METHODS: The cells were randomized into 4 groups, i.e., group
A: 10%
fetal bovine serum (FBS) group; group B:
hypoxia + 10% FBS group; group C: serum
starvation group; group D:
hypoxia + serum
starvation group; each group was further divided into three subgroups as blank control, treated with rh-
endostatin and
bevacizumab, respectively. Cell counting kit-8 (CCK-8) was used to assess the inhibition rate of cell growth induced by
endostatin and
bevacizumab, in order to determine the proper working concentration and time of the two drugs. Transwell assay was conducted to detect the cell invasion and migration in vitro. The expressions of c-Met and MMP-9 were detected by Western blot. The cells treated with rh-
endostatin or
bevacizumab under serum
starvation were tested by hybridization using Exiqon miBase 18.0 microarray. The
miRNAs which exibited significant differences (P < 0.05) in
miRNA hybridization were verified by real-time PCR assay.
RESULTS:
CCK-8 assay showed that the inhibition rates of MDA-MB-231 cells cultured with 800 mg/L rh-
endostatin for 48 h and 24 h were (32.2 ± 2.5)% and (27.0 ± 1.3)%, respectively, showing a significant difference (P = 0.023). The inhibition rates of MDA-MB-231 cells cultured with 80 mg/L
bevacizumab for 48 h and 24 h were (30.5 ± 1.4) % and (26.1 ± 2.4) %, respectively, showing also a significant difference (P = 0.015). The Transwell assay showed that in the
starvation blank group, the number of invaded and penetrated cells were 28.8 ± 2.2 and 31.4 ± 1.5, respectively, significantly different from that in the rh-
endostatin and
bevacizumab groups (P < 0.05). The relative expressions of c-Met and MMP-9 were 0.213 ± 0.017 and 0.542 ± 0.048, respectively, with a significant difference from those of the groups treated with each drug (P < 0.05 for both). The numbers of penetrated cells in the Transwell assay treated with rh-
endostatin in
hypoxia were 17.5 ± 2.1 and 16.5 ± 2.8, respectively, and the numbers of penetrated cells in the Transwell assay treated with
bevacizumab were 16.3 ± 3.5 and 17.5 ± 2.4, respectively, showing no significant difference among them (P > 0.05 for both). The ability of migration and invasion of MDA-MB-231 cells and the expression of c-Met and MMP-9 were not impacted by
hypoxia (P > 0.05). Real-time PCR assay showed that only the levels of miR-2355 and miR375 were significantly and stably decreased in the cells which had increased ability of invasion and migration. The relative expression levels of miR375 and miR-2355 in the serum
starvation blank group were 0.550 ± 0.036 and 0.852 ± 0.121, respectively, significantly lower than that in the groups treated with rh-
endostatin or
bevacizumab (P<0.05). In the serum
starvation group, the expression levels of miR375 and miR-2355 of cells treated with rh-
endostatin were 0.295 ± 0.012 and 0.253 ± 0.011, and the expression levels of cells treated with
bevacizumab were 0.234 ± 0.020 and 0.309 ± 0.022, respectively, (P > 0.05 for all). Compared with the serum
starvation blank group, the expression levels of miR2355 and miR375 were significantly decreased when cells were treated with rh-
endostatin/
bevacizumab under serum
starvation, but no significant difference was found between the two drugs (P > 0.05). However,
hypoxia did not affect the expressions of miR2355 and miR375 (P > 0.05).
CONCLUSIONS: