The Effect On MDA-MB-231 Cell Line In Response To LAPTM4B

Background: The occurrence and development of breast cancer is a very complex process,which is the result of a variety of abnormal genes.Lysosome-associated protein transmembrane 4 beta(LAPTM4B)is a potential oncogene.This gene can promote the growth and proliferation,migration and invasion of tumor cells,resistance to apoptosis and induce drug resistance.The LAPTM4B-35 protein is highly expressed in various types of solid tumors.The results which confirmed by literature show that the gene may be involved in the occurrence and development of breast cancer,affecting the prognosis of patients suffering breast cancer,and may provide reference value for the diagnosis and prognosis of breast cancer.It is suggested that LAPTM4 B may be a potential target for the treatment of breast cancer.P27 is a tumor suppressor gene,whose protein is a kind of cyclin dependent kinase inhibitor protein(CKIs).Through a variety of ways to participate in the regulation of cell cycle,to prevent the cell from G1 phase to S phase of the transition,inhibit cell division,proliferation,promote cell differentiation and apoptosis.A large number of research results show that,P27 expression was abnormal in many tumors,and may be of value in hormone therapy,radiotherapy and chemotherapy in patients suffering breast cancer.However,there are few reports about the relationship between LAPTM4 B and P27 in breast cancer.Therefore,studying on the development of LAPTM4 B and P27 in breast cancer,then guiding the clinical treatment of breast cancer is very valuable.Objective: TCGA database is used to investigate the expression of LAPTM4 B inbreast cancer and normal breast tissue,as well as in various subtypes of breast cancer.RT-PCR and Western Blot are used to detect the expression of LAPTM4 B in breast cancer cell lines.The effects of silencing LAPTM4 B on proliferation,apoptosis and migration of human breast cancer cell line MDA-MB-231 are observed by using small interfering RNA(si RNA)mediated gene silencing.In addition,the possible relationship between the two genes was explored by silencing LAPTM4 B and P27 gene,so as to provide a theoretical basis for the LAPTM4 B Gene as a target for the treatment of breast cancer.Methods: TCGA database was used to analyze the expression of LAPTM4 B in breast cancer and normal breast tissues,and its expression in various molecular subtypes of breast cancer.The si RNA-LAPTM4 B sequences and si RNA-P27 sequences were synthesized by chemical synthesis.The MDA-MB-231 cells transfected with si RNA-LAPTM4 B served as experimental group,The MDA-MB-231 cells transfected with si RNA-NC served as control group,cell proliferation was detected by MTT assay;Cell migration was measured by scratch test;Cell apoptosis was detected by flow cytometry.The si RNA was transfected into MDA-MB-231 cells by Lipofectamine 2000 Transfection Reagent.The expression of LAPTM4 B and P27 in m RNA and protein level were detected by RT-PCR and Western Blotting after transfection for 48 hours.Results:1.LAPTM4 B was highly expressed in breast cancer,and was highest in triple negative breast cancer.2.MTT assay indicated that the cell proliferation rate in si RNA-LAPTM4 B transfected group was notable decreased than blank control group.3.Flow cytometry revealed that the cell apoptosis rate in si RNA-LAPTM4 B transfected group was notable increased than blank control group.4.Wound healing test suggested that compare with the blank control group,the migration ability of si RNA-LAPTM4 B transfected group was clearly weakened.5.RT-PCR results show that the expression of LAPTM4 B m RNA in silent LAPTM4 B group was significantly lower than that in control group,while theexpression of P27 m RNA was lower than control groups.The expression of P27 m RNA in silent P27 group was significantly lower than that in control group,while the expression of LAPTM4 B m RNA was lower than control groups.6.Western Blot results show that the expression of LAPTM4B-35 protein in silent LAPTM4 B group was significantly lower than that in control group,while the expression of P27 protein was lower than control groups.The expression of P27 protein in silent P27 group was significantly lower than that in control group,while the expression of LAPTM4B-35 protein was lower than control groups.Conclusion:1.LAPTM4 B was highly expressed in breast cancer,and was highest in triple negative breast cancer.2.Silent LAPTM4 B can inhibit the proliferation,migration and promote apoptosis of human breast cancer MDA-MB-231 cell line.This reveals that LAPTM4 B gene might regulates the apoptosis,migration and proliferation via certain mechanism.3.Silencing of LAPTM4 B can reduce the expression of P27 in breast cancer MDA-MB-231 cells,and when P27 gene silencing,the expression of LAPTM4 B also decreased.This suggests that LAPTM4 B and P27 gene may regulate each other through certain signaling pathways or some genes.