Study Of Two CCNY Transcripts Differentially Expressed In NSCLC And Clinical Significance

Background:CyclinY (CCNY) is a newly identified cell cycle proteins, which include two isoform:CCNY isoform1(CCNY1) and to CCNY isoform of2(CCNY2). Comparing withCCNY2,CCNY1has more N-terminal1-54amino acid, the rest is identical. The currentresearch is focused on the CCNY1. The study found that CCNY knockdown by siRNAcan significantly inhibit the tumor cells proliferation in the gliomas. CCNY is highexpression in mRNA and protein level in Non-small-cell lung cancer (NSCLC), whichcan promote tumor cells proliferation. However, due to the two CCNY isoform are highlysimilar, previous studies CCNY RNAi sequence and probers detecting CCNY mRNA andprotein expression can not distinguish CCNY1and CCNY2. For both the expression andfunction in lung cancer remain unclear. Therefore,we design two primers specifictargeting CCNY isoform1N-terminus and the shared part by CCNY isoform1andCCNY isoform2to investigate to both expression in NSCLS,which provide preliminaryclues for two isoform function. Methods:We design CCNY1-primer and CCNY2-primer specific targeting CCNY1N-terminusand the shared part by CCNY1and CCNY2respectively. And on this basis weinvestigate the two cyclins expression in nine lung cancer cells and92paired of patientssamples using real time PCR. Moreover we analyze the relation between two cyclinsexpression and clinical pathologic parameters.Results:on the basis of designing two primers which can distinguish CCNY1and CCNY2,weexamined two cyclins expression in lung cancer cell lines and tissue.the result showedthat CCNY1expression in lung cancer cell lines and NSCLC tissue was lower than thatin normal lung cell and tissue, while CCNY2expression in NSCLC tissue was higherthan that in normal lung tissues. There was no significant correlation between CCNY1expression and patients’ age, gender, pathological type, lymph node metastasis, T stage,except differentiation.；Whereas There was no significant correlation between CCNY2expression and patients’ age, gender, pathological type, lymph node metastasis, T stage,and differentiation.furthermore,CCNY2was high expression in samples of NSCLC withlow differentiation,which indicated that CCNY2exert important role in NASCLC.Conclusion:On the base of establishing the primers to distinguish CCNY1CCNY2successfullywe,for the first time,reported that two CCNY expression in lung cancer: CCNY isoform2,not CCNY isoform1, is up-regulation in lung cancer cell lines and tissue sample,whichmay help to understand the two cyclins function in NSCLC.Moreover, CCNY2was highexpression in samples of NSCLC with low differentiation, suggesting that CCNY2play akey role in lung cancer. Further researches on CCNY and CCNX role in NASCLC areimportant. It could not only help elucidate the mechanism of tumorgenesis but alsoprovide a new perspective on the control of cancer. Background:cyclinY (CCNY) is a recently discovered class of cell cycle proteins. Studies have shownthat CCNY gene is highly expressed in non-small cell lung cancer tumor tissue and lungcancer cell lines; CCNY gene expression downregulation could significantly inhibit thetumor cells proliferation. but the mechanism is unclear. In this study, using siRNAtechnology to knock down CCNY two isoform expression cell lines H1299(H1299KD)and nonsense siRNA transfected cell lines H1299(H1299NC) as control, to explore twocell lines different gene expression by microchip analytical procedure, and furtherchoosing some different gene related with tumor development screened from geneexpression profiles,to validate the results by real-time PCR.Methods:The total RNAs were isolated from H1299KD/H1299NC cells. The equal amounts ofRNAs were both reversely transcribed to cDNAs and labeled with Cy5and Cy3fluorescence as probe respectively.Labeled cDNA were hybridized with cDNAmicroarray. After scanning and image processing,the different gene expression profilingof two cell lines was investigated.Results:1.Globally,267differentially expressed genes were screen out, among which up-anddown-regulated genes were175and92, respectively. The CCNY two isoformknockdown resulted in the altered expression of many genes related t o the occurrence and progression of tumor, which mainly involved in cell proliferation, apoptosis, invasionand metastasis, and cell cycle regulation;2. real-time PCR detect the expressionp21CIP1、DKK1、KIF18A、PCNA and PLK1mRNA levels.Compared with the controlgroup, the results have the same directional chip test results.Conclusion:1.By gene chip analysis many genes expression happen to change after CCNY twoisoform knockdown, some of which are involved in tumor-related genes and importanttumor signaling pathway.2. Using real time PCR to detect the genes change chose by gene expression profiling, theresult is in good agreement with the gene chip,which provides a solid basis for thefollowing research on CCNY functional mechanisms of carcinogenesis.3.CCNY1and CCNY2may have different regulatory pathways in tumors.Study CCNYtwo isoform function in tumor may provide new strategy for cancer therapy.