The LncRNA MAFG-AS1 Upregulated By Estrogen Promotes Progression Of ER Positive Breast Cancer Through MiR-339-5p/CDK2 Axis And Confers Tamoxifen Resistance

Objective: Breast cancer(BC)is the most common malignant tumor in women,and its incidence rate ranks first among women.Hormone receptor(HR)-positive breast cancer patients account for about two-thirds of all breast cancer patients,belonging to the hormone-sensitive breast cancer subtype.The estrogen receptor(ER)-mediated signaling pathway plays an important role in its occurrence and development.In addition to chemotherapy,endocrine therapy is an effective treatment method.Tamoxifen is a classic anti-estrogen.The drug has become the standard adjuvant medication for patients with HR-positive breast cancer.Compared with other types of breast cancer,HR-positive breast cancer patients have a better prognosis,but the majority of patients who die of breast cancer are still HR-positive because of their large proportion and primary or secondary endocrine resistance.One-third of breast cancer patients who respond to adjuvant tamoxifen therapy will eventually relapse due to endocrine resistance.However,HR-positive breast cancer does not have ideal prognostic indicators.Therefore,it is urgent to find new biomarkers for prognostic analysis,and it is expected to become a target for treatment,and even a predictor of the efficacy of endocrine therapy.In recent years,studies have found that one of the mechanisms of endocrine resistance is crosstalk of the ER signaling pathway and the cell cycle pathway.CDK4 / 6 inhibitors have been used in patients with progressive endocrine therapy.However,there is little research on cyclinE-CDK2,another important cyclin-CDK complex in the G1 / S phase of the cell cycle.Moreover,studies have shown that resistance to CDK4 / 6 inhibitors may be related to the activation of cyclinE-CDK2,but the relationship between endocrine resistance and cyclinE-CDK2 in ER-positive breast cancer is unclear.Long non-coding RNAs(lncRNAs)are a class of RNA molecules whose transcripts are more than 200 nucleotides in length.They are similar in structure to mRNA,but they do not encode proteins,but in the form of RNA regulating gene expression at multiple levels(epigenetic regulation,transcriptional regulation,and post-transcriptional regulation,etc.).High-throughput sequencing data from breast cancer patients show that lncRNAs expression is highly subtype-specific.For ER-positive breast cancer,estrogen-activated ER dimers can be used as transcription factors for lncRNAs.Activation of the ER signaling pathway affects cell proliferation,differentiation and programmed death.The crosstalk between intracellular and extracellular pathways and ER signals indicates that endocrine resistance in ER-positive breast cancer occurs at multiple levels,such as the cell cycle and the PI3 K / AKT / mTOR pathway.In this crosstalk,lncRNAs may play an important role.However,the mechanisms of estrogen-induced lncRNAs in cell cycle progression and tamoxifen resistance remain unclear.Therefore,it is necessary to explore the regulatory role of lncRNA in the ER signaling pathway and CDK2-regulated cell cycle crosstalk.In this study,we performed a meta-analysis of the survival-based GEO dataset and combined gene expression to screen for lncRNA MAFG-AS1,which is highly expressed in ER-positive breast cancer and has a poor prognosis.ER? binds directly.Existing research shows that MAFG-AS1 can promote the invasion and metastasis of colon cancer and non-small cell lung cancer,but the mechanism of action in ER-positive breast cancer is not yet clear.Whether it is related to tamoxifen resistance is also worth further exploration.This study explored the mechanism of ceRNA network composed of MAFG-AS1 / miR-339-5p / CDK2 to promote the proliferation of ER-positive breast cancer and confirmed its correlation with tamoxifen resistance.Crosstalk between MAFG-AS1 and CDK2-mediated ER signaling and cell cycle pathways indicates that MAFG-AS1 may be a biomarker and therapeutic target for ER-positive breast cancer,while CDK2 inhibitors are likely to be used for endocrine resistance treatment.In summary,this article provides new prognostic markers and therapeutic targets for ER-positive breast cancer,and provides a new research direction for endocrine resistance.Methods: Part I: 1.Meta-analysis using six breast cancer datasets in the GEO database and external verification by Kaplan-Meier Plotter,and at the same time verify the expression of cancer and adjacent normal tissues on the GEPIA online database website to screen for luminal subtype breast cancer target lncRNA MAFG-AS1 that is significantly associated with high risk of overall survival(OS)/ relapse-free survival(RFS)/ distant metastasis free survival(DMFS)and is highly expressed in breast cancer;2.Collect fresh cancerous and adjacent normal tissue samples from breast cancer afteroperation,and use qRT-PCR and in situ hybridization(ISH)to detect MAFG-AS1 expression in breast cancer and adjacent normal tissues.Spearman correlation analysis was used to calculate the correlation between the expression of MAFG-AS1 and clinicopathological parameters of breast cancer;3.qRT-PCR was used to detect the expression level of MAFG-AS1 in different breast cancer cell lines and normal breast epithelial cells;4.Use qRT-PCR to detect the expression level of MAFG-AS1 after estrogen induction on wild-type and ER knockdown type ER-positive breast cancer cells;5.Screen for transcription factors on MAFG-AS1 promoter region in the Jaspar and PROMO databases;6.Chromatin immunoprecipitation(CHIP)was used to verify the binding of ER? to the MAFG-AS1 promoter region after estrogen action;7.MTT and colony formation experiments were used to detect MAFG-AS1 action on ER-positive breast cancer cell proliferation activity;8.Detection of the effect of MAFG-AS1 on cell cycle and apoptosis of ER-positive breast cancer cells by flow cytometry;9.Using female immunodeficiency mice to perform in situ tumor formation experiments after stable transfection of MAFG-AS1 knockdown lentivirus;10.Using RNA nuclear plasma separation assay to detect nuclear and plasm distribution of MAFG-AS1 in ER positive breast cancer cells;11.Predict microRNAs that may bind to MAFG-AS1 on the starbase online database website,and perform qRT-PCR at the cellular level to verify expression correlation;12.Use qRT-PCR to verify miR-339-5p expression level of breast cancer and normal tissues,verify the correlation between miR-339-5p and MAFG-AS1 in cancer tissues;13.Double-luciferase reporter gene assay and RIP to verify the binding of MAFG-AS1 and miR-339-5p;14.Use MTT,colony formation,and flow cytometry to detect cell cycle and apoptosis for miR-339-5p and MAFG-AS1 rescue experiments;15.Use starbase to predict target genes downstream of mi R-339-5p,Online website enrichment of target genes;16.Use qRT-PCR to verify the expression of CDK2 in cancer and adjacent normal tissues and the correlation between miR-339-5p and CDK2;17.Double-luciferase reporter gene assay to verify the combination of miR-339-5p and CDK2;18.Use MTT,colony formation and flow cytometry to detect cell cycle and apoptosis for miR-339-5p and CDK2 rescue experiments;19.Use immunohistochemical experiments to verify the correlation between MAFG-AS1 and CDK2,Ki-67;20.Use qRT-PCR and western blot detect the ceRNA network relationship between MAFG-AS1,miR-339-5p and CDK2;21.Use Western blot to detect the correlation between MAFG-AS1 and CDK2 and other cell cycle related factors,and explore the apoptosis-related mechanism according to action of CDK2 on FOXO1;22.qRT-PCR and western blot were used to detect changes in ER? after MAFG-AS1 was knockdown.Part II: 1.Screen the patients treated with tamoxifen monotherapy on Kaplan-Meier Plotter online website,analyze the relationship between the expression of MAFG-AS1 and survival,and further discover whether MAFG-AS1 is related to tamoxifen resistance;2.qRT-PCR was used to detect the expression of MAFG-AS1 and ESR1 in MCF-7parental cells and MCF-7 tamoxifen-resistant cells;3.qRT-PCR was used to detect MAFG-AS1 expression after tamoxifen action for 12 h and 24h;4.Use MTT to detect whether it is susceptible to tamoxifen action in MCF-7 tamoxifen-resistant cells after MAFG-AS1 knockdown;5.Screen the patients treated with tamoxifen monotherapy on the Kaplan-Meier Plotter online website,analysis of the relationship between CDK2 expression and survival,whether CDK2 is related to tamoxifen resistance;6.MTT was used to detect whether it is sensitive to the action of tamoxifen in MCF-7 tamoxifen resistant cells after CDK2 knockdown;7.Explore whether MAFG-AS1 overexpression in T47 D cells to confer resistance to tamoxifen;8.Use subcutaneous tumor formation experiments in nude mice to verify Whether develops tamoxifen resistance after MAFG-AS1 overexpression.Results: Part I:1.The lncRNA MAFG-AS1 that is highly expressed in breast cancer and associated with the poor prognosis of luminal breast cancer is screened by bioinformatics;2.The expression of MAFG-AS1 in breast cancer tissues is higher than adjacent normal tissue,and the high expression of MAFG-AS1 is significantly associated with breast cancer size,ki-67;3.MAFG-AS1 expression in breast cancer cell lines is higher than normal breast epithelium cell,and higher expression in ER-positive breast cancer cell lines;4.Estrogen can promote the expression of MAFG-AS1 in wild-type ER-positive breast cancer cells,but the change of MAFG-AS1 is not statistically significant after ER knockdown;5.Both online database websites predict that ER? is MAFG-AS1 transcription factor;6.CHIP experiments show that after the action of estrogen,ER?binds more in the MAFG-AS1 promoter region;7.MAFG-AS1 can promote the proliferation of ER-positive breast cancer cells;8.MAFG-AS1 can Promote the G1 / Sphase of the cell cycle and inhibit apoptosis;9.MAFG-AS1 promotes tumor formation in situ in nude mice;10.MAFG-AS1 is mainly distributed in the cytoplasm in ER-positive breast cancer cells;11.After screening and verification,miR-339-5p binding with MAFG-AS1 was obtained;12.The expression level of miR-339-5p in breast cancer tissues was significantly lower than that in adjacent normal breast tissues,and the expression was negative correlative with MAFG-AS1;13.Double luciferase reporter gene assay and RIP verified that MAFG-AS1 binds to miR-339-5p;14.Rescue experiment results show that overexpression of miR-339-5p can partially reverse the effect of overexpression of MAFG-AS1 on the proliferation and inhibition of apoptosis of ER-positive breast cancer cells;15.Online database prediction and pathway enrichment interaction to obtain CDK2 as the target gene of miR-339-5p;16.CDK2 expression level in breast cancer tissue higher than adjacent normal breast tissue,and negatively correlated with miR-339-5p;17.Double luciferase reporter gene assay proves that miR-339-5p binds to CDK2;18.The rescue experiment results show that miR-339-5p knockdown can partially reverse the inhibitory effect of CDK2 knockdown on ER-positive breast cancer cells in inhibiting proliferation and promoting apoptosis;19.immunohistochemistry confirmed that samples with high expression of MAFG-AS1,CDK2 and ki-67 also have high expression;20.At the mRNA and protein level,overexpression of mi R-339-5p can reverse the increase in CDK2 caused by overexpression of MAFG-AS1;21.MAFG-AS1 is positively correlated with CDK2 at the mRNA and protein levels,MAFG-AS1 is positively correlated with cyclinE,Rb at the protein level and negatively correlated with the transcription inhibitor FOXO1 regulated by CDK2,but not with CDK4,CDK6,and cyclinD1.MAFG-AS1 was negatively correlated with PARP,cl-caspase8,and cl-caspase3 in the apoptosis-related pathway;22.The mRNA and protein levels showed that the expression of p-ER? was down-regulated after MAFG-AS1 knockdown.Part II: 1.In patients treated with tamoxifen monotherapy,high expression of MAFG-AS1 is associated with poor DMFS and RFS;2.The expression of MAFG-AS1 and ESR1 in tamoxifen-resistant MCF-7 cells was higher than that of parent MCF-7 cells;3.MAFG-AS1 expression decreased after 12 hours of tamoxifen action and returned to its original level at 24 hours;4.The knockdown of MAFG-AS1 in tamoxifen-resistantMCF-7 cells became sensitive to the effect of tamoxifen and restored the effect of tamoxifen on inhibiting cell proliferation;5.In patients treated with tamoxifen monotherapy,high expression of CDK2 is associated with poor DMFS;6.The knockdown of CDK2 in tamoxifen-resistant MCF-7 cells became sensitive to the effect of tamoxifen and restored the effect of tamoxifen on inhibiting cell proliferation;7.Overexpression of MAFG-AS1 in T47 D cells increased tamoxifen resistance;8.Overexpression of MAFG-AS1 increased tamoxifen resistance of tumor formation in situ in nude mice.Conclusion:1.MAFG-AS1 is highly expressed in ER-positive breast cancer and its high expression is significantly associated with poor prognosis.2.MAFG-AS1 is induced by estrogen and the transcription factor ER? binds to the promoter region of MAFG-AS1 after estrogen action.3.MAFG-AS1 promotes the proliferation of ER-positive breast cancer cells and the transition of the cell cycle G1 / S through the ceRNA network of mi R-339-5p / CDK2,and inhibits apoptosis.4.Crosstalk of ER signaling pathway and cell cycle pathway mediated by MAFG-AS1 and CDK2 promotes tamoxifen resistance in ER-positive breast cancer.5.Positive feedback loop formed by ER?-MAFG-AS1-CDK2-ER? promotes tamoxifen resistance in hormone receptor-positive breast cancer.