miR‐193b is an epigenetically regulated putative tumor suppressor in prostate cancer

miRNAs have proven to be key regulators of gene expression and are differentially expressed in various diseases, including cancer. Our aim was to identify epigenetically dysregulated genes in prostate cancer. We performed miRNA expression profiling after relieving epigenetic modifications in 6 prostate cancer cell lines and nonmalignant prostate epithelial cells. Thirty‐eight miRNAs showed increased expression in any prostate cancer cell line after 5‐aza‐2′‐deoxycytidine (5azadC) and trichostatin A (TSA) treatments. Six of these also had decreased expression in clinical prostate cancer samples compared to benign prostatic hyperplasia. Among these, miR‐193b was methylated in 22Rv1 cell line at a CpG island ～1 kb upstream of the miRNA locus. Expressing miR‐193b in 22Rv1 cells using pre‐miR‐193b oligonucleotides caused a significant growth reduction (p < 0.001) resulting from a decrease of cells in S‐phase of the cell cycle (p < 0.01). In addition, the anchorage independent growth was partially inhibited in transiently miR‐193b‐expressing 22Rv1 cells (p < 0.01). Altogether, our data suggest that miR‐193b is an epigenetically silenced putative tumor suppressor in prostate cancer.     

Cluster microRNAs miR‐194 and miR‐215 suppress the tumorigenicity of intestinal tumor organoids

Tumor stem cells with self‐renewal and multipotent capacity play critical roles in the initiation and progression of cancer. Recently, a new 3‐D culture system known as organoid culture has been developed, allowing Lgr5‐positive stem cells to form organoids that resemble the properties of original tissues. Here we established organoids derived from intestinal tumors of Apc^min/+ mice and normal intestinal epithelia of C57BL/6J mice and investigated the roles of microRNA (miRNA) in intestinal tumor organoids. The results of microarray analyses revealed that expression of the cluster miRNAs, miR‐194 and miR‐215 was markedly suppressed in intestinal tumor organoids in comparison with organoids derived from normal intestinal epithelia. Enforced expression of miR‐194 resulted in inhibition of E2f3, a positive regulator of the cell cycle and growth suppression of intestinal tumor organoids. In addition, enforced expression of miR‐215 suppressed the cancer stem cell signature through downregulation of intestinal stem cell markers including Lgr5. These findings indicate that the miRNA cluster including miR‐194 and miR‐215 plays important roles in suppressing the growth and attenuating the stemness of intestinal tumor organoids.     

Tumor‐suppressive microRNA‐135a inhibits cancer cell proliferation by targeting the c‐MYC oncogene in renal cell carcinoma

Recently, many studies have suggested that microRNAs (miRNAs) are involved in cancer cell development, invasion, and metastasis of various types of human cancers. In a previous study, miRNA expression signatures from renal cell carcinoma (RCC) revealed that expression of microRNA‐135a (miR‐135a) was significantly reduced in cancerous tissues. The aim of this study was to investigate the functional significance of miR‐135a and to identify miR‐135a‐mediated molecular pathways in RCC cells. Restoration of mature miR‐135a significantly inhibited cancer cell proliferation and induced G₀/G₁ arrest in the RCC cell lines caki2 and A498, suggesting that miR‐135a functioned as a potential tumor suppressor. We then examined miR‐135a‐mediated molecular pathways using genome‐wide gene expression analysis and in silico analysis. A total of 570 downregulated genes were identified in miR‐135a transfected RCC cell lines. To investigate the biological significance of potential miR‐135a‐mediated pathways, we classified putative miR‐135a‐regulated genes according to the Kyoto Encyclopedia of Genes and Genomics pathway database. From our in silico analysis, 25 pathways, including the cell cycle, pathways in cancer, DNA replication, and focal adhesion, were significantly regulated by miR‐135a in RCC cells. Moreover, based on the results of this analysis, we investigated whether miR‐135a targeted the c‐MYC gene in RCC. Gain‐of‐function and luciferase reporter assays showed that c‐MYC was directly regulated by miR‐135a in RCC cells. Furthermore, c‐MYC expression was significantly upregulated in RCC clinical specimens. Our data suggest that elucidation of tumor‐suppressive miR‐135a‐mediated molecular pathways could reveal potential therapeutic targets in RCC.     

MicroRNA‐125b suppresses the development of bladder cancer by targeting E2F3

Increasing evidence has suggested that dysregulation of certain microRNAs (miRNAs) may contribute to tumorigenesis. microRNA‐125b (miR‐125b) was implicated to have close relationship with cell proliferation and differentiation, and downregulation of miR‐125b was observed in various types of cancers. However, the biological function of miR‐125b in bladder tumorigenesis is still unknown. In our study, we showed that the expression of miR‐125b was significantly decreased in bladder cancer tissues and four bladder cancer cell lines. Moreover, miR‐125b could suppress bladder cancer cells to form colonies in vitro and to develop tumors in nude mice. E2F3, which was critical for G1/S transition and was overexpressed in most of poor‐differentiated bladder cancers, was identified as a target of miR‐125b by luciferase assay. The E2F3 mRNA and protein expression levels were detected in bladder cancer tissues and cell lines, and interestingly, inverse correlations between miR‐125b and E2F3 protein level were found in bladder cancer tissues and four E2F3 nonamplified cell lines. Introduction of miR‐125b could reduce the expression of E2F3 protein but not the E2F3 mRNA. In addition, we observed that transfection of miR‐125b could inhibit the expression of Cyclin A2, one of the E2Fs‐responsive genes involved in G1/S transition. These results suggest that miR‐125b may regulate G1/S transition through the E2F3–Cyclin A2 signaling pathway. Taken together, miR‐125b may act as a tumor suppressor in bladder urothelium, and downregulation of miR‐125b may contribute to the tumorigenesis of bladder cancer.     

Downregulation of miR‐29 contributes to cisplatin resistance of ovarian cancer cells

Drug resistance is an obstacle to the treatment of ovarian cancer. Using a unique cell model, we have proven previously that a subpopulation of ovarian cancer cells is more resistant to cisplatin than are the original cells. MicroRNAs (miRNAs), small noncoding RNAs, are involved in many biological events in cancer cells. In our study, we explored whether miRNAs are involved in cisplatin resistance of ovarian cancer cells. Cisplatin‐resistant cells expressed a lower level of miR‐29a/b/c. Manipulation of microRNA‐29 (miR‐29) expression modulated cisplatin sensitivity of CP70, HeyC2, SKOV3 and A2780 ovarian cancer cells. Knockdown of miR‐29a/b/c increased the ability of cells to escape cisplatin‐induced cell death partly through upregulation of collagen type I alpha 1 (COL1A1) and increased the activation of extracellular signal‐regulated kinase 1/2 and inactivation of glycogen synthase kinase 3 beta. When combined with cisplatin treatment, knockdown of miR‐29 decreased the amount of the active form of caspase‐9 and caspase‐3. Ectopic expression of miR‐29 alone or in combination with cisplatin treatment efficaciously reduced the tumorigenicity of CP70 cells in vivo. Our data show that downregulation of miR‐29 increases cisplatin resistance in ovarian cancer cells. Taken together, these data suggest that overexpression of miR‐29 is a potential sensitizer to cisplatin treatment that may have therapeutic implications.     

PVT1‐derived miR‐1207‐5p promotes breast cancer cell growth by targeting STAT6

Accumulating evidence indicates that ectopic expression of non‐coding RNAs are responsible for breast cancer progression. Increased non‐coding RNA PVT1, the host gene of microRNA‐1207‐5p (miR‐1207‐5p), has been associated with breast cancer proliferation. However, how PVT1 functions in breast cancer is still not clear. In this study, we show a PVT1‐derived microRNA, miR‐1207‐5p, that promotes the proliferation of breast cancer cells by directly regulating STAT6. We first confirm the positive correlated expression pattern between PVT1 and miR‐1207‐5p by observing consistent induced expression by estrogen, and overexpression in breast cancer cell lines and breast cancer patient specimens. Moreover, silence of PVT1 also decreased miR‐1207‐5p expression. Furthermore, increased miR‐1207‐5p expression promoted, while decreased miR‐1207‐5p expression suppressed, cell proliferation, colony formation, and cell cycle progression in breast cancer cell lines. Mechanistically, a novel target of miR‐1207‐5p, STAT6, was identified by a luciferase reporter assay. Overexpression of miR‐1207‐5p decreased the levels of STAT6, which activated CDKN1A and CDKN1B to regulate the cell cycle. We also confirmed the reverse correlation of miR‐1207‐5p and STAT6 expression levels in breast cancer samples. Therefore, our findings reveal that PVT1‐derived miR‐1207‐5p promotes the proliferation of breast cancer cells by targeting STAT6, which in turn controls CDKN1A and CDKN1B expression. These findings suggest miR‐1207‐5p might be a potential target for breast cancer therapy.     

Molecular pathogenesis of pancreatic ductal adenocarcinoma: Impact of passenger strand of pre‐miR‐148a on gene regulation

We previously used RNA sequencing to establish the microRNA (miRNA) expression signature of pancreatic ductal adenocarcinoma (PDAC). We found that both strands of pre‐miR‐148a (miR‐148a‐5p: the passenger strand and miR‐148a‐3p: the guide strand) were downregulated in cancer tissues. Ectopic expression of miR‐148a‐5p and miR‐148a‐3p significantly inhibited cancer cell migration and invasion, indicating that both strands of pre‐miR‐148a had tumor‐suppressive roles in PDAC cells. In silico database and genome‐wide gene expression analyses identified a total of 15 genes that were putative targets regulated by these miRNAs. High expression of miR‐148a‐5p targets (PHLDA2, LPCAT2 and AP1S3) and miR‐148a‐3p targets (SMA, ENDOD1 and UHMK1) was associated with poor prognosis of patients with PDAC. Moreover, knockdown of PHLDA2 expression inhibited cancer cell aggressiveness, suggesting PHLDA2 acted as an oncogene in PDAC cells. Involvement of the passenger strand of pre‐miR‐148a (miR‐148‐5p) is a new concept in cancer research. Novel approaches that identify tumor‐suppressive miRNA regulatory networks in lethal PDAC might provide new prognostic markers and therapeutic targets for this disease.     

Impact of novel oncogenic pathways regulated by antitumor miR‐451a in renal cell carcinoma

Recent analyses of our microRNA (miRNA) expression signatures obtained from several types of cancer have provided novel information on their molecular pathology. In renal cell carcinoma (RCC), expression of microRNA‐451a (miR‐451a) was significantly downregulated in patient specimens and low expression of miR‐451a was significantly associated with poor prognosis of RCC patients (P = .00305) based on data in The Cancer Genome Atlas. The aims of the present study were to investigate the antitumor roles of miR‐451a and to identify novel oncogenic networks it regulated in RCC cells. Ectopic expression of miR‐451a significantly inhibited cancer cell migration and invasion by RCC cell lines, suggesting that miR‐451a had antitumor roles. To identify oncogenes regulated by miR‐451a in RCC cells, we analyzed genome‐wide gene expression data and examined information in in silico databases. A total of 16 oncogenes and were found to be possible targets of miR‐451a regulation. Interestingly, high expression of 9 genes (PMM2, CRELD2, CLEC2D, SPC25, BST2, EVL, TBX15, DPYSL3, and NAMPT) was significantly associated with poor prognosis. In this study, we focused on phosphomannomutase 2 (PMM2), which was the most strongly associated with prognosis. Overexpression of PMM2 was detected in clinical specimens and Spearman's rank test indicated a negative correlation between the expression levels of miR‐451a and PMM2 (P = .0409). Knockdown of PMM2 in RCC cells inhibited cancer cell migration and invasion, indicating overexpression of PMM2 could promote malignancy. Analytic strategies based on antitumor miRNAs is an effective tool for identification of novel pathways of cancer.     

MiRNA‐362‐3p induces cell cycle arrest through targeting of E2F1, USF2 and PTPN1 and is associated with recurrence of colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of cancer deaths in Western countries. A significant number of CRC patients undergoing curatively intended surgery subsequently develop recurrence and die from the disease. MicroRNAs (miRNAs) are aberrantly expressed in cancers and appear to have both diagnostic and prognostic significance. In this study, we identified novel miRNAs associated with recurrence of CRC, and their possible mechanism of action. TaqMan^® Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosas and 46 microsatellite stable CRC tumors. Four miRNAs (miR‐362‐3p, miR‐570, miR‐148 a* and miR‐944) were expressed at a higher level in tumors from patients with no recurrence (p<0.015), compared with tumors from patients with recurrence. A significant association with increased disease free survival was confirmed for miR‐362‐3p in a second independent cohort of 43 CRC patients, using single TaqMan^® microRNA assays. In vitro functional analysis showed that over‐expression of miR‐362‐3p in colon cancer cell lines reduced cell viability, and proliferation mainly due to cell cycle arrest. E2F1, USF2 and PTPN1 were identified as potential miR‐362‐3p targets by mRNA profiling of HCT116 cells over‐expressing miR‐362‐3p. Subsequently, these genes were confirmed as direct targets by Luciferase reporter assays and their knockdown in vitro phenocopied the effects of miR‐362‐3p over‐expression. We conclude that miR‐362‐3p may be a novel prognostic marker in CRC, and hypothesize that the positive effects of augmented miR‐362‐3p expression may in part be mediated through the targets E2F1, USF2 and PTPN1.     

Elevated expression of microRNAs 155, 203, 210 and 222 in pancreatic tumors is associated with poorer survival

Pancreatic cancer is the eighth most common cancer and has an overall 5‐year survival rate lower than 10%. Because of their ability to regulate gene expression, microRNAs can act as oncogenes or tumor‐suppressor genes and so have garnered interest as possible prognostic and therapeutic markers during the last decade. However, the prognostic value of microRNA expression in pancreatic cancer has not been thoroughly investigated. We measured the levels of miR‐155, miR‐203, miR‐210, miR‐216, miR‐217 and miR‐222 by quantitative RT‐PCR in a cohort of 56 microdissected pancreatic ductal adenocarcinomas (PDAC). These microRNAs were chosen as they had previously been shown to be differentially expressed in pancreatic tumors compared to normal tissues. The possible association of microRNA expression and patients' survival was examined using multivariate Cox's regression hazard analyses. Interestingly, significant correlations between elevated microRNA expression and overall survival were observed for miR‐155 (RR = 2.50; p = 0.005), miR‐203 (RR = 2.21; p = 0.017), miR‐210 (RR = 2.48; p = 0.005) and miR‐222 (RR = 2.05; p = 0.035). Furthermore, tumors from patients demonstrating elevated expression levels of all 4 microRNAs possessed a 6.2‐fold increased risk of tumor‐related death compared to patients whose tumors showed a lower expression of these microRNAs. This study provides the first evidence for an oncogenic activity of miR‐155, miR‐203, miR‐210 and miR‐222 in the development of pancreatic cancer as has been reported for other tumor types. Furthermore, the putative target genes for these microRNAs suggest a complex signaling network that can affect PDAC tumorigenesis and tumor progression.     

Retinoblastoma gene‐independent G1 phase arrest by flavone,phosphatidylinositol 3‐kinase inhibitor,and histone deacetylase inhibitor

In most human malignant tumors, retinoblastoma tumor‐suppressor gene (RB) product is inactivated by phosphorylation. Therefore, cancer preventive agents or molecular‐targeting agents can inhibit the tumor growth at G₁ phase through RB reactivation. However, little is known about the effectiveness of RB reactivating agents against malignancies with mutated RB. We report here that chemopreventive agent flavone, phosphatidylinositol 3‐kinase (PI3K) inhibitor LY294002, and histone deacetylase (HDAC) inhibitor trichostatin A (TSA) also induce G₁ phase arrest in malignant tumor cells with mutated RB. In human prostate cancer DU145 cells with mutated RB, flavone increased cyclin‐dependent kinase (CDK) inhibitors p21 and p27, and reduced cdk4 and cdk6, resulting in decrement of phosphorylated RB family proteins p130 and p107. LY294002 also dephosphorylated p107 and p130 proteins, whereas TSA dephosphorylated p130, but not p107. Furthermore, flavone induced G₁ phase arrest in both mouse embryo fibroblast (MEF) wild‐type and MEF RB ^{? / ?} cells, but did not do so in RB, p107, and p130 triple‐knockout MEF cells. These results suggested that p130 and p107 contributed to G₁ phase arrest by flavone in RB‐mutated cells. However, flavone induced tumor suppressor microRNA miR‐34a with reduction of E2F1 and E2F3, known to be downregulated by miR‐34a, raising the possibility that miR‐34a might partially contribute to G₁ arrest by flavone. These results raise the possibility that RB reactivating chemopreventive agents or molecular targeting agents might also be effective against a variety of malignant tumor cells with mutant RB.     

Delphinidin suppresses breast carcinogenesis through the HOTAIR/microRNA‐34a axis

Delphinidin, one of the main anthocyanidins, has potent anti‐cancer properties. In this study, we investigated the effect of delphinidin on 1‐methyl‐1‐nitrosourea (MNU)‐induced breast carcinogenesis on rats and the mechanism of delphinidin via negative regulation of the HOTAIR/microRNA‐34a axis. We found administration of delphinidin could effectively suppress MNU‐induced mammal breast carcinogenesis. Delphinidin downregulated the level of HOTAIR and upregulated miR‐34a in breast carcinogenesis. Western blot analysis confirmed that delphinidin treatment can significantly decrease the expression of β‐catenin, glycogen synthase kinase‐3β (Gsk3β), c‐Myc, cyclin‐D1, and matrix metalloproteinase‐7(MMP‐7) expression in breast cancer cells, and inhibition of miR‐34a significantly reduced the effect of delphinidin on c‐Myc, cyclin‐D1, and MMP‐7. HOTAIR overexpression also blocked the effect of delphinidin on miR‐34a and the Wnt/β‐catenin signaling pathway in MDA‐MB‐231 cells. RNA immunoprecipitation (RIP) assay and chromatin immunoprecipitation (ChIP) assay results showed that delphinidin upregulated miR‐34a by inhibiting HOTAIR, coupled with enhancement of the zeste homolog 2 (EZH2) and histone H3 Lys27 trimethylation (H3K27me3). This study indicated that delphinidin may potentially suppress breast carcinogenesis and exert its anti‐cancer effect through the HOTAIR/miR‐34a axis. These findings provided new evidence for the use of delphinidin in preventing breast carcinogenesis.     

Regulation of antitumor miR‐144‐5p targets oncogenes: Direct regulation of syndecan‐3 and its clinical significance

In the human genome, miR‐451a, miR‐144‐5p (passenger strand), and miR‐144‐3p (guide strand) reside in clustered microRNA (miRNA) sequences located within the 17q11.2 region. Low expression of these miRNAs is significantly associated with poor prognosis of patients with renal cell carcinoma (RCC) (miR‐451a: P = .00305; miR‐144‐5p: P = .00128; miR‐144‐3p: P = 9.45 × 10^?5). We previously reported that miR‐451a acted as an antitumor miRNA in RCC cells. Involvement of the passenger strand of the miR‐144 duplex in the pathogenesis of RCC is not well understood. Functional assays showed that miR‐144‐5p and miR‐144‐3p significantly reduced cancer cell migration and invasive abilities, suggesting these miRNAs acted as antitumor miRNAs in RCC cells. Analyses of miR‐144‐5p targets identified a total of 65 putative oncogenic targets in RCC cells. Among them, high expression levels of 9 genes (FAM64A, F2, TRIP13, ANKRD36, CENPF, NCAPG, CLEC2D, SDC3, and SEMA4B) were significantly associated with poor prognosis (P < .001). Among these targets, expression of SDC3 was directly controlled by miR‐144‐5p, and its expression enhanced cancer cell aggressiveness. We identified genes downstream by SDC3 regulation. Data showed that expression of 10 of the downstream genes (IL18RAP, SDC3, SH2D1A, GZMH, KIF21B, TMC8, GAB3, HLA‐DPB2, PLEK, and C1QB) significantly predicted poor prognosis of the patients (P = .0064). These data indicated that the antitumor miR‐144‐5p/oncogenic SDC3 axis was deeply involved in RCC pathogenesis. Clustered miRNAs (miR‐451a, miR‐144‐5p, and miR‐144‐3p) acted as antitumor miRNAs, and their targets were intimately involved in RCC pathogenesis.     

miR‐145 sensitizes ovarian cancer cells to paclitaxel by targeting Sp1 and Cdk6

Multidrug resistance (MDR) remains a major obstacle to effective chemotherapy treatment in ovarian cancer. In our study, paclitaxel‐resistant ovarian cancer patients and cell lines had decreased miR‐145 levels and expressed high levels of Sp1 and Cdk6. Introducing miR‐145 into SKOV3/PTX and A2780/PTX cells led to a reduction in Cdk6 and Sp1 along with downregulation of P‐gp and pRb. These changes resulted in increased accumulation of antineoplastic drugs and G1 cell cycle arrest, which rendered the cells more sensitive to paclitaxel in vitro and in vivo. These effects could be reversed by reintroducing Sp1 or Cdk6 into cells expressing high levels of miR‐145, resulting in restoration of P‐gp and pRb levels. Furthermore, we confirmed that both Cdk6 and Sp1 are targets of miR‐145. Intriguingly, demethylation with 5‐aza‐dC led to reactivation of miR‐145 expression in drug‐resistant ovarian cancer cell lines, which also resulted in increased sensitivity to paclitaxel. Collectively, these findings begin to elucidate the role of miR‐145 as an important regulator of chemoresistance in ovarian cancer by controlling both Cdk6 and Sp1.