The Mechanism Of C-MYC Promoting Cancer Progression By Regulating MiR-185-5p Targeting Truncated Neurokinin-1 Receptor In Breast Cancer

Objective: We have found that there are binding sites of mi R-185-5p and c-MYC in the 3’UTR and promoter of NK1R-Tr,respectively.Our study aims to discover the relations among mi R-185-5p,c-MYC and NK1R-Tr in vitro and in vivo,ultimately affecting the invasion and metastasis of breast cancer cells.We hope to provide more detailed evidence for breast cancer research,prevention,and the basis of the individual or predictable treatment.Methods: 1.Expression studies in tissues and cell lines 100 cases of breast cancer and paired adjacent normal tissues from Tianjin Medical University Cancer Institute and Hospital in 2014-2015 were collected,and these patients were all diagnosed with breast cancer through pathology.Analysis the expression of NK1 R,c-MYC and mi R-185-5p in the cancer tissue and paired adjacent normal tissues by Western blot/RT-PCR,independent sample t-test was used to analyze the difference between cancer tissue and paired adjacent normal tissues/ metastasis group and non-metastasis group/TNM stageⅠ~Ⅱand Ⅲ~Ⅳ.And the expression of NK1R-Tr/c-MYC protein and m RNA level in breast cancer cell lines(MDA-MB-231,MCF-7,T47 D and SK-BR-3)were detected by Western blot/RT-PCR.2.The regulation of c-MYC on NK1R-Tr We predicted a binding site of c-MYC in the promoter of NK1R-Tr through JASPAR,and the chromatin immunoprecipitation experiment was used to verify the finding.We found that c-MYC promote the transcription of NK1R-Tr by luciferase reporter gene;and then the expression of NK1R-Tr were detected by RT-PCR and Western blot after interfering the c-MYC.3.The influence of miR-185-5p on expression of NK1R-Tr and biological behavior We predict the binding sites of mi R-185-5p in the 3’UTR of NK1R-Tr through Target Scan(http://www.targetscan.org/),mi Randa(http://www.microrna.org/)and Pic Tar(http://pictar.mdc-berlin.de/),which was verified through luciferase reporter gene.mi R-185-5p mimic/negative control and mi R-185-5p inhibitor/ negative control were transfected into MDA-MB-231,T47 D and SK-BR-3,respectively.And then the m RNA level,protein level of NK1R-Tr and mi R-185-5p were detected by RT-PCR and Western blot.SP was used to stimulate NK1R-Tr,mi R-185-5p mimic/negative control were transfected into MDA-MB-231 and rescued by NK1R-Tr expression plasmid;mi R-185-5p inhibitor/ negative control were transfected into SK-BR-3 and rescued by NK1R-Tr si RNA.Western blot was used to detect the protein level of NK1R-Tr.In addition,CCK8 experiment,Clone formation assay,EDU proliferation assay,Cell cycle distribution detection,Transwell assay were used to verify whether the influence of mi R-185-5p on biological behavior is through NK1R-Tr under the stimulus of SP.4.c-MYC regulate NK1R-Tr indirectly through miR-185-5p We predicted several binding sites of c-MYC in the promoter of mi R-185-5p through JASPAR.RT-PCR was used to detect the level of mi R-185-5p after interfering the c-MYC in MDA-MB-231.The change of NK1R-Tr m RNA and protein level in cell lines that were treated with c-MYC si RNA and mi R-185-5p inhibitor was detected by RT-PCR/Western blot.5.c-MYC regulate the activation of Erk1/2 induced by SP SP(10-7M)was used to stimulate breast cancer cell lines with four different treatments(c-MYC si RNA/control,NK1 R antagonist/control),and then activation of Erk1/2 was detected by Western blot.6.miR-185-5p inhibit growth and metastasis of breast cancer cells in vivo MDA-MB-231 was incubated by mi R-185-5p agomir/agomir control,and SK-BR-3 was incubated by mi R-185-5p antagomir/antagomir control,injecting in the groin and caudal vein of SCID mice,then tumor and metastasis model were construct.The size of tumor was measured each week,and tumor were got out 5-7 weeks after injection.And we resect the lung tissue of mice and analyze of pulmonary metastasis after HE staining;Stripping subcutaneous tumor,measuring size of tumor and taking photos.Results:1.Expression studies in tissues and cell line Expression of NK1R-Tr/c-MYC in the adjacent normal tissues was lower than cancer tissue,and the expression of mi R-185-5p had opposite trend.Higher level of NK1R-Tr/c-MYC and lower level of mi R-185-5p in cancer tissue with metastasis as well as TNM stage in Ⅲ~Ⅳ.In addition,lower level of mi R-185-5p and higher level of NK1R-Tr/c-MYC in MDA-MB-231,a highly invasive human breast cancer cell line.But higher level of mi R-185-5p and lower level of NK1R-Tr/c-MYC in SK-BR-3,a lowly invasive human breast cancer cell line.2.The regulation of c-MYC on NK1R-Tr c-MYC can bind to the promoter of NK1R-Tr and promote the transcription of NK1R-Tr;and the m RNA or protein level of NK1R-Tr were decreased after interfering the c-MYC and increased after overexpression of c-MYC.3.The influence of mi R-185-5p on expression of NK1R-Tr and biological behavior mi R-185-5p can target 3’UTR of NK1R-Tr and inhibit its transcriptional activity;mi R-185-5p mimics can suppress the expression of NK1R-Tr in MDA-MB-231,and mi R-185-5p inhibitor can promote the expression of NK1R-Tr in SK-BR-3.Biological behavior experiment showed that under the stimulus of SP overexpressing mi R-185-5p,MDA-MB-231 cell line had a slower growth rate,a weaker ability of clone formation,a lower ratio of G2 and S,blocked in the G1 phase,a weaker ability of invasion and metastasis,which were recoverd after rescue of NK1R-Tr plasmid,and SK-BR-3 whose mi R-185-5p was inhibited had opposite results.4.c-MYC regulate NK1R-Tr indirectly through mi R-185-5p c-MYC inhibit the expression of mi R-185-5p and regulate NK1R-Tr indirectly through mi R-185-5p.5.c-MYC regulate the activation of Erk1/2 induced by SP c-MYC can target NK1R-Tr regulating the activation of Erk1/2 induced by SP,and then promote proliferation and invasion of breast cancer.6.mi R-185-5p inhibit growth and metastasis of breast cancer cells in vivo SCID mice with over-expressed mi R-185-5p had weak tumorigenic ability and low invasive and metastasis ability,on the contrary,tumorigenic ability and invasive and metastasis ability enhanced after inhibiting endogenous mi R-185-5p.Conclusion: The breast cancer tissue had higher level of NK1R-Tr/c-MYC and lower level of mi R-185-5p,NK1R-Tr/c-MYC and mi R-185-5p had negative correlation.c-MYC can regulate the expression of NK1R-Tr directly or indirectly through mi R-185-5p,and then activate Erk1/2 ultimately promote proliferation and invasion of breast cancer.mi R-185-5p can target NK1R-Tr to affect proliferation,cell cycle and invasion and metastasis as well as tumorigenic ability and metastasis ability in vivo.c-MYC NK1R-Tr and mi R-185-5p may form a loop which plays an important role in the development of breast cancer.