MicroRNA-30d Promotes Tumor Progression And Angiogenesis By Targeting MYPT1 In Human Prostate Cancer

Background and purpose:Prostate cancer (PCa) is the most common malignancy and the second highest cause of cancer death in men in Western countries. Despite the availability of an earlier diagnosis using serum prostate-specific antigen (PSA) and effective treatments, including hormone therapy, surgery, and radiation, many patients with PCa subsequently die following disease progression. Some highly aggressive cases may develop biochemical recurrence (BCR) or clinical metastasis shortly even though they have received radical therapy after early diagnosis. For this kind of patients, auxiliary chemotherapy or radiotherapy should be performed immediately after radical therapy, and active follow-up should also be carried out subsequently. However, most of the cases are indolent diseases, which progress slowly and even asymptomatically in the lifetime.A substantial number of patients demonstrate PSA elevation on long-term follow-up examinations, and onethird of these cases develop distant metastases within several years after the initial operation. The predictive markers for biochemical recurrence and metastasis include the patient’s PSA level, Gleason score, and radiological features. However, the outcomes of long-term followup examinations are diverse, and it is difficult to predict patient prognosis even among patients with equivalent PSA levels, Gleason scores, and pathological stages. Better prognostic markers are needed to stratify patients and design appropriately aggressive therapies.MicroRNA (miRNA) consists of 19-25 noncoding RNA molecules that bind to imperfect complementarity sites within the 3’-untranslated regions (3’-UTR) of the target mRNAs, resulting in translational repression or mRNA degradation [6]. MiRNA plays an important role in normal tissue development and cell differentiation, proliferation, and apoptosis. The altered expression of miRNA has been observed in a variety of human cancers, and it is widely accepted that miRNA is a key player in tumor progression. More than 50% of miRNA genes are located in fragile genomic regions that are prone to amplification, deletion, or rearrangement in human cancer cells. The aberrant expression of miRNA in cancer has been attributed to alterations in miRNA biogenesis, miRNA promoter methylation, and the transcription factors that regulate miRNA production. Some miRNAs are thought to act as oncogenes or tumor suppressor genes. Recent findings have shown that the expression patterns of miRNA are often altered in PCa and that miRNAs contribute to the progression of PCa by accelerating androgen-independent growth, migration, invasion, cell cycle arrest, and apoptosis.The biological function of miR-30d in tumor becomes more and more popular in the past years. miR-30d has been proven to be up-regulated in multiple tumors and acted as an tumor suppressor, including melanoma, lung cancer, hepatocellular carcinoma and malignant peripheral nerve sheath tumour. According to these studies, miR-30d promoted the progression of tumors and controlled the cellular cycle, cellular apoptosis, cellular migration, cellular invasion and angiogenesis by targeting its downstream genes. However, because the biological functions of miRNAs have highly tissue and cell specificity, miR-30d was found to inhibit tumor cells proliferation, migration and invasion by controlling TGF-β1. To date, miR-30d expression and molecular biological function in prostate cancer are still elusive. Here, we aim to investigate the role and targets of miR-30d in prostate cancer.Materials:1. For qRT-PCR and western blot analyses,18 pairs of primary PCa and self-matched adjacent non-cancerous frozen samples were obtained from the tissue bank at Guangzhou First People’s Hospital. The Taylor dataset is a publicly available dataset including 113 primary PCa patients with microarray expression data (1). For Human PCa tissue microarrays (TMA),225 consecutive PCa patients who underwent radical prostatectomy at the MGH Hospital from September 1993 to March 1995 were included in our study.2. Animal experiments in this study were performed in compliance with the guidelines of the Institute for Laboratory Animal Research at Guangzhou Medical University, P.R.China. Twenty BALB/c nude mice (4-5-week-old males) were purchased from Guangdong Medical Animal Center3. Cell culture:Normal human prostate epithelial cells (PREC) were purchased from Lonza Company and were cultured in PrEGM Bullet kit (Lonza, USA) with antibiotics. Human PCa cell lines, PC-3, LNCaP and DU145 were purchased from the American Type 6 Culture Collection (Manassas, VA, USA) and were cultured in RPMI 1640 medium (Hyclone, USA) supplemented with 10% fetal bovine serum (Gibico, USA),2 mM L-glutamine, and antibiotics. All cell lines were maintained at 37℃ in a humidified chamber supplemented with 5% CO2.Methods:1. DU145 and LNCaP cell lines were transfected with lentivectors to stably overexpressing or knocking down the miR-30d; Oligonucleotide and plasmid transfection were used to establish cell lines overexpressing MYPT1 or miR-30d/MYPT1.2. The publicly available Taylor dataset was used to investigate the correlations between miR-30d expression levels and clinicopathological features or MYPT1 expression levels of PCa patients.3. In vitro cell functional experiments including cell proliferation, wound healing, transwell and HUVEC tube formation, were performed to evaluate the effects by miR-30d in DU145 and LNCaP cell lines. Subcutaneous xenograft tumor model was established by injecting DU145 or LNCaP cell lines aberrantly expressing miR-30d into the subcutaneous of nude mices, evaluating the in vivo effects induced by miR-30d.4. Genomic expression profiling with gene expression profile chip, followed by IPA software and GO functional analysis were performed to investigate the differentially expressed protein induced by miR-30d and their involved pathways and molecular functions.5. Three online programs Target-Scan, miRWalk and miRanda were used to predict potential target genes for miR-30d. Besides, luciferase reporter assay was performed to identify the target genes of miR-30d.6. qRT-PCR was erformed to detect the expression levels of the candidate targets CALD1, TNPO1, ATP2B1, SEPT7, MYPT1, ZNF148, CEP350, KIF5B, STAG2 and GALNT1. qRT-PCR was performed to detect the mRNA expression level of miR-30d and MYPT1 in prostate cell lines PC3, DU145, LNCaP and Normal human prostate epithelial cells PREC, as well as 18 pairs of human prostate cancer and adjacent benign prostate tissues.7. Western blot was carried out to detect the protein levels of MYPT1 in the DU145 and LNCaP cell lines and xenografts with aberrant expression of miR-30d; MYPT1 protein level was detected by western blot to confirm the cell lines was successfully established. Western blot was also carried out to detect the protein levels of downstream effectors total-cJUN, phosphor-cJUN (ser63), phosphor-cJUN (ser73) and VEGF in DU145 and LNCaP cell lines overexpressing miR-30d or downregulating MYPT1.8. Immunohistochemistry (IHC) and immunofluorescence (IF) analysis was performed to evaluate the MYPT1 and CD31 expression pattern in tissue microarray including 225 human PCa samples and 25 adjacent benign prostate tissues; CD31 and MMP-9 expression pattern in xenograft tumors established by DU145 and LNCaP cell lines aberrantly expressing miR-30d were evaluated by IHC.Statistical analysis:The version 17.0 SPSS (SPSS Inc, IL, USA) software was employed for statistical analysis. Continuous variables were expressed as X±s. Statistical analysis was performed independently by two biostatisticians with Fisher’s exact test and Pearson χ2 test. Kolmogorov-Smirnov (K-S) test was used to test for normality of the distribution of miR-30d expression level. Statistical analyses of qRT-PCR, IHC, Western blot and in vitro cell functional experiments were conducted using two independent samples t test. Two independent samples t test and one-way ANOVA were performed to analyse the associations between mRNA expression level of miR-30d and clinicopathological characters of PCa patients in Taylor dataset. Kaplan-Meier method was used for the survival analysis and Cox regression analysis was used for the univariate and multivariate analysis. The Spearman correlation was calculated between the expression levels of miR-30d and MYPT1 also MYPT1 and CD31 in PCa tissues. Statistically significant differences were considered when the p value was less than 0.05.Results:1. The mRNA expression levels of miR-30d in three human PCa cell lines (LNCaP, DU145, and PC-3) (P=0.002,0.001 and 0.001, respectively) and PCa tissues (P=0.009) were respectively higher than those in a non-malignant prostate epithelial cell line (PrEC) and adjacent non-cancerous prostate tissues.2. Statistical analysis of Taylor dataset showed that miR-30d upregulation was frequently found in PCa tissues with high serum PSA level(P=0.003), Gleason score (P=0.01), clinical stage(P=0.007), pathological stage(P=0.004), positive metastasis failure (P=0.002) and biochemical recurrence (P<0.001). However, miR-30d expression was not correlated to survival. Kaplan-Meier and Log rank showed a significant difference in the BCR-free survival and overall survival between patients with high and low miR-30d expression (P<0.001 and 0.041, respectively). Univariate analysis showed that tumor pathological stage (P<0.001), Gleason score (P<0.001) and miR-30d expression level (P<0.001) had the potential to serve as predictors for the risk of BCR. However, age, serum PSA levels and tumor clinical stage could not be predictors. Multivariate analysis further revealed that up-regulation of miR-30d [P=0.008, HR (95% CI):3.510 (1.387-8.878)], high pathological statge[P<0.001, HR (95%CI):5.056 (2.089-12.239)] and high Gleason score [P<0.001, HR (95% CI):7.433 (2.941-18.784)] had the potential to serve as independent predictors for shorter BCR-free survival.3. Transwell assays clearly revealed that enforced expression of miR-30d significantly reduced the invasive activities of both DU145 and LNCaP cells compared with those of control cells. Wound-healing assays demonstrated that miR-30d upregulation markedly weakened the migratory abilities of both DU145 and LNCaP cells. HUVEC tube formation experiments showed that the medium conditioned by miR-30d upregulation is more active in inducing tube formation of HUVECs compared with that of control DU145 and LNCaP cells. Moreover, we also stably suppressed the miR-30d expression in LNCaP and DU145 cell lines via lentivectors transduction. Intriguingly, the knockdown of miR-30d expression with lentivectors in LNCaP and DU145 cell lines could dramatically enhance the abilities of cellular invasion, motility, proliferation and inducing tube formation of HUVECs.4. The LNCaP and DU145 cells stably expressing miR-30d formed significantly larger tumor nodules and remarkably accelerated tumor xenografts growth compared with the controls. On the other hand, we further found that the PCa cells that permanently suppressed the expression of miR-30d with lentivectors could slowed tumor growth compared with the controls. The expression level of CD31 and MMP-9 protein in the tumor xenografts established by LNCaP or DU145 cells stably expressing miR-30d was remarkably higher than that in the xenografts established by cells transfected with control vectors. Moreover, the tumor xenografts established by LNCaP or DU145 cells with high miR-30d expression presented significantly more CD31 and MMP-9 protein than the control xenografts. These results strongly demonstrated that miR-30d could significantly promote tumor growth, angiogenesis and invasion in vivo.5. A total of 1420 differentially expressed genes were identified by gene expression profile chip. Among all the genes quantified in at least two experiments, only 116 were differentially regulated with fold changes ≤-2 or ≥2 (miR-30d vs miR-NC) both in LNCaP and DU145 cells, including 143 down-regulated proteins and 3 up-regulated proteins. Pathway and biological functional enrichment analyses respectively based on Ingenuity Pathways Analysis (IPA) and GO annotation system showed that most of the downregulated genes were involved in the tumor-related pathways including Rho signaling, RAN signaling, HIF-la signaling and protein ubiquitination pathway. GO functional clustering analysis showed that the changed proteins induced by miR-30d significantly controlled many biological processes directly relevant to cancers, such as cellular growth and proliferation, tumor morphology, celluar development.6. Three programs all predicted CALD1, TNPO1, ATP2B1, SEPT7, MYPT1, ZNF148, CEP350, KIF5B, STAG2 and GALNT1 as candidate targets of miR-30d, which encodes the corresponding down-regulated genes according to the results of genomics analysis of miR-30d-induced changes in gene inhibition. qRT-PCR analysis was performed and the results showed that the endogenous MYPT1, CEP350, STAG2 and GALNT1 expression in cells and established tumors associated with LNCaP cells stably expressing miR-30d were all significantly reduced at mRNA levels. Luciferase activity assay further revealed that MYPT1 was the direct target of miR-30d (P<0.001). qRT-PCR and western blot confirm an adverse relationship between miR-30d and MYPT1 expression in PCa cell lines, xenograft tissues and human PCa tissues.7. Migration, invasion and HUVECs tube fomation assays all indicated that restoration of MYPT1 expression dramatically attenuated the effects induced by miR-30d. These results further implied that MYPT1 was a critical downstream mediator of miR-30d promotive effects in PCa progression.8. The IHC score of MYPT1 protein in PCa clinical samples was significantly lower than that in adjacent benign tissues (P<0.001). We analyzed 225 radical prostatectomy specimens represented in TMA from PCa patients. The results revealed that MYPT1 low-expression was significantly associated with high Gleason score(P=0.002), positive BCR (P<0.001) and positive metastasis (P=0.018) of PCa patients. Kaplan-Meier analysis was conducted to assess the prognostic value of MYPT1 expression in human PCa. We found that there were significant differences in the BCR-free survival (P=0.001), overall survival (P=0.005) and metastasis-free survival (P=0.049), between patients with high and low MYPT1 expression.Cox proportional hazards multivariate model indicated that only MYPT1 expression was independent predictors of BCR-free survival [P= 0.011, HR (95% CI):0.505 (0.299-0.854)] but not metastatsis-free survival in PCa patients.9. To assess the clinical relevance of MYPT1 expression in human PCa and its correlation with microvessel density in human PCa, immunostaining with anti-CD31 antibodies was also carried out on the same slides of HMS human prostate tissue microarrays. The expression levels of MYPT1 were negatively correlated with microvessel density determined by CD31 staining (Pearson correlation:R=-0.457, P<0.01). Kaplan-Meier analysis was conducted to assess the prognostic value of MYPT1 low-expression/CD31 high-expression in human PCa. We found that there were significant differences in the BCR-free survival (P=0.001), overall survival (P=0.017) and metastasis-free survival (P=0.008), between patients with MYPT1 low-expression/CD31 high-expression and MYPT1 high-expression/CD31 low-expression. Cox proportional hazards multivariate model indicated that only MYPT1 low-expression/CD31 high-expression was independent predictors of BCR-free survival [P= 0.005, HR (95%CI):0.366 (0.183-0.734)] but not metastatsis-free survival in PCa patients.10. Enforced expression of miR-30d and knockdown of MYPT1 protein in two PCa cell lines LNCaP and DU145 both significantly reduced the protein expression levels of phosphor-cJUN (ser63), phosphor-cJUN (ser73) and VEGF, but there was not change with the protein expression level of total-cJUN, implying the dephosphorylation of cJUN by MYPT1 which lead to aberrantly expression of VEGF might function as the downstream signaling of miR-30d-MYPT1 axis in human PCa cells and contributes to tumor angiogenesis.Conclusion:1. MiR-30d acts as an important tumor promotor in prostate cancer. Moreover, miR-30d enhances tumor progression by negatively regulating MYPT1 in human prostate cancer.2. MiR-30d and MYPT1 respectively could serve as an independent factor for predicting the risk of BCR after radical prostatectomy. Furthermore, MYPT1 could also be an independent predictor for the overall survival of PCa patients. In summary, miR-30d and MYPT1 expression levels in PCa tissues along with the traditional clinicopathological factors could more accurately stratify the PCa cases into high-risk and low-risk groups, optimizing the therapeutic strategy.3. The expression levels of MYPT1 were negatively correlated with microvessel density determined by CD31, the combination of both MYPT1 and microvessel density could predicted the clinical outcomes of PCa patients better. miR-30d promotes tumor angiogenesis by targeting MYPT1 in PCa.4. MiR-30d-MYPTl axis partially illustrates the molecular mechanism of PCa progression and tumor angiogenesis, represents a novel potential therapeutic target for PCa treatment.