Screening And Functional Investigation Of The Genes Associated With Prostate Cancer Development Using Affymetrix Microarray

Objective:The incidence of prostate cancer has been increasing in recent years in Chinese males,In the western countries,the incidence of prostate cancer has been remained in the first position over lung cancer for continuous 15 yeas in male malignant carcinomas,and prostate cancer has become the second leading cause of death in male malignant carcinomas.Prostate cancer is a kind of alloplastic disease derived from multiple nidus and developing in random mode.Effective therapeutic methods remain to be found for the advanced prostate cancer with metastasis, especially the androgen-independent and chemical and radio therapy resistant prostate cancer.Our investigation was designed to find novel molecular biomarkers, androgen-responsive genes and radiation induced genes using Cdna microarray, RT-PCR,Real time PCR and immunohistochemistry methods to provide clues and foundation for exploring the molocular mechanism mediating prostate cancer development to androgen-independence and radiation resistence and provide novel insight for clinical diagnosis and therapy.Methods and Results:1.1053 genes/transcripts were screened out to be differently expressed in LNCaP and C4-2 using U133A microarray chip from Affymetrix company.739 and 835 genes/transcripts were screened out to be expressed solely in LNCaP and C4-2 respectively.104 genes/transcripts were selected and 76 genes/transcripts differential expression were confirmed to be consistent with microarray results using RT-PCR.2.To screen out the molecular biomarker of PCa,RT-PCR was performed to examine the expression pattern of the 76 genes described above in BPH1,LNCaP,C4-2,C4-2B,PC-3,DU145,MCF-7,HEPG2,Hela,PC12,293T cell lines.26 genes expression were screened out to be positively associated with prostate cancer development and prostate tissue specific.17 genes expression were found out to be negatively associated with prostate cancer progression and possess prostate tissue specific characteristic.3.To screen out androgen responsive genes,1nM R1881 and 20μM Casodex were used to treat LNCaP and C4-2.RT-PCR were performed to examine the androgen responsive characteristics in the 76 genes described above.As the result,49 R1881 responsive genes were screened out and 42 genes were found to be responsive to Casodex in LNCaP;In C4-2 cell,16 genes were found to be responsive to R1881 and 15 responsive to Casodex.4.To investigate whether LNCaP and C4-2 are suitable to be radiation sensitive/resistant model cell lines for screening the radiation induced genes,MTT, soft agar colony formation and cell cycle assays were performed to detect the 5 Gy radiation response in LNCaP and C4-2 respectively.As the result,LNCaP and C4-2 were found to be radiation sensitive and resistant cell lines respectively. Real-time PCR were performed to examine the differential expression of p53,p21,Chek1,Chek2,Bcl-2,ATR,MRE11 in LNCaP and C4-2.As the result, expression of p53,Chek1,Bcl-2,ATR,MRE11 genes were found to be up-regulated in LNCaP cells compared with C4-2 cells.Then the radiation responses were examined in the 7 genes described above using 5 or 10 Gy radiation in LNCaP and C4-2.As the result,in LNCaP cell line,p21 expression was up-regulated in 24hs post-5Gy-radiation and the expression of Chek1,Chek2, Bcl-2,MRE11 were found to be down-regulated in the same condition.In 24hs post-10Gy-radiation p21 expression was up-regulated and the expression of Chek1,Chek2 were found to be down-regulated in the same condition.In C4-2 cell line,p53,p21,Chek1,Bcl-2,ATR,MRE11 expression were enhanced in 24hs post-5Gy radiation.The expression of p21,Chek1,Bcl-2,ATR,MRE11 were found to be increased in 24hs post-10Gy radiation.The genes expression differentiation provide the molecular foundation for the radiation sensitive LNCaP and radiation resistant C4-2.5.To screen out the radiation induced genes,LNCaP and C4-2 cells were treated with 0,2.5,5,10Gy Co60 radiation and RT-PCR were performed to examine the expression of the 76 candidate genes described above 6,12,24,72 hours post radiation.As the result,31genes/transcript were found to be responsive to the radiation.6.Base on the results of the screening ofbiomarker,androgen response and radiation response described above,25 genes/transcript were selected and real-time PCR were performed to confirm the differential expression pattern of these genes in LNCaP and C4-2.The results were demonstrated to be consistent with the microarray and RT-PCR assays.7.To confirm the reliability and practicality of the results from PCa molecular marker screening,immunohistochemistry were performed using the antibodies against AGR2 and RPS4Y1 respectively.The results revealed that AGR2 expression was positive in cytoplasma and in positive relation to the malignant level,which is consistent with the expression pattern analysis.Moreover,RPS4Y1 expression was also found to in cytoplasma and negatively associated with the malignant level,consistent with the expression pattern analysis.These results revealed that the molecular screened may be applied to the clinic as molecular marker for PCa. 8.To evaluate the role of the androgen and radiation responsive genes in the androgen independent and radiation resistant development of prostate cancer,the prostate cancer cell lines stably overexpressing ELF5-2 and ACADL respectively were constructed.The MTT,plate colony formation assay,soft agar colony formation assay and Casodex or radiation treatment were performed to investigate the effect of ELF5-2 and ACADL overexpression on the growth ability, anchorage-independent growth ability and androgen-dependence,radiation resistence and malignant progress in prostate cancer.The results demonstrated that ELF5-2 overexpression promoted the growth ability,colony formation and malignant progress in LNCaP and C4-2 and augmented the androgen-independence,Casodex and radiation resistence in LNCaP.Moreover, ACADL expression increased the growth ability,colony formation and malignant progress in C4-2,and promoted the androgen-independence,Casodex and radiation resistence in C4-2.In order to investigate the molecular mechanism mediating ELF5-2 and ACADL function,real-time PCR were performed to examine the expression changes of AR,PSA,p53,p21,Chek1,Chek2,Bcl-2, ATR,MRE11 mediated by ELF5-2 and ACADL overexpression.The results revealed that ELF5-2 expression up-regulated the expression of PSA,p53,,ATR,MRE11 and inhibited the expression of Chek1 in LNCaP.ELF5-2 overexpression in C4-2 increased the expression of AR,PSA,p21,Chek2,Bcl-2,ATR,MRE11. Moreover,ACADL expression in C4-2 promoted the expression of p21,Bcl-2,ATR,MRE11 and inhibited the expression of PSA,Chekl.These molecular results are consistent with the function of ELF5-2 and ACADL in promoting androgen-independence,Casodex and radiation resistence.These results also show the reliability of the screening assay,indicating that the androgen-independent and radiation resistant progress of PCa may be significantly associated with androgen and radiation responsive genes.Conclusions:1.1023 genes/transcripts were screened out to be differentially expressed between LNCaP and C4-2.690 genes/transcripts expression are up-regulated and 333 are down-regulated in C4-2.76 genes/transcripts expression differentiation were confirmed using RT-PCR,and 25 were confirmed using Real-time PCR.2.43 candidate molecular markers,62 androgen induced and 31 radiation induced genes/transcripts were screened out.3.C4-2 possesses radiation resistant property compared with parent LNCaP cell line. LNCaP and C4-2 prostate cancer progress model can be used not only for investigation of androgen-independent progress,but also for research of radiation resistence in prostate cancer. 4.AGR2 and RPS4Y1 were demonstrated to be positively and negatively associated with prostate cancer progress respectively.5.ELF5-2b overexpression promotes cell growth,survival and malignant progress, moreover induces androgen independence and radiation resistence in early stage of prostate cancer development.6.ACADL overexpression enhances cell growth,survival and malignant progress, moreover induces androgen independence and radiation resistence in late stage of prostate cancer development.