circGFRA1 affects the sensitivity of triple negative breast cancer cells to paclitaxel via the miR-361-5p/TLR4 pathway

2021 ◽  
Author(s):  
Shu-Rong Zheng ◽  
Qi-di Huang ◽  
Zhi-Hai Zheng ◽  
Zhong-Tao Zhang ◽  
Gui-Long Guo
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Luciferase Reporter Gene ◽  
Tnbc Cell ◽  
Gene Assay ◽  
Tnbc Cell Line

Abstract In recent years, the role of circular RNAs (circRNAs) in tumors has attracted widespread attention. Some circRNAs have been reported to play a role in triple negative breast cancer (TNBC), however, circRNAs have rarely been reported in terms of TNBC resistance. This study aimed to clarify that circGFRA1 affects the sensitivity of TNBC cells to paclitaxel (PTX) by the miR-361-5p/TLR4 pathway. Compared with the non-PTX-resistant TNBC cell line MDA-MB-231, the expression of circGFRA1 in the PTX-resistant TNBC cell line MDA-MB-231.PR was significantly increased. The small hairpin RNA-mediated circGFRA1 knockdown inhibited the resistance of TNBC cells to PTX. RNA pull-down assay and luciferase reporter gene assay confirmed the binding between circGFRA1 and miR-361-5p and between miR-361-5p and TLR4. It has been proven that circGFRA1 knockdown can inhibit the resistance of TNBC cells to PTX by promoting the expression of miR-361-5p, and subsequently reduce the expression of TLR4.

scholarly journals Suppression of Spry1 inhibits triple-negative breast cancer malignancy by decreasing EGF/EGFR mediated mesenchymal phenotype

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Qing He ◽  
Hongyu Jing ◽  
Lucy Liaw ◽  
Lindsey Gower ◽  
Calvin Vary ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Lung Metastases ◽  
Mesenchymal Phenotype ◽  
Matrigel Invasion ◽  
Tnbc Cell ◽  
Tnbc Cell Line ◽  
E Cadherin

Abstract Sprouty (Spry) proteins have been implicated in cancer progression, but their role in triple-negative breast cancer (TNBC), a subtype of lethal and aggressive breast cancer, is unknown. Here, we reported that Spry1 is significantly expressed in TNBC specimen and MDA-MB-231 cells. To understand Spry1 regulation of signaling events controlling breast cancer phenotype, we used lentiviral delivery of human Spry1 shRNAs to suppress Spry1 expression in MDA-MB-231, an established TNBC cell line. Spry1 knockdown MDA-MB-231 cells displayed an epithelial phenotype with increased membrane E-cadherin expression. Knockdown of Spry1 impaired MDA-MB-231 cell migration, Matrigel invasion, and anchorage-dependent and -independent growth. Tumor xenografts originating from Spry1 knockdown MDA-MB-231 cells grew slower, had increased E-cadherin expression, and yielded fewer lung metastases compared to control. Furthermore, suppressing Spry1 in MDA-MB-231 cells impaired the induction of Snail and Slug expression by EGF, and this effect was associated with increased EGFR degradation and decreased EGFR/Grb2/Shp2/Gab1 signaling complex formation. The same phenotype was also observed in the TNBC cell line MDA-MB-157. Together, our results show that unlike in some tumors, where Spry may mediate tumor suppression, Spry1 plays a selective role in at least a subset of TNBC to promote the malignant phenotype via enhancing EGF-mediated mesenchymal phenotype.

scholarly journals TMEPAI genome editing in triple negative breast cancer cells

2017 ◽  
Vol 26 (1) ◽  
pp. 14-8 ◽  
Author(s):  
Bantari W.K. Wardhani ◽  
Meidi U. Puteri ◽  
Yukihide Watanabe ◽  
Melva Louisa ◽  
Rianto Setiabudy ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Genome Editing ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
High Efficiency ◽  
Knock Out ◽  
Tnbc Cell ◽  
A Genome ◽  
Tnbc Cell Line

Background: Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) is a powerful genome editing technique. It consists of RNA-guided DNA endonuclease Cas9 and single guide RNA (gRNA). By combining their expressions, high efficiency cleavage of the target gene can be achieved, leading to the formation of DNA double-strand break (DSB) at the genomic locus of interest which will be repaired via NHEJ (non-homologous end joining) or HDR (homology-directed repair) and mediate DNA alteration. We aimed to apply the CRISPR/Cas9 technique to knock-out the transmembrane prostate androgen-induced protein (TMEPAI) gene in the triple negative breast cancer cell line.Methods: Designed gRNA which targets the TMEPAI gene was synthesized, annealed, and cloned into gRNA expression vector. It was co-transfected into the TNBC cell line using polyethylenimine (PEI) together with Cas9-GFP and puromycin resistant gene vector. At 24-hours post-transfection, cells were selected by puromycin for 3 days before they were cloned. Selected knock-out clones were subsequently checked on their protein levels by western blotting.Results: CRISPR/Cas9, a genome engineering technique successfully knocked-out TMEPAI in the Hs578T TNBC cell line. Sequencing shows a frameshift mutation in TMEPAI. Western blot shows the absence of TMEPAI band on Hs578T KO cells.Conclusion: TMEPAI gene was deleted in the TNBC cell line using the genomic editing technique CRISPR/Cas9. The deletion was confirmed by genome and protein analysis.

scholarly journals Knock-out transmembrane prostate androgen-induced protein gene suppressed triple-negative breast cancer cell proliferation

2017 ◽  
Vol 26 (3) ◽  
pp. 178-82 ◽  
Author(s):  
Bantari W.K. Wardhani ◽  
Meidi U. Puteri ◽  
Yukihide Watanabe ◽  
Melva Louisa ◽  
Rianto Setiabudy ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Proliferation ◽  
Cell Line ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Ki 67 ◽  
Knock Out ◽  
Tnbc Cell ◽  
Tnbc Cell Line ◽  
Mrna Expression Levels

Background: Triple negative breast cancer (TNBC) tends to grow more rapidly and has poorer prognosis compared to others. High expression of transmembrane prostate androgen-induced protein (TMEPAI) correlates with poor prognosis in TNBC patients. However, the mechanistic role of TMEPAI in tumorigenic remains unknown. This study aimed to knock-out TMEPAI in TNBC cell line to determine its function further in cells proliferation.Methods: CRISPR-Cas9 has been used previously to knock-out TMEPAI in Hs857T TNBC cell line. Hs587T TNBC parental cell line (wild-type/WT) and TMEPAI knock out Hs 586T cell lines were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and amphotericin B. Both cell lines were seeded in 24-well plates and counted every two days, then proliferation rates were plotted. Afterwards, total RNA were isolated from the cells and Ki-67, and TGF-β mRNA expression levels as proliferation markers were determined.Results: Cell proliferation rates as displayed in growth curve plots showed that WT-TMEPAI cell line grew more rapidly than KO-TMEPAI. In accordance, mRNA expression levels of  Ki-67 and TGF-β  were significantly decreased KO-TMEPAI as compare to TMEPAI-WT.Conclusion: Knock-out of TMEPAI attenuates cell proliferation in TNBC.

scholarly journals CircWAC induces chemotherapeutic resistance in triple-negative breast cancer by targeting miR-142, upregulating WWP1 and activating the PI3K/AKT pathway

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Lei Wang ◽  
Yehui Zhou ◽  
Liang Jiang ◽  
Linlin Lu ◽  
Tiantian Dai ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Luciferase Reporter ◽  
Akt Pathway ◽  
Circular Rnas ◽  
Chemotherapeutic Resistance ◽  
Repressive Effect

Abstract Background Chemotherapeutic resistance is the main cause of clinical treatment failure and poor prognosis in triple-negative breast cancer (TNBC). There is no research on chemotherapeutic resistance in TNBC from the perspective of circular RNAs (circRNAs). Methods TNBC-related circRNAs were identified based on the GSE101124 dataset. Quantitative reverse transcription PCR was used to detect the expression level of circWAC in TNBC cells and tissues. Then, in vitro and in vivo functional experiments were performed to evaluate the effects of circWAC in TNBC. Results CircWAC was highly expressed in TNBC and was associated with worse TNBC patient prognosis. Subsequently, it was verified that downregulation of circWAC can increase the sensitivity of TNBC cells to paclitaxel (PTX) in vitro and in vivo. The expression of miR-142 was negatively correlated with circWAC in TNBC. The interaction between circWAC and miR-142 in TNBC cells was confirmed by RNA immunoprecipitation assays, luciferase reporter assays, pulldown assays, and fluorescence in situ hybridization. Mechanistically, circWAC acted as a miR-142 sponge to relieve the repressive effect of miR-142 on its target WWP1. In addition, the overall survival of TNBC patients with high expression of miR-142 was significantly better than that of patients with low expression of miR-142, and these results were verified in public databases. MiR-142 regulated the expression of WWP1 and the activity of the PI3K/AKT pathway. It was confirmed that WWP1 is highly expressed in TNBC and that the prognosis of patients with high WWP1 expression is poor. Conclusions CircWAC/miR-142/WWP1 form a competing endogenous RNA (ceRNA) network to regulate PI3K/AKT signaling activity in TNBC cells and affect the chemosensitivity of cells.

scholarly journals Substance P Antagonism as a Novel Therapeutic Option to Enhance Efficacy of Cisplatin in Triple Negative Breast Cancer and Protect PC12 Cells against Cisplatin-Induced Oxidative Stress and Apoptosis

Cancers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 3871
Author(s):  
Emma Rodriguez ◽  
Guangsheng Pei ◽  
Sang T. Kim ◽  
Alexis German ◽  
Prema Robinson
Keyword(s):  
Breast Cancer ◽  
Flow Cytometry ◽  
Cell Line ◽  
Pc12 Cells ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Therapeutic Option ◽  
Neuronal Cell ◽  
Receptor Antagonism ◽  
Tnbc Cell

Although cisplatin is very effective as a treatment strategy in triple-negative breast cancer (TNBC), it has unwarranted outcomes owing to recurrence, chemoresistance and neurotoxicity. There is critically important to find new, effective and safe therapeutics for TNBC. We determined if SP-receptor antagonism in combination with cisplatin may serve as a novel, more efficacious and safer therapeutic option than existing therapies for TNBC. We used a neuronal cell line (PC12) and two TNBC cell lines (Sum 185 and Sum 159) for these studies. We determined that the levels of cells expressing the high-affinity SP-receptor (neurokinin 1 receptor (NK1R)), as determined by flow-cytometry was significantly elevated in response to cisplatin in all three cells. We determined that treatment with aprepitant, an SP-receptor antagonist decreased cisplatin-induced, loss of viability (studied by MTT assay), production of reactive oxygen species (by DCFDA assay) and apoptosis (by flow-cytometry) in PC12 cells while it was increased in the two TNBC cells. Furthermore, we demonstrated that important genes associated with metastases, inflammation, chemoresistance and cell cycle progression are attenuated by SP-receptor antagonism in the TNBC cell line, Sum 185. These studies implicate that SP-receptor antagonism in combination with cisplatin may possibly serve as a novel, more efficacious and safer therapeutic option than existing therapies for TNBC.

scholarly journals Long Non-Coding RNA ANRIL Promotes Doxorubicin Resistance in Triple-Negative Breast Cancer via Suppressing Glycolysis Through the MicroRNA-125a/ENO Pathway

2021 ◽  
Author(s):  
Jianli Ma ◽  
Wenhui Zhao ◽  
Han Zhang ◽  
Zhong Chu ◽  
Huili Liu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Drug Resistance ◽  
Cell Lines ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Active Role ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Tnbc Cell

Abstract BackgroundBreast cancer is the main cause of death among women worldwide. More and more long non-coding RNAs (lncRNAs) have been identified as oncogenes or tumor suppressors during cancer development. However, whether ANRIL is involved in drug resistance in triple-negative breast cancer (TNBC) has not been investigated. MethodsLuciferase reporter assay was conducted to verify the binding of miR-125a and ANRIL. RT-PCR and western blot were performed to detect the expression of miR-125a, ANRIL and ENO1. Gene silence and overexpression experiments as well as CCK-8 and colony formation assays on TNBC cell lines were performed to determine the regulation of molecular pathways. Glycolysis analysis was performed with Seahorse extracellular flux methodology. ResultsANRIL expression in TNBC patients and TNBC cells was examined and we found that ANRIL expression was upregulated in both TNBC patients and TNBC cell lines. Knockdown of ANRIL increased the cytotoxic effect of ADR and inhibited HIF-1α-dependent glycolysis in TNBC cells. In addition, we found that ANRIL negatively regulated miR-125a expression in TNBC cell lines. Besides, a dual-luciferase reporter assay proved ANRIL functioned as a sponger of miR-125a. Further investigation revealed that ENO1 was a target of miR-125a and positively regulated by ANRIL in TNBC cells. Additionally, ANRIL upregulation reversed miR-125-mediated inhibition on HIF-1α-dependent glycolysis in TNBC cells. More notably, 2-deoxy-glucose (2-DG) attenuated ANRIL-induced increase of drug resistance in TNBC cells. ConclusionsTaken together, our study was the first to identify that knockdown of ANRIL plays an active role in overcoming the drug resistance in TNBC by inhibiting glycolysis through the miR-125a/ENO1 pathway, which maybe serve useful for the development of novel therapeutic targets.

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