| Objective: Cholesteatoma of the middle ear is a benign squamous keratinizing hyperplastic lesion composed of squamous lamellar keratinocytes.It presents as an epidermoid cyst with fibroblasts and inflammatory cells.Cholesteatoma of the middle ear is characterized by excessive proliferation of cells,accumulation of keratin fragments and destruction of temporal bone and its surrounding bone structure.Cholesteatoma can lead to a variety of intracranial and extracranial complications,such as labyrinthitis,facial paralysis,sigmoid sinus thrombosis phlebitis,meningitis,brain abscess,in addition to hearing damage and otorrhea.The pathogenesis of cholesteatoma is complex with many influencing factors.RECK gene was a new transcriptional inhibitory gene discovered by Japanese scholar Takahashi et al in NIH3T3 cell line transfected with v-ki-ras in 1998.Widespread in its expression in normal tissue,but in many tumor cell lines and by a variety of cancer-causing gene transformation into fiber cell line detection,and the occurrence and development of many tumors and inflammatory diseases,invasion and metastasis are closely related,the inhibitory effect of tumor invasion and metastasis of several by inhibiting the expression of matrix metalloproteinases and implementation.Mmp-2 and 9are two of the most important of them.They are produced in the form of inactive precursors and have a wide range of functions.Degradable gelatin can degrade collagen,elastin,laminin A chain as well as interstitial collagen and matrix protein.Strict regulation of MMP gene is crucial to tissue destruction and reconstitution.If overexpressed,connective tissue destruction can lead to pathological conditions such as arthritis and periodontitis.RECK is low expressed in many inflammatory diseases at present,but the expression and biological function of RECK in the occurrence and development of cholesteatoma of the middle ear are still unclear.In this study,the expression of RECK in cholesteatoma of the middle ear and the mechanism of RECK in the progression of cholesteatoma of the middle ear were studied.Methods: 1.36 cases of cholesteatoma specimens of part of middle ear that were operated on in the department of otology,shengjing hospital from February 2017 to October 2017 were collected,and 29 cases of skin of external auditory canal(open mastectomy + tympanoplasty + small fragments of skin of external auditory canal that were discarded and not used during the operation)were collected as the control group.The mRNA expressions of RECK,MMP2 and MMP9 in the middle ear cholesteoma and the skin of the external auditory canal were detected by RT-PCR.Western Blot was used to detect the protein expressions of MMP2 and MMP9 in cholesteatoma of the middle ear and the skin of the external auditory canal.2,cultivate people immortalized HaCaT epidermis cells(human immortalized keratinocyte),cell culture for DMEM(including 2 ml-glutamine)+ 10% fetal bovine serum,culture conditions for 37 ? and 5% CO2,constant temperature incubator adherent culture,once every three days to extend,represented by 0.25% when the pancreatic enzyme digest 3min constant temperature incubator,heavy suspension cells by 1:2 batches.Silencing RECK gene and overexpressing RECK gene in HaCaT cells were constructed by plasmid transfection.Real-time quantitative PCR was used to detect the expression level of RECK mRNA in order to evaluate whether the silent RECK gene and the overexpressed HaCaT were successfully constructed.MTS assay was used to detect the effect of silencing and overexpression of RECK on the proliferation of HaCaT cells.Results: 1.RT-PCR results showed that the relative expression level of RECK mRNA in cholesteatoma of the middle ear was 0.723±0.099,which was lower than 1.180±0.121 in the skin of the external auditory canal(P=0.004).The relative expression levels of MMP2 and MMP9 mRNA in the middle ear cholesteatoma were 11.180 ± 3.158 and99.35 ± 31.92,respectively,which were significantly higher than that in the external auditory canal skin 2.013±0.495(P=0.012)and 1.522±0.529(P=0.007).Western Blot results showed that the relative expression levels of MMP2 and MMP9 in cholesteatoma of the middle ear were 4.105 ± 1.209 and 15.980 ± 6.520,respectively,which were significantly higher than that in normal skin of the external auditory meatus of 0.666±0.215(P=0.040)and 1.033±0.375(P=0.047).2.The relative expression levels of RECK mRNA in the si-RECK transfection group and the con non-transfection group were 0.581± 0.107 and 1.033 ± 0.033,respectively(P=0.016).The RECK mRNA expression inHaCaT cells of the si-RECK transfection group was significantly lower than that of the con non-transfection group,and the RECK gene was successfully silenced.The relative expression levels of RECK mRNA in HaCaT cells of the OE-RECK transfection group and con group were 70.040 ± 22.640 and 1.033 ± 0.033,respectively(P=0.038).The proliferation of HaCaT cells was detected by MTS method after down-regulation of RECK gene.The proliferation of HaCaT cells in con group,si-RECK NC group and si-RECK group at 24 h was 1.179±0.044,1.191±0.028,1.442±0.034.The proliferation of HaCaT cells in si-RECK transfection group was significantly higher than that in si-RECK NC group(P=0.005)and blank control group(P=0.009).At 48 h,the proliferation of HaCaT cells in con group,si-RECK NC group and si-RECK group were1.540 ± 0.088,1.548 ± 0.109,2.071 ± 0.116.The proliferation of HaCaT cells in si-RECK transfection group was significantly higher than that in si-RECK NC group(P=0.030)and blank control group(P=0.022).The proliferation of HaCaT cells in the con group,the si-RECK NC group and the si-RECK group at 72 h was 1.869±0.129,1.895 ± 0.136,3.242 ± 0.119.The proliferation of HaCaT cells in the si-RECK transfected group was significantly higher than that in the si-RECK NC group(P=0.001)and the blank control group(P=0.001),while there was no significant difference between the si-RECK NC group and the blank control group(P>0.05).MTS assay was used to detect the effect of up-regulated RECK gene on the proliferation of HaCaT cells:the proliferation of HaCaT cells in the con group,the OE-RECK NC group and the OE-RECK group at 24 h was 1.553 ± 0.042,1.579 ± 0.040,1.196 ± 0.035.The proliferation of HaCaT cells in the OE-RECK transfection group was significantly lower than that in the OE-RECK NC group(P=0.002)and the blank control group(P=0.002).At 48 h,the proliferation of HaCaT cells in the con group,the OE-RECK NC group and the OE-RECK group were 1.712±0.074,1.771±0.084,1.314±0.013,respectively.The proliferation of HaCaT cells in the OE-RECK transfection group was significantly lower than that in the OE-RECK NC group(P=0.005)and the blank control group(P=0.006),while there was no significant difference between the OE-RECK NC group and the blank control group(P>0.05).Conclusions: 1.The expression of RECK in cholesteatoma of the middle ear was lower than that in the skin of the external auditory canal,and the expression of MMP2 andMMP9 in cholesteatoma of the middle ear was higher than that in the skin of the external auditory canal.The expression patterns of RECK,MMP2 and MMP9 may play a certain role in promoting the progression of cholesteatoma in the middle ear,and may also help provide new ideas for the clinical treatment of cholesteatoma in the middle ear.2.Interfering with RECK gene can regulate the proliferation of HaCaT cells,silencing RECK promotes cell proliferation,and overexpression of RECK inhibits cell proliferation,suggesting that molecular diagnosis and gene therapy designed based on RECK gene may be applied to the clinical treatment of cholesteatoma in the middle ear. |
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