LncRNA-408 Promotes Human Breast Cancer Cell Migration By Regulating Lim Domain Kinase 1(LIMK1)

Objective: to study the effect of LncRNA-408 and its target gene LIMK1 on human breast cancer cell migration and the molecular mechanism among the process.Methods:(1)Bioinformatics analyze was used to screen differently expressed LncRNAs before and after the EMT transformation of MCF-7,and the expression of LncRNA-408 in clinical samples was verified by qRT-PCR.(2)The expression level of LncRNA-408 in different BC cell lines was detected by qRT-PCR.Cell lines with LncRNA-408 knockdown were established by lentivirus transfection of BT549 and HS578 T,and cell lines with LncRNA-408 overexpression were established by lentivirus transfection of MCF-7 and SKBr-3.Wound healing and Transwell cell migration assay was used to detect the cell migration ability in each group.(3)Extract RNA from separation of nuclear and cytoplasmic fractions of BC cells and detect the expression of LncRNA-408 by qRT-PCR.The expression of target microRNAs and LIMK1 in the LncRNA-408 knockdown/overexpression group was detected by qRT-PCR.The expression of LncRNA-408 and LIMK1 in HS578 T transfected with miRNA-654-5p NC/mimics and MCF-7 transfected with miRNA-654-5p NC/inhibitor were detected by qRT-PCR.WB method was used to detect LIMK1 protein expression in each treatment group.The correlation between LIMK1 and LncRNA-408 expression levels in 30 clinical samples was detected by qRT-PCR.The bindings between LncRNA-408 and miR-654-5p,miR-654-5p and LIMK1 were verified by luciferase reporting experiment,respectively.(4)Inquire the survival analysis of breast cancer patients in the group with high/low LIMK1 expression.qRT-PCR was used to detect the expression level of LIMK1 in breast cancer patients and BC cells of LIMK1 knockdown/ overexpression groups.The expressions of LIMK1,p-cofilin and cofilin were detected by WB.The cells of each group were stained by TRITC Phalloidin and their migration abilities were detected by Transwell assay.(5)The expression levels of LncRNA-408 and LIMK1 in HS578T(scramble sh+veh oe/ Lnc-408 sh+veh oe/ Lnc-408 sh+LIMK1 oe)and MCF-7(veh oe+scramble sh/ Lnc-408 oe+LIMK1 sh)were detected by qRT-PCR.The expressions of LIMK1,p-cofilin and cofilin were detected by WB.The cells of each group were stained by TRITC Phalloidin and their migration abilities were detected by Transwell assay.Results:(1)LncRNA-408 was highly expressed in EMT-treated breast cancer cells.The expression level of LncRNA-408 in the samples of patients with positive lymph node metastasis(N=19)was significantly higher than that without lymph node metastasis(N=11)and the expression level increased with the improvement of breast cancer grade and stage.(2)The expression level of LncRNA-408 increased with the enhancement of malignant degree of human breast cancer cell lines.The BT549 and HS578 T cell with LncRNA-408 knockdown and the MCF-7 and SKBr-3 cell with LncRNA-408 overexpression were successfully established.After LncRNA-408 was knocked down,the migration ability of cells was significantly weakened,but significantly enhanced after overexpression.(3)LncRNA-408 was mainly distributed in the cytoplasm of breast cancer cells.The expression of miR-654-5p was increased/ decreased with the knockdown/ overexpression of LncRNA-408.The expression of LncRNA-408 was decreased/increased after miR-654-5p was overexpression/ knockdown.LIMK1 expression(RNA and protein level)decreased/ increased after LncRNA-408 was knockdown/ overexpressed,and decreased/ increased after miR-654-5p was knockdown/ overexpressed.LncRNA-408 was positively correlated with the RNA expression level of LIMK1 in samples of BC patients.Luciferase reporting assay confirmed that LncRNA-408 was bound to miR-654-5p,and miR-654-5p was bound to LIMK1.(4)LIMK1 is associated with poor prognosis of BC patients.LIMK1 expression level in lymph node metastasis positive patients(N=19)specimens was significantly higher than those in the negative patients(N=11),that in stageⅡ(N=10)and stageⅢ(N=14)was significantly higher than that in stageⅠ(N=6),that in G2(N=13)and G3(N=9)were significantly higher than in the G1(N=8).HS578 T with LIMK1 knockdown and MCF-7 with LIMK1 overexpressed cell lines were successfully established.The expression of p-cofilin decreased/ increased after the knockdown/ overexpression of LIMK1.BC’s cytoskeleton was weakened and diffused/ stabilized after LIMK1 knockdown/ overexpression.The migration ability of BC cells was decreased/ enhanced after the knockdown/overexpression of LIMK1.(5)Successfully established HS578 T with LncRNA-408 knockdown +LIMK1 overexpression and MCF-7 with LncRNA-408 overexpression +LIMK1 knockdown.The expression of p-cofilin changed with the change of LIMK1.Both cytoskeleton status and migration capacity of breast cancer were recovered with the recovery of LIMK1 expression level.Conclusion:(1)The high expression of LncRNA-408 is associated with invasion and metastasis of human breast cancer.(2)The LncRNA-408 promotes BC cell migration.(3)The LncRNA-408 regulates the expression of LIMK1.(4)LIMK1 promotes BC cell migration by p-cofilin/F-actin pathway.(5)The LncRNA-408 enhances cell migration ability of BC cells by regulating the expression of LIMK1.