MicroRNA-195 Inhibits Tumor Progression By Targeting S6K1 In Human Prostate Cancer

Background and purpose:Recently, prostate cancer (PCa) is still a serious problem worldwide, which is the most common nondermatological cancer and has a second leading cause of cancer-related death in American men.PCa is difficult to predict due to its highly variable natural history. 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. Studies have demonstrated that 48 additional cases of prostate cancer would need to be treated to prevent one death from prostate cancer, because most of the cases would die from other diseases such as cardiovascular disease during the long history of PCa. Therefore, the low-risk cases usually have little impact on the life expectancy without any treatment. Introduction of PSA screening have lead to an increase of indiscriminate overtreatment, which becomes a serious problem in the past ten years. Radical treatment is associated with high morbidity, while conservative approach to treatment of prostate cancer is not without consequences because prostate cancer is already a common form of cancer death in men in developed countries. Furthermore, conservative management can lead to considerable anxiety, especially when the clinical outcome is uncertain. Therefore, accurately stratifying PCa patients into high-risk and low-risk groups is critical to the rational and effective management of PCa.Clinical variables like Gleason score, tumour stage, margin status, and PSA concentration, in various combinations, have been used in models for prediction of disease outcome. In the most favourable of clinical settings (ie, after surgical resection), these prognostic models are about 75-85% accurate, but are less accurate in conservatively treated patients. Therefore, clinicopathological factors could not provide an accurate prediction. It is urgent to investigate the molecular biological mechanism by which PCa has aggressive progression and metastasis. If the critical molecular signaling were identified, it would be promising to provide reliable evidences for target treatment.Recently, studies on the biological role of microRNA (miRNAs) in various tumors become more and more popular. miRNAs represent the best characterized class of small (19-25 nt in length) non-coding RNA transcripts. Mature miRNAs regulate a variety of gene expression by directly degrading mRNA or suppressing post-transcriptional protein translation by pairing with complementary nucleotide sequences in the 3’-UTR of specific target genes. The dysregulation of miRNAs have been observed in many types of human malignancies. miRNAs are involved in the progression and tumorigenesis of PCa via directly targeting coding genes. Moreover, miRNAs control more than 50% of the coding genes. Recently, many studies have proven that miRNAs also play an important role in prostate cancer, such as miR-34a, miR-373 and miR-520c. Therefore, getting deeper insight into the molecular functions of miRNAs is an indispensable part.MiR-195, which belongs to miR-15/107 family, biological function in tumor becomes more and more popular in the past years. miR-195 has been proven to be down-regulated in multiple tumors and acted as an tumor suppressor, including breast carcinoma, hepatocellular carcinoma, esophageal squamous carcinoma and adrenocortical carcinoma. According to these studies, miR-195 inhibited 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-195 was found to promote tumor cells proliferation, migration and invasion by controlling WEE1. To date, miR-195 expression and molecular biological function in prostate cancer are still elusive. Here, we aim to investigate the role and targets of miR-195 in prostate cancer.Materials:1. For quantitative real-time reverse transcriptase PCR (qRT-PCR) and western blot analyses,12 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 Massachusettes General 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 Laboratory Animal Center.3. 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-195; Oligonucleotide and plasmid transfection were used to establish cell lines overexpressing S6K1 or miR-195/S6K1.2. The publicly available Taylor dataset was used to investigate the correlations between miR-195 expression levels and clinicopathological features of PCa patients.3. In vitro cell functional experiments including cell proliferation, cell apoptosis, wound healing and transwell, were performed to evaluate the effects by miR-195 in DU145 and LNCaP cell lines. Subcutaneous xenograft tumor model was established by injecting DU145 or LNCaP cell lines aberrantly expressing miR-195 into the subcutaneous of nude mices, evaluating the in vivo effects induced by miR-195.4. Proteomic expression profiling with iTRAQ tagging, followed by IPA software and GO functional analysis were performed to investigate the differentially expressed protein induced by miR-195 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-195. Besides, luciferase reporter assay was performed to identify the target genes of miR-195.6. qRT-PCR was erformed to detect the expression levels of the candidate targets S6K1, SMAP2, DCUN1D1, SMAD4, IPO9, CAPZA2 and CPNE1.qRT-PCR was also performed to detect the expression level of miR-195 and S6K1 in prostate cell lines PC3, DU145, LNCaP and Normal human prostate epithelial cells PREC, as well as 12 pairs of human prostate cancer and adjacent benign prostate tissues.7. Western blot was carried out to detect the protein levels of S6K1 in the DU145 and LNCaP cell lines and xenografts with aberrant expression of miR-195; S6K1 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 MMP-9, VEGF, BAD and E-cadherin in DU145 and LNCaP cell lines overexpressing miR-195 or downregulating S6K1.8. Immunohistochemistry (IHC) analysis was performed to evaluate the S6K1 expression pattern in tissue microarray including 225 human PCa samples and 25 adjacent benign prostate tissues; CD31 and Vimentin expression pattern in xenograft tumors established by DU145 and LNCaP cell lines aberrantly expressing miR-195 were evaluated by IHC.Statistical analysis:The versionl3.0 SPSS for Windows (SPSS Inc, IL, USA) and SAS 9.1 (SAS Institute, Cary, NC) softwares were used for statistical analysis. Continuous variables were expressed as X±s. Statistical analysis was performed independently by two biostatisticians with Fisher’s exact test for any 2×2 tables and Pearson χ2 test for non-2×2 tables. Kolmogorov-Smirnov (K-S) test was used to test for normality of the distribution of miR-195 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 examine the associations between miR-195 expression 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-195 and S6K1 in PCa tissues. Differences were considered statistically significant when the p value was less than 0.05.Results:1. Expression levels of miR-195 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 lower 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-195 downregulation was frequently found in PCa tissues with high Gleason score (P=0.001), positive metastasis failure (P=0.011) and biochemical recurrence (P<0.001). However, miR-195 expression was not correlated to serum PSA level, clinical stage, pathological stage and survival. Kaplan-Meier and Log rank showed a significant difference in the BCR-free survival and non-metastatic BCR-free survival between patients with high and low miR-195 expression (P<0.001 and 0.009, respectively). However, the data revealed that there were no significant differences in overall survival and non-metastatic survival between high and low miR-195 expression group (P=0.721 and 0.806, respectively). Univariate analysis showed that tumor pathological stage (P<0.001), Gleason score (P<0.001) and miR-195 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 down-regulation of miR-195 [P=0.022, HR (95% CI):0.61 (0.41-0.93)] and high Gleason score [P<0.001, HR (95% CI):6.24(2.82-13.80)] had the potential to serve as independent predictors for shorter BCR-free survival.3. Transwell assays clearly revealed that enforced expression of miR-195 significantly reduced the invasive activities of both DU145 and LNCaP cells compared with those of control cells. Wound-healing assays demonstrated that miR-195 upregulation markedly weakened the migratory abilities of both DU145 and LNCaP cells. In contrast, the apoptotic rates of miR-195-transfected DU145 and LNCaP cells were significantly higher than those of control cells. Moreover, we also stably suppressed the miR-195 expression in LNCaP and DU145 cell lines via lentivectors transduction. Intriguingly, the knockdown of miR-195 expression with lentivectors in LNCaP and DU145 cell lines could dramatically enhance the abilities of cellular invasion, motility, proliferation and reduce cellular apoptosis.4. The LNCaP and DU145 cells stably expressing miR-195 formed significantly smaller tumor nodules and remarkably slowed 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-195 with lentivectors could enhance tumor growth compared with the controls. The expression level of CD31 and Vimentin protein in the tumor xenografts established by LNCaP or DU145 cells stably expressing miR-195 was remarkably lower than that in the xenografts established by cells transfected with control vectors. Moreover, the tumor xenografts established by LNCaP or DU145 cells with low miR-195 expression presented significantly more CD31 and Vimentin protein than the control xenografts. These results strongly demonstrated that miR-195 could significantly inhibit tumor growth, angiogenesis and invasion in vivo.5. A total of 3194 differentially expressed proteins were identified by iTRAQ. Among the 3194 proteins quantified in at least two experiments, only 78 were differentially regulated with fold changes ≤-1.5 or ≥1.5 (miR-195 vs miR-NC), including 50 down-regulated proteins and 28 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 upregulated genes were involved in the metabolic pathway, such as TCA cycle, Valine degradation, Alanine biosynthesis, Coenzyme biosynthesis and fatty acid oxidation. In contrast, most of the downregulated genes were involved in the tumor-related pathways including mTOR signaling, IL-8 signaling, AMPK signaling and IGF-1 signaling. GO functional clustering analysis showed that the changed proteins induced by miR-195 significantly controlled many biological processes directly relevant to cancers, such as cell cycle, cell invasion, cell morphology, cell assembly and organization, and cellular movement.6. Three programs all predicted S6K1, SMAP2, DCUN1D1, SMAD4, IPO9, CAPZA2, and CPNE1 as candidate targets of miR-195, which encodes the corresponding down-regulated proteins according to the results of proteomics analysis of miR-195-induced changes in protein synthesis. qRT-PCR analysis was performed and the results showed that the endogenous SMAP2, S6K1, IPO9 and DCUN1D1 expression in cells and established tumors associated with LNCaP cells stably expressing miR-195 were all significantly reduced at mRNA levels. Luciferase activity assay further revealed that S6K1 was the direct target of miR-195 (P<0.001). qRT-PCR and western blot confirm an adverse relationship between miR-195 and S6K1 expression in PCa cell lines, xenograft tissues and human PCa tissues.7. Proliferation, migration, invasion and apoptosis assays all indicated that restoration of S6K1 expression dramatically attenuated the effects induced by miR-195. Moreover, the knockdown of endogenous S6K1 expression could imitate the effects associated with miR-195 overexpression in PCa cells, including inhibiting the abilities of migration and invasion, as well as promoting cellular apoptosis. These results further implied that S6K1 was a critical downstream mediator of miR-195 suppressive effects in PCa progression.8. The positive expression rate of S6K1 protein in PCa clinical samples [174/225 (77.3%)] was significantly higher than that in adjacent benign tissues [10/25 (40%), (χ2=16.140, P<0.001)]. We analyzed 225 radical prostatectomy specimens represented in TMA from PCa patients. The results revealed that S6K1 positive expression was significantly associated with advanced pathological stage (χ2=4.396, P=0.036), positive surgical margin status (χ2=5.371, P=0.02), positive BCR (x2=5.696, P=0.017) and shorter overall survival (χ2=5.417, P=0.02) of PCa patients. Kaplan-Meier analysis was conducted to assess the prognostic value of S6K1 expression in human PCa. We found that there were significant differences in the BCR-free survival (P=0.011) and overall survival (P=0.022), but not in metastasis-free survival (P=0.058), between patients with positive and negative S6K1 expression.Cox proportional hazards multivariate model indicated that only S6K1 expression was independent predictors of BCR-free survival and overall survival in PCa patients [P= 0.024 and 0.039, respectively; HR (95% CI):2.041 (1.098-3.792) and 2.992 (1.058-8.462), respectively].9. Enforced expression of miR-195 and knockdown of S6K1 protein in two PCa cell lines LNCaP and DU145 both significantly reduced the protein expression levels of MMP-9 and VEGF, but increased those of E-cadherin and BAD, implying the four proteins might function as the downstream effectors of miR-195-S6K1 axis in human PCa cells.Conclusion:1. MiR-195 acts as an important tumor suppressor in prostate cancer. Moreover, miR-195 inhibits tumor progression by negatively regulating S6K1 in human prostate cancer.2. MiR-195 and S6K1 respectively could serve as an independent factor for predicting the risk of BCR after radical prostatectomy. Furthermore, S6K1 could also be an independent predictor for the overall survival of PCa patients. In summary, miR-195 and S6K1 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. MiR-195-S6K1 axis partially illustrates the molecular mechanism of PCa progression and represents a novel potential therapeutic target for PCa treatment.