Identification of BACH1-IT2-miR-4786-Siglec-15 immune suppressive axis in bladder cancer.

The sialic acid binding Ig like lectin 15 (Siglec-15) was previously identified as tumor immune suppressor gene in some human cancers with elusive molecular mechanism to be elucidated. The continuous focus on both clinical and basic biology of bladder cancer leads us to characterize aberrant abundance of BACH1-IT2 associating with stabilization of Siglec-15, which eventually contributes to local immune suppressive microenvironment and therefore tumor advance. This effect was evidently mediated by miR-4786-5p. BACH1-IT2 functions in this scenario as microRNA sponge, and competitively conceals miR-4786 and up-regulates cancer cell surface Siglec-15. The BACH1-IT2-miR-4786-Siglec-15 axis significantly influences activation of immune cell co-culture. In summary, our data highlights the critical involvements of BACH1-IT2 and miR-4786 in immune evasion in bladder cancer, which hints the potential for both therapeutic and prognostic exploitation.

microRNA-183 plays as oncogenes by increasing cell proliferation, migration and invasion via targeting protein phosphatase 2A in renal cancer cells.

The aim of this study was to investigate the function of miR-183 in renal cancer cells and the mechanisms miR-183 regulates this process. In this study, level of miR-183 in clinical renal cancer specimens was detected by quantitative real-time PCR. miR-183 was up- and down-regulated in two renal cancer cell lines ACHN and A498, respectively, and cell proliferation, Caspase 3/7 activity, colony formation, in vitro migration and invasion were measured; and then the mechanisms of miR-183 regulating was analyzed. We found that miR-183 was up-regulated in renal cancer tissues; inhibition of endogenous miR-183 suppressed in vitro cell proliferation, colony formation, migration, and invasion and stimulated Caspase 3/7 activity; up-regulated miR-183 increased cell growth and metastasis and suppressed Caspase 3/7 activity. We also found that miR-183 directly targeted tumor suppressor, specifically the 3'UTR of three subunits of protein phosphatase 2A (PP2A-Cα, PP2A-Cß, and PP2A-B56-γ) transcripts, inhibiting their expression and regulated the downstream regulators p21, p27, MMP2/3/7 and TIMP1/2/3/4. These results revealed the oncogenes role of miR-183 in renal cancer cells via direct targeting protein phosphatase 2A.

miR-200c inhibits invasion, migration and proliferation of bladder cancer cells through down-regulation of BMI-1 and E2F3.

BACKGROUND: MicroRNA-200c (miR-200c) is one of the short noncoding RNAs that play crucial roles in tumorigenesis and tumor progression. It also acts as considerable modulator in the process of epithelial-to-mesenchymal transition (EMT), a cell development regulating process that affects tumor development and metastasis. However, the role of miR-200c in bladder cancer cells and its mechanism has not been well studied. The purpose of this study was to determine the potential role of miR-200c in regulating EMT and how it contributed to bladder cancer cells in invasion, migration and proliferation. METHODS: Real-time reverse transcription-PCR was used to identify and validate the differential expression of MiR-200c involved in EMT in 4 bladder cancer cell lines and clinical specimens. A list of potential miR-200 direct targets was identified through the TargetScan database. The precursor of miR-200c was over-expressed in UMUC-3 and T24 cells using a lentivirus construct, respectively. Protein expression and signaling pathway modulation were validated through Western blot analysis and confocal microscopy, whereas BMI-1 and E2F3, direct target of miR-200c, were validated by using the wild-type and mutant 3'-untranslated region BMI-1/E2F3 luciferase reporters. RESULTS: We demonstrate that MiR-200c is down-regulated in bladder cancer specimens compared with adjacent ones in the same patient. Luciferase assays showed that the direct down-regulation of BMI-1 and E2F3 were miR-200c-dependent because mutations in the two putative miR-200c-binding sites have rescued the inhibitory effect. Over-expression of miR-200c in bladder cancer cells resulted in significantly decreased the capacities of cell invasion, migration and proliferation. miR-200c over-expression resulted in conspicuous down-regulation of BMI-1 and E2F3 expression and in a concomitant increase in E-cadherin levels. CONCLUSIONS: miR-200c appears to control the EMT process through BMI-1 in bladder cancer cells, and it inhibits their proliferation through down-regulating E2F3. The targets of miR-200c include BMI-1 and E2F3, which are a novel regulator of EMT and a regulator of proliferation, respectively.

Cation-free siRNA-cored nanocapsules for tumor-targeted RNAi therapy.

Cation-associated cytotoxicity limits the systemic administration of RNA delivery in vivo, demanding the development of non-cationic nanosystems. In this study, cation-free polymer-siRNA nanocapsules with disulfide-crosslinked interlayer, namely T-SS(-), were prepared via the following steps: 1) complexation of siRNA with a cationic block polymer cRGD-poly(ethylene glycol)-b-poly[(2-aminoethanethiol)aspartamide]-b-poly{N'-[N-(2-aminoethyl)-2-ethylimino-1-aminomethyl]aspartamide}, abbreviated as cRGD-PEG-PAsp(MEA)-PAsp(C=N-DETA), 2) interlayer crosslinking via disulfide bond in pH 7.4 solution, and 3) removal of cationic DETA pendant at pH 5.0 via breakage of imide bond. The cationic-free nanocapsules with siRNA cores not only showed great performance (such as efficient siRNA encapsulation, high stability in serum, cancer cell targeting via cRGD modification, and GSH-triggered siRNA release), but also achieved tumor-targeted gene silencing in vivo. Moreover, the nanocapsules loaded with siRNA against polo-like kinase 1 (siRNA-PLK1) significantly inhibited tumor growth without showing cation-associated toxicity side effects and remarkably improved the survival rate of PC-3 tumor-bearing mice. The cation-free nanocapsules could potentially serve as a safe and effective platform for siRNA delivery. STATEMENT OF SIGNIFICANCE: Cation-associated toxicity limits the clinical translation of cationic carriers for siRNA delivery. Recently, several non-cationic carriers, such as siRNA micelles, DNA-based nanogels, and bottlebrush-architectured poly(ethylene glycol), have been developed to deliver siRNA. However, in these designs, siRNA as a hydrophilic macromolecule was attached to the nanoparticle surface instead of being encapsulated. Thus, it was easily degraded by serum nuclease and often induced immunogenicity. Herein, we demonstrate a new type of cation-free siRNA-cored polymeric nanocapsules. The developed nanocapsules not only showed capacities including efficient siRNA encapsulation, high stability in serum, and cancer cell targeting via cRGD modification, but also achieved an efficient tumor-targeted gene silencing in vivo. Importantly, unlike cationic carriers, the nanocapsules exhibited no cation-associated side effects.

MicroRNA-200c suppresses cell growth and metastasis by targeting Bmi-1 and E2F3 in renal cancer cells.

The aim of the present study was to evaluate the functions of miR-200c in the regulation of tumor growth and metastasis in renal cancer cells, and to investigate the underlying mechanisms. In this study, miR-200c was up- and downregulated in two renal cancer cell lines, namely ACHN and A498, and the proliferation, colony formation, migration and invasion of the cells were measured. The expression levels of various mRNAs and proteins were then analyzed using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. It was found that miR-200c suppressed proliferation, migration and invasion of the renal cancer cells and, conversely, the inhibition of endogenous miR-200c resulted in increased cell proliferation and metastasis. Furthermore, a luciferase reporter assay revealed that miR-200c directly targeted the 3' untranslated regions of the oncogenes B-cell-specific Moloney murine leukemia virus insertion site 1 (Bmi-1) and E2F transcription factor 3 (E2F3) mRNAs, reduced the expression of Bmi-1 and E2F3 and regulated the expression of downstream genes, including E-cadherin, N-cadherin, vimentin, p14 and p16. These results indicate a tumor suppressor role for miR-200c in renal cancer cells via the direct targeting of Bmi-1 and E2F3.

MicroRNA-429 suppresses cell proliferation, epithelial-mesenchymal transition, and metastasis by direct targeting of BMI1 and E2F3 in renal cell carcinoma.

BACKGROUND: MicroRNA-429 (miR-429), a short noncoding RNA belonging to the miR-200 superfamily, plays a crucial role in tumorigenesis and tumor progression. It also acts as a modulator of epithelial-to-mesenchymal transition, a cell development regulating process that affects tumor development and metastasis. The aim of this study was to investigate the potential role of miR-429 in regulating growth and metastasis of renal cell carcinoma. METHODS: miR-429 expression was stably up-regulated or down-regulated in the renal cell carcinoma ACHN and A498 cell lines, and cell proliferation and metastasis were assessed. RESULTS: miR-429 overexpression inhibited cell proliferation, colony formation, migration, and invasion. Suppression of endogenous miR-429 promoted cell growth and metastasis. miR-429 was shown to directly target the 3' untranslated regions of B-cell-specific Moloney murine leukemia virus insertion site 1 (BMI1) and E2F transcription factor 3 (E2F3) transcripts, regulating their expression, as well as that of the downstream epithelial-to-mesenchymal transition markers E-cadherin, N-cadherin, vimentin, p14, and p16. CONCLUSIONS: These results revealed a tumor suppressive role for miR-429 in renal cell carcinoma through directly targeting BMI1 and E2F3.