The Role Of MiR-744 In Prostate Cancer Progression And Its Regulation Mechanisms

As the most common malignancy and the second leading cause of cancer-related death among men in developed countries, prostate cancer (PCa) has long been considered as a substantial healthcare challenge in USA. Recent years, with the economy improvement, the morbidity and mortality of PCa has also been steadily increased in China as well. Even though the majority of PCa patients initially respond well for androgen deprivation therapy, the biggest hindrance for treatment of PCa is that most PCa patients within two years will inevitably progress to the castration-resistant prostate cancer (CRPC), a more aggressive form of PCa and the most common cause for prostate cancer patient death. The lacking of effective therapeutics strategies is the major limitation for treatment of recurrent and metastatic PCa. Thus, it is urgent need to understand the molecular mechanisms underlying PCa progression and develop the novel promising therapeutic approaches.MicroRNAs (miRNAs) belong to the non-coding RNAs, they regulate gene expression in the posttranscriptional level. Hundreds of miRNAs has yet been discovered. Recent study found that more than 50% of the miRNA genes was in tumor related genomes areas or fragile site, not only played the role of proto-oncogenes or tumor suppressor genes, but also involved in the development of tumor by fine adjusting several important signaling pathway. Recently, miRNAs control about half of the protein-coding genes, control of apoptosis, cell proliferation, differentiation,metabolism, and individual development, as well as the occurrence and development of tumor.The present study aims to analyse the different expressed miRNA by research of clinical relevance in ADPC and CRPC, and miR-744 is an important miRNA which we interested in. Prostate cancer cell functional experiment was further performed to verify the effect of miR-744 on prostate cancer cells. MiR-744 was a few years later miR-744 has been shown to serve as a tumor suppressor in several cancers including breast cancer, cervical cancer, colon cancer, and hepatocellular carcinoma; on the other hand,miR-744 was highly expressed in head and neck cancer, pancreatic cancer, and nasopharyngeal carcinoma, and mediated the tumor-promotion effects on these cancers.Using the method of combinating bioinformatics and molecular biology, the molecularmechanism of promoting prostate cancer cell apoptosis by miR-744. Our fndings indicate that miR-744 acts as one of oncogenic factor in the progression of prostate cancer.Part?The differential expression of miR-744 in prostate cancer tissuesObjective: As populations age and progress of the diagnosis, the morbidity and detection retest of ptostate cancer increase in our country. In the initial stage, many of them are androgen-dependent prostate cancer (ADPC), which will transform into castration-resistant prostate cancer (CRPC), and there are not yet effective treatments currently. Researchs of molecular biology on prostate cancer have become focuses for detection, diagnosis and treatment to this disease. miRNAs can regulate the expression of genes to control the apoptosis, proliferation, differentiation and metabolism of cells,and the development, progression of tumor through post-transcription. Differential expression of miRNA was detected by miRNA chips in early ADPC and advanced CRPC.Methods: Early ADPC and advanced CRPC were collected from fresh samples according to the instruction of observing of frezon section and preserved in liquid nitrogen. The data were firstly re-analyzed to compare miR-744 expression between metastatic prostate cancer and non-metastatic prostate cancer obtained from Memorial Sloan Kettering Cancer Center (MSKCC) prostate cancer database. Then we analyzed the role of miR-744 in diagnosis of metastasis prostate cancer and the relationship between miR-744 expression and biochemical recurrence.Results: Both three cases from ADPC and CRPC were detected by microRNAs chips.Differential expression of miR-744 in early ADPC and advanced CRPC was detected by qRT-PCR and ISH ( P<0.001) . Gene chip data about the expression of miRNAs in 98 prostate cancer patients were obtained from Memorial Sloan Kettering Cancer Center (MSKCC) prostate cancer database. KaplanMeier analysis with the log-rank test revealed, after radical prostatectomy, that the biochemical relapse-free survival in the patients with low level of miR-744 was significant longer than that in the patients with high level of miR-744 (P < 0.0001). Cox regression analysis to confrm the variables of potential prognostic signifcance and the results suggested that the miR-744 was an independent prognostic factors for biochemical relapse-free survival in patients with PCa.Conclusion: miR-744 expression in CRPC was much higher than in ADPC, which indicated that miR-744 increased along with the progress of prostate cancer, and it may be a potential and important tumor promoter gene.Part ?Functional analyses of miR-744 in prostate cancerObjective: In part I study, we have reported that miR-744 was probably correlated to the development and progression of prostate cancer, and participated the transformation of prostate cancer cells from androgen dependent to castration resistance. MiR-744 expression in CRPC was much higher than in ADPC and we suggesred that miR-744 was an independent prognostic factors for biochemical relapse-free survival in patients with PCa. However, the exact biological function of miR-744 on development of human prostate cancer has not been reported. We intend to explore the effects of miR-744 in cell apoptosis, proliferation, migration, invasion and the ability of in vivo tumorigenesis via in vitro cell functional study and subtaneous tumorigenesis in nude mice.Methods: Differential expression of miR-744 in PCa cells was detected by qRT-PCR.The in vitro proliferation of the PCa cells was measured using the MTT assay and Colony formation assay after they were transformed with miR-744 mimics or anti-miR-744 oligos. Cell apoptosis was detected by flow cytometry. The ability of PCs cells'penetrating transwell was analyzed by transwell migration and invasion experiment.The in vitro effect of miR-744 on the formation of tumor was detected by subcutaneous tumorigenesis in nude mice. Ki-67, Caspase-3 and CD31/34 of implanted tumor were tested by immunohistochemistry to further verify its effect on cell proliferation and tumor microvessel formation.Results: The expression of miR-744 was signifcantly up-regulated in CRPC cell lines(PC3 and DU145) than ADPC cell lines (LNCAP) (P < 0.01). MTT assay showed that anti-miR-744 oligos (groups of anti-miR-744) suppressed growth rate in PC3 and DU 145 cells while miR-744 minics (groups of miR-744) promoted growth rate in LNCAP cells (P < 0.05). Colony formation assay indicated that colony number of PC3 and DU 145 cells transfected with anti-miR-744 oligos was lower than control, in contrast, the number of LNCAP transfected with miR-744 minics was higher than control. Cell apoptosis assay (P < 0.05). The consequence showed PC3 and DU 145 cells with anti-miR-744 oligos demonstrate a higher apoptosis than control, on the contrary, the apoptosis in LNCAP cells transfected with miR-744 minics was lower than control (P < 0.05). The results of Transwell assay showed that migration and invasion ability of anti-miR-744 oligos group was lower than negative control in PC3 and DU 145 cells, while cells with upregulated expression of mir-744 present a higher migration and invasion ability than control in LNCAP (P < 0.05). Subcutaneous tumors formed in nude mice by PC3 cells stably inhibition of miR744 or control (P < 0.05).Silencing miR-744 by its inhibitor obviously suppressed tumor growth as manifested by reduced tumor size and tumor weight. In contrast, when endogenous miR-744 was stably overexpressed using LV-miR744 that encodes miR- 744 mimic by infected LNCaP cells, the tumors were larger in size and had increased weight than those formed by corresponding negative control cells subcutaneous tumors formed in nude mice by LNCaP cells stably overexpression of miR-744 or control (P < 0.05).Immunohistochemical staining revealed that reduced Ki67-positive cells, CD31-positive cells and CD34-positive cells, and signifcantly increased caspase-3-positive cells in miR-744 inhibitor-overexpressing PC3 tumors. In the contrary immunohistochemical staining of Ki67 in the endpoint tumors revealed that reduced signifcantly increased Ki67-positive cells in miR-744 overexpressing LNCaP tumors.Conclusion: miR-744 can promote PCa cells proliferation, migration, invansion and promote cell apoptosis, thereby play a role as cancer promoter gene.Part ? Funtional analyses of miR-744 in prostate cancer Objective: In part?, we reported that miR-744 can promote PCa cells proliferation,migration, invansion and promote cell apoptosis, thereby play a role as cancer promoter gene. However, the mechanism of miR-744 on development of human prostate cancer has not been reported. We intend to investigate the molecular mechanisms through which miR-744 exerts its prostate cancer-promoting effects.Methods: We conducted Affmetrix human gene expression array analysis combined with three bioinformatics tools (TargetScan, miRANDA, RNA22-HSA) to search whether NKD1 is one of putative targets of miR-744 and verified by luciferase reporter assay and western blot assay. In addition, we also detected the relationship between miR-744 and NKD1 used ISH and IHC in PCa tissues and subcutaneous tumors. By employing KEGG pathways database implied that Wnt/p-catenin signaling might be one major pathway involved in the progression of PCa mediated by miR-744. Moreover,Western blot analysis revealed the relationship between miR-744 and NKD1, GSK3?,SFRP1 and TLE3 in protein level. As nuclear ?-catenin is the critical effector of Wnt pathway, we therefore performed TOPflash/FOPflash luciferase reporter assay to determine whether the transcriptional activities of ?-catenin will be enhanced in PCa cells when miR-744 is disturbed. Furthermore, we performed rescue experiment to defined that NKD1 is a critical downstream mediator of miR-744 effects prostate cancer progression.Results: The luciferase activity assay and Western blot analysis were detected that NKD1 is a direct target of miR-744 since the 3'-UTR of NKD1 gene contains a binding site that perfectly complements with the seed sequence of miR-744. We performed ISH and IHC staining on PCa tissues and subcutaneous tumors observed that NKD1 expression was inversely correlated with miR-744 level. We performed TOPflash/FOPflash luciferase reporter assay to determine the transcriptional activities of ?-catenin enhanced in PCa cells when miR-744 is overexpressed (P < 0.05).Knockdown NKD1 by siRNA moderately attenuated the inhibitory effects on cell proliferation, colony formation, migration and invasion of PCa cells induced by reduction of miR-744 level. Western blot analysis also demonstrated that siRNA-mediated downregution of NKD1 apparently antagonized the enhancement of NKD1 protein induced by anti-miR-744 in both Du 145 and PC3 cells (P < 0.05).Conclusion: miR-744 promotes prostate cancer progression through aberrantly activating Wnt/?-catenin signaling by NKD1 and NKD1 is a critical downstream mediator of miR-744 effects in prostate cancer progression.Part IV Functional analyses of NKD1 in prostate cancerObjective: In part III, we reported that microRNA-744 promotes prostate cancer progression through aberrantly activating Wnt/?-catenin signaling by NKD1 and NKD1 is a critical downstream mediator of miR-744 effects in prostate cancer progression. However, the exact biological function of NKD1 on development of human prostate cancer has not been reported. We intend to explore the effects of NKD1 in cell apoptosis, proliferation, migration, invasion and the ability of in vivo.Methods: Differential protein expression of NKD1 in PCa cells was detected by western blot assay. The in vitro proliferation of the PCa cells was measured using the MTT assay and Colony formation assay after they were transformed with overexpressing NKD1 vectors or siNKD1. Cell apoptosis was detected by flow cytometry. The ability of PCs cells' penetrating transwell was analyzed by transwell migration and invasion experiment.Results: Compared with ADPC cell lines (LNCAP), lower expressions of NKD1 protein were found in CRPC cell lines (PC3 and DU 145) by western blot. Reduction of NKD1 by siRNA knockdown in LNCAP cells markedly inhibited cell proliferation and colony formation as well as invasive abilities, compared to the siNC-treated cells (P <0.05). MTT assay showed that overexpressing NKD1 vectors suppressed growth rate in PC3 and DU145 cells (P < 0.05). Colony formation assay and Transwell assay showed that overexpression of NKD1 in PC3 and DU 145 cells markedly inhibited cell proliferation and colony formation as well as invasive abilities, compared to the control-treated cells (P < 0.05).Conclusion: NKD1 can inhibit PCa cells proliferation, migration, invansion and promote cell apoptosis, thereby play a role as cancer suppressor gene.