Abstract:Objective: To investigate the effect of circular RNA hsa_circ_0002185 on cell viability and invasion of breast cancer cells and its mechanism of action. Methods: Totally 20 pairs of breast cancer tissues and adjacent tissues were collected from the patients who were treated by radical operation in General Hospital of Ningxia Medical University from January 2018 to January 2020, and the mRNA expression levels of hsa_circ_0002185 and hsa-miR-1248 in human mammary epithelial cells (MCF10A) and human breast cancer cells (MCF-7, T47D, BT-474 and SK-BR-3) were detected by qRT-PCR. The T47D siCirc (hsa_circ_0002185 silencing) and T47D-NC (empty vector control) cell lines were constructed by lentivirus infection. The expression of hsa_circ_0002185 mRNA was detected by qRT-PCR with T47D as blank control. CCK-8 cell viability test, scratch test and Transwell test were used to detect the proliferation, migration and invasion ability of hsa_circ_0002185 to T47D cells. Immunofluorescence assay was used to detect the expression of E-cadherin and Vimentin. Bioinformatics website Circular RNA Interactome was used to predict the complementary miRNA of hsa_circ_0002185, and then the corresponding miRNA targeted binding genes were predicted according to Targetscan website. The expression of MAPK/ERK signal protein and E-cadherin and Vimentin was detected by Western blot. The recovery ex periment was carried out by down-regulation of hsa-miR-1248 to verify the effect of hsa_circ_0002185 on T47D cells. Results: The expression of hsa_circ_0002185 in four kinds of human breast cancer cells was higher than that in MCF10A cells, and the mRNA expression level in breast cancer tissues was higher than that in normal tissues. The expression level of hsa-miR-1248 was opposite. Compared with the blank control cell line and T47D-NC cell line, T47D-siCirc cell line significantly decreased cell viability, migration and invasion ability, increased E-cadherin expression and decreased Vimentin (P<0.05) . The results of Circular RNA Interactome website prediction showed that hsa_circ_0002185 could combine with hsa-miR-1248, and Targetscan website prediction analysis showed that hsa-miR-1248 and Raf1 had targeted binding sites. Western blot analysis showed that the expression of hsa-miR-1248 in T47D-siCirc group were significantly higher than that in blank control group and T47D-NC group, and Raf1, P-MEK1/2, MEK1/2, P-ERK1/2 and ERK1/2 in T47D-siCirc group were significantly lower than those in blank control group and T47D-NC group (P<0.05) . There was no significant difference in above indexes between the blank control group and T47D-NC group (P>0.05) . The reversion experiment showed that down-regulation of hsa-miR-1248 could reverse the effect of hsa_circ_0002185 silence on T47D cells. Conclusion: Circular RNA hsa_circ_0002185 is highly expressed in breast cancer. Overexpression of hsa_circ_0002185 may up-regulate Raf1 level by regulating hsa-miR-1248, thus activating MAPK/ERK signaling pathway and promoting the process of epithelial mesenchymal transition.
