| Background and purpose:Cholestatoma of the middle ear is a benign keratosis and hyperproliferative squamous epithelial lesion,which can lead to the destruction of nearby bone structures,hearing loss,facial nerve paralysis,retroauricular subperiosteural abscess,fistula,bacterial disasteritis,and intracranial complications such as brain abscess,sigmoid thrombophlebitis,etc.,which can be life-threatening in severe cases.There were still 30%patients who relapse after surgical resection.Until now,the exact cellular and molecular mechanisms of cholesteatoma have not been fully elucidated.To improve the treatment of cholesteatoma,clinically useful biomarkers are urgently needed.BMI1 is a member of the polycomb family of proteins.It has been found that the wild type of BMI1 in vitro can cause malignant transformation of HaCaT cells(human keratinocytes).Dysregulation of BMI1 expression leads to keratinocyte degeneration and tumorigenesis,which may be caused by shortening cell cycle and increasing cell mobility.In the upper cortex of cholesteatoma,keratinocytes account for 95%of the whole epithelial cells,which have strong proliferation and differentiation ability.Recent studies have shown that BMI1 is highly expressed in cholesteatoma,and it is crucial for the development of cholesteatoma,suggesting that BMI1 may provide a new target for the treatment of patients with cholesteatoma.MicroRNAs are short non-coding RNA molecules that play a role in posttranscriptional regulation of gene expression.They can initiate protein translation inhibition by binding to their targeted mRNA in the 3’ untranslated region.In recent years,due to the involvement of miRNA in a variety of cellular pathways,including cell proliferation,differentiation,apoptosis,migration and invasion,more and more researchers have begun to pay attention to the study of miRNA in cholesteatoma.In the early stage of this project,we used bioinformatics to predict miRNAs that had a targeting relationship with BMI1,and combined with previous literature to screen out two miRNAs related to cholesteatoma,miR-1297 and miR-26a-5p.Therefore,to explore whether miR-1297 and miR-26a-5p could participate in the occurrence and development of cholesteatoma through targeted regulation of BMI1,and further provide a theoretical basis for targeted therapy of cholesteatoma.Research purposes1.To study the expression of BMI1 in cholesteatoma tissues,and down-regulate the effect of BMI1 on the proliferation,migration and invasion,apoptosis,cell cycle and related proteins of cholesteatoma keratinocytes;2.Study the expression of miR-1297 and miR-26a-5p in cholesteatoma keratinocytes and verify that miR-1297 and miR-26a-5ptargets BMI1 to affect the biological behavior of cholesteatoma keratinocytesPart 1:The expression of BMI1 in cholesteatoma tissues and its function in cholesteatoma keratinocytesMethods:1.qRT-PCR and Western blot experiments were used to detect the expression of BMI1 in the tissues of patients with cholesteatoma.2.Cholesteatoma keratinocytes were extracted and cultured in serum-free medium.Observe the cell morphology under a microscope and perform keratin ocyte keratin antibody immunofluorescence chemical detection to identify the n ature of keratinocytes.3.Experimental grouping:si-NC group,si-BMI1#1 group and si-BMI1#2 group.4.Western blot experiment was used to detect the transfection efficiency of BMI1 siRNA.5.MTT,EDU and clone formation experiments were used to detect the proliferation ability of cholesteatoma keratinocytes.6.Transwell chamber experiment and scratch experiment were used to detect cell migration and invasion ability.7.Western blot was used to detect the expression of E-cadherin,N-cadherin and Vimentin,which are the marker proteins of epithelial-mesenchymal transition(EMT).8.Flow cytometry was used to detect cell cycle distribution and cell apoptosis rate.9.Western blot was used to detect the expression of Cyclin D1,CDK4,Bax,Bcl-2,MMP-2 and MMP-9 related to cell cycle,apoptosis,invasion and migration.Results:1.In clinical specimens,the expression levels of BMI1 mRNA and protein in cholesteatoma tissues were significantly higher than those of normal skin tissues behind the ear.2.A sufficient number of cholesteatoma keratinocytes could be obtained by culturing with a serum-free medium,and the purity of the cholesteatoma keratinocytes extracted by keratin antibody detection can reach more than 90%,which could be used for subsequent experiments.3.BMI1 siRNA transfection showed that the expression of BMI1 decreased significantly.The results of cell proliferation test showed that the OD value,EdU positive rate and the number of clonogenic cells of cholesteatoma keratinocytes decreased significantly after BMI1 siRNA transfection(P<0.05).4.The results of cell invasion and migration experiment showed that the number of migrating and invading cholesteatoma keratinocytes decreased significantly after transfection of BMI1 siRNA,and the wound healing rate decreased significantly(P<0.05).5.EMT test showed that the expression of E-cadherin was significantly increased in cholesteatoma keratinocytes after BMI1 siRNA transfection,while the expression of N-cadherin and vimentin was significantly decreased(P<0.05).6.Cell cycle results showed that after BMI1 siRNA transfection,the proportion of G0/G1 phase cells increased significantly,while the proportion of S phase cells decreased significantly,and the apoptosis rate increased significantly(P<0.05).7.Western blot showed that the expression of cyclin D 1,CDK4,Bcl-2,MMP-2 and MMP-9 was significantly down regulated in BMI1 siRNA transfected cholesteatoma keratinocytes,while the expression of Bax was significantly up-regulated(P<0.05).Part 2:miR-1297 targeting BMI1 affects the biological behavior of cholesteatoma keratinocytesMethods:1.Bioinformatics software prediction,dual luciferase reporter gene and RIP experiment were used to detect the targeting relationship between miR-1297 and BMI1.2.qRT-PCR was used to detect the expression of miR-1297 in the tissues of patients with cholesteatoma.3.Experimental groups:miR-NC group,miR-1297 group,miR-1297+pcDNA group and miR-1297+BMI1 group.4.Western blot was used to detect the effect of miR-1297 on the expression of BMIl.5.MTT,EDU and clone formation experiments were used to detect the proliferation ability of cholesteatoma keratinocytes.6.Transwell chamber experiment and scratch experiment were used to detect cell migration and invasion ability.7.Flow cytometry was used to detect cell cycle distribution and cell apoptosis rate.8.Western blot was used to detect the expression of E-cadherin,N-cadherin,Vimentin,Cyclin D1,CDK4,Bax,Bcl-2,MMP-2 and MMP-9 proteins in cells.Results:1.There was a targeting relationship between miR-1297 and BMI1.miR-1297 is low expressed in tissues of patients with cholesteatoma.2.The expression of BMI1 protein in cholesteatoma keratinocytes transfected with miR-1297mimics was significantly lower than that in the control group.At the same time,the expression of BMI1 protein in cholesteatoma keratinocytes transfected with miR-1297mimics and BMI1 overexpression vector plasmid almost returned to the level of the control group(P<0.05).3.In the cell proliferation experiment,the OD value,edu positive rate and the number of clonogenic cells in cholesteatoma keratinocytes were significantly reduced after transfection with miR-1297mimics(P<0.05);At the same time,the OD value,edu positive rate and the number of clone forming cells of cholesteatoma keratinocytes transfected with miR-1297mimics and BMI1 overexpression vector plasmids were significantly increased(P<0.05).4.In the experiment of cell migration and invasion,the number of migrating and invading cholesteatoma keratinocytes decreased significantly after transfection of miR-1297mimics,and the wound healing rate decreased significantly(P<0.05);At the same time,the migration and invasion of plasmid transfected with miR-1297mimics and BMI1 overexpression vectors increased significantly,and the scratch healing rate increased significantly(P<0.05).5.In the experiment of epithelial mesenchymal transformation,the expression of E-cadherin was significantly increased in cholesteatoma keratinocytes transfected with miR-1297mimics,while the expression of N-cadherin and vimentin was significantly decreased(P<0.05);At the same time,the expression of E-cadherin in cholesteatoma keratinocytes transfected with miR-1297mimics and Bmi-1 overexpression vector plasmids decreased significantly,while the expression of N-cadherin and vimentin increased significantly(P<0.05).6.Cell cycle results showed that after transfection with miR-1297mimics,the proportion of G0/G1 phase cells increased significantly,while the proportion of S phase cells decreased significantly,and the apoptosis rate increased significantly(P<0.05);After transfection of miR-1297mimics and Bmil overexpression vector plasmids,the proportion of G0/G1 phase cells decreased significantly,while the proportion of S phase cells increased significantly,and the apoptosis rate decreased significantly(P<0.05).7.Western blot showed that the expression of cyclin D 1,CDK4,Bcl-2,MMP-2 and MMP-9 in cholesteatoma keratinocytes transfected with miR-1297mimics was significantly down regulated,while the expression of Bax was significantly up-regulated(P<0.05);The expression of cyclin D 1,CDK4,Bcl-2,MMP-2 and MMP-9 in cholesteatoma keratinocytes was significantly up-regulated,while the expression of Bax was significantly down regulated(P<0.05).Part 3:miR-26a-5p targeting BMI1 affects the biological behavior of cholesteatoma keratinocytesMethods:1.Bioinformatics software prediction,dual luciferase reporter gene and RIP experiment to detect the targeting relationship between miR-26a-5p and BMI1.2.qRT-PCR was used to detect the expression of miR-26a-5p in the tissues of patients with cholesteatoma.3.Experimental groups:miR-NC group,miR-26a-5p group,miR-26a-5p+pcDNA group and miR-26a-5p+BMI1 group;4.Western blot was used to detect the effect of miR-26a-5p on the expression of BMI1.5.MTT,EDU and clone formation experiments were used to detect the proliferation ability of cholesteatoma keratinocytes.6.Transwell chamber experiment and scratch experiment were used to detect cell migration and invasion ability.7.Flow cytometry was used to detect cell cycle distribution and cell apoptosis rate.8.Western blot was used to detect the expression of E-cadherin,N-cadherin,Vimentin,Cyclin D1,CDK4,Bax,Bcl-2,MMP-2 and MMP-9 proteins in cells.Results:1.There was a targeted relationship between mir-26a-5p and Bmi-1,and the expression of mir-26a-5p was low in patients with cholesteatoma.2.The expression of BMI1 protein in cholesteatoma keratinocytes transfected with miR-26a-5p mimics was significantly lower than that in the control group.At the same time,the expression of BMI1 protein in cholesteatoma keratinocytes transfected with miR-26a-5p mimics and Bmi1 overexpression vector plasmid almost returned to the level of the control group(P<0.05).3.In the cell proliferation experiment,the OD value,edu positive rate and the number of clonogenic cells in cholesteatoma keratinocytes were significantly decreased after transfection of miR-26a-5p mimics(P<0.05);At the same time,the OD value,edu positive rate and the number of clonogenic cells of cholesteatoma keratinocytes transfected with miR-26a-5p mimics and BMI1 overexpression vector plasmids were significantly increased(P<0.05).4.In the experiment of cell migration and invasion,the number of migrating and invading cholesteatoma keratinocytes decreased significantly after transfection of miR-26a-5p mimics,and the wound healing rate decreased significantly(P<0.05);At the same time,the migration and invasion of plasmid transfected with miR-26a-5p mimics and BMI1 overexpression vector increased significantly,and the wound healing rate increased significantly(P<0.05).5.In the experiment of epithelial stromal transformation,the expression of E-cadherin was significantly increased,while the expression of N-cadherin and vimentin were significantly decreased after transfection of miR-26a-5p mimics(P<0.05);At the same time,the expression of E-cadherin in cholesteatoma keratinocytes transfected with miR-26a-5p MICs and BMI1 overexpression vector plasmids decreased significantly,while the expression of N-cadherin and vimentin increased significantly(P<0.05).6.The results of cell cycle showed that after transfection with miR-26a-5p mimics,the proportion of G0/G1 phase cells increased significantly,while the proportion of S phase cells decreased significantly,and the apoptosis rate increased significantly(P<0.05);After transfection of miR-26a-5p mimics and BMI1 overexpression vector plasmids,the proportion of G0/G1 phase cells decreased significantly,while the proportion of S phase cells increased significantly,and the apoptosis rate decreased significantly(P<0.05).7.Western blot results showed that the expression of cyclin D 1,CDK4,Bcl-2,MMP-2 and MMP-9 in cholesteatoma keratinocytes transfected with miR-26a-5p mimics was significantly down regulated,while the expression of Bax was significantly up-regulated(P<0.05);The expression of cyclin D 1,CDK4,Bcl-2,MMP-2 and MMP-9 in cholesteatoma keratinocytes was significantly up-regulated,while the expression of Bax was significantly down regulated(P<0.05).Conclusions:1.Compared with the normal skin tissue behind the ear,BMI1 was highly expressed in cholesteatoma,and miR-1297 and miR-26a-5p were low expressed.2.Knockdown of BMI1 inhibited the proliferation,cycle progression,EMT,migration and invasion of cholesteatoma keratinocytes,and induces cell apoptosis.3.miR-1297 and miR-26a-5p targeted and regulated BMI1 to inhibit the proliferation,cycle progression,EMT,migration and invasion of cholesteatoma keratinocytes,and induce cell apoptosis. |
PDF Full Text Request