| With the development of economy and society,the incidence of breast cancer is gradually increasing.The statistical report of the National Cancer Center of China in 2018 shows that breast cancer has become the most common malignant tumor among women,which causes heavy economic and psychological burdens on society and families.Therefore,it is urgent to explore the driving mechanism of the occurrence and progression of breast cancer,and to develop targeted therapies for improving the prognosis of breast cancer.Epithelial-mesenchymal transition(EMT)increases the expression of mesenchymal cell markers such as a-SMA and vimentin under certain inducements(such as TGF-?1),and decreases the expression of epithelial cell markers such as E-cadherin and cytokeratin.It also enhances the ability of motor invasion and obtains mesenchymal characteristics.During the transition,cell morphology changed from"irregular polygon" to "fibroblast-like spindle",and the ability of anti-apoptosis,migration and invasion was enhanced.EMT phenotypic transformation plays important roles in embryonic development,tissue reconstruction,chronic inflammation,invasion and metastasis of malignant tumors and fibrous diseases.EMT phenotypic transformation can be divided into three types:Type 1,2 and 3.Type 1 is mainly related to embryogenesis,including embryonic development,neural crest development and intestinal formation.Type 2 is mainly related to wound healing,tissue regeneration and repair.Type 3 is mainly related to the migration and invasion of malignant tumor cells.EMT phenotypic transformation is an important cause of metastasis and invasion of epithelial malignant tumors,and has been proved to be an important link and key event in the occurrence and metastasis of various solid tumors including breast cancer.Some studies has found that EMT phenotypic transformation can change the microenvironment around tumors,so as to facilitate the invasion of the surrounding tissues.In addition,EMT phenotypic transformation can also promote the formation of stem cell clones with mesenchymal phenotype and self-renewal ability,and further promote the spread and self-renewal potential of malignant tumor cells.However,the mechanisms of EMT during cancer progression are still unclear.Therefore,it is necessary to clarify the molecular mechanism for the capacities of migration and invasion,and it will provide a new strategy for the effective treatment of breast cancer.Homologous box genes are highly conserved evolutionary DNA sequences,which regulate target genes by combining the helix-turn-helix structural pattern with promoters or enhancers of genes.The homeobox gene family contains two genes,Prrx1 and Prrx2.The homeobox gene Prrx2 is located on human chromosome 9q34 and encodes a protein composed of 253 amino acids.The homeobox gene Prrx1 is considered as a regulatory transcription factor of EMT.Our previous studies confirmed that silencing the expression of Prrxlb can inhibit Wnt/?-catenin signaling pathway and inhibit the proliferation,invasion and migration of triple-negative breast cancer by reversing EMT phenotype.Recent studies have reported that Prrx2,as a synergistic factor of TGF-?1,is involved in the invasion and migration of breast cancer.Some studies have found that Prrx2 expression is related to acute myeloid leukemia.In gastric malignant tumors,Prrx2 can be used as a new transcription factor to promote the invasion and metastasis of tumors.However,the biological function and molecular mechanism of Prrx2 in breast cancer are still unclear.This study will focus on the role of Prrx2 in EMT phenotype transformation of breast cancer and the related molecular mechanisms.It will further increase the molecular pathological characteristics of breast cancer metastasis and provide new targets for precision treatment.Wnt/?-catenin signal transduction pathway is a classical Wnt pathway,which consists of Wnt protein,loose protein,crimp protein,?-catenin,APC complex(including Axin,GSK-3? and APC protein)and T lymphocyte factor/lymphoid enhanced transcription factor.?catenin is an important nuclear signal protein in the classical Wnt signaling pathway.Its expression level directly affects the activity of the Wnt/?-catenin signaling pathway.The regulation mechanism of Wnt/?-catenin pathway is complex.One of the main ways is to regulate the output of Wnt signaling pathway through the stability of P-catenin.The classical activation mode of Wnt/?-catenin signaling pathway is as follows:when the cells are stimulated by external signals,secretory glycoprotein Wnt can bind to cell surface receptor crimp proteins.Under the synergistic action of low density lipoprotein-related receptor proteins 5 and 6,loose proteins in cytoplasm are collected under the cell membrane and phosphorylated to disintegrate the degradation complex,and protein kinase A is phosphorylated and inactivated.The degradation of ?-catenin decreased because it could not be phosphorylated.When ?-catenin accumulates in the cytoplasm to a certain extent,the concentration gradient promotes ?-catenin enter the nucleus and form a complex with transcription regulator TCF/LEF in the nucleus,which initiates the transcription regulation of target genes such as Cyclin-D1,C-Myc,and plays the role of transcription factors.A thorough study of Wnt/?-catenin signaling pathway regulatory factors and mode of action is conducive to exploring the driving mechanism of tumorigenesis and progression,and is expected to provide new clues for the prevention and treatment of breast cancer.In this study,the expression of Prrx2 in breast cancer tissues and adjacent non-cancerous tissues will be detected,and the relationship between Prrx2 expression and clinicopathological characteristics and EMT of breast cancer will be analyzed.Then,through cell and animal experiments,the effects of silencing Prrx2 expression on proliferation,invasion,metastasis and EMT phenotype transformation of breast cancer cells will be clarified.The successful implementation of this study not only enriched the Prrx2 biological function in breast cancer,but also shed light on exploring the prevention and treatment of the invasion and metastasis of breast cancer.This research mainly includes the following three parts:Part ?.The expression of Prrx2 and its correlation with EMT inbreast cancerObjectiveThe purpose of this study was to detect the expression of homologous box gene Prrx2 in breast cancer,analyze its relationship with the clinicopathological characteristics and EMT phenotype transformation of breast cancer,and explore its influence on the occurrence and progression of breast cancer.Methods(1)The expression of Prrx2 protein was detected by immunohistochemistry in 133 paired breast cancer tissues and adjacent non-cancer tissues,and analyze the relationship between Prrx2 and the clinicopathological features.(2)EMT-associated proteins expression(vimentin and E-cadherin)were assessed by immunohistochemistry in 11 8 breast cancer tissues.(3)Analyze the relationship between Prrx2 expression and EMT phenotype transformation in breast cancer.Results(1)Immunohistochemistry showed that Prrx2 protein was mainly expressed in the cytoplasm and occasionally in the nucleus of breast cancer cells.And Prrx2 protein was significantly higher expressed in breast cancer tissues than in adjacent non-cancerous tissues(P<0.05).(2)The expression of Prrx2 protein in breast cancer tissues was correlated with tumor size,lymph node metastasis and TNM stage,and the differences were statistically significant(P<0.05).(3)Spearman correlation analysis showed that the expression of Prrx2 protein in breast cancer tissue was negatively correlated with E-cadherin expression(r=0.613,P=0.001),and positively correlated with vimentin expression(r=0.345,P=0.012).ConclusionThe Prrx2 protein expression in breast cancer tissues was significantly higher than that in adjacent non-cancerous tissues.Statistical analysis showed that Prrx2 protein expression was negatively correlated with E-cadherin expression,while positively correlated with vimentin expression.It was speculated that Prrx2 protein might associate with EMT phenotype transformation and participate in the invasion and metastasis of breast cancer.Part II.Effects of Prrx2 gene silencing on the proliferation,invasion,metastasis and EMT of breast cancerObjectiveThe aim of this study was to investigate the effects of silencing Prrx2 expression on the proliferation,invasion,metastasis and EMT of breast cancer cells.Methods(1)Construct stably silencing Prrx2 expression cell lines.Using human breast cancer cell lines MCF-7 and MDA-MB-231 as experimental materials,lentiviral interference vector and blank vector of Prrx2 constructed in vitro were transfected into breast cancer cells respectively,then stable silenced Prrx2 expressing cell lines were obtained.The Prrx2 protein level was verified by Western blot.(2)Observe the changes of EMT phenotype after silencing Prrx2 expression.Western blot was used to detect the expression of EMT-related markers E-cadherin,vimentin and alpha-SMA in breast cancer,and optical inversion microscopy was used to observe the morphological changes.(3)Analyze the effect of silencing Prrx2 expression on proliferation,migration and invasion of breast cancer in vitro.MTT assay was used to analyze the proliferation of breast cancer cells in vitro.Transwell and Matrigel assay were used to detect the migration and invasion ability of breast cancer cells.(4)Observe the effect of silencing Prrx2 expression on the growth and metastasis of tumors in vivo.Construct a subcutaneous transplanted tumor model in nude mice,and observe the effect of silencing Prrx2 gene expression on the growth of transplanted tumors in nude mice in vivo by drawing the growth curve of the tumor,Also construct a metastasis model in nude mice,and analyze the effect of silencing Prrx2 expression on distant metastasis of breast cancer by counting the number of macrometastasis in lung.Results(1)In this study,we successfully constructed lentivirus interference expression vector and established stable transfection of breast cancer MCF-7 and MDA-MB-231 cell lines.Western blot results showed that the expression level of Prrx2 in the transfected group was significantly lower than that in the control group,indicating that the interference vector could effectively inhibit the expression of Prrx2.(2)Western blot assay showed that the expression of E-cadherin was significantly higher in the silenced Prrx2 expression group than in the control group.The morphology of tumor cells changed from "long spindle" to "irregular polygon"after silencing Prrx2 expression under inverted microscope.(3)MTT assay showed that the proliferation of cells in silenced Prrx2 expression group was significantly inhibited compared with the control group.Nude mice transplanted tumors showed that the growth of transplanted tumors was slow after silencing Prrx2 expression,and the volume and weight of the tumors were smaller than those of the control group.This indicated that the growth of tumors was significantly reduced and the growth of tumors was inhibited after silencing Prrx2 gene expression in nude mice.(4)Transwell and Matrigel experiments showed that the number of migrating and invading cells in the silent expression group was significantly reduced compared with the control group.The results of metastasis in nude mice showed that the number of macrometastatic lung tumors in the silent Prrx2 expression group was less than that in the control group.The results indicated that the invasive and migratory ability of breast cancer was decreased and the metastatic ability of breast cancer was significantly inhibited after silencing Prrx2 gene expression.Conclusion(1)Lentiviral vectors carrying Prrx2 interference fragments stably transfected human breast cancer MCF-7 and MDA-MB-231 cells can effectively inhibit the expression of Prrx2 protein,and successfully construct cell lines that silence the expression of Prrx2 for subsequent experimental study.(2)Lentivirus interference vector-mediated silencing of Prrx2 gene expression can effectively inhibit EMT phenotype transformation in breast cancer.(3)In vitro experiments showed that silencing Prrx2 gene expression could significantly inhibit the proliferation,cloning,migration and invasion of human breast cancer cells MCF-7 and MDA-MB-231.(4)In vivo transplantation model showed that silencing Prrx2 gene expression could effectively inhibit the growth of transplanted tumors.The results of nude mice metastasis model indicated that the number of macrometastases in lung after silencing Prrx2 expression was significantly less than that in control group,which was consistent with the results in vitro.(5)Taken together,silencing Prrx2 expression can effectively inhibit EMT phenotypic transformation and reduce invasion and metastasis of breast cancer,suggesting that Prrx2 may be an important inducer of EMT transformation of breast cancer cells,and may become a new target for breast cancer prevrntion and treatment.Part ?.Effects of Prrx2 gene silencing on the Wnt/?-cateninsignaling pathway of breast cancerObjectiveTo investigate the effect of silencing Prrx2 expression on Wnt/p-catenin signaling pathway activity in breast cancer cells MCF-7 and MDA-MB-231,and the regulation of downstream target genes.Methods(1)Construct breast cancer cell lines MCF-7 and MDA-MB-231 stably silenced Prrx2 expression.(2)Western blot was used to analyze the expression of ?-catenin in the nucleus and cytoplasm of breast cancer cells MCF-7 and MDA-MB-231 after silencing Prrx2 expression and the effect of downstream target protein Cyclin-D1 expression.(3)Immunofluorescence was used to observe the changes of expression intensity and distribution of ?-catenin in breast cancer cells MDA-MB-231 after silencing Prrx2 expression.(4)The activity of ?-catenin/TCF transcription factor after silencing Prrx2 expression was analyzed by double luciferase assay.Results(1)Western blot showed that silencing Prrx2 expression inhibited the expression of ?-catenin in breast cancer nucleus,but did not affect the expression of ?-catenin in total protein of cancer cells.(2)Immunofluorescence assay showed that after silencing the expression of Prx2,the expression of ?-catenin decreased in the nucleus and increased in the cytoplasm,indicating that silencing the expression of Prrx2 prevented the movement of ?-catenin from the cytoplasm to the nucleus.(3)Western blot analysis showed that the downstream protein Cyclin-D1 of Wnt/?-catenin signaling pathway decreased significantly after silencing Prrx2 expression,suggesting that silencing Prrx2 expression could down-regulate the activity of Wnt/p-catenin signaling pathway.(4)Dual luciferase assay showed that silencing Prrx2 expression could down-regulate the activity of transcription factor P-catenin/TCF,further suggesting that silencing Prrx2 expression could down-regulate the activation of Wnt/?-catenin signaling pathway.ConclusionSilencing Prrx2 expression can inhibit the activity of Wnt/?-catenin signal transduction pathway in breast cancer.Our findings not only enrich the current understanding of Prrx2's regulation of the EMT but also suggest that it plays an important role in both maintaining the mesenchymal phenotype and participating in breast cancer dissemination.Thus,our data suggested that Prrx2 might be served as a target for new therapeutic strategies against breast cancer. |
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