| Breast cancer is the most commonly diagnosed malignancy among all cancers in women and the second most common cause of cancer-related deaths after lung Cancer.In2018,breast cancer caused 626,679 deaths,accounting for 6.6% of all cancer-related fatalities.In the same year,2088849 new cases of breast cancer were diagnosed,accounting for 11.6%.With the popularization of breast cancer screening programs and development of novel anti-cancer therapies,breast cancer incidence and mortality rates have dropped significantly.However,the breast cancer screening rate is still very low in developing countries,such as China.Breast cancer is a highly heterogeneous group of tumors with different pathogenesis and relatively different prognosis.Therefore,different clinical treatment options are required.Triple-negative breast cancer(TNBC)is a molecular subtype of breast cancer,compared with the other subtypes,which cannot benefit from endocrine therapy and HER2-targeted therapy due to lack of hormone receptors and HER2 expression.Therefore,TNBC patients generally have the worst prognosis.Research on the molecular pathogenesis of TNBC has revealed that a considerable number of molecules are involved in the disease,and the functional characteristics of these molecules determine whether they can be new targets for anti-TNBC therapies.Long non-coding RNAs(lnc RNAs)do not encode proteins,but they regulate cancer-related gene expression and participate in cancer biological processes.Therefore,the study of lnc RNA-related targeted therapy has attracted the attention of more and more researchers.Accumulating evidence also suggests that these noncoding transcripts are involved in a wide range of cellular processes,including cell proliferation,invasion,metastasis,apoptosis,differentiation,and immune responses,all of which are involved in tumorigenesis.Li et al.reported that the lnc RNA HEIH regulates cell proliferation and apoptosis through the miR-4458/SOCS1 axis in TNBC.Previous studies have also shown that lnc RNA GATA3-AS1 promotes TNBC progression and immune evasion by stabilizing PD-L1 protein and degrading GATA3 protein.However,the functions of most lnc RNAs in cancer biology remain unclear.A recent study showed that the lnc RNA SLC16A1-AS1 was downregulated in lung cancer tissues and became a predictor of good prognosis.We analyzed the TCGA database and found that the expression of lnc RNA SLC16A1-AS1 was also significantly downregulated in triple-negative breast cancer.From this,we speculate that the lnc RNA SLC16A1-AS1 is likely to be involved in the regulation of TNBC progression.Objective:This study aimed to analyze the biological processes involved in the regulation of lnc RNA SLC16A1-AS1 in TNBC,and the possible molecular mechanisms.Methods:1.The TCGA data were analyzed using the GEPIA2(https://gepia2.cancer.pku.cn/#index)database to predict the differential expression of lnc RNA SLC16A1-AS1 in TNBC.Potential target miRNAs of lnc RNA SLC16A1-AS1 were predicted using Reg RNA2 and miRanda databases.Potential target genes of miR-182 were predicted using the miRPath DB,miRDB,miRWalk and Target Scan databases.Inta RNA2.0(http://rna.informatik.uni-freiburg.de/Inta RNA/Input.jsp)was used to predict the possible complementary base pairs formed by lnc RNA SLC16A1-AS1/miR-182,miR-182/PDCD4 m RNA.2.Using Real Time Quantitative Polymerase Chain Reaction(RT-q PCR)to measure the expression levels of lnc RNA SLC16A1-AS1,miR-182 and PDCD4 in paired TNBC tumor tissues/paratumor tissues,nude mouse xenografts and TNBC cell lines.3.Using RNA fluorescence in situ hybridization(Fluoacence in situ hybridization,FISH)and nucleocytoplasmic separation experiments to clarify the sub-localization of lnc RNA SLC16A1-AS1 in TNBC cell line.4.The binding capacity of lnc RNA SLC16A1-AS1/miR-182 and miR-182/PDCD4 m RNA was verified by dual luciferase activity assay and RNA immunoprecipitation(RIP)experiment.5.Western Blot was used to detect the expression of PDCD4 in postoperative paired TNBC tumor tissue/paratumor tissue,nude mouse xenografts and TNBC cell lines;to verify the mutual regulatory relationship between lnc RNA SLC16A1-AS1,miR-182 and PDCD4;to verify the regulatory effect of lnc RNA SLC16A1-AS1 on epithelial-mesenchymal tansition(EMT);to verify the regulatory effect of PDCD4 on autophagy.6.The effects of lnc RNA SLC16A1-AS1,miR-182 and PDCD4 on TNBC cell cycle progression were detected by flow cytometry.7.Cell Counting Kit-8(CCK-8)(Dojindo,Japan)was used to detect the effects of lnc RNA SLC16A1-AS1,miR-182 and PDCD4 on the proliferation of TNBC cells and IC50 values of TNBC cells to three chemotherapy drugs.8.Colony formation assay was used to detect the effects of lnc RNA SLC16A1-AS1,miR-182 and PDCD4 on the proliferation of TNBC cells.9.The effects of lnc RNA SLC16A1-AS1,miR-182 and PDCD4 on the migration and invasion ability of TNBC cells were detected by Transwell(matrigel-free/matrigel-laying)assay.10.Immunohistochemical staining was used to detect PDCD4 expression in postoperative paired TNBC tumor tissue/paratumor tissue,nude mouse xenografts and Ki67 index in nude mouse xenografts.11.In vivo experiment(subcutaneous xenografts in nude mice)confirmed that lnc RNA SLC16A1-AS1 targeted miR-182/PDCD4 axis to promote TNBC proliferation.Results: 1.The expression of lnc RNA SLC16A1-AS1 was significantly down-regulated in TNBC,negatively correlated with tumor histological grade and regional lymph node metastasis,and correlated with good prognosis.2.Overexpression of lnc RNA SLC16A1-AS1 inhibits TNBC cell proliferation,migration,invasion,EMT,and induces cell cycle arrest in G0/G1 phase.3.miR-182 is a tumor-promoting gene of TNBC,and lnc RNA SLC16A1-AS1 acts as a molecular sponge of miR-182 to inhibit its tumor-promoting effect.4.PDCD4 is a tumor suppressor gene of TNBC.miR-182 targets to inhibit the expression of PDCD4.Lnc RNA SLC16A1-AS1 competitively occupies the binding site of PDCD4 and miR-182,weakening the inhibitory effect of miR-182 on PDCD4,thereby inhibiting TNBC progress.5.There is a negative regulatory relationship between PDCD4 and autophagy-related markers(LC3B and ATG5).PDCD4 may inhibit the progression of TNBC by inhibiting autophagy.6.The lnc RNA SLC16A1-AS1 targeting the miR-182/PDCD4 axis increases the sensitivity of TNBC cells to chemotherapeutic drugs.7.In vivo experiments(subcutaneous xenografts in nude mice)confirmed that lnc RNA SLC16A1-AS1 targeted miR-182/PDCD4 axis to suppress TNBC proliferation.Conclusion:The lnc RNA SLC16A1-AS1 targeted the miR-182/PDCD4 axis to inhibit TNBC proliferation,migration,invasion,induce cell cycle G0/G1 arrest,and improve its sensitivity to chemotherapeutics. |
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