| Background Breast cancer is the most commonly diagnosed cancer,with～2.261 million new cases,and～684,000 deaths,making breast cancer the fifth deadliest cancer,according to Global Cancer Statistics 2020.It is classified into molecular subtypes with different clinical and pathological features,treatment responses,and outcomes.Among them is triple-negative breast cancer(TNBC),which lacks the expression of progesterone,estrogen,and human epidermal growth factor receptor 2(HER-2).TBNC has the poorest survival pattern due to its aggressiveness and higher metastatic potential and chemoresistance than other molecular subtypes of breast cancer.Cancer metastasis is a sophisticated,disorganized stage in the progression of a tumor.It is a potentially deadly process and the leading cause of death(90%)in cancer patients.Consequently,metastasis is regarded as a highly investigative process for eliminating remote spread and lowering cancerrelated mortality.During metastasis,cancer cells lose their epithelial characteristics and gain the ability to migrate and invade,allowing them to spread from the primary site and enter the circulation.Tumor cells are detained in microvessels of other organs,extravagate,and form micrometastasis,leading to macrometastasis formation.Cancer is caused by chromosomal gains,losses,and rearrangements caused by genomic instability and tumor cell heterogeneity.Aberrant cell-cycle progression,telomere dysfunction marked by chromosomal instability and cytogenetic abnormalities,inhibition of DNA repair genes,and modified epigenetic control mechanisms can compromise DNA integrity.Notch signaling is a high evolutionally conserved pathway.Recent reports have underlined the implication of Notch signaling upregulation in the TNBC.Increased Notchl stimulates epithelial to mesenchymal transition(EMT)and induces tumor formation in TNB.In mammals,Notch signaling is controlled by glycosyltransferases known as fringes:lunatic fringe(LFNG),manic fringe(MFNG),and radical fringe(RFNG)through their fucose-specificβ-1,3-N-acetylglucosaminyltransferase activity.Fringes modulate Notch signaling by adding GlcNAc to the O-fucosylated Notch receptor EGF-like domains with potential fucose addition sites.Of the fringes,MFNG acts as a tumor suppressor in lung cancer by inhibiting the activation of Notch3.Conversely,MFNG is upregulated in kidney clear cell renal carcinoma(KIRC),which could be linked to kidney cancer progression,hence an oncogene.The MFNG controversy underlines its dual function depending on the cancer type.Previously,we reported that MFNG was highly expressed in claudin-low breast cancer(CLBC)and promoted cell migration,proliferation,and tumorigenicity through modulating Notch activation by directly targeting phosphoinositide kinase(PIK3CG).However,the involvement of MFNG in TNBC progression has not been previously reported,and the mechanism in which MFNG is regulated also remains unclear.GATA3,a zinc-finger transcription factor belonging to the GATA family,has been reported to suppress cancers by regulating their target genes.GATA3 inhibits prostate cancer progression by up-regulating miR-503 expression to repress the oncogenic activity of ZNF217 and abrogates breast cancer metastasis through regulating Semaphorin 3B expression.Based on recent studies,GATA3 expression is frequently reduced in TNBC cells;however,the aberrantly activated signaling which confers TNBC malignancy is still unknown.microRNA-205(miR-205)is found in the second intron of the LOC642587 locus on human chromosome 1(1q32.2).Several studies have shown that miR-205 is associated with the growth of multiple cancerous tumors in humans by influencing tumor cell proliferation,differentiation,invasion,and cell death.The ectopic expression of miR-205 in prostate cancer could reduce cell invasion and metastasis via epithelial-mesenchymal transition(EMT).However,miR-205 levels are elevated in lung cancer,while miR-205 expression was reduced in bladder cancer.Other studies have proved that miR205-5p could also inhibit cancer progression by suppressing the expression of oncogenes.Therefore,miR-205 has a dual function in cancer as it may act as an oncogene or a tumor suppressor.Aim Provided that MFNG has shown the oncogenic effect in claudin law breast cancer,future studies should emphasize elucidating the underlying mechanism of its action.The mechanistic regulation of MFNG is still obscure,so we aimed to explore how MFNG is regulated and its effect on TNBC progression.Overall,developing a mechanism-based treatment strategy for TNBC is very demanding to tackle this deadly disease.Experimental Design The human breast cancer cells MCF-7 and T47D(non-TNBC),BT-549,MDA-MB-231,MDA-MB-468,HCC-1806,and HS578-T(TNBC)were obtained from Zhiyong Luo’s lab(School of Life Sciences,Central South University,Changsha,China).The cells were grown in DMEM with 10%fetal bovine serum(FBS)and antibiotics 100 U/mL penicillin and 100 μg/mL streptomycin(P/S)and were maintained in an incubator at 37℃supplied with 5%CO2.All tissue specimens were collected from Xiangya Second Hospital,Central South University(Changsha,China).The effect of MFNG on TNBC cell viability and proliferation was analyzed by CCK-8 assay and manual cell counting.Two cell migration assays evaluated the anti-metastatic function of MFNG in TNBC:wound healing assay and transwell migration assay.Total RNA was extracted and prepared for quantitative RT-qPCR to scrutinize the effect of MFNG and its regulators(GATA3 and miR-205-5p).Also,the outcome and effect of MFNG treatment and its molecular mediator were studied by implementing treatment of cells with several chemical agents,immunofluorescence staining to visualize protein co-localization,western blotting,immunohistochemistry(IHC),luciferase assay,and chromatin immunoprecipitation(ChIP).Additionally,transfecting cells with shRNA and overexpression vectors performed gain-of-and loss-of-gene function studies.All the experimental assays and their related signaling pathway and metastasis hallmarks were also examined by western blotting.To validate the in vitro findings from our study,we carried out in vivo experiment.We prepared nanoparticles to drive miR-205-5p into tumors.Briefly,black phosphorus nanoparticles(BP)with a particle size of 30-40 nm were prepared by the liquid-phase exfoliation method,and the surface of nanoblack-phosphorus was modified with polyacetimide(PEI)to make it positively charged(PEI-BP).Then,the negatively charged miR-205-5p was loaded on the surface of BP nanoparticles by electrostatic adsorption to construct a miRNA delivery system(miR-205-5p-PEI-BP).All animal care and procedures were carried out following National Institutes of Health(NIH)guidelines and were approved by the Ethics Committee of the State Key Laboratory of Medical Genetics(NO.2016030901).The nude mice were purchased and kept in the CSU animal laboratory facility,and all experiments were performed under pathogen-free conditions.GraphPad Prism 7.0 and SPSS 17.0 were used for statistical analysis.Survival curves were plotted by the Kaplan-Meier method and analyzed by the log-rank test.Student t-tests analyzed the difference between groups.The mean± standard deviation(SD)of data from at least three experiments was displayed.The data were judged statistically significant at*P<0.05,**P<0.01,and***P<0.001.Results To evaluate the MFNG expression levels in TNBC and nonTNBC subgroups,we analyzed the clinical data obtained from the cBio Cancer Genomics Portal database(TCGA,nature 2012).The result showed that MFNG was expressed at a higher level in TNBC compared to non-TNBC.We also assessed MFNG expression in TNBC and non-TNBC tissues using IHC staining,and similar results were observed.To further confirm the above result,we investigated the MFNG expression in breast cancer cells and found that MFNG was highly expressed in TNBC cells.Moreover,using the same clinical data,we classified patients into MFNG high and low expression groups based on the expression levels.The correlation between MFNG expression and prognosis of TNBC patients was assessed,and the result indicated that high expression of MFNG was positively correlated with worse overall survival of TNBC patients,not significantly correlated with breast cancer and non-TNBC patients.We uncovered that MFNG was highly expressed in TNBC tissues,and higher MFNG expression predicted a poor prognosis for TNBC patients.The above observations prompted us to investigate the function of MFNG in the malignant behavior of TNBC.We overexpressed MFNG and knocked it down using shRNA sequences in TNBC cells.The effect of MFNG on cell growth and migration was examined through colony formation,transwell,and CCK-8 assays.We detected that forced expression of MFNG notably increased the cell viability and migration of TNBC cells.In contrast,the knockdown of MFNG remarkably suppressed the cell migration and growth of TNBC cells.Further,we examined the expression of the factors involved in growth and epithelial to mesenchymal(EMT)in stable cells overexpressing MFNG.The results indicated that the EMT markers Vimentin and SLUG were up-regulated,E-cadherin was downregulated,and growth-related marker βcatenin was increased following MFNG overexpression.It was reported that MFNG could promote the malignancy of breast cancer by activating Notch signaling.Therefore,we evaluated whether the ectopic expression of MFNG could activate Notch signaling.Notably,the MFNG up-regulated Notch target genes HES1 and HEY1 expression in TNBC cells.These results collectively suggest that MFNG enhanced cell growth and migration in TNBC by elevating Notch signaling.It has been previously reported that GATA3 expression is low in TNBC.To identify its functional role in TNBC progression,we established BT549 and MDA-MB-231 stable cells overexpressing and knockdown GATA3,which was verified by RT-qPCR and Western blot assays.We performed colony formation,transwell,CCK8,and wound-healing assays to assess the effect of GATA3 on growth and migration in TNBC cells.The result in indicated that cell growth was reduced in GATA3 overexpressed cells,while GATA3 knocked down cells showed a high growth rate compared to control cells.Compared to the control cells,the migration was restrained in cells with forced expression of GAT A3,while faster migration was observed in the GATA3 knocked-down cells.EMT and growth-related genes were investigated by western blot,and the result indicated that overexpression of GATA3 increased E-cadherin expression.In contrast,the expression of Vimentin and β-catenin decreased in the TNBC cells.These findings suggested that GATA3 inhibited the expression of factors involved in EMT and growth.In addition,Notch signaling activity was examined by analyzing the expression of HES1 and HEY1 by RT-qPCR;their expression was reduced upon GATA3 overexpression.These data demonstrated that GATA3 negatively regulated the TNBC malignancy by suppressing the cell migration and growth and notch signaling activation.The above findings illustrated that both MFNG and GAT A3 affected Notch signaling activity,implying that there may be a correlation between MFNG and GATA3.GAT A3 is a known transcription factor that controls the expression of several genes in breast cancer;therefore,we presumed that GATA3 could regulate the transcription of MFNG.To test this hypothesis,we performed an in silico analysis using the GEPIA2 database,and the result implied that GATA3 expression was negatively correlated with MFNG expression.In the clinical breast cancer tissues,IHC staining revealed a consistent expressing pattern in TNBC.Meanwhile,RT-qPCR and western blot results indicated that overexpression of GATA3 reduced the expression of MFNG at mRNA and protein levels in TNBC cells.These results confirmed the negative correlation between GATA3 and MFNG,suggesting that GATA3 might regulate the transcription of MFNG in TNBC cells.Further,we analyzed the MFNG promoter sequence and found a potential GATA3 binding site(A[GATA]G)at the-358/-363 region.The wildtype reporter(-510 to+100bp at MFNG promoter region)and mutant reporter(missing the binding site of GATA3)were created to examine the effect of GATA3 on the activity of the MFNG promoter.The luciferase assay revealed that GAT A3 significantly inhibited the activity of the wild-type MFNG promoter in the TNBC cells,but the mutant promoter activity remained unchanged.This result demonstrated that GATA3 mediated transcriptional inhibition of MFNG.In the meantime,the ChIP assay indicated that GATA3 remarkably enriched the promoter of MFNG.These findings clarified that GATA3 was directly bound to the promoter of MFNG and suppressed its transcription.Since the onco-suppressive role of miR-205 in TNBC through targeting oncoproteins is evident,we predicted the target genes of miR205-5p by Targetscan and miRDB database analysis.We found that MFNG was a potential target of miR-205-5p based on putative target sequences at the position 812-819 of the MFNG mRNA 3’ UTR.To investigate the regulatory mechanism of miR-205-5p on MFNG,we ectopically expressed miR-205-5p mimic in TNBC cells and observed that the mimic significantly reduced the mRNA and protein levels of MFNG.We created the reporters with the wildtype or mutant MFNG mRNA 3’ UTR region and performed a luciferase assay to verify this inhibitory activity.The result showed that miR-205-5p was bound to the wild-type 3’ UTR region of MFNG mRNA but not to the mutant 3’ UTR.These results confirmed that miR-205-5p abrogated the MFNG transcription and suppressed the malignancy of TNBC cells.Since GATA3 regulates microRNA expression in breast cancer,we sought to determine whether GATA3 could control miR-205-5p expression to cooperatively co-suppress MFNG expression.Therefore,we examined the expression of miR-205-5p in GATA3 overexpressed TNBC cells and found that miR-205-5p was upregulated.We predicted that the miR205-5p promoter sequence harbored three putative GATA3 binding sites using the bioinformatics tool mentioned in the materials and methods section.To confirm that miR2055p was a potential target of GAT A3,we constructed a wildtype reporter,mutant reporters of each GATA3 binding site(WT-Mut-S1,WT-Mut-S2,WT-Mut-S3),as well as combined mutant GAT A3 binding sites(WT-Mut-Combined).Luciferase assay was performed in TNBC cells and found that increased luciferase activity of WT-GATA3 reporter was observed when overexpressed GATA3,while decreased luciferase activity of each mutant site appeared compared to the WT-GATA3.Besides,GAT A3 did not activate the luciferase activity of the WT-Mut-Combined reporter compared to the control.Furthermore,the ChIP assay revealed a considerable enrichment of GATA3 on the miR205-5p promoter.To further validate that miR205-5p was downstream of GATA3,we examined whether miR205-5p could restore the phenotype of GATA3 in TNBC.Compared to control cells,miR205-5p mimics inhibited MFNG expression despite the absence of GATA3.Additionally,miR205-5p mimics rescued GATA3 inhibitory phenotype regarding the cell growth and migration in GATA3 knockdown cells by colony formation and transwell assays.In general,our results discovered that GATA3 was a direct modulator of miR205-5p transcription and that miR-205-5p mimic rescued the phenotype of GATA3 by inhibition of MFNG expression,suggesting that GATA3/miR-205-5p/MFNG form a novel feed-forward loop in the regulation of TNBC malignancy.Black phosphorus has a unique advantage for drug delivery.To evaluate the tumor-suppressive role of miR205-5p in TNBC,we linked the synthetic miR205-5p with polyetherimide-black phosphorus(PEI-BP)nanoparticles that could accumulate in breast cancer and successfully obtained the composite nanoparticles(PEI-BP-miR205-5p).After that,we investigated the effect of composite nanoparticles on TNBC cells in vitro and in vivo.We discovered that miR205-5p-PEI-BP exerted remarkable inhibition in cell growth of TNBC when compared to control.Meanwhile,the in vitro migration ability of TNBC cells was also significantly decreased by miR205-5p-PEI-BP.To further confirm the inhibitory effect on TNBC cells,the tumorigenesis of TNBC cells was performed.Compared to control,reduced growth and weight of tumors were observed under the treatment of the composite nanoparticles.Therefore,our results indicated that miR-205-5p had potential in TNBC therapy and provided a novel strategy for clinical TNBC treatment.Conclusion We showed that MFNG was highly expressed in TNBC and associated with poor prognosis through Notch signaling activation.We demonstrated that GAT A3 inhibited the transcription of MFNG by binding to the promoter and suppressed the mRNA expression.In addition,GATA3 activated miR-205-5p transcription,the letter binds to the 3’ UTR region of MFNG.These findings revealed the underlying mechanism of GATA3miR205-5p on inhibition of TNBC malignancy and the crucial role of GATA3/miR205-5p/MFNG axis.Interestingly,miR205-5p remarkably reduced tumorigenesis of TNBC when GATA3 attenuated.Therefore,our study suggests a novel feed-forward loop that regulates TNBC malignancy and provides potential new treatment strategies to combat triple negative breast cancer.There are 37 figures,9 tables,245 references... |
PDF Full Text Request