Mechanism Study Of LncRNA AFAP1-AS1 Affects Breast Cancer Proliferation And Migration By Regulating MiR-21/PTEN Axis

Objectives:Searching specific molecular markers of breast cancer is the key to diagnosis and treatment of breast cancer.As a new target,long non-coding RNA has attracted more and more attention.Through bioinformatics analysis,we found that AFAP1-AS1 and miR-21 are potential markers of breast cancer proliferation and migration.The purpose of this study was to investigate the effect of AFAP1-AS1 on the proliferation and migration of breast cancer cells,to explore whether AFAP1-AS1 has a targeted regulation effect on miR-21,and to clarify whether the mechanism of AFAP1-AS1 involved in the process of breast cancer is related to the regulation of miR-21/PTEN axis,to find new molecular markers and effective targets for breast cancer treatment.Methods:Using bioinformatics analysis to search novel long non-coding RNA markers of breast cancer;The mRNA levels were detected by qRT-PCR.The protein levels were detected by Western blot assay.LF-2000 was used to transfer several sequences for breast cancer cells.MTT,clone formation and EdU assays were used to detect the proliferation of breast cancer cells.Wound healing and transwell assay were used to detect the migration ability of breast cancer cells.RNA pull down experiment was used to study the binding of AFAP1-AS 1 to miR-21.Double luciferase reporter assay was used to study the targeting effect of miR-21 on PTEN.The xenograft tumor model of breast cancer was constructed in nude mice to study the effect of AFAP1-AS1 stable knockdown on the growth of breast cancer cells in vivoResults:1.Bioinformatics analysis results showed that AFAP1-AS1 and miR-21 were abnormally expressed in breast cancer,and cell and human tissue levels verified that they were highly expressed in breast cancer tissues and breast cancer cells.2.The silencing of AFAP1-AS 1 significantly reduced proliferation and migration capacity of breast cancer cells MCF-7 and MDA-MB-231.3.The proliferation and migration of breast cancer cells after transfection with miR-21 mimic were significantly improved;The proliferation and migration of breast cancer cells were significantly decreased after transfection with miR-21 inhibitor.4.The expression of miR-21 decreased after AFAP1-AS1 was silenced;RNA pulldown results showed that AFAP1-AS 1 directly bound to miR-21.Increased the expression of miR-21 after silencing AFAP1-AS1,the proliferation and migration of breast cancer cells were reversed.5.Western blot and qRT-PCR showed that miR-21 had a negative regulatory effect on the mRNA and protein levels of PTEN.The luciferase reporter gene assay showed that miR-21 had a targeted regulatory effect on PTEN.Increasing the expression of PTEN after overexpression of miR-21,the effect of overexpression of miR-21 on breast cancer cells was reversed.Decreasing the expression of PTEN after low expression of miR-21,the effect of low expression of miR-21 on breast cancer cells was reversed.6.Silencing AFAP1-AS1 increased the mRNA and protein levels of PTEN.Increasing the expression of miR-21 after silencing AFAP1-AS1,the regulatory effect of AFAP1-AS1 on PTEN was reversed 7.Stable knockdown of AFAP1-AS1 with shRNA significantly slowed the growth of breast cancer cells in mice.Conclusions:1.AFAP1-AS1 and miR-21 were highly expressed in breast cancer cells and human breast cancer tissues,the expression level of AFAP1-AS1 in human breast cancer was positive correlation to the age of patients,which is expected to be a molecular marker and clinical treatment target for early screening of breast cancer patients.PTEN was low expressed in breast cancer tissue and cell lines.2.AFAP1-AS1 has a targeted regulatory effect on miR-21,and influences the proliferation and migration of breast cancer cells through miR-21.3.miR-21 has a targeted regulatory effect on PTEN,and AFAP1-AS1 regulates the tumor suppressor gene PTEN through miR-21,and ultimately participates in the process of cancer cell proliferation and migration.