Inhibition Of Epithelial-mesenchymal Transition By Metformin In Prostate Cancer Cells And Correlative Mechanisms

Prostate cancer (PCa) ranks the second most common cancer in men after lung cancer in western countries. In recent years, the incidence of PCa grows rapidly in our country and PCa has become a serious threat to the health of old men. Clinically, one of the most challenging issues is how to choose effective but low toxic targeted drugs to treat the aggressive PCa, which can reduce the risk of recurrence and metastasis. Metformin (1,1-Dimethylbiguanide) as a biguanide antidiabetic drugs, is one of the most common drugs for treatment of type II diabetes mellitus. In recent years, more and more evidence suggests that metformin can inhibit the growth of cancer cells, including breast, pancreas, colon, ovary, endometrium. Notably, preliminary clinical studies suggest that metformin can inhibit the growth rate of PCa and reduce the risk of developing PCa. So far the antitumor mechanism of metformin is unclear.The main causes of PCa mortality are cancer invasion and metastasis. Recent studies indicated that epithelial-mesenchymal transition (EMT) is closely related to the invasion and metastasis of malignancy. Most recent study found in breast cancer that metformin may inhibit the EMT program induced by the TGF-p signaling pathway, manifested by disappearance of TGF-β mediated downregulation of E-cadherin and upregulation of Vimentin. The achievements provide some ideas and reference for further study. Because both PCa and breast cancer are hormone dependent tumors, we hypothesized that metformin may also inhibit EMT program in PCa cells. However, no relevant study has been reported thus far.In this study, Vcap cells treated with PBS were used as control group. RT-PCR and western blot were used to detect mRNA and protein expression levels of epithelium markers (β-catenin, E-cadherin) as well as mesenchymal marker (Vimentin, N-cadherin). RT-PCR was used to detect the expression levels of miR30a, miR143, miR185, miR196b and miR205.5mmol/L metformin significantly influenced cell cycle distribution and inhibited invasiveness and motility capacity of Vcap cells. Metformin upregulated the expression of E-cadherin (P<0.05) and β-catenin (P<0.05), but downregulated Vimentin (P<0.05) and N-cadherin (P<0.05) expression at mRNA and protein levels in Vcap cells. Significant upregulation of miR30a expression levels by metformin was identified (P<0.05) and further experiments confirmed that miR30a significantly inhibited proliferation and EMT of Vcap cells.In this study, Vcap cells were treated with different concentrations of metformin, and the influence of biological behavior of Vcap cells were evaluated by MTS assay, flow cytometry, wound healing assay and Boyden chamber assay; RT-PCR and Western blot were used to determine mRNA and protein expression level of epithelial markers (E-cadherin, p-catenin) and mesenchymal markers (Vimentin, N-cadherin) of EMT,. The expression levels of miRNA, was detected by RT-PCR to reveal the miRNA alterations in prostate tumor cell by metformin treatment.Method:1. MTS assay was used to determine cellular proliferation of Vcap cells treated24、48and72h by metformin with various concentrations(1、5、10、50mmol/L).2. Flow cytometric analysis was performed to detect cycle distributions of Vcap cells treated24h by5mmol/L metformin.3. Wound healing assay and matrigel invasion assay were performed to evaluate motility and invasive capacity of cancer cells treated by5mmol/L metformin.4. Being treated24h、48h by5mmol/L metformin, RT-PCR and western blot were used to detect mRNA and protein expression levels of epithelium markers (β-catenin, E-cadherin) as well as mesenchymal marker (Vimentin, N-cadherin).5. RT-PCR was used to detect the expression levels of miRNA after being treated 24h by5mmol/L metformin to elucidate the regulatory effect of metformin on Vcap cell miRNA.6. Determine cellular proliferation of Vcap cells treated24、48�'�72h by MiR30a analogues and miR30a inhibitor respectively.After Vcap cells were treated24h by miR30a, RT-PCR was used to detect mRNA levels of epithelium marker (E-cadherin) as well as mesenchymal marker (Vimentin) to clarify the effect of miR30a on Vcap cell EMT.Result:1.MTS result：Metformin significantly inhibited proliferation of Vcap cells in a dose-and time-dependent manner.2.The influence of metformin to Vcap cell cycle distribution: Vcap cells were treated48h by5mmol/L metformin. Compared with the control group, the percentage of G0/G1phase cells increased significantly (54.1%vs.77.2%), and S (13.8%vs.6.7%) and G2/M (32.1%vs.16.1%) cells were significantly reduced in proportion.3.Metformin inhibits the migration and invasion of Vcap cells:After Vcap cells being treated48h by5mmol/L metformin, wound healing rate was significantly lower than that of the control group, It suggests that the migration of tumor cells decreased significantly treated by metformin. Boyden chamber assay showed that5mmol/L metformin can inhibit the invasion ability of Vcap cell. Compared with the control group cells, the ability of5mmol/L to metformin inhibits cell invasion reached more than45%(P<0.01, n=3).4.Metformin inhibits Vcap cell EMT:EMT related epithelial markers and mesenchymal markers were changed significantly after Vcap cells being treated24h by5mmol/L metformin. Compared with the control group, the epithelial marker expression of E-cadherin (β-catenin and mRNA of the Vcap cells treated by metformin was significantly higher than that in the control group, respectively6.2times,1.2times (P=0.023)(P=0.034); the level of mRNA interstitial markers Vimentin and N-cadherin were significantly decreased, respectively, lower than that of the control group3.6times (P=0.002) and2.9times (P=0.013). After Vcap cells were treated48h by5mmol/L metformin, western blot shows the expression of E-cadherin and β-catenin in protein levels were significantly elevated, while the expression of Vimentin and N-cadherin protein levels were significantly decreased.5.Regulatory effect of metformin on Vcap cell miRNA:compared with the control group, the expression levels of miR30a、 miR143and miR196b were significantly increased in Vcap cells treated24h by5mmol/L metformin and the most significant changes are of miR30a (a change of3.5times), but the expression level of miR185and miR205in Vcap cells had no obvious change.6.Effect of miR30a on the proliferation of Vcap cells and EMT:Vcap cells were treated respectively24,48,72h by miR30a analogues or inhibitors, compared with the control group, the miR30a analogues can inhibit the proliferation of Vcap cells, and miR30a inhibitor can significantly promote the proliferation of Vcap cells (P<0.05). Further tests found the epithelial marker E-cadherin mRNA levels were significantly increased, while the mRNA level mesenchymal marker Vimentin decreased obviously after Vcap cells were treated24h by miR30a analogues.Conclusions:1.Metformin significantly inhibits proliferation of Vcap cells in a dose-and time-dependent manner.2. Metformin can block PCa cells into S phase,but stay at G0/G1phase.3. Metformin inhibits the migration and invasion ability of Vcap cells.4. Metformin inhibits EMT program in Vcap cells, which maybe relevant to the upregulation of miR30a expression by metformin.