Prorenin Receptor Promotes Invasion And Metastasis Of Breast Cancer MCF-7 Cell Line Via Regulating EMT

Breast cancer,the leading cause of cancer death in females,is the most common malignant tumor among women.It is also one of the three most common malignant tumors in the world.Currently,the consensus has been reached that breast cancer which can metastasis at an early staging is a systemic disease.Although the advances in therapeutic methods has improved the survival rate of breast cancer,the recurrence and metastasis are the main cause of cancer-related death.20%-30%of breast cancer patient will develop distant metastasis and cases with progressive stage only have a median two-year survival time.Triple negative breast cancers(TNBC)which lack expression of ER,PR,and HER2,present with higher grade,often contain mutations in TP53,and have a poor prognosis.As three receptors are negative,TNBC which is not suitable for endocrine therapy and anti-HER2 targeted therapy is usually treated with surgery and chemotherapy clinically.Nevertheless,the survival rate of TNBC is lower than other breast cancer types.To find more effective therapy for TNBC is especially important.Tumor metastasis is a complex process.Simply,It include 3 processes which are metastasis initiation process,metastasis progression process,and metastasis virulence process.EMT(epithelial-mesenchymal transition)is attributed to the initial process,when tumor cells lose their epithelial characteristics to free from the primary tumor,invade extracellular matrix,and enter the blood vessel.EMT is also beneficial to supporting the survival of tumor cells in the blood circulation and promoting extravasation from blood vessel at the progressive process.Therefore,EMT is of great importance in the process of tumor metastasis.EMT(epithelial-mesenchymal transition)is a process by which epithelial cells transit to mesenchymal cell morphologically in special physiologic and pathological circumstances.As a result epithlial cells gain migratory and invasive capacity.In pathological conditions,especially in tumors,EMT play a crucial role.Breast cance can obtain the ability of invasion and metastasis when EMT taking place.The hallmarks of EMT are loss of epithelial molecule E-cadherin and gain of mesenchymal markers,such as N-cadherin and vimentin.So,detecting the transformation of the hallmarks can testify the occurrence of EMT.Renin-angiotensin-syetem(RAS)is the key component of humoral regulation system.RAS,which consist of multiple peptide,enzyme,and receptor,play a major role in regulating fluid and electrolyte balance and adjusting blood pressure.Accumulating research have found that the functions of RAS extend beyond its role in sodium and potassium homeostasis and the regulation of blood pressure.RAS also exerts effects on cell proliferation,apoptosis,inflammation,angiogenesis and tumorigenesis.The core component of RAS are prorenin,renin,and(pro)renin receptor(PRR).There is a lot of evidence linking PRR with tumorigenesis and tumor angiogenesis.PRR,discovered and successfully cloned by Nguyen etal.,is a 350-amino acid protein with a single transmembrane domain.The PRR gene,named ATP6ap2/PRR,is located on the X chromosome(Xp11.4)in humans.It is reported that the PRR is highly expressed in breast cancer and Blocking PRR results in inhibition of the growth of breast cancer cells.Yet,the mechanism is unclear.From the above,we propose that whether PRR can accelerate the proliferation of breast cancer and promote invasion and metastasis of breast cancer via regulating EMT.Objective:To investigate the effects of PRR on the invasion and migration ability of MCF-7 cells as well as EMT.Methods:1..Breast cancer tissue and para-carcinoma tissue were detected by immunohistochemical assay to investigate the expression of PRR.immunofluorescence assay was used to investigate the location of PRR in breast cancer tissue.2.MCF-7 cells transfected with human-PRR-siRNA and negative control-siRNA.Then,according to the PRR status of expression in breast cancer cells,MCF-7 was divided into two groups.MCF-7 whose PRR was silenced was the experimental group(MCF-7-PRR-si),MCF-7 without silencing PRR was the control group(MCF-7-NC).3.CCk-8 assay were used to test the proliferation ability of MCF-7 cells.4.Transwell matrigel invasion assay and transwell migration assay are used to test the invasion and migration ability of MCF-7 cells.5.Expressions of EMT-related markers(E-cadherin,N-cadherin,Vimentin)in MCF-7 cell are detected via western-blot method and RT-PCR.6.p-catenin was detected between the breast cancer tissue and adjacent tissue via immunohistochemical assay.Results:1.Immunohistochemical assay implicated that PRR was expressed in both breast cancer tissue and adjacent normal tissue.But,the expression of PRR was higher in breast cancer tissue.Futher immunofluorescence assay implicated that PRR was expressed in breast cancer cells rather than breast cancer mesenchyma.2.CCK-8 assay showed that the value of OD of the experimental group was lower than that of control group under same conditions,which implicated that MCF-7 of the experimental group grew slower than that of control group.3.The transwell matrigel invasion assay showed that invasion ability of control group cells was significantly higher than experimental group(p<0.05).The transwell migration assay showed that migration ability of control group was significantly higher than experimental group(p<0.05).4.Western blot showed the level of E-cadherin was upregulated in experimental group upregulated(p<0.05),but the level of N-cadherin and Vimentin was down regulated(p<0.05).PCR showed that the mRNA level of E-cadherin in experimental group was upregulated(p<0.05),but the mRNA level of N-cadherin and Vimentin was down regulated(p<0.05).5.?-catenin was highly expressed in breast cancer tissue.Conclusion:PRR promotes invasion and metastasis of breast cancer MCF-7 cell line via regulating EMT.