Expression characteristics of CDK1 in breast cancer tissues and its effect on proliferation and apoptosis of breast cancer cells
Objective Research on the expression characteristics of cyclin-dependent kinase 1(CDK1)in breast cancer and its effects on the biological functions of breast cancer cell proliferation and apoptosis,with the aim of evaluating whether CDK1 can be a meaningful monitoring point for the diagnosis and treatment of breast cancer.Methods Collected 65 pairs of breast cancer and adjacent normal tissues from the Department of Surgery,Mudanjiang City Oncology Hospital from March 2009 to March 2019.Gene Expression Profiling Interactive Analysis(GEPIA)database analysis of the differential expression of CDK1 in tumor tissues and normal tissues;used immunohistochemistry to detect the localization and expression of phosphorylated CDK1(p-CDK1)in breast cancer and adjacent normal tissues,and performed correlation analysis with clinicopathological parameters;immunofluorescence and Western blot detected the expression of CDK1 and p-CDK1 in breast cancer cells(MCF-7 and MDA-MB-231 were human breast adenocarcinoma epithelial cell lines).MCF-7 cells were divided into untransfected wild-type group(MCF-7 group),transfected negative control group(control group),and transfected with CDK1 siRNA1,2,and 3 groups to further silence CDK1 expression,and then the cell counting kit-8(CCK8)assay,plate cloning assay,TUNEL assay were comprehensively analyzed for the proliferation ability of breast cancer cells and the espression of tumor protein 53(TP53),cleaved-caspase3(C-CASP3),B-cell leukemia(Bcl-2),Bcl-2 associated X protein(Bax)were detected by Western blotting.Results Results from the GEPIA database analysis show that the expression level of CDK1 is upregulated in various tumors,including bladder urothelial carcinoma,cervical squamous cell carcinoma,colon cancer,esophageal cancer,lung squamous cell carcinoma,and thymoma;the differences in expression were statistically significant(all P＜0.05).Compared with normal tissues,CDK1 expression is increased in breast cancer tissues,and the difference was statistically significant(P＜0.05).Immunohistochemistry results reveal that in breast cancer tissues and adjacent normal tissues,CDK1 and p-CDK1 were localized in the cytoplasm and nucleus of cells,with higher expression levels in the nucleus.CDK1 and p-CDK1 expression levels were higher in breast cancer compared to adjacent normal tissues,and the difference was statistically significant(x2=30.540,P＜0.001).The expression of p-CDK1 in the nucleus of breast cancer cells had no significant difference with age,histological type,histological grade,or lymph node metastasis(all P＞0.05),but it had significant difference with tumor diameter(x2=5.659,P=0.017).The expression of p-CDK1 in the cytoplasm of breast cancer cells had no significant difference with age,tumor diameter,histological type,histological grade,or lymph node metastasis(all P＞0.05).Immunofluorescence detection shows that CDK1 and p-CDK1 were highly expressed in MCF-7 cells.The protein expression levels of CDK1(1.46±0.12)and p-CDK1(1.37±0.10)in MCF-7 cells were significantly higher than those in MDA-MB-231 cells[(0.27±0.05),(0.46±0.03)],and the differences were statistically significant(t=15.794,15.646;both P＜0.01);subsequent experiments selected MCF-7 cells to construct a transient silencing cell line to study the related functions of CDK1.After routine transfection of MCF-7 cells,the CDK1 protein expression in the siRNA1 group and the siRNA3 group was significantly lower than that in the control group,and the differences were statistically significant(both P＜0.05);the difference in the siRNA2 group was not statistically significant(P＞0.05).The silencing efficiency of the CDK1 siRNA1 group was the highest,so the subsequent experiments selected the siRNA1 to silence CDK1 expression.The results of the CCK8 experiment showed that after knocking down CDK1,the proliferation ability of MCF-7 cells in the CDK1 siRNA group(0.87±0.05)significantly decreased compared to the control group(1.48±0.08),with a statistically significant difference(t=1 1.199,P＜0.01).The colony formation assay demonstrated that the colony formation rate of MCF-7 cells in the CDK1 siRNA1 group[(37.00±1.50)％]was significantly lower than that of the control group[(64.00±1.00)％],with a statistically significant difference(t=18.706,P＜0.01).Western blot results revealed that compared with the control group,the CDK1 siRNA1 group showed an increased apoptosis rate,upregulation of Bax and C-CASP3 protein expression and downregulation of Bcl2 protein expression,all of which had statistically significant differences(t=5.329,4.076,3.789,3.060;all P＜0.05).Conclusions CDK1 and p-CDK1 were upregulated in breast cancer tissues.Knocking down CDK1 could inhibit the proliferation of breast cancer cells and promote cell apoptosis.CDK1 could be used as a marker for evaluating the growth and proliferation ability of breast cancer.
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