环状RNA hsa_circ_0002185对乳腺癌细胞瘤细胞活力和侵袭能力的影响及其作用机制

Effects of circular RNA hsa_circ_0002185 on cell viability and invasion of breast cancer cells and its mechanism of action

摘要:

目的： 探讨环状RNA hsa_circ_0002185对乳腺癌细胞瘤细胞活力和侵袭能力的影响及其作用机制。方法： 收集2018年1月至2020年1月在宁夏医科大学总医院接受根治性手术治疗的20对乳腺癌组织及癌旁，通过qRT-PCR检测hsa_circ_0002185和hsa-miR-1248在人乳腺上皮细胞MCF10A和人乳腺癌细胞（MCF-7、T47D、BT-474和SK-BR-3）中mRNA的表达水平。构建T47D-siCirc（hsa_circ_0002185沉默）和T47D-NC（空载体对照）细胞株系，并以T47D作为空白对照，qRT-PCR检测hsa_circ_0002185 mRNA的表达水平；分别采用CCK-8细胞活性实验、划痕实验和Transwell实验检测hsa_circ_0002185对T47D细胞的增殖、迁移和侵袭能力；免疫荧光实验检测E-cadherin和Vimentin表达；使用生物信息学网站Circular RNA Interactome预测hsa_circ_0002185可互补结合的miRNA，再根据Targetscan网站预测相应miRNA可靶向结合的基因；Western blot检测MAPK/ERK信号通路蛋白和E-cadherin和Vimentin表达情况；回复实验通过下调hsa-miR-1248验证hsa_circ_0002185对T47D细胞的影响。结果： hsa_circ_0002185在4种人乳腺癌细胞的表达水平均比在人乳腺上皮细胞MCF10A中高，并且在乳腺癌组织中的mRNA表达水平比在正常组织高；hsa-miR-1248的表达水平呈相反情况；T47D-siCirc细胞系相比空白对照细胞系和T47D-NC细胞系，细胞的活性明显降低，迁移能力和侵袭能力显著下降，E-cadherin表达明显升高，Vimentin明显下降（P<0.05）；Circular RNA Interactome网站预测结果显示，hsa_circ_0002185可与hsa-miR-1248互补结合，Targetscan网站预测分析显示，hsa-miR-1248与Raf1存在靶向结合位点；Western blot检测结果显示T47D-siCirc组中hsa-miR-1248表达明显高于空白对照组与T47D-NC组，Raf1、P-MEK1/2、MEK1/2、P-ERK1/2和ERK1/2的表达明显低于空白对照组与T47D-NC组（P<0.05）；空白对照组和T47D-NC组中上述指标的表达量无统计学意义（P>0.05）；回复实验表明下调hsa-miR-1248可逆转hsa_circ_0002185沉默对T47D细胞的影响。结论： 环状RNA hsa_circ_0002185在乳腺癌中高表达，高表达hsa_circ_0002185可能通过调控hsa-miR-1248上调Raf1水平，进而激活MAPK/ERK信号通路并促进上皮间质转化过程。

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.