The Fundamental Research On The Role Of The ARPC2 In Breast Cancer And Its Impact On The Biology Behaviors In Breast Cancer Cell

Research background:The incidence of cancer in women with Breast cancer is the highest.In 2016,Cancer Statistics reported 1,685,210 new cases of breast cancer in the United States,with 595,690 deaths.The overall mortality rate was only second to lung cancer.Cancer Statistics in China data shows that the incidence of breast cancer in our country has been increasing year by year.Many cities and regions have become the leading cancer death rate for women.The main cause of death is due to the lack of effective treatment measures to lead to the continuous progress of cancer.With the improvement of the medical level,surgical treatments for breast cancer,endocrine therapy,chemotherapy,radiation therapy,targeted therapy,and biological therapy have made considerable progress,and mortality has declined,but there are still about half of breast cancers.Early disease progression of the disease can not be effectively controlled,eventually leading to treatment failure and death.And many treatments are not only ineffective for patients with recurrent and metastatic breast cancer,but also waste a lot of medical resources,which is not only a problem for clinicians,but also a hot issue in breast cancer research.According to ER,PR,and HER2 as the dividing standard,breast cancer is mainly divided into four types: HER2 overexpression type(ER-/ PR-/ HER2 +),luminal A,luminal B,PR-/ HER2-).Breast cancer is a heterogeneous disease,even in the same pathological type of patients at the molecular level there is also a big difference,the therapeutic target is not the same,the treatment effect is not the same.With the feedback of a large number of clinical treatment data,scholars have increasingly found the importance of targeted therapy.However,the effective treatment targets that have been discovered so far are very limited and the targeted treatment can not be taken on more patients,which leads to the failure of the final treatment of some patients,which urgently needs us to discover that it is related to the development of breast cancer of the potential targets of different mechanisms of action,especially with the prognosis of patients with very close biomarkers,so that we can early in the prognosis of patients to make judgments and take targeted therapies.ARPC2(Actin related protein 2/3 complex)is one of the seven subunits of the actinrelated protein 2/3(Arp2/3)complex and is found in most tissues.Actin is the most abundant eukaryotic protein.The cytoskeleton is mainly composed of actin monomer assembled into microfilament formation to maintain cell morphology,while participating in a variety of physiological functions such as cell division and cell motility contraction.Actin can also make such as phagocytic phagocytic particles,cytomegalovirus and intracellular parasites and other cells within the material displacement.Cells and intracellular subcellular structures and protein molecules play a more important role in the localization or displacement.How to assemble intracellular actin filaments? There are many factors involved in this replication assembly.Studies have shown that ATP,actinbinding proteins and some small intracellular molecules can regulate actin multimerization,but actin nucleation forms microfilaments The specific process has not yet been fully studied.The cytoplasm and membrane are expressed in the actin cytoskeleton assembly plays an important role.The actin-related protein 2/3 complex is mainly involved in the control of intracellular actin polymerization and is relatively evolutionarily conserved.To date,two alternative spliced variants have been discovered that play a key role in the nucleus pulposus.It may act through the actin-related protein 2/3 in the branch,that is,the adjacent two microfilaments are connected at a 70 degree angle,causing the change of the plasma membrane,thereby advancing the cells.ARPC2 was first found in Acanthamoeba,and was subsequently found in both eukaryotic and animal cells.Recent reports report that ARPC2 is highly expressed in gastric cancer cells and promotes the proliferation and invasiveness of gastric cancer cells.We found in the pre-experiment ARPC2 low expression in breast cancer tissues,significantly high expression in breast cancer cells.However,the role of ARPC2 in the biological function of breast cancer cells has not been reported.Epithelial-mesenchymal transition(EMT)is one of the important mechanisms in tumor metastasis.It upregulates interstitial markers vimentin by inhibiting the expression of epithelial markers such as E-cadherin.Cells lose epithelial polarity,access to better invasiveness and exercise capacity,so as to achieve distant metastasis of the tumor.Transforming growth factor beta(TGF-beta)is an important regulator of EMT and promotes tumor EMT through Smad-dependent and non-dependent pathways.TGF-? signaling pathway is closely related to breast cancer progression.So in the invasion and metastasis of breast cancer,whether high expression of ARPC2 can promote epithelialmesenchymal transition? Whether ARPC2 plays a part in regulating EMT through TGF-? signaling pathway? This topic will combine ARPC2 expression in breast cancer cell lines and clinical cases,artificially change ARPC2 expression levels in vitro and animal experiments observed breast cancer cell proliferation and apoptosis and tumor invasion and metastasis changes;the use of biological information Prediction and molecular experiments to verify whether ARPC2 affects the metastasis of breast cancer through the involvement of TGF-?-mediated epithelial-mesenchymal transition.By exploring the role of ARPC2 in the pathogenesis of breast cancer,it provides new clues for the diagnosis and clinical treatment of breast cancer.To investigate the expression of ARPC2 in breast cancer cell lines and breast cancer patients and to explore its role in the pathogenesis of breast cancer.We found that ARPC2 was lowly expressed in adjacent tissues of breast cancer,and it was highly expressed in breast cancer tissues,and it was closely related to EMT marker proteins.In this study,we overexpressed or knocked down the level of ARPC2 expression in breast cancer cell lines,and used cell function experiments to observe the biological function changes of breast cancer cells,such as proliferation,invasion,apoptosis,cycle,etc.,and EMT indicators.Expression of situation.It was found that breast cancer cells could be induced to proliferate to obtain interstitial cell characteristics,enhance cell migration and invasiveness;using co-immunoprecipitation and mass spectrometry(IPMS)screening,query differential protein GO enrichment analysis and KEGG pathway analysis and found ARPC2 is involved in the TGF-?/Smad signaling pathway.To investigate the signal transduction and interaction mechanism of ARPC2 and TGF-?/Smad signaling pathways in EMT of breast cancer,Western-blot was used to detect the EMT parameters of TGF-? inhibitor(SB43)and TGF-? stimulated cells.The expression of ARPC2 was found to enhance the TGF-? signaling pathway and thus promote the occurrence of EMT.At the same time,TGF-? induces EMT in mammary epithelial cells and upregulates ARPC2 expression,and up-regulation of ARPC2 is required for TGF-? induced EMT.This study found that ARPC2 involved in breast cancer cell EMT transformation process promote breast cancer invasion and migration,and participate in breast cancer cell TGF-?/Smad signaling pathway.This finding provides an important basis for ARPC2 as a marker for predicting breast cancer prognosis and as a target for breast cancer targeted therapy.Research content: Part 1 Expression of ARPC2 in Breast CancerA total of 172 breast cancer tissues and 68 breast adenocarcinoma tissues conserved after radical mastectomy were collected from the First Affiliated Hospital of Anhui Medical University and serum samples of 46 breast cancer patients before and 50 normal physical examinations.All patients with breast cancer who were enrolled in the study had not been treated with biological therapy and radiotherapy and chemotherapy before surgery.There was no significant difference in the age of women in physical examination between the two groups.Immunohistochemistry,Western Blot and q RT-PCR were used to detect blood ARPC2 m RNA levels in specimens and histological specimens of protein expression.At the same time,the clinical and pathological data of breast cancer patients were collected and sorted out,including the tumor size,clinical stage,grade,prognosis,etc.The correlation between ARPC2 levels and clinicopathological parameters was analyzed by statistical correlation analysis.In addition,the expression of ARPC2 gene was detected by Western blotting and q RT-PCR in MCF-10 A and breast cancer cell lines MCF7,BT474,T47 D and MB-MDA-231.The expression of ARPC2 in breast cancer tissues and adjacent non-cancerous tissues was analyzed by from Oncomine microarray database and Bio Portal for Cancer Genomics(http://www.cbioportal.org/).To explore the relationship between the expression of ARPC2 and clinicopathological features of breast cancer.Clarify the expression status and clinical value of ARPC2 in breast cancer.Analyze the differential expression of ARPC2 gene in different breast cancer cell lines.Part 2 The ARPC2-mediated invasion of breast cancer preliminary mechanism studyTo investigate the effect of ARPC2 on the malignant phenotype of breast cancer cells by transfecting the plasmid with low expression of ARPC2 gene and low expression of ARPC2 gene in breast cancer cells by interfering with RNA method and then carrying out cytological experiments.To study the biological function of ARPC2 by transfecting the breast cancer cell line MCF-7 with plasmids with high expression of ARPC2 gene and the empty plasmid by liposome method,followed by CCK-8 method to detect cell proliferation and draw Growth curve;Flow cytometry was used to detect cell cycle changes;Annexin V/PI method was used to detect cell line apoptosis;Transwell/Matrigel method was used to observe cell line migration and invasion ability;Soft agar semi-solid colony formation method was observed.Cell independence Non-anchored growth ability.The effect of high expression of ARPC2 on the proliferation and invasiveness of breast cancer cells was demonstrated.The si RNA was used to inhibit the expression of ARPC2 in breast cancer cells.Subsequently,the cell proliferation was measured by MTT.The cell cycle was detected by flow cytometry.Cell apoptosis was detected by Annexin V/PI assay.Transwell/Matrigel assay was used to observe cell migration and invasion.The MCF-7 cell line over-expressed with ARPC2 plasmid was injected subcutaneously into female nude mice.We observed the tumor growth rate,weight,lung metastasis and the expression of Ki67 six weeks later.Affinity Purification and Mass Spectrometry(AP-MS)was used to screen the downstream pathways which can affect the biological behavior of breast cancer cells.Western blot and proteomics were used to verify that breast cancer cells were pulled down by specific ARPC2 antibodies.By investigating EMT transformation and possible downstream pathways in breast cancer cells through GO enrichment and KEGG pathway analysis,we include that ARPC2 induced EMT transformation through TGF-? signaling pathway in breast cancer cells.In order to find out whether ARPC2 can be a marker to predict the prognosis of breast cancer,and provide a basis for targeted therapy of breast cancer.Research result: Part 1(1)The public online database showed that the expression of ARPC2 m RNA in breast cancer tissues was higher than that in normal breast tissues(P <0.05).(2)The results of q RT-PCR in serum samples of 16 breast cancer patients and 16 corresponding healthy female patients showed that ARPC2 was highly expressed in breast cancer patients(P<0.05);(3)Clinical Immunohistochemistry results showed that the expression of ARPC2 protein in breast cancer tissues preserved in 172 cases of radical mastectomy was higher than that in 68 cases of benign breast disease(P<0.05);(4)The positive expression of ARPC2 was positively correlated with tumor size,histological stage and lymphatic metastasis,but not with other pathological features such as HER2 expression,ER and PR status.(5)Survival analysis showed that ARPC2 was correlated with the prognosis of breast cancer patients;(6)The results of RT-PCR and Western Blot showed that the expression of ARPC2 in breast cancer cell lines was higher than that in normal breast epithelial cell line MCF10A(P <0.05).Part 2(1)si RNA and ARPC2-si RNA were transfected into MDA-MB-231 cells,pc DNA3.1-ARPC2 and empty plasmid were transfected into MCF7 cells,and the corresponding ARPC2 protein levels were decreased or increased;(2)ARPC2 si RNA transfection reduced ARPC2-induced cell proliferation,cloning and invasion and migration inhibition,promote apoptosis;(3)The growth rate of tumor in nude mice injected with MCF-7 cells overexpressing ARPC2 was significantly higher than that of control plasmid(P <0.05).(4)Proteomic analysis showed that ARPC2 expression was highly correlated with the expression of adhesion and invasion related genes;(5)Overexpression of ARPC2 can promote the transformation of EMT and activate the TGF-? / Smad signaling pathway.(6)ARPC2 can be blocked by TGF-? receptor kinase inhibitor SB431542 reversal of TGF-? / Smad signaling pathway EMT transformation.