Effects of over expression or gene silencing of COX-2 on proliferation and morphology of Het-1A and BAR-T cells
Cell proliferation was assessed by MTS, as shown in Fig. 1. On the second day and the third day after over expression or silencing of COX-2 gene, the proliferation rate of COX-2 group was significantly higher than that of control group (P < 0.05), while cellular proliferation of siCOX-2 group was significantly lower than that of control group (P < 0.05). Meanwhile COX-2 was over expressed in Het-1A cells for 3 days, and the increase of microvilli on the cell surface was observed by electron microscopy, and the adenoid cavity structure was observed, suggesting intestinal metaplasia of the cells, while siRNA of COX-2 showed no such intestinal metaplasia. Nuclear abnormality and autophagosome were observed after COX-2 over expression in BAR-T cells for 3 days, suggesting atypia of the cells. siRNA of COX-2 induced no such changes(Fig. 2A,2B).
Effects of COX-2 over expression and gene silencing on COX-2, CDX-2, BMP-4, p-P65, P65, muc-2 and c-myb in Het-1A and BAR-T cells
Protein expression levels of COX-2, P65, p-P65, CDX-2, and BMP-4 were assessed by western blot on the second day after COX-2 over expression or gene silencing in the two cell lines. As showed in Fig. 3, COX-2 over expression or knockdown effects were achieved in both cell lines. Expressions of BMP-4, p-P65 and CDX-2 were positively correlated with COX-2, while the expression levels of P65, MUC2, and c-myb remained unchanged.
Effects of acid, bile salts, and their mixture on the proliferation of Het-1A and BAR-T cells
Different concentrations of bile salts were tested on both cell lines (Het-1A: 0µmol/L, 400µmol/L, 800µmol/L, 1200µmol/L; and BAR-T: 0µmol/L, 800µmol/L, 1200µmol/L, and 1600µmol/L). MTS test results were shown in Fig. 4A. In this experiment, bile salts concentrations of 1200µmol/L were selected for both cells, and treatment time was set at 0, 30, 60, 90min. For the experiment presented in Fig. 4B. The concentration of 1200µmol/L of bile salts was selected, and the treatment time was set as 30min, 60min and 90min to detect the COX-2 protein expression level. The detection results were presented in Fig. 4C: when HET-1A was treated with bile salts for 90min and BAR-T was treated with bile salts for 60min and 90min, the COX-2 expression was substantially up-regulated.
The PH value of the medium was adjusted with hydrochloric acid, and the cells were cultured in the medium with PH values of 4.0, 5.0, and 6.0. In the blank control group, medium was not treated with hydrochloric acid. Cell viability was measured by MTS, as shown in Fig. 5A, and COX-2 protein expression level was detected by western blot, as shown in Fig. 5B. We found that after incubation with hydrochloric acid at PH6.0, 5.0, and 4.0 for certain time, and the cell activity and COX-2 expression were both up-regulated. Based on the above experimental results, PH6.0 was selected for the treatment of both cell lines with Het-1A being treated for 30min and BAR-T for 60min.
According to the above experimental results, four groups (bile salts and hydrochloric acid) were set, namely, the control group (0µmol/L, PH7), the bile salts group (1200µmol/L, PH7), the hydrochloric acid group (0µmol/L, PH6), the hydrochloric acid, and bile salts mixed group (1200µmol/L, PH6). Cell proliferation was detected by MTS after 30min treatment of Het-1A and 60min treatment of BAR-T. As shown in Fig. 6, acid, bile salts and the mixture of the both inhibited the proliferation of the two cell lines, but the inhibitory effect of bile salts + hydrochloric acid was stronger than bile salts or hydrochloric acid treatments.
Effects of acid, bile salts, and their mixture on COX-2, CDX-2, BMP-4, and p-P65 expression in Het-1A and BAR-T cells
According to the above groups, protein expression levels of COX-2, P65, p-P65, CDX-2, and BMP-4 were detected after 30min treatment of Het-1A and 60min treatment of BAR-T. As shown in Fig. 7, the protein expressions of COX-2, CDX-2, BMP-4, and p-P65 in each group were increased compared with the normal group, and the expression of these proteins in bile salts and hydrochloric mixed group was strongest. However, the expression of P65 was not changed in all groups.
Effects of COX-2 gene silencing on the proliferation of Het-1A and BAR-T cells after acid and bile salts treatment
The cells were transfected with COX-2 siRNA, and treated with hydrochloric acid PH6.0 and bile salts of 1200µmol/L for 48h before sample collection. Het-1A cells were treated for 30min and BAR-T for 60min. Cell proliferation was detected by MTS, and the results are presented in Fig. 8. COX-2 siRNA silencing further enhanced the inhibitory effect of acid and bile salts mixture on the proliferation of Het-1A and BAR-T cells.
Effects of COX-2 gene silencing on expression of COX-2, CDX-2, BMP-4, and p-P65 in Het-1A and BAR-T cells after acid and bile salts treatment
The cells were transfected with COX-2 siRNA, and treated with hydrochloric acid (PH6.0) and bile salts (1200µmol/L) for 48h before sample collection. Het-1A cells were treated for 30min and BAR-T cells were treated for 60min. Protein expression levels of COX-2, P65, p-P65, CDX-2, and BMP-4 were identified by western blot. As shown in Fig. 9, the expressions of COX-2, CDX-2, BMP-4 and p-P65 proteins were up-regulated after treatment with acid and bile salts mixture, while they were down-regulated after COX-2 siRNA was transfected. The expression of P65 was not changed.
Effects of acid and bile salts mixture on the morphology of Het-1A and BAR-T cells before and after COX-2 gene silencing
After the mixture of acid and bile salts acted on the cells, nuclear inclusion bodies, autophagosome-like structures, and other cellular morphological manifestations were observed in Het-1A cells. Due to the damage of the cells induced by acid and bile salts, changes such as incomplete capsule, formation of vacuolar structure in cytoplasm, mitochondrial swelling, cavitation, and disappearance of the chute, and the intestinal metaplasia of the cells were not obvious. Heteromorphic changes such as nuclear heteromorphism was found in BAR-T cells. After gene silencing of COX-2 followed by treatment with a mixture of acid and bile salts, no such changes were seen in the two cell lines(Fig. 10A,10B).