MicroRNA-34a functions as an anti-metastatic microRNA and suppresses angiogenesis in bladder cancer by directly targeting CD44

Abstract

Background: Metastasis have considered as an important clinical obstacle in the treatment of human cancer including bladder cancer. Post-transcriptional regulation has emerged as robust effectors of metastasis. MiRNAs are involved in cancer development and progression, acting as tumor suppressors or oncogenes. In this study, we focus on it that microRNA-34a functions as an anti-metastatic microRNA and suppress angiogenesis in bladder cancer by directly targeting CD44.

Methods: The expression of mir-34a was detected by quantitative real-time PCR. Oligonucleotide and lentivirus were used to overexpress miR-34a. Tube formation assay and transwell assay were used to examine the effect on bladder cancer tube formation, migration and invasion in vitro. Animal models were used to examine the effect on metastasis and angiogenesis in vivo. Luciferase assay was carried out to verify the precise target of miR-34a.

Results: We not only proved that mir-34a was significantly downregulated in bladder cancer tissues and cell lines but also that circulating miR-34a levels are reduced in bladder cancer, and their levels were positively relevance. Gain-of-function experiments investigated that increased mir-34a expression suppressed tube formation and reduced cell migration and invasion. In vivo metastasis, assays also demonstrated that overexpression of mir34a markedly inhibited bladder cancer metastasis. CD31, an endothelial cell-specific marker which stained in T24 tumors to evaluate for blood vessel density, the immunohistochemistry results showed that blood vessel quantification reduced dramatically in the T24 tumors over-expressing mir-34a. Combining with our previous studies and bioinformatics analysis, we expected that CD44 gene was a direct target of mir-34a, siRNA-mediated knockdown of CD44 partially phenocopied mir-34a overexpression suggesting that the pro-apoptotic role of mir-34a may be mediated primarily through CD44 regulation, whereas restoring the expression of CD44 attenuated the function of mir-34a in bladder cancer cells. Additionally, we identified that EMT (epithelial-mesenchymal transition) related proteins could be regulated by mir-34a which indicated that mir-34a could partially reserve EMT.

Conclusion: Our study defines a major metastasis and angiogenesis suppressive role for mir-34a, a microRNA functions as a tumor suppressor in bladder cancer by directly targeting CD44, which would be helpful as a therapeutic approach to block bladder cancer metastasis.

Figure 1

mir-34a was downregulated in bladder cancer and circulatingmiR-34a levels are reduced in bladder cancer. A, relative expression of mir-34a expression levels were evaluated by qPCR in bladder cancer.U6 small nuclear RNA was used as an internal control. B, relative expression of circulating mir-34a expression levels were evaluated by qPCR in bladder cancer. U6 small nuclear RNA was used as an internal control. C, The expression of mir-34a in blood stream positively correlates with the expression in bladder tissues (R2 = 0.3036 P = 0.0411, Pearson’s correlation). D, relative expression of CD44 expression levels were evaluated by qPCR in bladder carcinoma cell lines. C, miR-34a expression after mimics transcription was detected by qPCR. E, MiR-34a inversely correlates with CD44 expression (R2 = 0.5988 P = 0.0412, Pearson’s correlation). F, relative expression of CD44v6 expression levels were evaluated by qPCR in bladder carcinoma cell lines. G, CD44v expression pattern in human bladder cancer cell lines and bladder cancer tissues. Data are plotted as the mean ± SEM of 3 independent experiments. **, P < 0.01.

Figure 2

Overexpression of mir-34a inhibits bladder cancer cell invasion and migration and tube formation. A, migration and invasion assay for renal cancer cells. Representative photographs were taken at × 200 magnification. B, number of migrated and invaded cells were quantified in 4 random images from each treatment group. Results are the mean ± SEM from 2 independent experiments plotted as percent (%) migrating and invading cells relative to NC treatment. C, representative bioluminescent images of lungs in nude mice at the 30th days after IV injection. D, quantification analysis of fluorescence signal from captured bioluminescence images. E, Tube formation of HUVECs was determined by assaying the numbers of branch nodes after 6 h of culture under a phase contrast microscope. F, number of branch point was quantified. HUVECs were cultured in the following media: Culture Media of 5637 and T24 cells transfected with mir-34a and NC. G, metastatic nodules in the liver. H, number of nodules was quantified. I, EMT related proteins and VEGF were determined by immunoblot analysis. J, the density of blots was quantified to show the expression level of EMT related proteins and VEGF, GAPDH was used as control. NC, Negative Control. ^*, P < 0.05; ^**, P < 0.01.

Figure 3

Overexpression of miR-34a attenuated the metastasis and angiogenesis of bladder cancer in vivo. A, hematoxylin/eosin (HE) and immunohistochemical staining revealed that stable transfection of miR-34a precursor resulted in decreased expression of VEGF, CD44 and CD31 within tumors. The mean vessel density within tumors decreased after stable overexpression of miR-34a. B, T24 cells were injected into the tail vein of nude mice (5 × 10⁶cells per mouse). Tumor cells stably infected with miR-34a lentivirus established significantly fewer metastatic colonies. C, Representative photographs of immunofluorescence were taken at × 400 magnification.

Figure 4

mir-34a targets CD44 in bladder cancer cells and the validation of transfection/infection. A and B, mir-34a expression was detected by qPCR after infection of miR-34a lentivirus and corresponding negative control in 5637 and T24 for 4 days. C and D, mir-34a expression was detected by qPCR after infection of miR-34a mimics or inhibitor and corresponding negative control in 5637 and T24 for 48 hours. CD44 protein expression was inhibited in miR-34a transfected bladder carcinoma cells. E, CD44 expression was detected by western blot after overexpression of miR-34a and corresponding negative control i 5637 and T24. F and G, The seed regions of the miR-34a target sites in CD44 and the luciferase activity assay. *, P < 0.05; **, P < 0.01.

Figure 5

The anti-angiogenesis functions of miR-34a were mediated by reducing the production of CD44. Knockdown of CD44 by siRNA inhibits tube-formation and increased CD44 expression could efficiently reverse the effect of anti-angiogenesis of miR-34a in bladder cancer cells A, EMT related factors and VEGF was detected by qPCR. Data are plotted as the mean ± SEM of 3 independent experiments. B, western blotting indicated that transfection of si-CD44, into 5637 and T24 cells resulted in decreased CD44 expression when compared with negative control. Western blotting indicated that transfection of CD44 restored the downregulation of CD44 induced by stable miR-34a overexpression. C and D,the tube formation of endothelial HUVEC cells was suppressed by treatment with the medium preconditioned by CD44-knockdown 5637 and T24 cells, when compared with that of control cells. E and F, increased CD44 expression could efficiently reverse the effect of anti-angiogenesis of miR-34a in bladder cancer cells. *, P < 0.05; **, P < 0.01. G, exchange of EMT-driving transcription factors, LEF1 and Axin-2 in T24/5637-mir-34s.

Figure 6

The anti-metastatic functions of miR-34a were mediated by reducing the production of CD44. Knockdown of CD44 by siRNA inhibits bladder cancer migration and invasion. Increased CD44 expression could efficiently reverse the effect of anti-metastatic of miR-34a in bladder cancer cells. A, matrigel invasion assay indicated the decreased invasion capabilities of CD44-knockdown 5637 and T24 cells than those of control cells. B, Transfection of CD44 rescued the angiogenic capabilities of miR-34a-overexpressing cells. *, P < 0.05; **, P < 0.01.