Oncogenic miRNA-182-5p Targets Smad4 and RECK in Human Bladder Cancer

Onco-miR-182-5p has been reported to be over-expressed in bladder cancer (BC) tissues however a detailed functional analysis of miR-182-5p has not been carried out in BC. Therefore the purpose of this study was to: 1. conduct a functional analysis of miR-182-5p in bladder cancer, 2. assess its usefulness as a tumor marker, 3. identify miR-182-5p target genes in BC. Initially we found that miR-182-5p expression was significantly higher in bladder cancer compared to normal tissues and high miR-182-5p expression was associated with shorter overall survival in BC patients. To study the functional significance of miR-182-5p, we over-expressed miR-182-5p with miR-182-5p precursor and observed that cell proliferation, migration and invasion abilities were increased in BC cells. However cell apoptosis was inhibited by miR-182-5p. We also identified Smad4 and RECK as potential target genes of miR-182-5p using several algorithms. 3′UTR luciferase activity of these target genes was significantly decreased and protein expression of these target genes was significantly up-regulated in miR-182-5p inhibitor transfected bladder cancer cells. MiR-182-5p also increased nuclear beta-catenin expression and while Smad4 repressed nuclear beta-catenin expression. In conclusion, our data suggests that miR-182-5p plays an important role as an oncogene by knocking down RECK and Smad4, resulting in activation of the Wnt-beta-catenin signaling pathway in bladder cancer.     

The microRNA -23b/-27b Cluster Suppresses the Metastatic Phenotype of Castration-Resistant Prostate Cancer Cells

MicroRNAs (miRs) are small, endogenous, non-coding RNAs that regulate the stability and/or translation of complementary mRNA targets. MiRs have emerged not only as critical modulators of normal physiologic processes, but their deregulation may significantly impact prostate and other cancers. The expression of miR-23b and miR-27b, which are encoded by the same miR cluster (miR-23b/-27b), are downregulated in metastatic, castration-resistant tumors compared to primary prostate cancer and benign tissue; however, their possible role in prostate cancer progression is unknown. We found that ectopic expression of miR-23b/-27b in two independent castration-resistant prostate cancer cell lines resulted in suppression of invasion and migration, as well as reduced survival in soft agar (a measure of anoikis). However, there was no effect of miR-23b/-27b on cell proliferation suggesting that these miRs function as metastasis (but not growth) suppressors in prostate cancer. Conversely, inhibition of miR-23b/-27b in the less aggressive androgen-dependent LNCaP prostate cancer cell line resulted in enhanced invasion and migration also without affecting proliferation. Mechanistically, we found that introduction of miR-23b/-27b in metastatic, castration-resistant prostate cancer cell lines resulted in a significant attenuation of Rac1 activity without affecting total Rac1 levels and caused increased levels of the tumor suppressor E-cadherin. Inhibition of these miRs had the opposite effect in androgen-dependent LNCaP cells. These results suggest that miR-23b/-27b are metastasis suppressors that might serve as novel biomarkers and therapeutic agents for castration-resistant disease.     

The MicroRNA-23b/27b/24 Cluster Promotes Breast Cancer Lung Metastasis by Targeting Metastasis-suppressive Gene Prosaposin

MicroRNAs (miRNAs) have been shown to function as key regulators of tumor progression and metastasis. Recent studies have indicated that the miRNAs comprising the miR-23b/27b/24 cluster might influence tumor metastasis, although the precise nature of this regulation remains unclear. Here, expression of the miR-23b/27b/24 cluster is found to correlate with metastatic potential in mouse and human breast cancer cell lines and is elevated in metastatic lung lesions in human breast cancer patients. Ectopic expression of the miRNAs in the weakly metastatic mouse 4TO7 mammary tumor cell line had no effect on proliferation or morphology of tumor cells in vitro but was found to increase lung metastasis in a mouse model of breast cancer metastasis. Furthermore, gene expression profiling analysis of miRNA overexpressing 4TO7 cells revealed the direct targeting of prosaposin (PSAP), which encodes a secreted protein found to be inversely correlated with metastatic progression in human breast cancer patients. Importantly, ectopic expression of PSAP was able to suppress the metastatic phenotype in highly metastatic 4T1 and MDA-MB-231 SCP28 cells, as well as in cells ectopically expressing miR-23b/27b/24. These findings support a metastasis-promoting function of the miR-23b/27b/24 cluster of miRNAs, which functions in part through the direct inhibition of PSAP.     

Colorectal Cancer Migration and Invasion Initiated by microRNA-106a

MicroRNAs have been implicated in the regulation of several cellular signaling pathways of colorectal cancer (CRC) cells. Although emerging evidence proves that microRNA (miR)-106a is expressed highly in primary tumor and stool samples of CRC patients; whether or not miR-106a mediates cancer metastasis is unknown. We show here that miR-106a is highly expressed in metastatic CRC cells, and regulates cancer cell migration and invasion positively in vitro and in vivo. These phenotypes do not involve confounding influences on cancer cell proliferation. MiR-106a inhibits the expression of transforming growth factor-β receptor 2 (TGFBR2), leading to increased CRC cell migration and invasion. Importantly, miR-106a expression levels in primary CRCs are correlated with clinical cancer progression. These observations indicate that miR-106a inhibits the anti-metastatic target directly and results in CRC cell migration and invasion.     

MicroRNA-216b targets HK2 to potentiate autophagy and apoptosis of breast cancer cells via the mTOR signaling pathway

Patients suffering from breast cancer (BC) still have a poor response to treatments, even though early detection and improved therapy have contributed to a reduced mortality. Recent studies have been inspired on the association between microRNAs (miRs) and therapies of BC. The current study set out to investigate the role of miR-216b in BC, and further analyze the underlining mechanism. Firstly, hexokinase 2 (HK2) and miR-216b were characterized in BC tissues and cells by RT-qPCR and Western blot assay. In addition, the interaction between HK2 and miR-216b was analyzed using dual luciferase reporter assay. BC cells were further transfected with a series of miR-216b mimic or inhibitor, or siRNA targeting HK2, so as to analyze the regulatory mechanism of miR-216b, HK2 and mammalian target of rapamycin (mTOR) signaling pathway, and to further explore their regulation in BC cellular behaviors. The results demonstrated that HK2 was highly expressed and miR-216b was poorly expressed in BC cells and tissues. HK2 was also verified as a target of miR-216b with online databases and dual luciferase reporter assay. Functionally, miR-216b was found to be closely associated with BC progression via inactivating mTOR signaling pathway by targeting HK2. Moreover, cell viability, migration and invasion were reduced as a result of miR-216b upregulation or HK2 silencing, while autophagy, cell cycle arrest and apoptosis were induced. Taken together, our findings indicated that miR-216b down-regulates HK2 to inactivate the mTOR signaling pathway, thus inhibiting the progression of BC. Hence, this study highlighted a novel target for BC treatment.     

MicroRNA-135b/CAMK2D Axis Contribute to Malignant Progression of Gastric Cancer through EMT Process Remodeling

There is a continued need for investigating the roles of microRNAs (miRNAs) and their targets on the progression of gastric cancer (GC), especially metastasis. Here, we performed an integrated study to identify dysregulated miRNAs critical for GC development and progression. miR-135b was determined as a promising biomarker for GC. The expression level of miR-135b was increased among GC cell lines, patient tumor tissues, serum samples, and correlation with aggravation of the GC patients. The in vitro functional assays demonstrated overexpression of miR-135b promoted cell proliferation, migration and invasion in GC, while miR-135b inhibition led to the opposite results. CAMK2D was found to be the direct target of miR-135b, serving as a tumor suppressor in GC cells. Based on our and public datasets, we confirmed the attenuation of CAMK2D expression in GC tissues. And, the expression levels of miR-135b and CAMK2D were closely associated with prognosis of GC patients. Ectopic expression of miR-135b resulted in the down-regulation of CAMK2D. Additionally, CAMK2D was a prerequisite for miR-135b to promote GC cells proliferation and migration by regulating the EMT process, which was confirmed by the in vivo experiments. Importantly, in vivo injection of miR-135b antagomir significantly repressed the tumor growth and metastasis of xenograft models, which suggested that the miR-135b antagomir were promising for clinical applications. Taken together, these results indicate that miR-135b/CAMK2D axis drives GC progression by EMT process remodeling, suggesting that miR-135b may be utilized as a new therapeutic target and prognostic marker for GC patients.     

Axl-altered microRNAs regulate tumorigenicity and gefitinib resistance in lung cancer

The involvement of Axl kinase in non-small cell lung cancer''s (NSCLC) acquired resistance to tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib has been identified recently, but the mechanism by which Axl contributes to TKI resistance is largely unknown. MicroRNAs (miRNAs) repress gene expression and their critical role in tumorigenesis has been implicated. To investigate the role of miRNAs in the Axl-mediated acquired gefitinib resistance, we examined the Axl-mediated miRNA changes in gefitinib-resistant lung cancers. A panel of Axl kinase-altered miRNAs was identified. In this study, we validate and report that miR-374a and miR-548b modulated by Axl have essential roles in cell cycle arrest, gefitinib-induced apoptosis, epithelial-to-mesenchymal transition, migration and tumorigenesis of gefitinib-resistant lung cancer cells in vitro and in vivo by targeting Wnt5a and CCNB1 genes, respectively. Of clinical significance, high expression of Axl and miR-374a and low expression of miR-548b are associated with poor disease-free survival postoperatively. These findings indicate that the modulation of specific miRNAs may provide a therapeutic target to treat or reverse gefitinib resistance in NSCLC with high expression of Axl in the future.     

Breast cancer stem cell-derived extracellular vesicles transfer ARRDC1-AS1 to promote breast carcinogenesis via a miR-4731-5p/AKT1 axis-dependent mechanism

ObjectivesDeregulation of long non-coding RNAs (lncRNAs) has been frequently reported in breast cancer (BC). This goes to show the importance of understanding its significant contribution towards breast carcinogenesis. In the present study, we clarified a carcinogenic mechanism based on the ARRDC1-AS1 delivered by breast cancer stem cells-derived extracellular vesicles (BCSCs-EVs) in BC.MethodsThe isolated and well characterized BCSCs-EVs were co-cultured with BC cells. The expression of ARRDC1-AS1, miR-4731-5p, and AKT1 was determined in BC cell lines. BC cells were assayed for their viability, invasion, migration and apoptosis in vitro by CCK-8, Transwell and flow cytometry, as well as tumor growth in vivo after loss- and gain-of function assays. Dual-luciferase reporter gene, RIP and RNA pull-down assays were performed to determine the interactions among ARRDC1-AS1, miR-4731-5p, and AKT1.ResultsElevation of ARRDC1-AS1 and AKT1 as well as miR-4731-5p downregulation were observed in BC cells. ARRDC1-AS1 was enriched in BCSCs-EVs. Furthermore, EVs containing ARRDC1-AS1 enhanced the BC cell viability, invasion and migration and glutamate concentration. Mechanistically, ARRDC1-AS1 elevated the expression of AKT1 by competitively binding to miR-4731-5p. ARRDC1-AS1-containing EVs were also found to enhance tumor growth in vivo.ConclusionCollectively, BCSCs-EVs-mediated delivery of ARRDC1-AS1 may promote the malignant phenotypes of BC cells via the miR-4731-5p/AKT1 axis.     

miR-195 Inhibits EMT by Targeting FGF2 in Prostate Cancer Cells

Prostate cancer (PCa) is one of the leading causes of deaths in America. The major cause of mortality can be attributed to metastasis. Cancer metastasis involves sequential and interrelated events. miRNAs and epithelial-mesenchymal transition (EMT) are implicated in this process. miR-195 is downregulated in many human cancers. However, the roles of miR-195 in PCa metastasis and EMT remain unclear. In this study, data from Memorial Sloan Kettering Cancer Center (MSKCC) prostate cancer database were re-analysed to detect miR-195 expression and its roles in PCa. miR-195 was then overexpressed in castration-resistant PCa cell lines, DU-145 and PC-3. The role of miR-195 in migration and invasion in vitro was also investigated, and common markers in EMT were evaluated through Western blot analysis. A luciferase reporter assay was conducted to confirm the target gene of miR-195; were validated in PCa cells. In MSKCC data re-analyses, miR-195 was poorly expressed in metastatic PCa; miR-195 could be used to diagnose metastatic PCa by measuring the corresponding expression. Area under the receiver operating characteristic curve (AUC-ROC) was 0.705 (P = 0.017). Low miR-195 expression was characterised with a shorter relapse-free survival (RFS) time. miR-195 overexpression suppressed cell migration, invasion and EMT. Fibroblast growth factor 2 (FGF2) was confirmed as a direct target of miR-195. FGF2 knockdown also suppressed migration, invasion and EMT; by contrast, increased FGF2 partially reversed the suppressive effect of miR-195. And data from ONCOMINE prostate cancer database showed that PCa patients with high FGF2 expression showed shorter RFS time (P = 0.046). Overall, this study demonstrated that miR-195 suppressed PCa cell metastasis by downregulating FGF2. miR-195 restoration may be considered as a new therapeutic method to treat metastatic PCa.     

Over-Expression of miR-106b Promotes Cell Migration and Metastasis in Hepatocellular Carcinoma by Activating Epithelial-Mesenchymal Transition Process

Hepatocellular carcinoma (HCC) is one the the most fatal cancers worldwide. The poor prognosis of HCC is mainly due to the developement of distance metastasis. To investigate the mechanism of metastasis in HCC, an orthotopic HCC metastasis animal model was established. Two sets of primary liver tumor cell lines and corresponding lung metastasis cell lines were generated. In vitro functional analysis demonstrated that the metastatic cell line had higher invasion and migration ability when compared with the primary liver tumor cell line. These cell lines were subjected to microRNA (miRNAs) microarray analysis to identify differentially expressed miRNAs which were associated with the developement of metastasis in vivo. Fifteen human miRNAs, including miR-106b, were differentially expressed in 2 metastatic cell lines compared with the primary tumor cell lines. The clinical significance of miR-106b in 99 HCC clinical samples was studied. The results demonstrated that miR-106b was over-expressed in HCC tumor tissue compared with adjacent non-tumor tissue (p = 0.0005), and overexpression of miR-106b was signficantly correlated with higher tumor grade (p = 0.018). Further functional studies demonstrated that miR-106b could promote cell migration and stress fiber formation by over-expressing RhoGTPases, RhoA and RhoC. In vivo functional studies also showed that over-expression of miR-106b promoted HCC metastasis. These effects were related to the activation of the epithelial-mesenchymal transition (EMT) process. Our results suggested that miR-106b expression contributed to HCC metastasis by activating the EMT process promoting cell migration in vitro and metastasis in vivo.     

Calcium sensing receptor stimulates breast cancer cell migration via the Gβγ-AKT-mTORC2 signaling pathway

Calcium sensing receptor, a pleiotropic G protein coupled receptor, activates secretory pathways in cancer cells and putatively exacerbates their metastatic behavior. Here, we show that various CaSR mutants, identified in breast cancer patients, differ in their ability to stimulate Rac, a small Rho GTPase linked to cytoskeletal reorganization and cell protrusion, but are similarly active on the mitogenic ERK pathway. To investigate how CaSR activates Rac and drives cell migration, we used invasive MDA-MB-231 breast cancer cells. We revealed, by pharmacological and knockdown strategies, that CaSR activates Rac and cell migration via the Gβγ-PI3K-mTORC2 pathway. These findings further support current efforts to validate CaSR as a relevant therapeutic target in metastatic cancer.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00662-y.