| Objective: Head and neck squamous cell carcinoma(HNSCC)is one of the most common type of cancer in the world,but the prognosis and quality of life of the patients are not well guaranteed and the five-year survival rate of less than 50% because of the wide range of lesions,cancer cells deep immersion,surgical excision area and so on.Therefore,it is very important to study the molecular biology mechanism of HNSCC development,invasion and transfer.Although a large number of studies have explored HNSCC biological markers and found some molecular biology mechanisms related to HNSCC,there is no specific HNSCC biological markers used to guide clinical treatment and prognostic evaluation,and it is difficult to explain the occurrence,development and transfer mechanisms of HNSCC through a unique molecule.More reliable experimental models are needed to better understand the molecular mechanisms of HNSCC progression in order to develop more effective therapeutic strategies.Based on high-volume sequencing techniques,this study obtained circRNA,miRNAs,and mRNAs expressed differently in HNSCC tissues.According to the theory of bioinformatics and competitive endogenous RNA(competing endogenous RNA,ceRNA),the circRNA-miRNA-mRNA regulatory network was constructed to study the role of circRNA＿101744 in human HNSCC cell proliferation,apoptosis and migration,and to explore the effects of circRNA＿101744/miR-489-3p/RAN axis on the biological behavior of human HSNCC cells.Methods:1.Five HNSCC samples without preoperative chemotherapy were selected,cancer and cancer sideline tissue were tested by high-throughput sequencing technology.CircRNA,miRNA and mRNA were obtained by differential expression,and RAN was selected as mRNA of the circRNA-miRNA-mRNA network for in-depth study through qRT-PCR verification and TCA database comparison.2.Using the qRT-PCR method,30 expressions of RAN in HNSCC and cancerous tissue were detected,and the expression of RAN in head and neck scale cancer cell line(PCI-37 B,SCC-9)and human embryonic renal cell strain(HaCaT)was detected by qRT-PCR and Western blot.Retrospective analysis of 94 cases of HNSCC and follow-up for more than five years,with 20 cases of normal oral mucosa tissue as control,the use of paraffin section and immunohistochemical staining to detect RAN in HNSCC and normal mucosa tissue expression.The relationship between RAN’s expression and HNSCC clinical pathological factors were evaluated using Kruskal-Wallis H and Mann-Whitney U test.The relationship between RAN’s expression and HNSCC survival time was evaluated using Kaplan-Meier test and log-rank test.3.The siRNA of RAN were constructed and then transfected into PCI-37 B and SCC-9cells by transfection technology.The influence of RAN on the biological behavior of proliferation,apoptosis and migration of HNSCC cells were detected by CCK-8 assay,FCM(flow cytometry,FCM)and transwell technology.4.The regulatory network of circRNA-miRNA-mRNA was constructed according to bioinformation and the theory of ceRNA,meanwhile the circRNA＿101744/miR-489-3p/RAN axis was determined as the subject of the study.The target relationship between circRNA＿101744 and miR-489-3p,miR-489-3p and RAN were detected by luciferase experiments.5.The plasmid of overexpression of RAN and miR-489-3p mimics were constructed and transfected by co-transfection technique.qRT-PCR and Western blot were used to detect the expression of RAN and MET in cells after transfection,and the effects of miR-489-3p on the proliferation,apoptosis and migration capacity of HNSCC cells by CCK-8experiments,FCM and transwell.6.The siRNA of circRNA＿101744 and miR-489-3p inhibitor were constructed and transfected by co-transfection technique.The expression of RAN and MET in transfection was detected using qRT-PCR and Western blot experiments.The effects of circRNA＿101744 on the proliferation,apoptosis and migration of HNSCC cells by regulating miR-489-3p were detected by CCK-8 assay,FCM and transwell techniques.Results:1.By comparing the results of high-throughput gene microarray with TCGA database,RAN was selected as a further study object and mRNA to build ceRNA network.2.Ran was closely associated with HNSCC development.High expression of RAN was significantly associated with tumor grade,lymph node metastasis and recurrence(p<0.05).While there was no significant correlation between RAN expression with gender,age and differentiation(p>0.05).Kaplan–Meier method showed that Ran level had a closely relationship with the patient’s survival rate in HNSCC.A lower RAN expression had a longer survival than those with a higher RAN expression(p< 0.01).Immunohistochemical results confirmed that RAN was mainly located in cellular plasma.The positive expression of RAN accounted for 43.62% in 94 HNSCC slices and the expression of RAN was negative in normal mucous membrane tissue.It was proved that the expression of RAN was closely related to the development of HNSCC and the high expression related to tumor stage,lymph node metastasis and recurrence(p<0.05),but not significant relation with the patient’s sex,age and the degree of tumor differentiation(p>0.05)through the analysis of various clinical pathological factors.The statistical results showed that RAN level had a closely relationship with the patient’s survival time in HNSCC,and the patient’s survival time with the high expression of RAN was significantly shorter than the patients with the low expression(p<0.01).3.qRT-PCR verified that the expression of RAN in HNSCC was significantly higher than that of cancerous tissue(p<0.05),and the expression of RAN in PCI-37 B and SCC-9 cells were significantly higher than that in HaCaT by qRT-PCR and Western blot(P<0.05).Transfected siRNA of RAN reduced the expression of RAN in HNSCC cells,as well as the expression of MET protein.The results of CCK-8 showed that transfected siRNA of RAN could significantly inhibit the proliferation capacity of PCI-37 B and SCC-9 cells(p<0.05),and significantly promote the apoptosis of PCI-37 B and SCC-9 cells by FCM(p<0.05).The results of transwell confirmed that transfected siRNA of RAN could significantly inhibit the migration capacity of PCI-37 B and SCC-9 cells(p<0.05).4.On the basis of biometric analysis and the results of ceRNA network construction,it is predicted that the presence of circRNA＿101744/miR-489-3p/RAN axis in HNSCC will affect the progression of HNSCC.Luciferase activity test confirmed that miR-489-3p can be combined with circRNA＿101744 specific area bits,and overexpression miR-489-3p can inhibit the relative luciferase activity of the circRNA＿101744-WT group.Mi R-489-3p can be associated with RAN by combining specific area bits,and overexpression miR-489-3p inhibits the activity of the RAN-WT group relative to luciferase,thus confirming the targeted relationship between the three proteins and providing a basis for further circRNA＿101744/miR-489-3p/RAN axis in the role of HNSCC.5.qRT-PCR verified that the expression of miR-489-3p in the tissues and cells of HNSCC was significantly lower than the expression in pericarcinomatous tissue and HaCaT cells(p<0.05).The transfection of miR-489-3p mimic could significantly reduce the expression of RAN and MET in PCI-37 B and SCC-9 cells by qRT-PCR and Western blot(p<0.05).Cell functional experiments confirmed that transfection of miR-489-3p mimic could significantly inhibit the proliferation and migration capabilities of PCI-37 B and SCC-9 cells and enhanced the apoptosis capacity of cells(p<0.05).These results were significantly reversed by co-transfection of miR-489-3p mimic and RAN overexpression plasmid(p<0.05).6.qRT-PCR verified that the expressions of circRNA＿101744 in HNSCC tissues and cells were significantly higher than that in pericarcinomatous tissues and HaCaT cells(p<0.05).qRT-PCR and Western blot confirmed that transfection of si-circRNA＿101744could significantly reduce the expression of RAN and MET in PCI-37 B and SCC-9 cells(p<0.05).Cell function experiments confirmed that transfection of si-circRNA＿101744could significantly inhibit the proliferation and migration of PCI-37 B and SCC-9 cells,enhance the ability of apoptosis of cells(p<0.05),and significantly reduce the migration of PCI-37 B and SCC-9 cells.Co-transfection with si-circRNA＿101744 and miR-489-3p inhibitor could significantly reverse the above results(p<0.05).Conclusion:1.RAN was highly expressed in HNSCC tissues and cells,and was closely related to tumor grade,recurrence,and survival time.The survival time of patients with high expression of RAN was significantly shorter than who with low expression.A higher RAN expression had a shorter survival than those with a lower RAN expression.2.The expression of miR-489-3p was low in HNSCC tissues and cells,and the overexpression of miR-489-3p could significantly inhibit the proliferation and migration ability of HNSCC cells,enhance the apoptosis ability,and inhibit the expression of RAN and MET.Mi R-489-3p inhibited the growth and migration of HNSCC cells by RAN.3.CircRNA＿101744 was highly expressed in HNSCC tissues and cells,and decreasing circRNA＿101744 could reduce the expression of RAN and MET in cells,significantly inhibit the proliferation and migration of HNSCC cells,and enhance the apoptosis ability.CircRNA＿101744 promotes the growth and migration of HNSCC cells by competitively binding miR-489-3p.4.CircRNA＿101744 can act as a ceRNA of HNSCC and as a molecular "sponge" of miR-489-3p to play an adsorption role.Combining with bioinformatics theory,circRNA＿101744 affected the expression of RAN by competitive binding miR-489-3p,and the effect of circRNA＿101744/miR-489-3p/ RAN axis on proliferation,apoptosis and migration of HNSCC cells was confirmed. |
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