doi: 10.1111/1759-7714.13283.
Epub 2020 Jan 4.
Knockdown of long noncoding RNA TP73-AS1 suppresses the malignant progression of breast cancer cells in vitro through targeting miRNA-125a-3p/metadherin axis
Affiliations
- PMID: 31901156
- PMCID: PMC6996984
- DOI: 10.1111/1759-7714.13283
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Knockdown of long noncoding RNA TP73-AS1 suppresses the malignant progression of breast cancer cells in vitro through targeting miRNA-125a-3p/metadherin axis
Thorac Cancer.
2020 Feb.
Abstract
Background: TP73 antisense RNA 1 (TP73-AS1) is a long noncoding RNA which has been shown to be involved in the progression of multiple malignant tumors. Previous studies have demonstrated the oncogenic role of TP73-AS1 in breast cancer. However, its molecular mechanism remains largely unknown in breast tumorigenesis.
Methods: Expression of TP63-AS1, miRNA-125a-3p (miR-125a) and metadherin (MTDH) was detected by real-time quantitative PCR and western blotting. The malignancy was evaluated by cell counting kit 8 (CCK-8), transwell assays, flow cytometry and western blotting. The target binding was confirmed by dual luciferase reporter assay. Xenograft tumor model was performed to detect tumor growth in vivo.
Results: Expression of TP73-AS1 was higher in breast cancer tissues and cell lines. Biologically, its knockdown could promote cell apoptosis rate, and inhibit proliferative capacity, migration and invasion ability in HCC-70 and MB231 cells, accompanied with higher cleaved caspase 3 level and lower Ki67, N-cadherin and Vimentin level. Moreover, TP73-AS1 downregulation restrained the tumor growth of HCC-70 cells in vivo. Mechanically, TP73-AS1 functioned as a molecular "sponge" for miR-125a to modulate MTDH, a downstream target of miR-125a. Intriguingly, both miR-125a overexpression and MTDH silencing exerted a tumor-suppressive effect in the malignant progression of HCC-70 and MB231 cells, which was counteracted by TP73-AS1 upregulation and miR-125a downregulation, respectively.
Conclusion: Knockdown of TP73-AS1 inhibited cell proliferation, migration and invasion, but facilitated apoptosis in breast cancer cells in vitro through targeting miR-125a and upregulating MTDH, suggesting a novel TP73-AS1/miR-125a/MTDH pathway in the malignant progression of breast cancer.
Keywords: Breast cancer; MTDH; TP73-AS1; malignant progression; miR-125a.
© 2019 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.
Figures
Expression of lncRNA TP73 antisense RNA 1 (TP73‐AS1) in breast cancer tissues and cell lines. (a) RT‐qPCR detected TP73‐AS1 expression level in paired tumor tissues (Cancer) and adjacent normal tissues (Normal) from breast cancer patients (n = 45). Fold change was analyzed using the formula 2−ΔΔCT. (b) RT‐qPCR estimated TP73‐AS1 level in human breast cancer cell lines (HCC‐70 and MB231) and the normal breast cell line MCF‐10A. Data represent mean ± standard error of the mean (SEM) and *P < 0.05.
The effect of TP73‐AS1 knockdown on the malignant progression of breast cancer cells in vitro. HCC‐70 and MB231 cells were transfected with siRNA against TP73‐AS1 (si‐TP73‐AS1) or its control (si‐control). (a) RT‐qPCR was used to analyze TP73‐AS1 level after transfection for 24 hours. (b, c) Cell counting kit 8 (CCK‐8) was utilized to determine cell proliferative capacity after transfection at 0 hour, 24 hours, 48 hours and 72 hours. (C) Flow cytometry was conducted to examine apoptosis rate after transfection at 24 hours. The percentage of apoptotic cells in quadrants of Annexin V+/PI− and Annexin V+/PI+ was statistically recorded. (d, e) Transwell assays were performed to evaluate cell migration and invasion abilities at 24 hours. The number of migrated cells and invaded cells was statistically recorded. (f) Western blotting was implemented to test protein expression of Ki67, cleaved caspase‐3, N‐cadherin and Vimentin after transfection at 24 hours. Protein bands of western blotting were quantified by densitometry and presented as fold changes with normalization to β‐actin. Data represent mean ± SEM and *P < 0.05. (a) (
) si‐control and (
) si‐TP73‐AS1. (b) HCC (
) si‐control and (
) si‐TP73‐AS1. MB231 (
) si‐control and (
) si‐TP73‐AS1. (c–e) (
) si‐control and (
) si‐TP73‐AS1. (f) HCC‐70 (
) si‐control and (
) si‐TP73‐AS1. MB231 (
) si‐control and (
) si‐TP73‐AS1.
) si‐control and () si‐TP73‐AS1. (b) HCC () si‐control and () si‐TP73‐AS1. MB231 () si‐control and () si‐TP73‐AS1. (c–e) () si‐control and () si‐TP73‐AS1. (f) HCC‐70 () si‐control and () si‐TP73‐AS1. MB231 () si‐control and () si‐TP73‐AS1.
TP73‐AS1 regulated miRNA‐125a‐3p (miR‐125a) expression by targeting. (a) Prediction of binding sites between TP73‐AS1 and miR‐125a (in the box) were shown according to starbase Tools. The corresponding mutant of TP73‐AS1 (MUT‐TP73‐AS1) was presented as well. (b) Luciferase activity of wild‐type of TP73‐AS1 (WT‐TP73‐AS1) and MUT‐TP73‐AS1 was confirmed by dual luciferase reporter assay in HCC‐70 and MB231 cells when cotransfected with miR‐125a mimic (miR‐125a) or its control (miR‐control). HCC (
) miR‐control and (
) miR‐125a. MB231 (
) miR‐control and (
) miR‐125a. (c) RT‐qPCR measured level of miR‐125a in breast cancer tissues (n = 45) compared with the paired normal tissues. (d) Spearman's rank correlation analysis clarified the association between miR‐125a and TP73‐AS1 expression in breast cancer tissues (n = 45). (e) RT‐qPCR measured miR‐125a level in breast cancer cell lines (HCC‐70 and MB231) comparing to the normal cell line MCF‐10A. (f) RT‐qPCR determined the transfection efficiency of pIRES2‐EGFP empty vector (vector) and recombinant vector containing TP73‐AS1 (TP73‐AS1) in HCC‐70 and MB231 cells. (
) Vector and (
) TP73‐AS1. (g) RT‐qPCR detected miR‐125a expression level in HCC‐70 and MB231 cells when transfected with si‐TP73‐AS1, si‐control, TP73‐AS1 and vector. Data represent mean ± SEM and *P < 0.05.
) miR‐control and () miR‐125a. MB231 () miR‐control and () miR‐125a. (c) RT‐qPCR measured level of miR‐125a in breast cancer tissues (n = 45) compared with the paired normal tissues. (d) Spearman's rank correlation analysis clarified the association between miR‐125a and TP73‐AS1 expression in breast cancer tissues (n = 45). (e) RT‐qPCR measured miR‐125a level in breast cancer cell lines (HCC‐70 and MB231) comparing to the normal cell line MCF‐10A. (f) RT‐qPCR determined the transfection efficiency of pIRES2‐EGFP empty vector (vector) and recombinant vector containing TP73‐AS1 (TP73‐AS1) in HCC‐70 and MB231 cells. () Vector and () TP73‐AS1. (g) RT‐qPCR detected miR‐125a expression level in HCC‐70 and MB231 cells when transfected with si‐TP73‐AS1, si‐control, TP73‐AS1 and vector. Data represent mean ± SEM and *P < 0.05.
The influence of TP73‐AS1 upregulation on the role of miR‐125a in breast cancer cell malignancy in vitro. (a) RT‐qPCR determined the transfection efficiency of miR‐125a and miR‐control in HCC‐70 and MB231 cells. (
) miR‐control and (
) miR‐125a. (b–f) HCC‐70 and MB231 cells were transfected with miR‐125a or miR‐control, and cotransfected with miR‐125a and either TP73‐AS1 or vector. (b) CCK‐8 determined cell proliferative capacity after transfection at 0 hour, 24 hours, 48 hours and 72 hours. HCC‐70 (
) miR‐control, (
) miR‐125a, (
) miR‐125a+vector and (
) miR‐125a+TP73‐AS1. MB231 (
) miR‐control, (
) miR‐125a, (
) miR‐125a+vector and (
) miR‐125a+TP73‐AS1. (c) Flow cytometry examined apoptosis rate after transfection at 24 hours. (
) miR‐control, (
) miR‐125a, (
) miR‐125a+vector and (
) miR‐125a+TP73‐AS1. (d, e) Transwell assays were performed to evaluate cell migration and invasion abilities at 24 hours. (
) miR‐control, (
) miR‐125a, (
) miR‐125a+vector and (
) miR‐125a+TP73‐AS1. (f, g) Western blotting tested protein expression of Ki67, cleaved caspase‐3, N‐cadherin and Vimentin after transfection at 24 hours. (f) HCC‐70 (
) miR‐control, (
) miR‐125a, (
) miR‐125a+vector and (
) miR‐125a+TP73‐AS1. (g) MB231 (
) miR‐control, (
) miR‐125a, (
) miR‐125a+vector and (
) miR‐125a+TP73‐AS1. Data represent mean ± SEM and *P < 0.05.
) miR‐control and () miR‐125a. (b–f) HCC‐70 and MB231 cells were transfected with miR‐125a or miR‐control, and cotransfected with miR‐125a and either TP73‐AS1 or vector. (b) CCK‐8 determined cell proliferative capacity after transfection at 0 hour, 24 hours, 48 hours and 72 hours. HCC‐70 () miR‐control, () miR‐125a, () miR‐125a+vector and () miR‐125a+TP73‐AS1. MB231 () miR‐control, () miR‐125a, () miR‐125a+vector and () miR‐125a+TP73‐AS1. (c) Flow cytometry examined apoptosis rate after transfection at 24 hours. () miR‐control, () miR‐125a, () miR‐125a+vector and () miR‐125a+TP73‐AS1. (d, e) Transwell assays were performed to evaluate cell migration and invasion abilities at 24 hours. () miR‐control, () miR‐125a, () miR‐125a+vector and () miR‐125a+TP73‐AS1. (f, g) Western blotting tested protein expression of Ki67, cleaved caspase‐3, N‐cadherin and Vimentin after transfection at 24 hours. (f) HCC‐70 () miR‐control, () miR‐125a, () miR‐125a+vector and () miR‐125a+TP73‐AS1. (g) MB231 () miR‐control, () miR‐125a, () miR‐125a+vector and () miR‐125a+TP73‐AS1. Data represent mean ± SEM and *P < 0.05.
Metadherin (MTDH) was a downstream target gene of miR‐125a. (a) Prediction of binding sites between miR‐125a and 3′ UTR of MTDH (in the box) were shown according to TargetScan tools. The corresponding mutant of MTDH 3′ UTR (MUT‐MTDH) was also presented. (b) Luciferase activity of wild‐type of MTDH (WT‐MTDH) and MUT‐MTDH was confirmed by dual luciferase reporter assay in HCC‐70 and MB231 cells when cotransfected with miR‐125a or miR‐control. HCC‐70 (
) miR‐control and (
) miR‐125a. MB231 (
) miR‐control and (
) miR‐125a. (c) RT‐qPCR measured MTDH mRNA level in breast cancer tissues (n = 45) compared with the paired normal tissues. (d) Spearman's rank correlation analysis clarified the association between miR‐125a and MTDH expression in breast cancer tissues (n = 45). (e, f) RT‐qPCR and western blotting measured MTDH levels in breast cancer cell lines (HCC‐70 and MB231) comparing to MCF‐10A. (g) RT‐qPCR determined the transfection efficiency of miR‐125a inhibitor (anti‐miR‐125a) and its control (anticontrol) in HCC‐70 and MB231 cells. (
) anticontrol and (
) anti‐miR‐125a. (h, i) RT‐qPCR and western blotting detected MTDH expression levels in HCC‐70 and MB231 cells when transfected with miR‐125a, miR‐control, anti‐miR‐125a and anticontrol. (
) miR‐control, (
) miR‐125a, (
) anticontrol and (
) anti‐miR‐125a. Data represent mean ± SEM and *P < 0.05.
) miR‐control and () miR‐125a. MB231 () miR‐control and () miR‐125a. (c) RT‐qPCR measured MTDH mRNA level in breast cancer tissues (n = 45) compared with the paired normal tissues. (d) Spearman's rank correlation analysis clarified the association between miR‐125a and MTDH expression in breast cancer tissues (n = 45). (e, f) RT‐qPCR and western blotting measured MTDH levels in breast cancer cell lines (HCC‐70 and MB231) comparing to MCF‐10A. (g) RT‐qPCR determined the transfection efficiency of miR‐125a inhibitor (anti‐miR‐125a) and its control (anticontrol) in HCC‐70 and MB231 cells. () anticontrol and () anti‐miR‐125a. (h, i) RT‐qPCR and western blotting detected MTDH expression levels in HCC‐70 and MB231 cells when transfected with miR‐125a, miR‐control, anti‐miR‐125a and anticontrol. () miR‐control, () miR‐125a, () anticontrol and () anti‐miR‐125a. Data represent mean ± SEM and *P < 0.05.
The impact of miR‐125a silencing on the role of MTDH downregulation in breast cancer cell malignancy in vitro. (a, b) RT‐qPCR and western blotting determined the transfection efficiency of siRNA against MTDH (si‐MTDH) and si‐control in HCC‐70 and MB231 cells. (
) si‐control and (
) si‐MTDH. (c–g) HCC‐70 and MB231 cells were transfected with si‐control or si‐MTDH, and cotransfected with si‐MTDH and either anticontrol or anti‐miR‐125a. (c) CCK‐8 determined cell proliferative capacity after transfection at 0 hour, 24 hours, 48 hours and 72 hours. HCC‐70 (
) si‐control, (
) si‐MTDH, (
) si‐MTDH+anticontrol and (
) si‐MTDH+anti‐miR‐125a. MB231 (
) si‐control, (
) si‐MTDH, (
) si‐MTDH+anticontrol and (
) si‐MTDH+anti‐miR‐125a. (d) Flow cytometry examined apoptosis rate after transfection at 24 hours. (
) si‐control, (
) si‐MTDH, (
) si‐MTDH+anticontrol and (
) si‐MTDH+anti‐miR‐125a. (e, f) Transwell assays were performed to evaluate cell migration and invasion abilities at 24 hours. (
) si‐control, (
) si‐MTDH, (
) si‐MTDH+anticontrol and (
) si‐MTDH+anti‐miR‐125a. (g) Western blotting tested protein expression of Ki67, cleaved caspase‐3, N‐cadherin and Vimentin after transfection at 24 hours. HCC‐70 (
) si‐control, (
) si‐MTDH, (
) si‐MTDH+anticontrol and (
) si‐MTDH+anti‐miR‐125a. MB231 (
) si‐control, (
) si‐MTDH, (
) si‐MTDH+anticontrol and (
) si‐MTDH+anti‐miR‐125a. Data represent mean ± SEM and *P < 0.05.
) si‐control and () si‐MTDH. (c–g) HCC‐70 and MB231 cells were transfected with si‐control or si‐MTDH, and cotransfected with si‐MTDH and either anticontrol or anti‐miR‐125a. (c) CCK‐8 determined cell proliferative capacity after transfection at 0 hour, 24 hours, 48 hours and 72 hours. HCC‐70 () si‐control, () si‐MTDH, () si‐MTDH+anticontrol and () si‐MTDH+anti‐miR‐125a. MB231 () si‐control, () si‐MTDH, () si‐MTDH+anticontrol and () si‐MTDH+anti‐miR‐125a. (d) Flow cytometry examined apoptosis rate after transfection at 24 hours. () si‐control, () si‐MTDH, () si‐MTDH+anticontrol and () si‐MTDH+anti‐miR‐125a. (e, f) Transwell assays were performed to evaluate cell migration and invasion abilities at 24 hours. () si‐control, () si‐MTDH, () si‐MTDH+anticontrol and () si‐MTDH+anti‐miR‐125a. (g) Western blotting tested protein expression of Ki67, cleaved caspase‐3, N‐cadherin and Vimentin after transfection at 24 hours. HCC‐70 () si‐control, () si‐MTDH, () si‐MTDH+anticontrol and () si‐MTDH+anti‐miR‐125a. MB231 () si‐control, () si‐MTDH, () si‐MTDH+anticontrol and () si‐MTDH+anti‐miR‐125a. Data represent mean ± SEM and *P < 0.05.
MTDH expression was positively regulated by TP73‐AS1 through sponging miR‐125a. RT‐qPCR and western blotting determined MTDH expression levels in (a, b) HCC‐70 cells and (c, d) MB231 cells transfected with si‐TP73‐AS1 alone or together with anti‐miR‐125a or anticontrol. MTDH expression levels in (e, f) HCC‐70 cells and (g, h) MB231 cells transfected with TP73‐AS1 alone or combined with miR‐125a or miR‐control were also measured. Data represent mean ± SEM and *P < 0.05.
Knockdown of TP73‐AS1 restrained tumor growth of breast cancer cells in vivo. HCC‐70 cells were stably expressed shRNA against TP73‐AS1 (sh‐H TP73‐AS1) or its negative control (sh‐NC), and were then subcutaneously injected into the right flanks of BALB/c nude mice (n = 6). (a) Tumor volume was measured every week after inoculation, and tumor growth curve was drawn. (
) sh‐NC and (
) sh‐TP73‐AS1. (b) Tumor weight was recorded on the last week. (c–e) RT‐qPCR analysis testified the relative expression of TP73‐AS1, miR‐125a and MTDH in xenograft tumors. (f) Western blotting determined MTDH protein expression in randomly selected one xenograft tumor. Data represent mean ± SEM and *P < 0.05.
) sh‐NC and () sh‐TP73‐AS1. (b) Tumor weight was recorded on the last week. (c–e) RT‐qPCR analysis testified the relative expression of TP73‐AS1, miR‐125a and MTDH in xenograft tumors. (f) Western blotting determined MTDH protein expression in randomly selected one xenograft tumor. Data represent mean ± SEM and *P < 0.05.Similar articles
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