Study Of The Correlation Between SALL4 And The Biological Behavior Of Breast Cancer And The Mechanism Of Drug Resistance Of Adriamycin

Background: Breast cancer is one of the most common malignancies of women around the world,and is a major malignant disease leading to the death in women.The diagnosis and comprehensive treatment strategies of breast cancer today include surgery,radiotherapy,chemotherapy,endocrine therapy,targeted therapy and immunotherapy has been significantly improved.However,local recurrence and distant metastasis of breast malignancies still occur frequently,leading to poor prognosis in some patients with breast cancer,and their survival quality and survival time.Therefore,it is necessary to identify the relevant factors of the biological characteristics such as the occurrence,development,metastasis,invasion,proliferation and drug resistance of breast cancer,and find the molecular mechanism to participate in the development of breast cancer.It is still urgent to explore new therapeutic targets and treatment methods.The Sal-Like 4(SALL4),which is a gene of the protooncogene found in recent years.The SALL gene family has four members of SALL1-SALL4.SALL4 is located in the area of human chromosome 20q13.13-q13.2.It is possible to encode three isomer protein SALL4 A,SALL4B and SALL4 C.SALL4 plays an important role in the early development of the embryo,organ formation and the proliferation,pluripotency maintenance and self-renewal of embryonic stem cells,and it is related to the development of hereditary diseases,leukemia,lymphoma and other diseases.More and more evidence indicates that SALL4 is highly expressed in a variety of tumors such as breast cancer,ovarian cancer,gastric cancer,hepatocarcinoma,colorectal cancer,renal blastoma,endometrial carcinoma,and germ cell tumor.It plays important roles in the occurrence and development of malignant tumor.In recent years,literatures on the structure,function,biological characteristics and relations to the non-tumor and tumor diseases of SALL4 have been reported extensively.There are a lot of researches on the relationship among the blood system,the congenital disease,the germline tumor,but relationship between SALL4 and breast cancer is still lacking.The role of SALL4 in breast cancer has not yet been elucidated.The purpose of this study is to investigate the correlation between SALL4 and the biological behavior of breast cancer and the mechanism on drug resistance of Adriamycin.Methods: In 75 cases of breast cancer tissues,the correlation between the expression level of SALL4 and the clinical pathological parameters in breast cancer is detected by immunohistochemical analysis.The expression of SALL4 in breast cancer tissues and paired noncancerous tissues is detected by real time-PCR and Western blot.In the loss-of-function assays,SALL4 is knockdown in breast cancer cell lines.The correlation between SALL4 and the proliferation activity of breast cancer cells is detected by Colony forming assay,MTT assay,cck-8 assay and vivo xenografts.Influence of SALL4 on apoptosis and apoptosis-related protein of breast cancer cell are detected by Flow cytometer and Western blot.Influence of SALL4 on cell cycle and cell cycle-related protein of breast cancer cell are detected by Flow cytometer and Western blot.The effect of SALL4 on breast cancer cell migration is detected by Wound-healing assay.The effect of SALL4 on invasion of breast cancer cells is detected by Transwell invasion assay.The effect of SALL4 on the resistance of Adriamycin in breast cancer cells is detected by MTT assay.After knockdown of SALL4,the expressions of PTEN,Notch1 and Wnt3 a are detected by Western blot to screen the possible drug resistance pathways of SALL4.Create shSALL4-MCF-7/ADR cell line through knockdowning SALL4.Add different concentrations of Adriamycin to MCF-7,MCF-7/ADR,shsall4-MCF-7/ADR cells,and then detecte cell proliferation activity by MTT assay and calculate the IC50 value of each group.Flow cytometry detecte cells apoptosis and intracellular Adriamycin content of each group;Using RT-PCR and Western blot to detect the change of drug-resistant genes ABCB1,ABCG2 and c-myc in each group.Using RTPCR and Western blot to detect the expression of PTEN in each group.Using Western blot to detect the change of p-AKT、AKT、p-mTOR、mTOR in each group.Add PTEN inhibitor to MCF-7/ADR 、 shSALL4-MCF-7/ADR cells after Adriamycin treatment,then use flow cytometry detecte the cell apoptosis rate.Using Western blot to detect the expression of ABCB1、c-myc、ABCG2 in each group.Results: SALL4 expression is higher in the BC tissues than that of the paired noncancerous tissues,and it is correlated with lymphatic metastasis(p<0.05).There is no correlation between SALL4 expression and the patient’s age,pathological type,histological grade,tumor size,ER,PR,Her-2,Ki67 expression level and molecular subtype.The tumor size of SALL4 positive group is larger than that of SALL4 negative group(p>0.05).Knockdown SALL4 successfully to create the breast cancer cell lines MDA-MB-435、MDA-MB-468、MCF-7.Knockdown of SALL4 in breast cancer cells suppress cell proliferation,inhibit tumor growth in vivo,induce cell cycle arrest in the G0/G1 phase,promote cell apoptosis,as well as inhibite the migratory and invasive abilities of breast cancer cells,reduce the drug resistance of breast cancer cells to adriamycin.Knockdown SALL4 successfully to create the breast cancer cell line shSALL4-MCF-7/ADR.SALL4 expression is higher in MCF-7/ADR than that of MCF-7、shSALL4-MCF-7/ADR.After Silencing of SALL4,the IC50 values of all the cells treated with Adriamycin decrease,the apoptosis rate and the intracellular Adriamycin content increase significantly,ABCB1,ABCG2 and c-myc expression decrease significantly.Silencing of SALL4 reduce the drug resistance of McF-7 /ADR to Adriamycin,can reduce the expression level of drug resistance genes.The expression of PTEN in each group significantly increase,the expression of p-AKT、p-mTOR significantly decrease after silencing SALL4.The apoptosis rate of sh SALL4-MCF-7 /ADR cells is significantly higher than that of MCF-7 /ADR cells,drug-resistant gene ABCB1,c-myc and ABCG2 expression decrease significantly after Adriamycin treatment.In addition,the apoptosis rate of each group with PTEN inhibitor does not increase significantly,and the expressions of ABCB1,c-myc and ABCG2 do not decrease significantly.It is suggested that SALL4 affects the resistance of Adriamycin in breast cancer cells through PTEN/AKT/mTOR pathway.Conclusions: SALL4 expression is higher in the breast cancer tissues than that of the paired noncancerous tissues,and it is correlated with lymphatic metastasis.Knockdown of SALL4 in breast cancer cells suppress cell proliferation,inhibit tumor growth in vivo,arrest cell cycle,promote cell apoptosis,as well as inhibite the migratory and invasive abilities of breast cancer cells,reduce the drug resistance of breast cancer cells to adriamycin.SALL4 affects the resistance of adriamycin in breast cancer cells through PTEN/AKT/mTOR pathway.This study provides a new theory for understanding the development and molecular mechanism of breast cancer,provides a potential new therapeutic target for breast cancer treatment.