| Objective:In this study,the expression levels of CCAT1,miR-130a-3p and KDM2A in human clear cell renal cell carcinoma(ccRCC)tissues and adjacent normal renal tissues were detected,and the correlation between the expression levels and clinical indicators and pathological stages was studied to explore the clinical significance of the expression of CCAT1,miR-130a-3p and KDM2A in ccRCC.Secondly,the expression of CCAT1,miR-130a-3p and KDM2A in 786-O cells and HK-2 cells were detected,and the expression of CCAT1,miR-130a-3p and KDM2A were interfered respectively,to study the regulation of the expression level and the effect on biological behavior of ccRCC cell.Finally,the binding of CCAT1 to miR-130a-3p and the binding of KDM2A to miR-130a-3p was verified by dual luciferase reporter assays.Methods:The 40 groups of tissue specimens were collected from patients with ccRCC who underwent radical nephrectomy in the First Affiliated Hospital of China Medical University from September 2016 to June 2018.The average age was 58.98±9.41 and the median age was 58 years.There were 23 males and 17 females.Each group consisted of ccRCC tissues and adjacent tissues,of which the adjacent tissues were taken from 5 cm away from the tumors,and those with larger tumors and less than 5 cm from the remaining adjacent tissues were taken from more than 3 cm away from the tumors,which were confirmed by pathology as non-cancer renal tissues.Inclusion criteria:1.All patients underwent radical nephrectomy;2.All the excised tumors were proved to be ccRCC by pathology;3.No preoperative radiotherapy,chemotherapy,immunotherapy,targeted therapy,interventional embolization,and other adjuvant treatment;4.No history of other malignant tumors was found in the patients;5.The clinical and pathological data of the patients were complete.Exclusion criteria:1.Patients did not undergo radical resection;2.Preoperative radiotherapy,chemotherapy,immunotherapy,targeted therapy,interventional embolization and other adjuvant treatment;3.History of other malignant tumors;4.Incomplete clinical and pathological data.All patients had informed consent.The experiment was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University.Human ccRCC 786-O cell line and human renal proximal convoluted tubular epithelial cell HK-2 cell line were used in this study.Firstly,the expression of CCAT1,Mi-130a-3p and KDM2Ain ccRCC tissues and adj acent tissues were detected by qRT-PCR,and the correlation between the expression level of CCAT1,Mi-130a-3p and KDM2A and clinical indicators of ccRCC were analyzed.The expression of KDM2A protein in ccRCC tissues and adjacent tissues were detected by immunohistochemistry.Secondly,the expression of CCAT1,Mi-130a-3p and KDM2A in 786-O cells and HK-2 cells were detected by qRT-PCR,and the expression of KDM2A protein in 786-O cells and HK-2 cells were detected by Western Blot assay,respectively.The 786-O cells were transfected:non-transfected group(control group),negative control group(si-NC group)and si-CCAT1 group were set up for the first transfection;control group,negative control group(miR-NC group),miR-130a-3p mimic group were set up for the second transfection;control group,negative control group(si-NC group)and si-KDM2A group were set up for the third transfection.After three transfections,the expression of CCAT1,miR-130a-3p and KDM2A were detected by qRT-PCR,and the expression of KDM2A protein was detected by Western Blot,and cells were detected by CCK-8 assay,flow cytometer and Transwell assay were used to detect cell proliferation,apoptosis and migration.Finally,the target binding of CCAT1 to miR-130a-3p and KDM2A to miR-130a-3p were verified by the dual luciferase reporter assay,respectively.All statistical analysis was carried out using SPSS Statistics 23 and Prism 7.The counting data were expressed by examples and rates or constituent ratios.The measurement data were expressed by mean ± standard deviation.The normality was tested by Kolmogorov-Smirnov method.The chi-square test was used to compare the rates of the two samples.The two-tailed Student’s t-test or One-way ANOVA was applied to evaluate statistical differences.P<0.05 was considered to be statistically significant.Results:1.The expression of CCAT1 was detected by qRT-PCR.The results showed that the expression of CCAT1 in ccRCC tissues was higher than that in adjacent tissues(P<0.05).The high expression of CCAT1 was correlated with tumor size and TNM stage(P<0.05).2.The expression of miR-130a-3p was detected by qRT-PCR.The results showed that the expression of miR-130a-3p in ccRCC tissues was lower than that in adjacent tissues(P<0.05).The low expression of miR-130a-3p was correlated with tumor size and TNM stage(P<0.05).3.The expression of KDM2A was detected by qRT-PCR.The results showed that the expression of KDM2A in ccRCC tissues was higher than that in adjacent tissues(P<0.05).The high expression of KDM2A was correlated with tumor size and TNM stage(P<0.05).4.Immunohistochemistry was used to detect the expression of KDM2A protein.The results showed that the positive expression rate of KDM2A protein in ccRCC tissues was higher than that in adjacent tissues(P<0.05).5.The expression of CCAT1,miR-130a-3p and KDM2A in 786-O cells and HK-2 cells were detected by qRT-PCR.The results showed that the expression of CCAT1 and KDM2A in 786-O cells was higher than that in HK-2 cells,and the expression of miR-130a-3p in 786-O cells was lower than that in HK-2 cells(P<0.05).6.Western Blot assay was used to detect the expression of KDM2A protein in 786-O cells and HK-2 cells.The results showed that the expression of KDM2A protein in 786-O cells was higher than that in HK-2 cells(P<0.05).7.After transfection of 786-O cells with si-CCAT1,the results of qRT-PCR showed that the expression of CCAT1 was decreased,the expression of miR-130a-3p was increased,the expression of KDM2A was decreased in si-CCAT1 group compared with those in control group and si-NC group(P<0.05),the Western Blot assay showed that the expression of KDM2A protein in si-CCAT1 group was decreased(P<0.05).The CCK-8 assay showed that the proliferation of cells in the si-CCAT1 group was weakened(P<0.05).The flow cytometry showed that the apoptotic rate of cells in the si-CCAT1 group was increased(P<0.05).The Transwell assay showed that the invasive ability of cells in the si-CCAT1 group was weakened(P<0.05).8.After transfection of 786-O cells with miR-130a-3p mimic,the results of qRT-PCR showed that the expression of miR-130a-3p was increased,the expression of CCAT1 and KDM2A was decreased in miR-130a-3p mimic group compared with those in control group and miR-NC group(P<0.05),the Western Blot assay showed that the expression of KDM2A protein in miR-130a-3p mimic group was decreased(P<0.05).The CCK-8 assay showed that the proliferation of cells in miR-130a-3p mimic group was weakened(P<0.05).The flow cytometry showed that the apoptotic rate of cells in miR-130a-3p mimic group was increased(P<0.05).The Transwell assay showed that the invasive ability of cells in miR-130a-3p mimic group was weakened(P<0.05).9.After transfection of 786-O cells with si-KDM2A,the results of qRT-PCR showed that the expression of KDM2A was decreased,the expression of CCAT1 was decreased and the expression of miR-130a-3p was increased in si-KDM2A group compared with those in control group and si-NC group(P<0.05),the Western Blot assay showed that the expression of KDM2A protein in si-KDM2A group was decreased(P<0.05).The CCK-8 assay showed that the proliferation of cells in the si-KDM2A group was weakened(P<0.05).The flow cytometry showed that the apoptotic rate of cells in the si-KDM2A group was increased(P<0.05).The Transwell assay showed that the invasive ability of cells in the si-KDM2A group was weakened(P<0.05).10.The dual luciferase reporter assay confirmed that CCAT1 was a direct target of miR-130a-3p,KDM2A was a direct target of miR-13 0a-3p.Conclusions 1.CCAT1 was overexpressed,microRNA-130a-3p was underexpressed and KDM2A was overexpressed in ccRCC,which was correlated with TNM stage and tumor size.2.The positive expression rate of KDM2A protein in ccRCC tissues was higher than that in adjacent tissues.3.786-O cells showed higher expression of CCAT1,lower expression of miR-130a-3p and higher expression of KDM2A compared with HK-2 cells.4.KDM2A protein was higher expressed in 786-O cells compared with HK-2 cells.5.Downregulation of CCAT1 in 786-O cells could up-regulate the expression of miR-130a-3p,down-regulate the expression of KDM2A,and inhibit cell proliferation,invasion and promote cell apoptosis.6.Upregulation of miR-130a-3p in 786-O cells could down-regulate the expression of CCAT1 and KDM2A,and inhibit cell proliferation,invasion and promote cell apoptosis.7.Downregulation of KDM2A in 786-O cells could down-regulate the expression of CCAT1 and up-regulate the expression of miR-130a-3p,and inhibit cell proliferation,invasion and promote cell apoptosis.8.CCAT1 was a direct target of miR-130a-3p,KDM2A was a direct target of miR-130a-3p.In conclusion,the CCAT1/miR-130a-3p/KDM2A axis could regulate the biological behavior of ccRCC cells and play a role in its development.This study may provide new ideas for the diagnosis and treatment of ccRCC. |
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