ABSTRACT
Objective The method for detecting expression of human CDK14 gene with Real-time quantitative PCR was developed.Methods To establish a method for detecting expression of human CDK14 gene with Real-time quantitative PCR by designing and synthesis of the primers of CDK14 target gene andβ-Actin reference gene and extracting total RNA from different lung cancer cell lines.Then the specificity,detection range and repeatability of this method were evaluated.At last,the expression level of CDK14 gene in different cell lines,which were with or without siRNA interference,were carried out by using this method.Results The method for detecting expression of human CDK14 gene with Real-time quantitative PCR,which had good specificity,good repeatability (CV=7.3 ％) and wide detection range (Ct value range of CDK14 and β-Actin amplification curve were 22.47～32.96 and 15.14～ 27.55 respectively,r2 =0.9844),was developed and it was verified by electrophoresis analysis,melting curve,PCR product sequencing.And CDK14 gene expression level,which was detected by this method,increased in HCC827 D5,H1650 and number 1 siRNA segment was effective interference segment.Conclusion The method for detecting expression of human CDK14 gene with Real-time quantitauve PCR was established successfully.
ABSTRACT
Background:CDK14 is a novel cyclin-dependent kinase,which is overexpressed in a variety of cancer and related to their malignant behavior. Aims:To investigate the effect of CDK14 on proliferation of human esophageal carcinoma cells and its possible mechanism. Methods:Expressions of CDK14 and two cell proliferation markers,PCNA and Ki-67 were estimated in 8 fresh-frozen specimens of esophageal squamous cell carcinoma(ESCC),96 paraffin-embedded specimens of ESCC,and human ESCC cell line Eca-109 by Western blotting and immunohistochemistry. Correlations of CDK14 expression with the clinicopathological characteristics and prognosis of ESCC were analyzed. Serum starvation and release assay was performed to evaluate the relationship between CDK14 expression and cell cycle progression in Eca-109 cells. Furthermore,Eca-109 cells were transiently transfected with shRNA-CDK14 to reduce CDK14 protein level,and then the phosphorylation of tumor suppressor protein Rb,cell cycle progression and proliferation capability of Eca-109 cells were determined. Results:CDK14 was highly expressed in both ESCC tissue and cell line,which was paralleled with the expressions of PCNA and Ki-67 and correlated significantly with the tumor size,histological grade,invasiveness and metastasis of ESCC(P < 0. 05). The overall survival was poor in patients with high CDK14 expression than those with low CDK14 expression(P < 0. 05). Serum starvation and release assay showed that the expression of CDK14 was cell cycle-dependent. Knockdown of CDK14 reduced the expression level of phosphorylated Rb,induced significant G1 phase arrest and resulted in less colony formation in Eca-109 cells(P all < 0. 05). Conclusions:CDK14 is highly expressed in ESCC. It may promote cell cycle progression by phosphorylating downstream Rb protein,thus enhancing the proliferation of tumor cells,and ultimately participating in the occurrence and development of ESCC.