A Preliminary Study On The Regulatory Mechanisms Of P53 And MiR-449a In Breast Cancer

Background: The morbidity and mortality of breast cancer which are threatening human health rank first in female malignant tumors.Especially the triple negative breast cancer(TNBC)accounts for 10–20% of breast cancers and leads poor prognosis because of the high malignancy and lacking of effective treatment.Early diagnosis and precise molecular subtype would confer the targeted therapy,also lead the vital achievement not only for breast cancer,also benefits to other malignancies.The initiation and progression of tumor is a multistage process which involves a series of genetic alterations,such as oncogene activation and tumor suppressor gene inactivation.P53,PTEN and c-Myc act as cancer suppressor genes or oncogenes are closely associated with the tumorigenesis and development of many cancers.Especially,in TNBC the P53 mutation frequency is very high,and about 80% of cancers harboring P53 mutations which make the treatment more complicated due to the malignant characteristics.However,it is still poorly understanding about the expression of P53 in Peripheral blood from breast cancer.Peripheral blood is easily obtained compared with tumor tissue and some studies have found that the expression of genes in peripheral blood of tumor patients showed onvious different pattern compared with healthy people.Therefore,in the first part of our study,we detected the mRNA level of P53,PTEN and c-Myc in peripheral blood leukocytes from breast cancer patients,and explored their correlations with the clinicopathologic characteristics,aiming to provide potential andefficient approaches for the early and noninvasive detection of breast cancer.micro RNAs(miRNAs)are a class of small non-coding RNA molecules(containing about 22 nucleotides)that function in RNA silencing and post-transcriptional regulation of gene expression by binding to the target genes of mRNAs,inhibiting the translation and promoting the degradation of mRNAs.Previous researches have confirmed the important regulatory role of miRNAs which act as oncogenes or cancer suppressor genes in the initiation and progression of tumors.To investigate the role of miRNAs in tumors and the related mechanisms will be critical in exploring potential tumor markers and therapeutic targets.The latest studies have showed that miR-449 a was significantly down-regulated in tissue from breast cancer patients with P53 mutation,and also showed in the basal-like breast cancer which is the most malignant type of TNBC.In other studies miR-449 a was confirmed to act as cancer suppressor gene by inhibiting the invasion,migration and proliferation abilities in some type of tumors,such as lung cancer,liver cancer,prostate cancer and so on.Whereas,the regulatory mechanisms of P53 and miR-449 a in breast cancer,especially in TNBC embedding mutant P53 has not been elucidated and poorly understood.Here we’re trying to work on this point,aiming to find potential tumor markers and therapeutic strategics.Purpose: 1.To detect the expression of the tumor related gene P53,PTEN and c-Myc in peripheral blood leukocytes from breast cancer patients and explore their interaction with the clinicopathologic characteristics,then discuss the potential clinical applications.2.To clarify the role of miR-449 in regulating mutant P53 in TNBC and the involved mechanisms.Methods: 1.Detect the mRNA level of P53,PTEN and c-Myc inperipheral blood leukocytes from breast cancer patients,benign breast diseases patients and healthy women by Real-time PCR.2.Measured the mRNA level of miR-449 a,P53,E-cadherin,Vimentin,Twist and Snail by Real-time PCR,and the protein level of P53,AKT1,p-AKT,p-mTOR,Bcl-2,caspase-3,Cleaved-caspase-3 were checked by Western Blot after transfecting retrotiviral shRNA,pSuper-Retropuro-sh-P53(sh-P53)and pSuper-Retro-puro-Vector(Vector)in MDAMB-468.3.Detected the mRNA level of P53,E-cadherin,Vimentin,Twist,Snail and miR-449 a by Real-time PCR and checked the protein level of P53,AKT1,p-AKT,p-mTOR,Bcl-2,caspase-3,Cleaved-caspase-3 by Western Blot after transfecting MDA-MB-468 with miR-449a-mimic and miR-449a-NC for 24 h or 72 h respectively.4.The growth and proliferation abilities of the manipulated MDA-MB-468 cells were determined by the growth curve through the crystal violet staining method.5.The migration ability of the manipulated MDA-MB-468 cells was measured by scratch-wound healing test and Transwell migration assay.Results: 1.The mRNA level of P53 and PTEN in peripheral blood leukocytes from breast cancer patients and benign breast diseases patients were both decreased significantly.2.The down-regulation of PTEN mRNA level is closely related to the lymph node metastasis and the pathological stage of breast cancer.3.Knockdown of P53 didn’t affect the level of miR-449 a in MDA-MB-468 cells.4.The proliferation and migration of MDA-MB-468 were suppressed byknocking down the expression of P53.5.After knockdown of mutant P53 in MDA-MB-468 cells,the PI3K/AKT/mTOR signaling pathway and and the downstream targets were inhibited,along with EMT suppression,decreased Bcl-2 level and activated caspase-3 as well.6.The proliferation and migration of MDA-MB-468 were inhibited by up-regulated the expression of miR-449 a.7.Elevating the level of miR-449 a initiated the decline of P53 and Bcl-2in MDA-MB-468 cells,also mediated the PI3K/AKT/mTOR pathway and EMT inhibition.Conclusions: 1.The mRNA level of P53 and other cancer-related genes in peripheral blood leukocytes from breast cancer patients were significant difference with them from healthy women and patients of breast benign disease,and correlated with the clinicopathologic characteristics,which indicated the detection of cancer-related genes in peripheral blood leukocytes would be a potential non-invasive approach for the early diagnosis and targeted therapy for cancer patients;2.Down-regulation of mutant P53 could suppress the proliferation and migration of MDA-MB-468,also could reduce its malignancy.However miR-449 a was not affected by the decrease of P53 level;3.Down-regulation of mutant P53 could suppress PI3K/AKT/ mTOR pathway and EMT,as well as inducing apoptosis4.miR-449 a could inhibit the proliferation and migration of MDAMB-468 by down-regulating the expression of mutant P53,suppressing PI3K/AKT/mTOR pathway and EMT,as well as inducing apoptosis;5.miR-449 a could act as a cancer suppressor gene in MDA-MB-468 through regulating the mutant P53 and its related signaling pathways.