The Functions And Mechanisms Of Long Non-coding RNA-ARHGAP5-AS1 In Breast Cancer Metastasis

Breast cancer is the most common malignant cancer in women.Metastasis is the leading cause of high mortality of breast cancer.Recent studies have shown that non-coding RNAs play an essential role in the initiation and progression of cancer.Our study focus on the non-coding RNAs involved in breast cancer metastasis.In the first part,we used RNA high-throughput sequencing to detect the transcriptome of triple-negative breast cancer cell line MDA-MB-231 and its high pulmonary metastasis subtype MDA-MB-231-LM2 cells.In the first selective method,MCF-7 cells were detected as a control,we identified 14 up-regulated LncRNAs in LM2 cells.By using a second selective method,we successfully identified 7down-regulated lncRNAs and 8 up-regulated lncRNAs for the following in vitro functional verification.The cell migration assay was used to detect the effect of the above 15 lncRNAs on the cell motility.The down-regulated lncRNA-ARHGAP5-AS1?NR₀27263?inhibited cell migration,so we studied the molecular mechanisms underlying the effects of ARHGAP5-AS1 on cell motility.In the second part,we detected diverse cellular function of Lnc-ARHGAP5-AS1,it inhibited cell migration and the formation of stress fibers in breast cancer cells.However,it had no effect on cell proliferation and colony formation.Then,we used Huprot^TM human protein array to identify the protein SMAD7,interacting with Lnc-ARHGAP5-AS1.The interaction between Lnc-ARHGAP5-AS1 and SMAD7was once again verified by RNA pull down assay and RNA immunoprecipitation assay.Knockdown or over-expression of Lnc-ARHGAP5-AS1 had no effect on the mRNA level of SMAD7.However,the protein level of SMAD7 was influenced by Lnc-ARHGAP5-AS1,suggesting that Lnc-ARHGAP5-AS1 could affect the degradation of SMAD7.Then,we used protein synthesis inhibitor CHX and protease inhibitor MG132 treated with cells to prove that Lnc-ARHGAP5-AS1 stabilizes SMAD7.Moreover,overexpression of Lnc-ARHGAP5-AS1 inhibited ubiquitination of SMAD7.Whether knockdown or over-expression of Lnc-ARHGAP5-AS1 has no effect on the cellular distribution of SMAD7,but it can influence the activation of TGF-?signaling.Knockdown of SMAD7 could simulate the function of knockdown of Lnc-ARHGAP5-AS1,which inhibits cell migration and the formation of stress fibers,suggesting that Lnc-ARHGAP5-AS1 might present its biological function via SMAD7.