| Background and Objectives:Breast cancer is one of the most common malignant tumors in women.There are about 1 million new cases and 400,000 deaths in the world every year,and about 270,000 new cases and 70,000 deaths in China every year.Breast cancer has become the first killer threatening women's health.Once metastasis occurs,the existing treatment methods are difficult to control the progress of the disease.Metastasis has become the main reason for the failure of clinical treatment of malignant tumors.Breast cancer is extremely aggressive.Bone is one of the most common distant metastatic organs of breast cancer.It can cause severe pain,pathological fracture,spinal cord compression,hypercalcemia and other skeletal-related events(SREs),which seriously affect the life quality of patients,and bring heavy financial burden to clinical treatment and family nursing.The median survival of patients with bone metastasis is about 2-3 years,and only 20% of patients survive for more than 5 years.Bone metastasis is the "watershed" to accelerate the process of death.At present,the clinical treatment of breast cancer bone metastasis is facing many difficulties: First,the lack of effective monitoring and diagnostic indicators.Monitoring and early diagnosis of bone metastasis of malignant tumors are very difficult.Most patients come to the hospital after obvious pain,neurological dysfunction or even paralysis caused by metastasis of tumors.At the time of diagnosis,these patients may suffer multiple metastases,which brings great difficulties to clinical treatment.Secondly,it is difficult for the current treatment of bone metastasis of breast cancer to make a qualitative change in clinical prognosis.The main objective of clinical treatment is to control the progress of disease.Whether traditional radiotherapy and chemotherapy or endocrine therapy or targeted therapy,the control of bone metastases is still unsatisfactory.Thirdly,there is a lack of in-depth understanding of the clinical characteristics of bone metastasis of tumors,and the regulatory mechanism of bone metastasis of tumors needs to be further explored.According to the expression patterns of estrogen receptor(ER),progesterone receptor(PR)and growth hormone receptor(Her2)on the surface of cancer cells,breast cancer can be divided into Luminal,Her2 and triple negative subtypes.Tumor cells have heterogeneous characteristics.Cell subsets with different gene expression patterns of the same tumor form different metastases,while cells with the same gene expression characteristics tend to transfer to the same target organs.A large number of studies suggest that different types of breast cancer have different clinical prognosis.Luminal breast cancer is more prone to form bone metastasis,but its specific regulatory mechanism is not clear.Starting from the clinical issues,this project attempts to have an in-depth understanding of breast cancer at the molecular level,screen the key molecules regulating bone metastasis of Luminal breast cancer,and clarify the regulatory mechanism of bone metastasis,so as to provide a new theoretical basis for finding the diagnostic markers and new targeted therapeutic drugs for tumor bone metastasis.Methods:Firstly,we conducted cluster analysis on the sequencing results of normal control and breast cancer tissue samples by using TCGA(The Cancer Genome Atlas)database.We screened specific high expression genes in Luminal breast cancer and compared them with bone metastasis related genes reported in literatures.We found that NAT1 might play an important regulatory role in bone metastasis of Luminal breast cancer.Subsequently,we collected tissue samples of primary breast cancer and bone metastasis of breast cancer.Detected the expression level of NAT1 by immunohistochemistry,and compared the difference of NAT1 expression between primary breast cancer and bone metastasis.The expression of NAT1 in different types of breast cancer cell lines was determined by qRT-PCR and Western Blot.To further clarify the regulatory role of NAT1 in Luminal breast cancer,we constructed stable cell lines with knockout or overexpression of NAT1.Transwell assay,clonal formation assay,tumor conditioned medium induced osteoblasts and osteoclast differentiation assay were used to explore the regulatory role of NAT1 in Luminal breast cancer in vitro.Moreover,tumor metastasis model by left ventricular injection and bone injury model by tibial intramedullary injection were used for further verifying the function of NAT1 in vivo.Finally,we used secretory protein chip assay to screen differentially expressed proteins.KEGG and GO analysis were used to enrich the differential expression of proteins,and to explore the specific molecular mechanism of NAT1 regulating Luminal breast cancer bone metastasis.The differentially expressed protein IL-1B was used in rescue experiments to further verify its important role in bone metastasis of tumors.Result:Through bioinformatics analysis,we found that NAT1 was highly expressed in Luminal breast cancer and was closely related to bone metastasis.Primary tumors with high expression of NAT1 were more prone to form bone metastasis.The expression of NAT1 in bone metastases was significantly higher than that in primary breast cancer.The expression of NAT1 in Luminal breast cancer cell lines BT474,T47 D and MFC7 was relatively high,while that in triple negative breast cancer cell lines BT549,HS578 T and MDA-MB-231 was low.We successfully constructed NAT1-knockdown cell lines in T47 D and MCF7 cells and overexpressed NAT1 in BT549 cells.In vitro experiments showed that the migration and colony formation ability of tumor cells were significantly weakened after NAT1 knockout,osteoclast differentiation was weakened and the ratio of RANKL/OPG of osteoblasts was down-regulated by conditioned medium of tumor cells.The corresponding ability after over-expression of NAT1 was significantly enhanced.Animal experiments showed that after knocking out NAT1,tumor metastasis and bone injury were significantly reduced.Using secretory protein microarray,we found that the expression of IL-1B was significantly down-regulated and the NF-?B signaling pathway was obviously inhibited after NAT1 knockout.We further confirmed that NAT1 could regulate the expression of IL-1B through the signal pathway of NF-?B by experiments of luciferase reporter gene,Western Blot and immunofluorescence.At the same time,we confirmed that the phenotypes of tumor cells which knock out NAT1 could be rescued after exogenous supplementation of IL-1B.Conclusions:Based on the above findings,we concluded that NAT1 may be a key regulator of bone metastasis in Luminal breast cancer.High expression of NAT1 in cancer cells can promote tumor migration,colonization and modification of bone microenvironment,and ultimately promote bone metastasis.NAT1 can regulate the expression of IL-1B through NF-?B signaling pathway.On one hand,IL-1B can promote the migration and colonization of tumor cells by a paracrine manner.On the other hand,IL-1B may regulate the differentiation and function of osteoblasts and osteoclasts by endocrine or paracrine manner,participating in the regulation of pre-metastatic niche and the transformation of bone microenvironment after metastasis.Targeting NAT1 or its downstream IL-1B may be a new therapeutic target for bone metastasis of breast cancer,and IL-1B may also be a clinical predictor and disease monitoring indicator for bone metastasis of breast cancer. |
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