| Objectives :The middle ear cholesteatoma is a well-defined cystic lesion that is formed by abnormal growth of squamous epithelium in temporal bone,and the cholesteatoma otitis media is a common disease in otolaryngology.Although many researches about the cholesteatoma pathological mechanism have produced a lot of controversies,but there isn't a theory to be able to explain all the clinical characteristics of cholesteatoma completely.The highly proliferative activity of Cholesteatoma epithelium is regarded as the basic characteristics of this disease.MiRNAs are involved in a series of important life process,including proliferation,differentiation,apoptosis and migration and have extensive and important biological functions.Especially many miRNAs have an inhibitory effect on the growth and invasion of tumors,which play a role in cancer suppression.Mi R-203 a with epithelial tissue specificity,affects the epithelial cell growth,differentiation and function,and plays a role of cancer suppressor.In the epithelial tissue,miR-203 a regulates keratinocyte cell proliferation and directional differentiation;it can inhibit cell stemness,stop proliferation and start directional differentiation.Through bioinformatic prediction,human Bmi1 gene is a predicted target gene of miR-203 a.Bmi1 belongs to the Polycomb group genes(PCG)family,which is a transcription inhibitor with genetically modified function.It can silence the expression of related genes mainly through changing the structure of the chromosomes.Human Bmi1 gene is expressed in most organizations and many malignant tumors,and plays an important role in maintaining stemness and self-renewal of both normal cells and cancer cells.Cholesteatoma has many similar characteristics of malignant tumor,such as the abnormal proliferation and invasion and so on,so we speculate that the expression of miRNAs which suppress the tumors could be changed in cholesteatoma.At present,the reports about miRNAs in cholesteatoma are extremely rare,especially the report about miR-203 a and the predict target gene Bmi1 in the cholesteatoma is never been seen.By detecting the expression of miR-34 a,miR-125 a and miR-203 a,we hope to screen out a miRNA which expresses abnormally in cholesteatoma and retroauricular normal skin.After detection,we concluded that there was a significant difference in miR203 a expression in the cholesteatoma compared with the corresponding retroauricular normal skin.Then we would analyze the correlation between miR-203 a expressive quantity and clinical features.We tried to find a targeted gene by using the Target Scan and miRanda predictive software,then the "stem" gene Bmi1 was found.By detecting the expression and distribution of the Bmi1 in cholesteatoma and retroauricular normal skin,we would analyze whether there is a difference between them,and analyze the correlation between Bmi1 and miR-203 a,then verify the negative regulation relationship in human keratinocyte cells.Then we would test whether mi R-203 a could regulate the keratinocyte cell biological behavior including proliferation,apoptosis,cell cycle regulation and migration by targeting Bmi1.There are many reports about that Bmi1 can regulate the PI3K/Akt signaling pathway in many tumors and involves in the development of tumors.At the same time the existed researches confirmed that the PI3K/Akt pathway involves in the pathological development of cholesteatoma.So we will detect the expression of p-Akt protein in cholesteatoma and the correlation with Bmi1.At last we will validate whether the Bmi1 could regulate the activity of PI3K/Akt signaling pathway in keratinocyte,and this change could be reversed by miR-203 a.The reports about miR-203 a and Bmi1 expression in cholesteatoma have not been found right now.We hope this research could provide certain helps for further exploration of cholesteatoma pathogenesis and provide a new direction for prevention and treatment of this disease in the future.Methods:A total of 56 cases of cholesteatoma specimens and 28 cases of retroauricular normal skin specimens including 20 cases of cholesteatoma specimens with self control were collected.The clinical information of patients including gender,age,and course of the disease,clinical classification,recurrence and complications were collected.The experimental cell is human immortalized keratinocyte(HaCaT cell),cultured in DMEM sugar medium containing 10% fetal bovine serum,under the conditions of 37 ? and 5%CO2.Experimental method: 1.The real-time fluorescent quantitative PCR was used for detecting of miR-203 a,mi R-34 a and miR-125 a in cholesteatoma and the corresponding retroauricular normal skin.2.The real-time fluorescent quantitative PCR and western blotwere used for miR-203 a and Bmi1 detection in cholesteatoma and the corresponding retroauricular normal skin.3.Immunohistochemical staining was used to observe the expression and distribution of Bmi1 in cholesteatoma and the corresponding retroauricular skin.4.The culture of human immortalized keratinocyte cells(HaCaT cells).5.Using transient transfection of NC miR-mimics,miR-203 a mimics,NC miR-inhibitor and miR-203 a inhibitor into HaCaT cells respectively,then detected the Bmi1 mRNA and protein expression level by using the real-time PCR and western blot in the cells of transfection.6.The dual luciferase reporter assay was used to analyze the relationship between miR-203 a and Bmi1.7.The NC miR-inhibitor,miR-203 a inhibitor,Bmi1 si RNA,NC si RNA and miR-203 a inhibitor+Bmi1 siRNA were transient transfected into HaCaT cells,the western blot was used for detecting the transfection efficiency,and cultivate 24 to 48 hours for follow-up study.8.The colony formation experiment was used for testing the colony formation ability.9.The MTS experiment was used for testing the ability of cell proliferation.10.Using flow cytometry instrument to detect the cell cycle process.11.Using flow cytometry instrument to detect the cells apoptosis rate.12.The application of Transwell cell migration experiment was to test the cell migration ability.13.Western blot was used to detect the p-Akt protein expression in cholesteatoma and the corresponding retroauricular skin.14.The application of immunohistochemical staining for detecting the p-Akt protein expression and distribution in cholesteatoma and the corresponding retroauricular skin.15.Western blot was used to detect the expression of Bmi1,total-Akt and p-Akt protein in cells.Results : 1.The result of real-time PCR detection showed that the expression of miR-203 a in cholesteatoma was significantly lower than the corresponding retroauricular normal skin,the difference was statistically significant(P<0.05).There were no significant differences of miR-34 a and miR125 a expressions in cholesteatoma and the corresponding retroauricular normal skin.2.The expression level of miR-203 a in cholesteatoma was not significant correlation with the patient's gender,age,course of the disease,clinical classification,recurrence and complications(P > 0.05).3.The expression of miR-203 a in 20 cases of self control cholesteatoma were significantly lower than the corresponding retroauricular normal skin;On the contrary,the expression of Bmi1 protein in cholesteatoma was significantly higher than the corresponding skintissue.The result of person correlation analysis showed that there was an obvious negative correlation between the miR-203 a and Bmi1 in cholesteatoma(P<0.05).4.Immunohistochemistry showed that Bmi1 mainly stained in nuclei.It stained in almost all layer of the cholesteatoma epithelium and the degree was strong.But in the corresponding skin tissues it was stained only in the basal layer cells and the degree was weak.The degree and area of Bmi1 staining in cholesteatoma were greater than the corresponding retroauricular skin,and the comparison of staining positive rate between two groups was statistically difference(P<0.05).5.In the HaCaT cells,compared with the control group,overexpression of miR203 a could reduce the expression of Bmi1 mRNA and protein significantly;On the contrary,down-regulated expression of miR203 a could increase the expression of Bmi1 mRNA and protein significantly(P < 0.05).6.Through the target gene prediction and validation,we found that Bmi1 is a downstream target gene of miR-203 a.7.In the HaCaT cells,inhibiting the expression of mi R-203 a could improve the expression of Bmi1 significantly,but the Bmi1 protein was almost down to the level of control cells when the cells co-transfected miR-203 a inhibitor and Bmi1 siRNA.8.The result of cell proliferation MTS experiment showed that inhibiting miR-203 a could significantly promote HaCaT cells proliferation;when co-transfecting miR-203 a inhibitor and Bmi1 siRNA,cell proliferation ability was almost down to the control group level.9.Colony formation experiments showed that the inhibition of miR-203 a expression could promote cell colony formation obviously;when co-transfected miR-203 a inhibitor and Bmi1 siRNA,the colony forming cells decreased obviously.10.The result of Cell cycle detection showed that inhibiting miR-203 a could increase the percentage of S phase cells,and reduce the percentage of G1 phase cells;when co-transfected miR-203 a inhibitor and Bmi1 siRNA,S and Gl phase cells percentage was almost back to the control group level.11.Apoptosis detection result showed that the inhibition of miR-203 a expression could significantly reduce the proportion of early apoptotic cells;when co-transfected miR-203 a inhibitor and Bmi1 siRNA,the number of apoptotic cells was almost back to the control group level.12.Cell migration experiment result showed that the silencing miR-203 a could effectively promote the HaCaT cells migration;when co-transfected miR-203 a inhibitor and Bmi1 siRNA,cell migration ability was almost back to the control group level.13.The resultof western blot showed that the expression of p-Akt in cholesteatoma epithelium is higher than that in the corresponding retroauricular skin,the difference was statistically significant(P<0.05).And there was a positive correlation between the p-Akt and Bmi1 in the cholesteatoma.14.Immunohistochemical staining result showed that the p-Akt was cytoplasmic staining,and mainly showed in the basal layer of normal skin.The staining degree was moderate to mild.On the contrary,the staining was almost in all layers,and the staining degree was deeper in cholesteatoma.The staining positive rate between the two groups was statistically significant(P< 0.05).15.There was a significant positive correlation between Bmi1 and p-Akt protein staining in cholesteatoma(P <0.05).16.Western blot detection result showed that Bmi1 and p-Akt expressions were significantly decreased in the HaCaT cells which were transfected Bmi1 siRNA;when co-transfected Bmi1 siRNA and miR-203 a inhibitor,the expressions of Bmi1 and p-Akt were restored.Conclusions:1.We found that the expression of miR-203 a in cholesteatoma decreased significantly compared to the corresponding retroauricular skin,and has statistical significance.2.The expressive level of miR-203 a in cholesteatoma has no obvious correlation with the clinical features of the patients.3.The expression of Bmi1 protein in cholesteatoma increases obviously,and has a significantly negative correlation with miR-203 a.4.Through the target gene prediction and validation,we found that Bmi1 is a downstream target gene of miR-203 a,and accept the negative regulation of mi R-203 a.5.The lack of expression of mi R-203 a in cholesteatoma leads to the inhibition of Bmi 1decreased,which in turn increases the expression of Bmi1 and result in excessive proliferation of cholesteatoma keratinocytes.6.Lower expression of miR-203 a promoted the proliferation,colony formation and migration,and inhibited the apoptosis by up-regulating Bmi1 in HaCaT cells.7.Compared to the corresponding retroauricular normal skin p-Akt present an over-expression in cholesteatoma,and has a positive correlation with Bmi1.8.In HaCaT cells miR-203 a can regulate Bmi1 and then influence the expression of p-Akt. |
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