The Regulation Function Of MiR-615-3p And Its Target Gene B4GALT1 On The Proliferation And Invasion Abilities Of Human Breast Cancer Cells

Objectives 1 This study aims to detect the expression of miR-615-3p in triple negative and non-triple negative human breast cancer and investigate the effects of miR-615-3p level on proliferation and invasion abilities of human breast cancer cells. 2 This study aims to identify miR-615-3p’s target gene, as well as the function of the target gene in regulation of proliferation and invasion abilities of human breast cancer cells.Methods 1 RT-q PCR was used to analyze miR-615-3p level in triple negative breast cancer cell MDA-MB-231, non-triple negative breast cancer cell MCF-7 and breast cancer specimens(triple negative breast cancer and non-triple negative breast cancer). 2 The miR-615-3p was knockdowned or overexpressed in MCF-7 or MDA-MB-231 cells for 24 hours respectively. Then, CCK-8, flat cloning experiment, transwell migration and invasion assay were used to evaluate the effects of miR-615-3p on the activity, proliferation and invasion of human breast cells. 3 The candidate target genes of miR-615-3p were screened by bioinformatics, and the regulation of miR-615-3p on gene was futher verified by RT-q PCR, Western blot and double luciferase report assay. Morever, RT-q PCR, Western blot and Immuno histochemistry were used to detect the m RNA and protein levels of the target gene in breast cancer cells and breast cancer specimens. 4 The target gene of miR-615-3p was knockdowned or overexpressed in MCF-7 or MDA-MB-231 cells for 24 hours, respectively. Then, CCK-8, flat cloning experiment, transwell migration and invasion assay were applied to detect the effects of it on the activity, proliferation and invasion of human breast cancer cells.Results 1 The miR-615-3p level was significantly higher in non-triple negative breast cancer cell MCF-7 than that in triple negative breast cancer cell MDA-MB-231(P<0.05). The miR-615-3p level was markedly higher in non-triple negative breast cancer specimens than that in triple negative breast cancer specimens(P<0.05). 2 Compared with the control group, the activity of miR-615-3p-knockdowned MCF-7 cells was significantly enhanced(P<0.05), and the number of clones and cells passing through the transwell chamber(with matrigel or without matrigel) were notably increased in miR-615-3pknockdowned MCF-7 cells(P<0.01). On the other hand, compared with the control group, the activity of miR-615-3p-overexpressed MDA-MB-231 cells was significantly reduced(P<0.05), and the number of clones and cells passing through the transwell chamber(with matrigel or without matrigel) were significantly reduced in miR-615-3p-overexpressed MDA-MB-231 cells(P<0.05). The results showed that the hypoexpression of miR-615-3p could promote, whereas the hyperexpression of miR-615-3p could inhibit the proliferation and invasion of breast cancer cells. 3 Based on bioinformatics analysis, beta-1,4-galactosyltransferase(beta-1,4-galactosyltransferase 1, B4GALT1) was identified as a target gene of miR-615-3p. The direct regulating function of miR-615-3p on B4GALT1 was confirmed by double luciferase report assay. Knockdown of miR-615-3p siganificantly decreased the m RNA and protein levels of B4GALT1 in MCF-7 cells(P<0.05). And overexpression of miR-615-3p markedly increased the m RNA and protein levels of B4GALT1 in MDA-MB-231 cells(P<0.05). These results suggested that B4GALT1 was a direct target gene of miR-615-3p in breast cancer cells, and it was positively regulated by miR-615-3p. 4 The m RNA and protein levels of B4GALT1 were all significantly higher in non-triple negative breast cancer cell MCF-7 than those in triple negative breast cancer cell MDA-MB-231(P<0.05). The protein level of B4GALT1 was markedly higher in non-triple negative breast cancer specimens than that in triple negative breast cancer specimens(P<0.05). 5 Compared with the control group, the activity of B4GALT1-knockdowned MCF-7 cells was significantly reduced(P < 0.05), and the number of clones and cells passing through the transwell chamber(with matrigel or without matrigel) were notably reduced in B4GALT1-knockdowned MCF-7 cells(P<0.05). On the other hand, compared with the controls, the activity of B4GALT1-overexpressed MDA-MB-231 cells was significantly reduced(P<0.01), and the number of clones and cells passing through the transwell chamber(with matrigel or without matrigel) were significantly reduced in B4GALT1-overexpressed MDA-MB-231 cells(P<0.05). The results indicated that hypoexpression or hyperexpression of B4GALT1 could inhibit the proliferation and invasion of breast cancer cells.Conclusions 1 The expression levels of miR-615-3p and B4GALT1 in triple negative breast cancer are significantly lower than those in non-triple negative breast cancer, respectively. 2 B4GALT1 is a direct target gene of miR-615-3p in both triple negative and non-triple negative breast cancer and is positively regulated by miR-615-3p. 3 The miR-615-3p negatively regulates the proliferation and invasion abilities of breast cancer(triple negative and non-triple negative) cells. 4 B4GALT1 negatively regulates the proliferation and invasion ability of triple negative breast cancer cells, whereas positively regulates the proliferation and invasion ability of non-triple negative breast cancer cell.