br showed that mRNA level of c
showed that mRNA level of c-myc was increased after BBP treatment in both LNCaP and PC-3 cell lines (p < 0.05, Fig. 3B). The western blotting results also showed that BBP increased the protein level of c-myc (Fig. 3C). Above data suggested that miR-34a may be involved in BBP-induced cell proliferation of prostate cancer cells.
3.4. miR-34a inhibited the proliferation of prostate cancer cells
In order to verify the role of miR-34a in prostate cancer cell growth, miR-34a mimic and miR-34a inhibitor were transfected into PC-3 cells to up-regulated and down-regulated the level of miR-34a, respectively. MTT assay showed that transfection of miR-34a mimic significantly decreased the viability of PC-3 cells compared with the control mimic group (p < 0.05), while transfection of miR-34a inhibitor increased the viability of PC-3 cells (p < 0.05, Fig. 4B). Meanwhile, we detected the protein levels of c-myc, cylinD1, PCNA and p21 by western blotting. The results indicated that upregulation of miR-34a decreased the pro-tein levels of c-myc, cylinD1, PCNA, and increased p21 level. Mean-while, downregulation of miR-34a up-regulated c-myc, cylinD1 and PCNA expression levels, and down-regulated p21 level (Fig. 4C). These data demonstrated that miR-34a modulated 58-58-2 related proteins, and then suppressed the proliferation of prostate cancer cells.
3.5. BBP promoted cell proliferation through miR-34a/c-myc axis in prostate cancer cells
The above findings demonstrated that BBP down-regulated miR-34a and promoted the growth of prostate cancer cells. To verify the role of miR-34a/c-myc axis in BBP-induced cell proliferation of prostate cancer cells, we transfected miR-34a mimic and miR-34a inhibitor in BBP-treated PC-3 cells. MTT assay and western blotting showed that BBP treatment up-regulated cyclin D1 and PCNA, down-regulated p21 and promoted growth of prostate cancer cells, which were consistent with our previously finding. However, in miR-34a over-expressed cells, BBP treatment did not alter the expression level of cell cycle related gene nor promoted cell growth of prostate cancer cells in comparison with the control group (p < 0.05, Fig. 5A and 6A). In cells transfected with miR-34a inhibitor, the results also suggested that the cell proliferation-promoting effect of BBP was mediated through miR-34a (p < .05, Fig. 5B and 6B). Next, we detected the change of miR-34a target gene c-myc in protein level by western-blotting. BBP treatment increased the expression of c-myc; however, BBP-induced upregulation of c-myc was diminished after miR-34a mimic transfection compared with BBP alone treatment group in PC-3 cells (Fig. 6). Together, these data revealed that the promotive effect of BBP on prostate cancer cell growth was mediated through miR-34a/c-myc axis.
Fig. 2. BBP altered the expression of cell cycle genes in LNCaP and PC-3 cells. Cells was treated with 10−7 and 10−6 mol/L of BBP for 6 days, and quantitative real-time PCR was used to detected the mRNA level of cylinD1, PCNA and p21 (A); Western blotting was used to analyze cell cycle related protein levels of cylinD1, PCNA and p21 (B). Data are expressed as mean ± SD. *p < 0.05, **p < 0.01; compared with control group. All experiments were performed in triplicate.
Phthalate esters have been defined as environmental endocrine disruptors, which possess the ability to disturb sexual maturation, pregnancy loss, female reproductive tract and mammary gland devel-opment, male testicular development and sperm production (Piche et al., 2012; Kay et al., 2013; Hauser et al., 2015). The most adverse effects of phthalate esters have been noted in the reproductive system. Animal studies have showed that phthalate esters decreased testicular testosterone production and insulin-like 3 hormone expression, which also affected male reproductive tract, puberty, semen quality and fer-tility (Howdeshell et al., 2008; Kay et al., 2014). In female animals, reproductive effects from phthalate esters include altered serum estra-diol levels, advanced or delayed onset of puberty, increased ovarian and uterine weights, and deficits in growing follicles and corpora lutea (Yaghjyan et al., 2016). Human studies have showed that increased phthalate esters concentrations up-regulated kisspeptin level, which may promote the onset of puberty in girl (Chen et al., 2013). Gaspar FW et al. reported that 82–89% of children in California had phthalate esters exposure estimates exceeding reproductive health benchmarks (Gaspar et al., 2014). Higher concentration of phthalate esters were found in women with endometriosis (Reddy et al., 2006).
Several studies have also shown that phthalate esters exhibit po-tential role in promoting cancer development and have been listed as cancer promoting agents (Doull et al., 1999). Oshida K et al. reported that DEHP increased the incidence of liver cancer through PPARα ac-tivation (Oshida et al., 2015). Chen FP et al. demonstrated that low dose of DEHP, BBP and DBP induced the proliferation of estrogen