Inhibitory effect of miR-29a on the viability of EC cells
In order to study the effect of miR-29a on the activity of EC cells, miR-29a inhibitors (miR-29a specific siRNA) or mimics were transfected into EC cell lines, with non-specific siRNA or mimics as negative controls. From the qPCR results, miR-29a inhibitors significantly inhibited miR-29a levels compared with inhibitor controls, while miR-29a mimics significantly increased miR-29a levels compared with simulated controls (Fig. 1A). The survival rate of cells in each group was detected by CCK8 and clone formation assay. As shown in Fig. 1B, compared with the inhibitors control group, the OD value of miR-29a inhibitor transfection group increased significantly at 24 h, 48 h and 72 h after transfection, and decreased 72 h after miR-29a mimetic transfection compared with the mimics control group. The clone formation assay provided consistent results. Compared with the inhibitor control group, the colony number of miR-29a inhibitor was significantly increased, while the colony number of miR-29a mimic was significantly lower than that of the mimic control group (Fig. 1C). These results suggested that miR-29a inhibited the viability of EC cells.
miR-29a induced apoptosis in EC cells.
24 hours after transfection, apoptosis was analyzed by flow cytometry. As shown in Fig. 1D, the percentage of apoptotic cells decreased after transfection of miR-29a inhibitor compared with the inhibitor control group, while the percentage of apoptotic cells increased after transfection of miR-29a mimics compared with the mimics control group. It suggested that miR-29a could enhance apoptosis of EC cells. Previous studies have confirmed that miR-29a inhibits the progression of hepatocellular carcinoma by negatively regulating PTEN expression and activating Akt/mTOR signaling pathway. In this study, we also explored the progress of miR-29a influencing EC through AKT/mTOR pathway. As shown in Fig. 2, miR-29a inhibitors significantly increased the phosphorylation and P70 levels of p-AKT/AKT and p-mTOR; miR-29a inhibitors also decreased the levels of p53, Bax and C-caspasse3, while increased the levels of T-caspase-3 and Bcl-2. miR-29a simulated the opposite effect. The results showed that miR-29a inhibited the growth of EC cells by inhibiting the activation of AKT/mTOR signal pathway.
miR-29a inhibited the expression of STAT3 by binding to the 3 'untranslated region (3' untranslated region) of STAT3.
In order to further study the action mechanism of miR-29a, we analyzed the binding sites of miR-29a on TargetScan data sets. The results showed that there were two binding sites between the 3' region of STAT3 and miR-29a (Fig. 3A). Then, we examined the regulatory effect of miR-29a on STAT3 expression. As shown in Figs. 3B and C, the expression of STAT3 increased significantly after transfection of miR-29a inhibitor, while the expression of STAT3 decreased significantly after transfection of miR-29a mimic, indicating that miR-29a inhibited the expression of STAT3 in EC cells. It was speculated that miR-29a can inhibit the growth of tumor cells by targeting STAT3 in EC cells.
STAT3 promoted the viability and inhibited apoptosis of EC cells.
In order to study the specific effect of STAT3 on EC cells, EC cells was transfected with shRNA or high expression plasma to construct STAT3 gene knockdown and overexpression cell lines. The results of qPCR and western blot showed that miR-29a inhibitors could promote the expression of STAT3, while miR-29a mimics could inhibit the expression of STAT3 (Fig. 4A and Fig. B). The cell viability was detected by CCK8 and clone formation assay. Compared with NC group, the OD value of EC cells in STAT3-OE group increased and that of EC cells decreased in STAT3-KD group (Fig. 4C). Similar results were observed in clone formation assay. Compared with NC cells, the number of colonies in STAT3-OE group increased, while that in STAT3-KD group decreased (Fig. 4D). Apoptosis was analyzed by flow cytometry. As shown in Fig. 4E, the percentage of apoptotic cells decreased after overexpression of STAT3, while the percentage of apoptotic cells increased when STAT3 was knocked down. It is proved that STAT3 can promote cell viability and inhibit apoptosis of EC cells.
STAT3 activated AKT/mTOR signal pathway in EC cells.
Since STAT3 was the target of miR-29a, we further studied its effect on AKT/mTOR signal pathway. Western blot results showed that overexpression of STAT3 could activate the phosphorylation of p-AKT/AKT and p-mTOR, increase the expression of P70, T-caspase-3 and Bcl-2, and decrease the expression of p53, Bax and C-Caspasse3. Knockdown of STAT3 gene showed the opposite effect. Our results suggested that STAT3 may promote the growth of EC cells by activating AKT/mTOR signal pathway. (Fig. 5).
miR-29a/STAT3/AKT/mTOR inhibited the growth of human EC cells.
Then, we verified the functional axis of miR-29a/STAT3/AKT/mTOR by overexpressing STAT3 and miR-29a (STAT3-OE + miR-29a mimic) in EC cells. As shown in Figs. 4 and 5, overexpression of miR-29a significantly blocked the increase of cell viability induced by overexpression of STAT3 and the inhibition of apoptosis induced by overexpression of STAT3. In addition, miR-29a mimic can reverse the effect of STAT3-OE activating AKT/mTOR signal pathway. (Fig. 5).