Study On LncRNA DARS-AS1 Binding With PCBP1 In The Positive Feedback Regulation Of HIF Pathway To Promote Breast Cancer Metastasis And Cell Function Of ALYREF In Breast Cancer

This paper aims to study the molecular mechanism of breast cancer and find potential molecular targets for breast cancer treatment.We analyzed the breast cancer hypoxic expression profile and screened hypoxia related lncRNAs.The analysis results showed that lncRNA DARS-AS1 and lncRNA MIR210 HG were significantly upregulated under hypoxia.qRT-PCR assay verified that lncRNA DARS-AS1 and lncRNA MIR210 HG were expressed in breast cancer and other tumor cells,and were significantly upregulated under hypoxia indeed.We used RACE technology to get the end sequences of lncRNA DARS-AS1 and lncRNA MIR210 HG.Full length of lncRNA DARS-AS1 and lncRNA MIR210 HG was amplified by PCR assay,and their full length are 1095 nt and 2303 nt.We analyzed the TCGA database about RBPs in breast cancer,and ALYREF caught our attention.We found that lncRNA DARS-AS1,lncRNA MIR210 HG and ALYREF were associated with progression of breast cancer and other tumors by analyzing the data in Kaplan Meier plotter database and TCGA database.We silenced target gene by RNAi technology and overexpressed the target gene with lentivirus to conduct cellular functional experiments,so that we can observe the changes of migration,invasion and proliferation of tumor cell.We found that the silencing lncRNA DARS-AS1 significantly inhibited the migration and invasion ability of tumor cells under hypoxia,and overexpression of lncRNA DARS-AS1 significantly promoted the migration and invasion ability of tumor cells under hypoxia by using Transwell assay.We found that silencing ALYREF significantly inhibited the proliferation of breast cancer cells by using Clonal formation assay and CCK-8 assay.Flow analysis showed that silencing ALYREF significantly increased apoptosis and inhibited cell cycle of breast cancer.Western blot and qRT-PCR revealed that lncRNA DARS-AS1 and HIF-1α were dose-dependent.HIF-1α was found to bind the promoter region of lncRNA DARS-AS1 by using ChIP-qPCR assay.Therefore,HIF-1α promoted the expression of lncRNA DARS-AS1.We found that silencing lncRNA DARS-AS1 downregulated HIF-1α protein.The nuclear/cytoplasm fractionation assay and qRT-PCR assay showed that lncRNA DARS-AS1 entered the nuclear and functioned under hypoxia.RNA pull-down assay and mass spectrometry revealed that lncRNA DARS-AS1 binded PCBP1 under hypoxia.By using western blot assay,we found that lncRNA DARS-AS1 indeed binded PCBP1 under hypoxia.We concluded that lncRNA DARS-AS1,lncRNA MIR210 HG and ALYREF may be new targets for the treatment of breast cancer.lncRNA DARS-AS1 binding PCBP1 regulates HIF pathway to promote the metastasis of breast cancer,and it is positive feedback.It provides a new direction for studying the molecular mechanism of breast cancer.