| Background: Hypopharyngeal squamous cell carcinoma(HSCC)is a malignant tumor derived from hypopharynx,which accounts for 3 to 5% of all head malignancies.Usually,because the location of HSCC is hidden,the initial symptoms are not significant and lack of typical features,it is difficult to diagnose HSCC early in clinic.When patients have swallowing obstruction or pain go to the hospital for treatment,the tumors often have invaded the laryngeal cavity,tongue root or cervical esophagus.70% of HSCC patients were pathologically diagnosed as poorly differentiated squamous cell carcinoma with high malignancy.In addition,HSCC is easy to spread to submucosa and metastasis to cervical lymph nodes.Currently,the treatment of HSCC is limited to surgery,radiotherapy,chemotherapy and immunotherapy,while the exact pathogenesis of HSCC is still unclear,lacking specific therapeutic targets.In order to make early diagnosis,early treatment and improve the cure rate and survival rate of SHCC,we need to study the mechanism of occurrence,development,invasion and metastasis of HSCC from the perspective of molecular biology,and find a new method to treat HSCC,which will be of great significance.miRNA is a kind of non-coding single-stranded small molecule RNA that widely exists in animals and plants.It plays an important role in cell growth,proliferation,cell cycle and apoptosis by regulating the expression of target gene.Studies have shown that miRNAs are involved in the development and progression of tumors.miR-137 is a member of the miRNA family,the current study has confirmed its abnormal expression in glioma,gastric cancer,non-small cell lung cancer,ovarian cancer and other malignant tumors abnormally reduced,it may have a similar tumor suppressor gene effect.Studies have shown the miR-137 promoter methylation in oral cancer,indicating that miR-137 promoter methylation is the main way to silence.miR-137 DNA methylation is associated with a lower survival rate in patients with head and neck squamous cell carcinoma.At present,the relationship between promoter methylation of miR-137 and hypopharyngeal cancer has been rarely reported.The aim of this study was to investigate the mechanism of miR-137 DNA methylation up-regulation CDK6 in the malignant process of HSCC.Understanding the biological role of miR-137 DNA methylation in hypopharyngeal cancer will be of great significance in improving the diagnosis and treatment of hypopharyngeal cancer.Materials and Methodsa: 1.Clinical specimens of hypopharyngeal cancer and its adjacent specimens,human hypopharyngeal squamous cell carcinoma cell line Fadu and normal laryngeal mucosa cells were used as experimental materials.All human microRNAs with a sequence of less than 3000 bp were searched on the Microbase website,and then microRNAs with methyl island in the 1K gene fragment of 5'-UTR were found in the microRNAs mentioned above.The most significant difference in DNA methylation level between human hypopharyngeal cancer and adjacent human cancers was detected by MSP.The methylation levels of microRNA-137 DNA in 50 normal hypopharyngeal mucosa tissues and 50 cancer tissues of hypopharyngeal cancer patients were detected by MSP,and the relationship between microRNA-137 methylation and clinicopathological parameters of HSCC patients was analyzed.The methylation level of microRNA-137 and the expression of microRNA-137 in Fadu and normal laryngeal mucosa cells were detected by MSP and fluorescence quantitative PCR,respectively.The effect of DNA methylation inhibitor 5-AZA-CdR on the expression of microRNA-137 in Fadu cells was analyzed.CCK-8 method was used to detect the proliferation activity of Fadu cells,Transwell chamber method was used to detect the invasive ability of Fadu cells,cell scratch test was used to detect the migration ability of Fadu cells,and its effects on the proliferation,invasion and migration of Fadu cells were analyzed.2.Bioinformatics software was used to predict the target gene of miR-137,and the target gene CDK6 was locked according to the literature.Immunohistochemical staining was used to detect the positive expression of CDK6 in hypopharyngeal carcinoma.The expression of CDK6 in human hypopharyngeal cancer specimens was detected by PCR and WB.The control group was used for detection.MiR-137 mimics negative control group(mimics-NC),miR-137 mimics transfection group(miR-137 mimics),Cotransfection group of over-expression plsmids of miR-137 mimics + CDK6(miR-137 mimimics + CDK6),CDK6 overexpression control group(no load),CDK6 overexpression group(CDK6 overexpression plasmid).CCK-8 method and Transwell chamber method were used to detect the changes of cell proliferation and invasion in each transfection group.The effect of miR-137 on the proliferation and invasion of Fadu cells by regulating CDK6 was analyzed.The downstream target gene EGR1 was identified according to the literature.Immunohistochemical staining was used to detect the expression of EGR1 in hypopharyngeal carcinoma.The expression of EGR1 mRNA and protein in HSCC tissues was detected by fluorescence quantitative PCR and Western blot,respectively.CDK6 overexpression plasmid was transfected into Fadu cells to detect the expression of EGR1 mRNA and protein.CCK-8 method was used to detect the proliferation of Fadu cells and Transwell chamber method was used to detect the invasive ability of Fadu cells.To verify the effect of CDK6 on the proliferation and invasion of Fadu cells by regulating Egr1,Mir-137 mimic was transfected into hypopharyngeal Fadu cells to detect the expression of EGR1 mRNA and protein.mir-137 mimic and EGR1 overexpression plasmids were transfected into Fadu cells.Hsa-mir-137 affects the proliferation and invasion of hypopharyngeal Fadu cells by regulating Egr1.Result: 1.Compared with normal pharynx and larynx tissues,the level of miR-137 DNA methylation in HSCC tissues was significantly higher(P < 0.01).MiR-137 DNA methylation was not related to age,sex and smoking of HSCC patients,but related to tumor size,lymph node metastasis and clinical stage of HSCC patients.The methylation level of miR-137 DNA in Fadu cells was significantly higher than that in human normal laryngeal mucosa cells(P < 0.05),while the expression of miR-137 in Fadu cells was lower than that in human normal laryngeal mucosa cells(P < 0.05).Using DNA methylation inhibitor 5-AZA-CdR as the intervention of demethylation,the DNA methylation level of miR-137 in Fadu cells was significantly lower than that in control group(P < 0.05),while the expression of miR-137 was significantly higher than that in control group(P < 0.05).In Fadu cells,compared with control group,the proliferation activity of 5-AZA group decreased significantly(P < 0.05),and the number of invasive cells decreased significantly(P < 0.05).2.Bioinformatics software analysis showed that CDK6 is a target gene of miR-137,and the binding site of miR-137 on CDK6 is located in the 3'UTR region.Compared with the control group,the expression levels of CDK6 protein and mRNA in the miR-137 mimic group were significantly decreased(P < 0.05).Compared with the control group,the proliferation activity of the cells in the miR-137 mimic group was significantly decreased(P < 0.05),while the proliferation activity of the cells in the miR-137 mimic + CDK6 group was not significantly different from that in the control group(P > 0.05).Compared with the control group,the number of invasive cells in the miR-137 mimic group decreased significantly(P < 0.05),while the number of invasive cells in the miR-137 mimic + CDK6 group had no significant difference(P > 0.05).3.Compared with normal pharynx and larynx mucosa,the expression levels of Egr1 mRNA and protein in HSCC were significantly lower(P < 0.05).Compared with NP69 cells,the expression levels of Egr1 mRNA and protein in Fadu cells were significantly lower(P < 0.05).Compared with control group,the expression levels of Egr1 mRNA and protein in CDK6 cells were significantly lower(P < 0.05).Compared with control group,the proliferation activity and the number of invasive cells in CDK6 group increased significantly(P < 0.05).The proliferation activity and invasive cell number of CDK6 + Egr1 group did not change significantly at each time point(P > 0.05).Compared with the control group,the expression levels of Egr1 mRNA and protein in the miR-137 mimic group were significantly higher(P < 0.05).Compared with the control group,the proliferative activity of the cells in the miR-137 mimic group decreased significantly(P < 0.05).The number of invasive cells decreased significantly(P < 0.05).The proliferative activity of cells in the miR-137 mimic + Egr1 group was significantly lower than that in the miR-137 mimic group(P < 0.05).The number of invasive cells decreased significantly(P < 0.05).4.Immunohistochemical results: CDK6 was expressed in hypopharyngeal cancer cells,but EGR1 was negative in hypopharyngeal cancer cells.CDK6 and EGR1 were negative in adjacent tissues.Conclusion: 1.The DNA methylation level of miR-137 in HSCC tissues was significantly higher than that in adjacent normal tissues,and was related to the size of tumors,lymph node metastasis and clinical stage of HSCC patients.2.The expression level of miR-137 decreased after DNA methylation,which promoted the proliferation,invasion and migration of HSCC cells,and ultimately promoted the malignant process of HSCC.3.miR-137 through target inhibits the expression of CDK6,promotes the production of EGR1 and ultimately inhibits the proliferation and invasion of Fadu cells. |
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