MicroRNAs Alterations Associated With The Prostate Cancer LNCaP Cell Progression To The Androgen-independence

MicroRNAs alterations associated with the prostate cancer LNCaP cell progression to the androgen-independenceBackgroundSince androgen ablation was administrated to treat late-stage prostate cancer(Pca) and gained significant therapeutic efficacy by Huggins in 1941, hormone therapy had been an important therapy for PCa. However, with the spreading administration of hormone therapy, people found the effect of androgen ablation remained only 12-16 months, and then PCa cell continued to grow and deteriorate. After that, only about half of the patients could survive beyond 1 year. This situation was called clinically as androgen-independent prostate cancer (AIPC). The study of AIPC had been a significant topic of urology.Currently, the study of AIPC focus on certain ascpects such as the androgen receptor, tumor cell apoptosis and the regulation of cell cycle, polypeptide growth factor (PGF), angiopoiesis of tumor and genes strongly related with AIPC etc.Although those studies have provided us lots of valuable information from different aspects of the pathogenesis of AIPC, the molecular biological change from ADPC to AIPC remains unclear. The major obstacle is lacking of ideal cell model that can imitates the transition from ADPC to AIPC in vitro. An ideal Pca cell line shall fulfill following criteria such as androgen-dependent,express a wild-tye AR and would progress to androgen independence after chronic androgen withdrawal. Among the known PCa cell lines,neither PC-3 nor DU-145 expresses AR,while CWR22R expresses a mutant AR.Only LNCaP cell remains androgen dependent characteristics and express AR. LNCaP cell also secretes PSA and PSMA and grow in the presence of steroid hormone. Recently some stuidies demonstrated that LNCaP could be transformed from androgen dependent status to androgen independent status under the circumstance of androgen ablation. This kind of PCa cell model could imitate the process from ADPC to AIPC during endocrine therapy of PCa. Because LNCaP-AI was developed from LNCaP cell inferred, it could be compared with LNCaP cell. The cell model could be used to observe the molecular biological change from ADPC to AIPC and is an ideal model to study the pathogenesis of AIPC.MicroRNAs (miRNAs) are endogenous short noncoding RNA molecules (20 - 23 nucleotides) that regulate cell differentiation, cell proliferation, andapoptosis through post-transcriptional suppression of gene expression by binding to the complementary sequence in the 3' untranslated region (3'UTR) of target messenger RNAs (mRNAs). Recently, It has been revealed that the change of miRNA expressions contributes to the initiation and progression of cancer. Some recent studies indicate that miRNAs can function as tumor suppressors and oncogenes,and the miRNAs expression profiling of human malignancies has identified signatures involving in cancer development, progression, diagnosis and prognosis. Using miR-15a and miR-16 to control the B-cell chronic lymphocytic leukemia (CLL) recently lead to a new era of novel cancer therapies.Up to date the miRNAs relating prostate cancer showing differential expression were either not the same ones or showed the opposite results of up-regulation or down-regulation. This discrepancy may be due to the fundamental methodologic differences used in the studies. It is well known and widely predicted that the relationship between microRNAs and target mRNAs is not a "one to one" connection, as the same mRNA can be regulated by more than one miRNA, and that the choice of how many and which miRNAs target one 3'UTR is strongly determined by the specific cellular environment. An miRNA that regulates targets playing opposite roles in the control of cell proliferation may act as a tumor suppressor in some cancers and as an oncogene in others, depending on which targets are driving tumorigenesis in that specific cellular milieu. And it still is known that the functional of miRNA dysregulation in cancer has not been well understood.Those aspects contribute to the discrepancy of the microRNAs relating the prostate cancer by now.As there is no systematically study on the alteraions between the LNCaP and LNCaP-AI cell lines by today and also there are still much microRNAs have not been elucidated in the progression of Pca,it is important to identify how many microRNAs functions in development of the Pca and the transition of androgen independence. The change of microRNAs associated with prostate cancer progression may reveal some aspect of the androgen independence biological behavior,providing a new strategy for controlling and treating prostate cancer.ObjectiveTo construct an androgen independent LNCaP cell line ,we imitated the process from ADPC to AIPC by androgen ablation and investigated the pathogenesis of this process. The cell model provided us a reliable platform for further investigating the pathogenesis of AIPC. By comparing the microRNAs between the LNCaP and LNCaP-AI cell lines the mechanism of androgen independence can be further elucidated in some aspects.Methods1. Culturing LNCaP cells in the medium with the steroids deprived step by step for 10 days and then culturing the cells in the medium with 10% FBS depleted of steroids by charcoal stripped for 3 months and gain LNCaP-AI cell line that can accommodate the circumstance without hormone.2. Verify the reproductive activity of LNCaP and LNCaP-AI cell lines in the absence of hormone by CCK-8 method. Detect PSA level secreted by using immun fluorescence method. Detect AR expression by using RT-PCR.3.The microRNA alterations between the LNCaP and LNCaP-AI cell lines were detected by Agilent's microassay.The microRNAs alteration were verificated by RT-PCR.The EPO,EPOR expression were detected in the LNCaP and LNCaP-AI cell lines using RT-PCR and the c-kit expression by PE anti-human antibody immunofluorescent staining with flow cytometric analysis.4.The functions of microRNAs alterations during the transition of androgen independence were eludicated by a search with miRBase software (http://microrna.sanger.ac.uk)Results1.Initially,the proliferation of LNCaP cells were suppressed rapidly in the absence of hormone and laid in autocrine status after about 1 week. Three months later, the cells gradually adapted for the no hormone circumstance and began to proliferate. At this time the cells become androgen independent LNCaP-AI cell line.2. LNCaP-AI cells proliferated rapidly in the no hormone circumstance; In contrast, LNCaP cells were suppressed significantly in the no hormone circumstance. PSA level in LNCaP-AI cell culture supernatant increased with time. However, the PSA expression of LNCaP-AI cell was suppressed as of that in LNCaP cell. The expression level of AR gene level in LNCaP-AI cell inecreased significantly comparing with LNCaP.3.The Aglint's microRNA microassay showed there were 11 microRNAs upreglulations and 37 microRNAs downregulations during the LNCaP progression into the androgen independence.The verification of the microRNAs changes between the two cells by RT-PCR indicated the Agilen's microassay chips was reliable.4. There was no significant difference of the EPO and EPOR between LNCaP and LNCaP-AI cell. Expression of c-kit protein elevated in LNCaP-AI cell comparing to LNCaP cell by the PE anti-human antibody immunofluorescent staining with flow cytometric analysis.5.By looking through the regulating targets of microRNAs using the miRBase software (http://microrna.sanger.ac.uk).It shows those downregulation of the microRNAs prompt the upregulation of the genes including AR,MMP9,bcl-2,c-kit,EGFR and the multiple genes mediating androgen metabolism which involved in the androgen independent transition.Conclusions1.Androgen independent LNCaP-AI cell line can be induced from parental cell line LNCaP in the absence of hormone.The resulted cell line imitates the process from ADPC to AIPC during endocrine therapy of PCa. The direct comparasion between LNCaP and its parental strain LNCaP can provide us invaluable insights regarding to the molecular biological change from ADPC to AIPC and the pathogenesis of AIPC.2. Those alterations of the microRNAs play roles in the progression of LNCaP into androgen independence. The increase in the expression of c-kit may be a new reasons that androgen-stimulated PCa gained the ability of resistance to androgen ablation.