The Mechanism Of Circ-MTCL1 In Affecting The Biological Behavior Of Laryngeal Cancer Cells

Laryngeal cancer is one of the common tumors of the head and neck,with 177,422 new cases and 94,771 deaths worldwide every year.Although the incidence of laryngeal cancer has shown a gradual decline in recent years,the five-year survival rate for laryngeal cancer has declined significantly over the past 40 years,from 66% to 63%,which is relatively rare among cancer diseases.In addition,according to statistics,about40% of patients with laryngeal cancer were diagnosed with advanced stage(stage III or IV),or even accompanied by metastasis,which seriously affected the prognosis of patients with laryngeal cancer.Laryngeal cancer can be caused by a series of genetic changes caused by smoking,drinking alcohol,papillomavirus infection and exposure to harmful dust.However,the specific pathogenesis of laryngeal cancer is still unclear,and there is a lack of ideal biomarkers in diagnosis,treatment and prognosis.Therefore,the screening of pathogenic genes and the indepth exploration of the molecular mechanism of the disease are of great significance for optimizing the diagnosis of laryngeal cancer and exploring potential therapeutic targets.The transcriptomic analysis of laryngeal cancer using second-generation high-throughput sequencing can help to explore pathogenic genes related to laryngeal cancer,and further explain the mechanism of laryngeal cancer occurrence and development.Sequencing analysis of human genome showed that DNA with coding function only accounted for 2% of the total genome sequence,most of which were non-protein-coding DNA,and the transcriptional products of these DNA were non-coding RNA.CircRNAs are single-stranded,closed-loop non-coding RNAs formed by the reverse splicing of the 3 ’-5’ end of pre-m RNA(pre-m RNA),which exist in large numbers and stablely in and outside cells,and are endowed with highly conserved,high expression abundance and tissue-specific characteristics of circRNA.Recent studies have reported that circRNA is involved in the occurrence and development of human neuropsychiatric diseases,cardiovascular diseases and other diseases,especially tumors.These evidences suggest that circRNA is expected to be an ideal biomarker for some diseases,even laryngeal cancer.Therefore,total transcriptome analysis,screening of differentially expressed circRNAs,verification and mechanism study will contribute to the diagnosis,treatment and prognosis of laryngeal cancer.This study is divided into the following three parts.Part Ⅰ Transcriptome sequencing analysis of laryngeal cancer Objective: In this study,the second-generation high-throughput sequencing technology was used to analyze the differentially expressed circRNA profiles in cancer tissues and paracancerous tissues of 5 patients with laryngeal cancer,identify new transcripts,screen disease-related genes,and provide specific markers for the diagnosis,treatment and prognosis of laryngeal cancer.Methods: Collect resection of laryngeal cancer and tissue adjacent to carcinoma specimens,Trizol method to extract RNA,Agilent2100 and integrity,agarose gel electrophoresis to detect RNA using biotin labeling specific probe purified RNA,build sequencing library and quality control,using the Illumina Hi Seq X Ten sequencing,in quantitatively,circRNA build gene expression profile,using DEGseq genetic variations and DEseq2 method of screening,and sources of circRNA NR gene,GO and KEGG database annotation.The GO function analysis,KEGG Pathway enrichment and Pathway enrichment analysis were performed.The co-expression network was mapped using Pearson correlation to construct the ceRNA regulatory network.Bioinformatics software Target Scan,miRwail and Circ Iteractome were used to analyze the sequencing results.Finally,2 circRNAs,2 lnc RNAs and 1 m RNA were screened.Real-time quantitative PCR was used to verify the expression levels of the 5 screened genes and the corresponding co-expressed genes.Results: The RNA integrity of the tissue samples included in this study was good,and the quality control was qualified.A total of 153676 transcripts were detected by sequencing,including 22580 circRNAs,86424 lnc RNAs,and 44,672 m RNAs.The GO functions of differentially expressed genes were enriched,and the results showed that the functions of differentially expressed genes concentrated on cell cycle,cell adhesion,organelle synthesis,catalytic activity,molecular function regulation,nucleic acid binding transcription factor activity.KEGG pathway analysis showed that the pathways of differential gene enrichment were mainly apoptosis,mitogen-activated protein kinase pathway,ubiquitin-mediated protein degradation pathway,adenylate activated protein kinase pathway,m TOR signaling pathway,and FOXO signaling pathway.According to the correlation greater than 0.99 or less than-0.99,relevant RNAs were selected to draw the circRNA-miRNA-m RNA interaction network,and the top 20 nc RNAs in differential expression ranking were selected to draw the ceRNA network.The real-time fluorescence quantitative PCR results showed that the expression trend of the screened genes was basically consistent with the sequencing results.The expression trend of m RNA co-expressed with SNHG29 was consistent with or opposite to that of SNHG29.Conclusions: Through transcriptome sequencing,we found a new transcript,which can be used as an effective biomarker for the diagnosis and prognosis of laryngeal cancer.Functional enrichment analysis,co-expression analysis and ceRNA network of differential genes contribute to the study of the pathogenesis of laryngeal cancer,and provide scientific basis for precise treatment.Part Ⅱ Detection of circ-RANBP9 expression in laryngeal cancer and its clinical significance Objective: Given the highly conserved,stable and tissue-specific circRNAs,differentially expressed circRNAs contribute to the diagnosis,treatment and prognosis of laryngeal cancer,and play an important role in the occurrence and development of laryngeal cancer.One circRNA with differential expression was screened,and the differential expression levels of these circRNAs in laryngeal cancer tissues were detected by RT-qPCR,and their clinical significance was analyzed.Methods: Laryngeal cancer and paracancillary tissues of 47 patients were collected,RNA was extracted by Trizol method,RNA integrity was detected by RNA agarose gel electrophoresis,and RT-qPCR was performed to verify the expression level of circ-RANBP9.The clinical correlation of circ-RANBP9 was also analyzed by one-way analysis of variance.Furthermore,CCK-8 test,EdU test,scratch test and Transwell test were used to verify the effect of circ-RANBP9 on the proliferation,invasion and migration of laryngeal cancer.Pearson correlation was used to map the co-expression network and construct the ceRNA regulatory network.Then,GO and KEGG enrichment analysis was performed on the co-expression analysis and ceRNA regulatory network to determine the possible role of circ-RANBP9 in laryngeal cancer.Results: The expression of circ-RANBP9 was significantly lower in laryngeal cancer(P<0.001),and its low expression was closely related to T stage(P=0.018),clinical stage(P=0.003),tumor differentiation(P=0.031)and lymph node metastasis(P=0.046),which is expected to be a biological marker for early diagnosis of laryngeal cancer(AUC:0.716,sensitivity: 97.9%,specificity: 55.3%).Bioinformatics analysis revealed that circ-RANBP9 may act on JAK3,FOXN1,MYC,and APC2 through miR-4746-3p,miR-4757-5p,miR-3131,and miR-611,thereby regulating extracellular matrix(ECM)-receptor interactions,c AMP,calcium,and wnt signaling pathways.Conclusions: The expression trend of circ-RANBP9 screened in laryngeal cancer tissues is consistent with the sequencing results.The low expression of circ-RANBP9 in laryngeal cancer tissue shows good diagnostic sensitivity and specificity,which is helpful for the early diagnosis and prognosis of laryngeal cancer.Circ-RANBP9 may play a role through external matrix(ECM)-receptor interactions,c AMP,calcium,and wnt signaling pathways.Part Ⅲ The mechanism of circ-MTCL1 in laryngeal cancer Objective: Because circ-MTCL1 is highly expressed in laryngeal cancer tissues,we speculated that circ-MTCL1 plays a role in promoting cancer during the occurrence and development of laryngeal cancer.Thus,we further studied the specific mechanism of action of circRNA in laryngeal cancer through the analysis of ring forming characteristics,functional experiments and tumor formation experiments in nude mice.Methods: The ring formation characteristics of circ-MTCL1 were analyzed by Sanger sequencing and exonuclease digestion assay.The subcellular localization of circ-MTCL1 was performed by fluorescence in situ hybridization and nucleo-cytoplasmic separation.Tu212 and LCC cell lines were selected to interfere with the expression of circ-MTCL1,and EdU experiments were performed to determine the effect of circ-MTCL1 on the proliferation level of laryngeal cancer cells.Transwell assay was used to detect the effect of circ-MTCL1 on the invasion level of laryngeal cancer cells.The effect of circ-MTCL1 on the migration of laryngeal cancer cells was detected by scratch test.Pulldown assay was used to verify whether circ-MTCL1 was involved in the ceRNA mechanism and detect proteins directly bound to circ-MTCL1.The RIP assay verified the binding of circ-MTCL1 to the protein.Co-IP assay verified the binding effect of the protein with the downstream protein.The effect of circ-MTCL1 on tumor growth was determined in tumor formation of nude mice.Results: Circ-MTCL1 was cleaved and cyclified by MTCL1 gene,located on CHR18:8718421-8720494,with a length of 384 nt and shear point of GC.It was located in the cytoplasm of laryngeal cancer cells and was resistant to exonuclease digestion.Silencing circ-MTCL1 inhibited laryngeal and laryngeal cancer cell viability,invasion,and migration.Overexpression of circ-MTCL1 enhanced laryngeal cancer cell viability,invasion ability,and migration ability.Circ-MTCL1 is positively correlated with the expression of C1 QBP in tissues and cell lines.Circ-MTCL1 can increase the expression of C1 QBP by inhibiting the degradation of C1 QBP,and activate the wnt/β-catenin pathway by binding with C1 QBP,thereby affecting the proliferation,invasion,and migration of laryngeal cancer cells.Circ-MTCL1 affects the growth and metastasis of xenograft tumors in nude mice.Conclusions: Circ-MTCL1 is an effective biomarker for early diagnosis and prognosis of laryngeal cancer.The wnt/β-catenin pathway can be affected by C1 QBP.Circ-MTCL1 plays an important role in the proliferation,invasion,and migration of laryngeal cancer.