548c-3p may promote the progression of hypopharyngeal cancer through downstream target genes.miR-548c-3p negatively regulates ErbB and Hippo pathway genes.7.Analysis of TCGA database showed that the expression of miR-548c-3p was higher in cancer samples than in normal samples,and the survival rate of patients with high expression of miR-548c-3p was lower.8.The expression of miR-548c-3p in cancer tissues of HSCC patients was significantly higher than that in adjacent normal tissues.The 5-year survival rate of patients with low expression of miR-548c-3p was lower.Compared with normal human oral mucosal epithelial cells(HOMEC),the expression of miR-548c-3p was down-regulated in HSCC cell lines(FaDu,HSC2,HSC3,D562).9.miR-548c-3p mimic transfected FaDu cells inhibited the proliferation,cloning,migration and invasion of FaDu cells,and promoted the apoptosis of FaDu cells.10.TP53BP2 is a candidate target gene of miR-548c-3p.Luciferase reporter gene detection showed that co-transfection of miR-548c-3p mimic and TP53BP2 3'UTR wt vector significantly inhibited the luciferase activity of FaDu cells.Transfection of miR-548c-3p mimic and TP53BP2'UTR mut vector had no significant effect on the luciferase activity.Transfection of miR-548c-3p mimic significantly inhibited the expression of TP53BP2.11.The tumor volume and weight of mice in the miR-548c-3p mimic group were significantly smaller than those in the control group.The expression of TP53BP2 in the miR-548c-3p mimic group was significantly lower than that in the control group by qRT-PCR.12.miR-548c-3p mimic and pcDNA-TP53BP2 co-transfection of the vector reversed the effects of miR-548c-3p mimic on the proliferation,cloning,migration,invasion and apoptosis of FaDu cells.Conclusions:1.Differential expression of CircRNA in hypopharyngeal cancer tissues and adj acent non-cancerous normal tissues;CircRNA(hsa-circ-0008287 and hsa-circ-0005027)/miRNAs(miR-548c-3p)/mRNA axis may play an important role in the progression of hypopharyngeal cancer through the Hippo pathway.2.The expression of miR-548c-3p in hypopharyngeal cancer tissues and cell lines is low,which inhibits FaDu cell proliferation,cloning,migration and invasion,and promotes cell apoptosis.The expression pattern of miR-548c-3p conforms to the expression pattern of tumor suppressor genes.3.miR-548c-3p targeting combined with TP53BP2,and the effect of the molecular axis of miR-548c-3p/TP53BP2 on the biological functions of hypopharyngeal cancer cells such as colonization,cloning,circuitous migration,invasion,cycle and apoptosis.This study provides a new way to find specific tumor markers for hypopharyngeal cancer,and also lays a good theoretical foundation for the diagnosis and treatment of hypopharyngeal cancer.