LncRNA DLX6-AS1 Contributes to Epithelial-Mesenchymal Transition and Cisplatin Resistance in Triple-negative Breast Cancer via Modulating Mir-199b-5p/Paxillin Axis

Abstract

Triple-negative breast cancer (TNBC) is one of the most aggressive cancer types with high recurrence, metastasis, and drug resistance. Recent studies report that long noncoding RNAs (lncRNAs)-mediated competing endogenous RNAs (ceRNA) play an important role in tumorigenesis and drug resistance of TNBC. Although elevated lncRNA DLX6 antisense RNA 1 (DLX6-AS1) has been observed to promote carcinogenesis in various cancers, the role in TNBC remained unclear. In this study, expression levels of DLX6-AS1 were increased in TNBC tissues and cell lines when compared with normal tissues or breast fibroblast cells which were determined by quantitative real-time PCR (RT-qPCR). Then, CCK-8 assay, cell colony formation assay and western blot were performed in CAL-51 cells transfected with siRNAs of DLX6-AS1 or MDA-MB-231 cells transfected with DLX6-AS1 over expression plasmids. Knock down of DLX6-AS1 inhibited cell proliferation, epithelial-mesenchymal transition (EMT), decreased expression levels of BCL2 apoptosis regulator (Bcl-2), Snail family transcriptional repressor 1 (Snail) as well as N-cadherin and decreased expression levels of cleaved caspase-3, γ-catenin as well as E-cadherin, while up regulation of DLX6-AS1 had the opposite effect. Besides, knockdown of DLX6-AS1 in CAL-51 cells or up regulation of DLX6-AS1 in MDA-MB-231 cells also decreased or increased cisplatin resistance of those cells analyzed by MTT assay. Moreover, by using dual luciferase reporter assay, RNA immunoprecipitation and RNA pull down assay, a ceRNA which was consisted by lncRNA DLX6-AS1, microRNA-199b-5p (miR-199b-5p) and paxillin (PXN) was identified. And DLX6-AS1 function through miR-199b-5p/PXN in TNBC cells. Finally, results of xenograft experiments using nude mice showed that DLX6-AS1 regulated cell proliferation, EMT and cisplatin resistance by miR-199b-5p/PXN axis in vivo. In brief, DLX6-AS1 promoted cell proliferation, EMT, and cisplatin resistance through miR-199b-5p/PXN signaling in TNBC in vitro and in vivo.

Keywords: DLX6-AS1; ceRNA; miR-199b-5p; paxillin; triple-negative breast cancer.

Fig. 1.

Expression levels of DLX6-AS1 are increased in TNBC tissues and cells. (A) Expression levels of DLX6-AS1 in 47 TNBC tissues (tumor) and 28 adjacent normal tissues (normal) were determined by RT-qPCR. (B) Expression levels of DLX6-AS1 in different cell lines were determined by RT-qPCR (*, P < 0.05; **, P < 0.01). DLX6-AS1: DLX6 antisense RNA 1; RT-qPCR: quantitative real-time polymerase chain reaction; TNBC: triple-negative breast cancer.

Fig. 2.

Long noncoding RNA DLX6-AS1 regulates cell proliferation, epithelial–mesenchymal transition, and cell apoptosis in TNBC cells. (A) Expression levels of DLX6-AS1 in TNBC cells transfected with siRNAs or overexpression plasmids of DLX6-AS1 were determined by RT-qPCR. (B) Cell growth of CAL-51 cells transfected with siRNA (siDLX6-AS1) or control of siRNA (siNC) and MDA-MB-231 cells transfected with DLX6-AS1 overexpression plasmids (pcDNA3.1-DLX6-AS1) or control (pcDNA3.1-NC) were determined by Cell Counting Kit-8 assay. (C) Cell colony formation was performed. Representative pictures of cell colony are shown. Numbers of cell colony were counted. (D) Protein levels of Bcl-2, cleaved caspase-3, γ-catenin, Snail, E-cadherin, and N-cadherin in cells transfected with siRNA or overexpression plasmids of DLX6-AS1 were determined by western blot. Gray values of bands were analyzed by Image J and normalized to β-actin. (**, P < 0.01). DLX6-AS1: DLX6 antisense RNA 1; RT-qPCR: quantitative real-time polymerase chain reaction; TNBC: triple-negative breast cancer.

Fig. 3.

Long noncoding RNA DLX6-AS1 regulates cisplatin resistance of triple-negative breast cancer cells. (A) IC₅₀ of CAL-51 cells transfected with siDLX6-AS1 to cisplatin were determined by MTT assay. (B) Half-maximal inhibitory concentration (IC₅₀) of MDA-MB-231 cells transfected with DLX6-AS1 overexpression plasmids to cisplatin were determined by MTT assay. DLX6-AS1: DLX6 antisense RNA 1; IC₅₀: half-maximal inhibitory concentration; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Fig. 4.

ncRNA DLX6-AS1 binds with miR-199b-5p and regulates expression levels of miR-199-5p. (A) Predicted binding sites of miR-199b-5p on lncRNA DLX6-AS1 are shown. (B) Dual luciferase reporter assay was performed. Relative luciferase activities of the cells transfected with indicated lncRNA and mimics of miRNA were measured. (C) RNA immunoprecipitation was performed using anti-ago2 or anti-IgG primary antibodies. Relative expression levels of miR-199b-5p or lncRNA DLX6-AS1 in the immunoprecipitated complex were determined by RT-qPCR. (D) Expression levels of miR-199b-5p were determined by RT-qPCR in the complex pulled down by the biotin-labeled DLX6-AS1 probes. (E) Expression levels of miR-199b-5p were determined by RT-qPCR in the cells transfected with siDLX6-AS1 or siNC (**, P < 0.01). DLX6-AS1: DLX6 antisense RNA 1; IgG: immunoglobulin G; lncRNA: long noncoding RNA; miRNA: microRNA; MUT: mutated; RT-qPCR: quantitative real-time polymerase chain reaction; WT: wild type.

Fig. 5.

miR-199b-5p binds to 3′ UTR of PXN and regulated expression levels of PXN. (A) Predicted binding sites of miR-199b-5p on 3′ UTR of PXN were shown. (B) Dual luciferase reporter assay. Relative luciferase activities of the cells transfected with indicated plasmids and mimics of miRNA were measured. (C) Expression levels of miR-199b-5p were determined by RT-qPCR in CAL-51 cells transfected with mimics of miR-199b-5p or control (miR-NC). (D) mRNA levels of PXN were determined by RT-qPCR in the CAL-51 cells transfected with mimics of miR-199b-5p or miR-NC. (E) Protein levels of PXN were determined by western blot in the CAL-51 cells transfected with mimics of miR-199b-5p or miR-NC. Gray values of bands were analyzed by Image J and normalized to β-actin (**, P < 0.01). miRNA: microRNA; PXN: paxillin; RT-qPCR: quantitative real-time polymerase chain reaction; UTR: untranslated region; WT: wild type.

Fig. 6.

Long noncoding RNA DLX6-AS1 regulated cell proliferation, epithelial–mesenchymal transition, and cisplatin resistance through miR-199b-5p/PXN axis. (A) Cell growth was determined by Cell Counting Kit-8 assay. (B) Cell colony formation was performed. Representative pictures of cell colony are shown. Numbers of cell colony were counted. (C) Protein levels of PXN, Bcl-2, cleaved caspase-3, γ-catenin, Snail, E-cadherin, and N-cadherin in cells transfected with siRNA or inhibitor of miR-199b-5p were determined by western blot. Gray values of bands were analyzed by Image J and normalized to β-actin. (D) Transfection siRNAs or inhibitor of miR-199b-5p into cells affected cell viability in response to cisplatin (*, P < 0.05; **, P < 0.01). DLX6-AS1: DLX6 antisense RNA 1; PXN: paxillin; siRNA: .

Fig. 7.

Long noncoding RNA DLX6-AS1 regulated xenografted tumor growth in vivo. (A) Representative images of tumor xenografts and tumor growth curves are shown. Ad-shNC+PBS: negative control; Ad-shDLX6-AS1: tumors injected with adenovirus expressing shRNA of DLX6-AS1; Ad-shNC+DPP: applying with cisplatin; Ad-shDLX6-AS1+DPP: injected with adenovirus expressing shRNA of DLX6-AS1 combined with applying with cisplatin. (B) Protein levels of paxillin in xenografts were determined by western blot. Gray values of bands were analyzed by Image J and normalized to β-actin. (C) Representative images of ki67 immunohistochemical staining of xenografts are shown (magnification, 100× or 400×) (**, P < 0.01). DLX6-AS1: DLX6 antisense RNA 1; PBS: phosphate-buffered saline; shRNA: .