CircRAD18 Promote The Proliferation Of Triple-Negative Breast Cancer By Regulating CDK6 Expression

Objective: Triple negative breast cancer is an aggressive tumor of all breast cancer.Due to absence of the expression of estrogen receptor and the amplification of HER1/neu gene,patients with TNBC have to choose chemotherapy.However,because of the resistance to chemotherapeutic drugs,patients with TNBC generally have the poor prognosis,high recurrence rate and metastatic ability.Recent studies have found that circRNAs play an important role in tumorigenesis,but the role of circRNAs in TNBC is not yet clear.Method:High throughput CircRNAsMicroarray analysis was used to screen the circRNAs of three negative adult breast cancer patients,non-cancer tissues and TNBC cell lines MDA-MB-231,MDA-MB-468,BT549,normal breast epithelial cells-MCF-10 A.Design circRAD18 specific primers paired with bcak-splicing junction,Sanger sequencing verified the sequence.bioinformatics predicted the possible miRNAs targeted the circRAD18.Targetscan,miRBase predicted miR-1299,mi R-498 co-target genes.Dual luciferase reporter gene assay was used to verify targeting relationship of mi R-1299,miR-498 and CDK6.qRT-PCR detecte the expression of circRAD18,CDK6,mi R-1299,miR-498 RNA.EdU proliferation assay is used to detect cell proliferation.Apoptosis Apoptosis assay detecte the apoptosis of the cells.Colony formation assay detect the ability of cell colony formation.The invasiveness of TNBC cell is verified by Transwell assay.Cell cycle assay was used to detect the change of the cycle.Western blot is used to detect the expression of CDK6,cyclinD1 and pRb.RESULTS: Microarray analysis screened the differentially expressed circRNAs in three negative breast cancer tissues and cells,and circRAD18 was upregulated in TNBC tissues and cell lines(differentiation >2)and then wo named it circRAD18.qRT-PCR results showed that compared to normal breast cancer cell MCF-10 A and non-TNBC cell lines(BT474,MCF-7,and Skbr3),circRAD18 was significant upregulated in TNBC cell line(HCC38,MDA-MB-468,BT549,MDA-MB-231,p<0.05,n=3).we use the qRT-PCR to detect the the highest interference efficiency of the three interfering sequences of circRAD18 and fined that si-circRAD-s1.In TNBC cell lines,loss of the expression of circRAD18 is significantly decreased cell proliferation,colony formation,and invasion and induced apoptosis.Bioinformatics analysis found that circRAD18 had higher binding rates with miR-1299 and mi R-498.qRT-PCR found that knockdown of circRAD18 can reduce the expression of miR-1299 and mi R-498.The target binding site between miR-1299/miR-498 and CDK6 was predicted by Targetscan and miRbase are used to predicted the target and found that they could co-target CDK6.The dual luciferase reporter gene assay confirm that miR-1299 and miR-498 could target CDK6,and co-transfected the mimics of miR-1299 and miR-498 with the better effect.qRT-PCT showed that the expression of CDK6 was increased after transfecting the inhibitors of mi R-1299 and miR-498,and the expression of CDK6 was decreased after transfecting the mimics miR-1299 and miR-498.The effect of co-transfection with mi R-1299 and miR-498 was more significant.Next,following the co-transfection of inhibitors or mimics of miR-1299 and miR-498,Ed U proliferation experiments revealed that inhibit the expression of miR-1299 and mi R-498 can promote the proliferation of TNBC cells,and over-expression the miR-1299 and miR-498 inhibited the proliferation of TNBC cells.The cell cycle showed?inhibiting the expression of miR-1299 and mi R-498 can promote cell cycle progression to G2/S phase,and overexpression can block cell cycle in G1 phase.Western blot reveal that expression of CDK6,cyclinD1,and pRb elevated after miR-1299/miR-498 inhibition And over-expression inhibited the expression of CDK6,cyclinD1,and pRb proteins.Then co-transfected with si-circRAD18,the inhibitors of mi R-1299/miR-498,qRT-PCR found that co-transfected si-circRAD18 and the inhibitors of miR-1299/miR-498 can restore si-circRAD18-induced down-regulation of CDK6 mRNA level.EdU proliferation experiments showed that co-transfection of si-circRAD18 and the inhibitors of mi R-1299/miR-498 can restore inhibition of TNBC cell proliferation induced by si-circRAD alone.Cell cycle experiments found that co-transfection of si-circRAD18 and the inhibitors of miR-1299 and miR-498 were able to restore cell cycle arrest induced by si-crcRAD18 in G1 phase.Western blot experiments showed that co-transfected with si-circRAD18 and the inhibitors of miR-1299/miR-498 could restore tne low expression level of CDK6,cyclinD1,and pRb caused by si-circRAD18.Conclusions: 1.circRAD18 is up-regulated in TNBC tissues and cells;2.CircRAD18 was highly expressed in STNBC cell lines,but there was no difference in normal breast cells and other breast cancer cells.3.The silence of circRAD18 can inhibit the proliferation,colony formation and invasive ability of TNBC,and increase the apoptosis of cells.4.CircRAD18 can down-regulate the expression of miR-1299 and mi R-498;5.Mi R-1299,miR-498 can inhibit cell cycle progression by co-targeting CDK6;6.CircRAD18 regulates CDK6 thereby promoting the proliferation of TNBC cells by down-regulating the expression of miR-1299 and miR-498.