The Regulation Of MiR-34 On Neurokinin 1receptor Expression In Breast Cancer

Objective: Exploring the expressions of miR-34 and neurokinin 1 receptor(NK1R)in breast cancer tissues and cell lines,and the effect of miR-34 and NK1 R on cell proliferation and invasion,so that to provide the evidence of breast cancer prevention.Methods: 1.Fifty pairs of primary breast cancer and matched adjacent normal tissues were obtained between February and May 2013 at Tianjin Cancer Hospital.Clinical and clinicopathological classifications and stage were determined according to the American Joint Committee on Cancer(AJCC)criteria.Realtime-polymerase chain reaction(RT-PCR)and Pearson correlation coefficient experiments were conducted to analyze the expression and correlation of miR-34b/c-5p and NK1R-Tr.Western blotting and RT-PCR tests were detected miR-34b/c-5p and NK1 R and expression.2.Western blotting and luciferase assay were conducted to detect the regulation of miR-34 on NK1 R.3.CCK-8,colony formation,wound healing and Transwell tests were conducted to identify the effects of miR-34b/c-5p and NK1 R on cell proliferation and invasion.4.Flow cytometry test was conducted to detect the regulation of miR-34b/c-5p and NK1 R on cell cycle and apoptosis.Western blotting was conducted to detect the expression of cell cycle related proteins,and Caspase-3 activity was investigated the regulation of miR-34b/c-5p and NK1 R on cell apoptosis.5.The expression levels of miR-34b/c-5p and NK1 R were detected after using different concentrations of TGF-β1 and at different time points.6.NK1R-Tr expression was detected after co-transfected with miR-34b/c-5p and c-myc-si RNA.7.MDA-MB-231 cells transfected with miR-34b/c-5p angimor and control,three days later,cells were injected to mice.Twenty seven days later,the mice tumors were extracted and measured and weighed.Results: 1.Mi R-34b/c-5p was significantly overexpressed in normal breast tissues,but NKIR-Tr was the opposite.Mi R-34b/c-5p and NK1R-Tr expression were associated with malignant potential.Pearson correlation analyses revealed a significant positive correlation between the miR-34b-5p and miR-34c-5p expression levels.Mi R-34b/c-5p expression was inversely correlated with NK1R-Tr expression.NK1R-Tr was significantly overexpressed in estrogen receptor(ER)-and progesterone receptor(PR)-positive tumors.2.NK1R-fl and NK1R-Tr expression was significantly downregulated after overexpression miR-34b/c-5p.Transient co-transfection with the NK1R-FL reporter plasmid and pre-miR-34b-5p or pre-miR-34c-5p significantly reduced luciferase activity compared with the negative controls in HEK-293 T and MDA-MB-231 cells.However,the activity of the reporter vector containing a mutant 3’UTR sequence was unaffected by simultaneous transfection with pre-miR-34b-5p or pre-miR-34c-5p.3.Overexpression miR-34b/c-5p and NK1 R silencing inhibited cell proliferation,invasion,and induced cell cycle phase arrest and apoptosis,and the growth inhibitory effect was enhanced in combination with aprepitant.4.NK1R-Tr expression was no change in MDA-MB-231 cells after c-myc silencing and overexpression miR-34b/c-5p comparing with the control group.5.TGF-β1 downregulated miR-34b/c-5p expression,and upregulated NK1R-Tr expression.6.The tumor volumes and weights were significant impaired after transfected miR-34b/c-5p angimor in vivo.Conclusions: In breast cancer tissues,miR-34b/c-5p and NK1R-Tr expression were closely related to cancer progression,and miR-34b/c-5p and NK1R-Tr are negatively correlated.Mi R-34b/c-5p could bind to the 3’UTR of NK1R-fl,and c-myc involved in the regulation of miR-34b/c-5p on NK1R-Tr.Mi R-34b/c-5p can reverse the promoting effect of TGF-β1 on NK1R-Tr.Mi R-34b/c-5p inhibited cell proliferation,invasion and induced cell G2/M phase arrest mainly through the regulation on NK1R-Tr.Mi R-34b/c-5p and NK1 R may serve as diagnostic and therapeutic targets for breast cancer.