| BackgroundProstate cancer(PCa), as a kind of male malignant tumor, is very common in Europe and America. In recent years, with the improvement of China’s economic and living standards, the development of diet and culture, the aging of the society and the popularity of the physical examination, the incidence of prostate cancer in China is increasing year by year. The high death rate of prostate cancer patients is closely related to its high metastasis rate (the most common for bone metastasis). Therefore, the treatment point at metastasis of prostate cancer patients has become one of the hot spots in the current medical research. Epithelial-mesenchymal transformation (EMT) has been proved to be an important role in the metastasis of prostate cancer, and its regulation mechanism is guided by a number of signaling pathways, including TGF-β, Notch, Wnt and ERK/MAPK. The TGF-β/SMAD4 pathway is closely related to the development of EMT, especially in the late stage of cancer. SMAD4 plays the role of "ferry" in the TGF- β/SMAD4 signaling pathway. When inhibiting the expression of TGF-β, it can inhibit the SMAD4 signaling pathway and prevent the epithelial-mesenchymal transformation (EMT) in tumor cells. In recent years, the discovery of miRNA is a group of non coding small RNA, which involves many physiological and pathological processes, mainly to inhibit the translation of mRNA or induce its degradation, which changes the expression level of target protein. Study on miRNA has become the hot spot, a large number of studies have shown that miRNA in different tumors have different expression, and play an important role:such as part of miRNA on tumor cells in EMT play a role, and some of the miRNA inhibitory effect on tumor cells in EMT. Therefore, the future treatment of cancer through research aimed at miRNA targeted therapeutic drugs have great prospects for development.After a large number of studies show that Valproic acid (VPA) can inhibit Prostate cancer cells and down regulated expression of SMAD4 protein. However, VPA on prostate cancer cells SMAD4 expression in the presence of miRNA in the process control effect, which relates to the specific mechanism is unclear, if found by the experiments of core miRNA SMAD4 expression, we can study the mechanisms of drug treatment for miRNA related, will have important significance this treatment for prostate cancer.ObjectiveOur previous studies have demonstrated that sodium valproate (VPA) can inhibit prostate cancer cell metastasis (PCa) and can decrease the expression level of SMAD4. Through this experiment, we studied valproate (VPA) reduced the expression level of SMAD4 protein in what way, looking for the existence of related miRNA and its regulation are closely related, and to explore the possibility of finding that miRNA only through the regulation of the miRNA level, the expression change of SMAD4.MethodsWe identifed miRNAs that can complementarily bind to SMAD4 mRNA using www.targetscan.com and searched PUBMED to identify miRNAs related to VPA. And correlated with both SMAD4 and VPA miRNA were chosed as our research objectIn the experiment design, two kinds of prostate cancer cell lines (PC3, LNCaP) were cultured. After the treatment of VPA, the protein and mRNA levels of SMAD4 were detected, and whether the changes were statistically significant. To use VPA to deal with prostate cancer cell line again, to observe whether the expression level of miRNA as the research object is affected. Select the application of VPA treatment after the change is statistically different miRNA for the next experiment. The effect of miRNA on the expression of SMAD4 in prostate cancer cells was detected by plasmid transfection technique, respectively. Select miRNA as the ultimate goal of SMAD4 expression with statistical significance. Finally, through the corresponding expression of miRNA inhibitor selected target miRNA inhibition, observe the effect of VPA on SMAD4 can be eliminated, to assess whether the inhibition in VPA expression plays a key role in the process of SMAD4.RseultsBy using the www.targetscan.com search can be combined with miRNAs and SMAD4 complementary PubMed in search and review of the literature to determine the VPA associated with the miRNA and then,5 kinds of miRNA and SMAD4 and VPA at the same time, we will be related as our research object. In the experiment, including miR-20a,34a,124a,144 and 449a.The results showed that the mRNA and protein levels of SMAD4 in prostate cancer cell line were significantly down regulated after VPA treatment, and had obvious concentration dependence. After we treated with VPA prostate cancer cell lines,5 miRNA from the preliminary screening, found that the expression level of miR-20a,34a and 449a after VPA treatment increased significantly and statistically significant, while the miR-124a, miR-144 expression did not change significantly. So we will miR-20a,34a and 449a as the further research objectives.We through plasmid transfection, in two prostate cancer cell lines (LNCaP, PC3, cells) were implanted with 34a transfected miR-20a,449a precursor and miR-20a,34a and 449a inhibitors, found that the decreased expression of SMAD4 by transfection of pre-miR-34a or miR-449a, and transfection of miR-20a increased the expression of SMAD4, and there were statistically the expression changes of SMAD4 and difference; by transfection of miR-34a inhibitor, increased the expression of SMAD4 and miR-20a; transfection or miR-449a inhibitors were reduced the expression of SMAD4. In the prostate cancer cell line LNCaP, the change of SMAD4 expression after transfection of miR-34a inhibitor was statistically significant, and there was no significant difference in the expression of SMAD4 after transfection of miR-20a inhibitor or miR-449a inhibitor. In the prostate cancer cell line cells SMAD4, the changes of PC3 expression after transfection of pre-miR-34a inhibitor, pre-miR-20a inhibitor and pre-miR-449a inhibitor were statistically significant. Through the experiments, we found that miR-34a can inhibit the expression of SMAD4 in two prostate cancer cell lines, while miR-20a and 449a can promote the expression of SMAD4 in a prostate cancer cell line. Through the experiments we found that the transfection of pre-miR-34a and inhibitors can significantly affect the expression levels of SMAD4 protein and mRNA, miR-34a in the VPA effect of SMAD4 expression should play an important role in the process. So we chose MiR-34a as the ultimate goal.In two prostate cancer cell lines (LNCaP, PC3, cells) by transfection of pre-miR-34a inhibitor to increase the expression level of SMAD4 protein, and then combined with VPA treatment, and no obvious change of SMAD4 protein level and no significant difference. At the same time in the two prostate cancer cell lines (PC3, cells LNCaP), the application of pre-miR-34a inhibitors after the application of VPA treatment, the level of mRNA SMAD4 changes are not statistically significant difference. The results showed that the inhibition effect of VPA on SMAD4 could be eliminated by reducing the expression of miR-34a. That is, up regulation of miR-34a levels can simulate the effect of VPA is inhibited by SMAD4, while the level of miR-34a is reduced by eliminating the effect of VPA on prostate cancer cells.ConclusionTransfection of pre-miR-20a or pre-miR-449a induces the up-regulation of SMAD4 protein levels, which may be mainly due to indirect effects. VPA induced up-regulation of miR-34a reduced the expression of SMAD4, and the effect of miR-20a and miR-449a on the expression of SMAD4. The expression of miR-449a in miRNA after VPA treatment was significantly up-regulated, which suggested that miR-449a might be involved in other mechanisms of VPA, which provided a potential prospect for further study. MiR-34a is a key regulator of the expression of VPA in SMAD4. VPA inhibits the expression of SMAD4 by up regulating the expression of miR-34a. |
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