Preliminary Study On The Function And Regulatory Mechanism Of PRR1-SKA2 Gene Pair In Breast Cancer

PRR11(proline-rich 11)and SKA2(spindle and kinetochore associated complex subunit 2)are two newly identified tumor-associated genes that are closely located on chromosome 17q22 and constitute a classic head-to-head gene pair driven by a unique bidirectional promoter.Our previous study indicated that,PRR11 and SKA2 play an important role in both cell cycle regulation and tumorigenesis,and the expression of them can be regulated by NF-Y and p53 in lungh cancer cell lines.In lung cancer tissues,PRR11 and SKA2 were upregulated and the expression was also negatively associated with prognostic conditions.In lung cancer cells,the deletion of PRR11 and SKA2 can lead to the inhibition of cellular proliferation,migration and invasion.In this paper which is based on the conclusion mentioned above,the following parts were designed to investigate the function and regulatory mechanism of PRR11-SKA2 gene pair in breast cancer.(1)The relevance between the expression level of PRR11-SKA2 gene pairs and the prognostic level in breast cancerWe analyzed,by bioinformatic approachs,the prognostic value of PRR11 and SKA2 in breast cancer.Microarray and patient survival data were downloaded from the GEO database(GSE3493 and GSE4944).The results indicated that,the low expression level of PRR11 or SKA2 is more significantly correlated with better prognosis of patients with breast cancer.(2)si RNA-mediated PRR11 or SKA2 silencing suppressed the proliferation,migration and invasion of breast cancer cellsPRR11-targeted si RNA and SKA2-targeted si RNA was transiently transfected alone or combinedly into breast cancer cells including MCF-7 and MDA-MB-231 respectively.The phenotypic analysis of the transfected cells revealed that,compared with control groups,the ability of proliferation,migration and invasion in single or simultaneous silencing cells was significantly inhibited,especially in combined silencing cells.The results suggested that both PRR11 and SKA2 play an important role in tumorigenesis and the development of breast cancer,and the gene pair might not only coexist but also function in a complementary manner.q PCR analysis revealed that the expression of several genes relating to proliferation,migration and invasion varied in different extent after si RNA mediated silencing.These findings suggested that both PRR11 and SKA2 might be implicated in tumorigenesis and progression of breast cancer by the expression of genes above-mentioned.(3)Transcriptional analysis of NF-Y in the regulation of PRR11 and SKA2 in breast cancerWe have constructed various deletion mutants of luciferase reports corresponding to the intergenic region between PRR11 and SKA2 and cotransfected them with NFYB expression vector into MCF-7 cells.Luciferase activity analysis showed that PRR11 and SKA2 can be promoted bidirectionally.In addition,NFYB can transactivate the expression of PRR11,while it did not have the ability of regulating the trancription of SKA2.Further investigation by q PCR can provide an apparent evidence.Bioinformatic analysis found that,NFYB was correlated with PRR11 expression but showed no relevance with the expression of SKA2.(4)Transcriptional analysis of p53 in the regulation of PRR11-SKA2 gene pairs and the cilinical significance of p53 in breast cancerPreviously constructed various deletion mutants of luciferase reports corresponding to the intergenic region between PRR11 and SKA2 were cotransfected with p53 expression vector into MCF-7 cells.The results showed that wild type p53 can inhibit the activity of PRR11-SKA2 promoters in breast cancer.Meanwhile bioinformatic analysis revealed that wild type p53 show a negative relevance with the expression of PRR11 and SKA2.In addition,survival analysis revealed that breast cancer patients with low expression level of PRR11 or SKA2 and wild type p53 exhibited a better survival outcomes compared with patients with p53 mutation and higher expression of either PRR11 or SKA2.In summary,our present study demonstrated that the expression level of PRR11-SKA2 is negatively correlated with the prognosis of patients with breast cancer,which can serve as an important predictor for prognosis.In addition,PRR11 and SKA2 played an important role in the development of breast cancer,and the expression of them can be regulated by NF-Y and p53 in different manners.This study not only establishes a theoretical basis for further exploring the regulatory mechanism of PRR11-SKA2 gene pairs and their role in tumorigenesis,but also provides a potential significance for the diagnosis and prognosis analysis in breast cancer.