| Objective:In recent years,increasing studies have shown that long non-codingRNAs（lncRNAs）are closely related to the occurrence,development,and treatment of various tumors and play a key role in these processes.However,the biological effects of a typical lncRNA,LncRNA EGFR antisenseRNA 1（EGFR-AS1）,in BC（BC）and the underlying mechanisms are still unclear.EGFR-AS1 was highly expressed in BC tissues and cell lines based on TCGA and Star Base databases.It could promote the proliferation,migration,and invasion of BC cells in vitro and in vivo and increase the sensitivity of BC cells to docetaxel.FISH and nucleocytoplasmic separation experiments confirmed that EGFR-AS1 was mainly located in the cytoplasm of BC cells.Mechanistically,EGFR-AS1 was found to act as a molecular sponge for microRNAs to participate in the regulation of ceRNAs,and results established that the EGFR-AS1/mi R-149-5p/ELP5 axis can promote BC progression.Furthermore,ELP5was found to activate the PI3K/AKT pathway by mediating EGFR affects,with LY294002 normalizing activation of the pathway,and ELP5 was determined to be essential for the post-transcriptional regulation of EGFR via EGFR-AS1.2.Cell Culture:The human normal breast cell line MCF-10A cells were cultured in1:1 Dulbecco’s modified Eagle’s medium（DMEM）/F12（Gibco,Waltham,MA,USA）supplemented with 5%horse serum,10μg/m L insulin（Sigma-Aldrich Co,St.Louis,MO,USA）,and 20ng/m L epidermal growth factor（EGF）.MCF-7 and SK-BR-3 cell lines were cultured in DMEM（Gibco）supplemented with 10%FBS.MDA-MB-231and MDA-MB-453 cells were cultured in L15（Gibco）supplemented with 10%fetal bovine serum（FBS）.BT549 cells was cultured in Roswell Park Memorial Institute（RPMI）1640（Gibco）supplemented with 10%FBS.Cells were incubated at 37°C in a humidified atmosphere containing 5%CO2.3.Cell Transfection and Interference:Breast cancer cell lines MCF-7 and MDA-MB-231 were homogeneously seeded in six-well plates and the cells were cultured overnight at 37°C in a humidified environment with 5%CO2.LncRNAEGFR-AS1 siRNA,si ELP5,mi R-149-5p mimics and inhibitors were purchased from Guangzhou Ruibo Biotechnology.Plasmids p CMV6-ddk-myc and p CMV6-ddk-myc-ELP5 were purchased from Origene（Rockville,MD,USA）.Transfection was performed using Lipofectamine 3000 reagent（Invitrogen）according to the manufacturer’s instructions.After 24-48 hours,breast cancer cells were harvested.4.Screening of stable cell lines:shRNA-EGFR-AS1 plasmid was purchased from Zimmer Gene Company.Using MCF-7 cells into 24-well plates with different gradients of puromycin（1-11μg/ml）in order to determine the optimal cell tolerance concentration.After 48 hours,add puromycin-containing culture medium（puromycin is 6μg/ml）,and change the medium DMEM every 1-2 days.After about 4 weeks,a cell line stably expressing the target gene was obtained.5.Real-time PCR:The totalRNA was isolated from BC tissues and cells using the TRIzol Reagent Kit（Invitrogen,Carlsbad,CA,USA）.PartsRNAs was then used for reverse transcription into c DNA（Ta Ka Ra,RR036A,Japan）.The expression of lncRNAs and genes transcription level was measured by SYBR-Green q PCR（Takara,Otsu,Shiga,Japan）（Step One Plus;Applied Biosystems,USA）.miRNAs levels were assayed by（Takara,047A Shiga,Japan）.An Applied Biosystems 7500 Real-Time PCR system（Applied Biosystems,Foster City,CA）was also utilized.Meanwhile,GAPDH and U6 snRNA were used to normalize the relative amount of lncRNA,miRNA and mRNA,separately.The 2-ΔΔCT method was used to quantify the gene expression.6.Western blot:Total protein was extracted using a lysis buffer（Thermo Fisher Scientific）,quantified with the Bradford method.eightyμg of the total protein samples were separated by 10%SDS-PAGE and then transferred to polyvinylidene difluoride membranes（PVDF;Millipore,Billerica,MA,USA）.After blocking with 5%BSA in TTBS buffer for 2h,the membrane was incubated at 4°C overnight with the appropriate primary antibody previously.Afterwards,the membrane was washed 4times with TTBS buffer for 5min and then incubated with horseradish peroxidase-labeled secondary antibody at 37°C for 2 hours.After washing with TTBS buffer.Finally,visualized with ECL on a Bio Imaging System（UVP Inc.,Upland,CA,USA）.Protein levels were analyzed using the Image J software.7.Cell proliferation assay:The breast cancer cell lines MCF-7 and MDA-MB-231were evenly seeded in six-well plates,cultured overnight for the expression of each index of transfection or interference,and the number of cells was measured about 24hours.After counting,they were evenly spread in sterile 96-well plate and 6-well plate,and then MTT,clone formation experiment and EDU detection were carried out.8.Cell migration and invasion ability assay:The breast cancer cell lines MCF-7 and MDA-MB-231 were evenly seeded in six-well plates.After transfection or interference,the number of cells was measured about 24 hours.After counting,200ul of 10%concentration medium was added to the 24-well plate and then added to the transwell chamber.Evenly,in the upper chamber,each group was blown and added to the same number of cells and 2%concentration medium to form a concentration gradient.24 hours pasted,the cells were washed,fixed.And the cells on the surface of the upper chamber were wiped with a cotton swab and then photographed with an upright fluorescence microscope to determine the number of cells that passed through the chamber.9.Cellular fractionation assay ofRNA:PARISTM kit（Ambion,AM1921）was used to carry out the separation of nuclear and cytoplasmicRNA according to the manufacturer’s instructions.The expression of U6,β-actin was used as cytoplasmic control and nuclear control by real-time PCR.10.RNA pull-down assays:EGFR-AS1 and mi R-149-5p probe were designed,lysed from breast cancer cells and incubated with 3μg of purified biotinylated transcripts for 1 hour at 25°C;streptavidin agarose Beads（Invitrogen,Karlsruhe）were used to separate complexes.RNA present in the pull-down material was purified using phenol:chloroform:isoamyl alcohol（125:24:1 p H=4.3）and detected by real-time PCR analysis.11.Dual-luciferase reporter assay:Wild-type or mut-lnc EGFR-AS1 and wild-type or mut-ELP5 fragment of the 3′-UTR was constructed a using the Dual Luciferase Reporter System Kit（E1910,Promega,USA）.Firefly and renilla luciferase activities were measured,and renilla luciferase was used as control.Afterwards,we co-transfected the mi R-149-5p mimic with the reporter gene for 48h by using Lipofectamine 3000（Invitrogen Co.,Carlsbad,CA,USA）into MCF-7 and MDA-MB-231 cells.12.FISH:EGFR-AS1 FISH probe design and synthesis,breast cancer cells MCF-7and MDA-MB-231 cells were washed twice with phosphate-buffered saline（PBS）and fixed in 4%formaldehyde15 minute.Fixed cells were membrane punched with Triton X-100 and dehydrated with increasing concentrations of ethanol.Cells were then incubated with 50 nmol probe in hybridization buffer followed by incubation at73°C for 5 minutes.Cells were hybridized for 14 hours at 37°C,washed and dehydrated.After adding DAPI,cells were scanned and imaged by confocal microscopy.13.Immunohistochemistry（IHC）:4μm thick tissue sections were deparaffinized continuously with xylene,fixed by 10%neutral formalin and embedded in paraffin.Immunohistochemistry（IHC）staining using the standard streptavidin–peroxidase complex method..Tissue sections were then incubated with ELP5 antibody（1:300;ORGIN,Cambridge,UK）at 4°C overnight,After washing,followed by the biotinylated goat anti-Rabbit Ig G secondary antibody,developed with a horseradish peroxidase-labeled streptavidin reagent,and then stained for 3–5 min with 3,3′-diaminobenzidine（DAB）.The nuclei were counterstained with hematoxylin.14.coimmunoprecipitation（CO-IP）:Breast cancer cells were lysed,12000r/20mins centrifugation to extract protein lysate supernatant.After two hours of blocking effect by adding magnetic beads,the supernatant was centrifuged and EGFR antibody was added.The magnetic beads are added to the 4°shaker overnight and then shaken for6-8 hours.After the end of incubation,the magnetic beads were centrifuged at1000r/5mins and the loading samples were added,and subsequent the protein immunoprecipitation experiment was performed to incubate the ELP5 antibody.To detect the presence of an interaction between the two proteins.15.Half-life assay:The breast cancer cells were evenly spread in a sterile 6-well plate to interfere with EGFR-AS1 and set up 5 sub-wells.Actinomycin D was added after 0,2,4,8 and 12 hours,respectively,and the cells were collected to extractRNA.The stability of itsRNA level was determined by real-time PCR.16.RIP:Breast cancer cells were lysed and incubated with anti-EGFR Ig G for shaking12 hours at 4°C.And then,30μl of Protein A/G beads were added.After 3-6 hours,the beads are washed and put TRIzol reagent and subjected toRNA isolation.Finally,immunoprecipitatedRNA was subjected to real-time PCR analysis using specific primers to confirm the presence of EGFR-AS1 interaction with EGFR.At the same time,it was resolved by agarose gel electrophoresis and developed by silver staining.17.Tumor xenografts in nude mice:1×107cells stably transfected cell line MCF-7 in nude mice on the right flank（each 5 per group）were injected subcutaneously in the logarithmic growth phase were collected.Subcutaneous tumor volume was measured starting from the fourth day after cell injection,The calculation formula is as follows:every 3 days 0.5×length×width2.After 21 days,resection of the tumor,the tumor volume measured and weighed,and snap frozen at-80°C for subsequent analysis.18.Database analysis:In this study,TCGA,GESA,Star Base,mi RBase and RegRNA2.0 database software were used to obtain the expression and prognosis of indicators in breast cancer tissue,and the analysis of binding sites between indicators.19.Statistical analysis:Data were presented as means±standard deviation（SD）,and each experiment was performed in triplicate.The statistical software SPSS 22.0（SPSS,Chicago,IL,USA）was used for all analyses.The Pearson’s Chi-square test was used to assess possible correlations between ELP5 and clinic pathological features of breast cancer.Student’s t-test was used to compare the significant differences of two groups.Spearman’s correlation analysis was used to identify the association.Mann-Whitney U test was used for the image analysis of western blot results and the invasive assay results.P<0.05 was considered as statistically significant.Results:1.TCGA,Starbase and 33 pairs of matched BC tissues by real-time PCR revealed that the level of EGFR-AS1 was upregulated in BC tissues compared with normal tissues.The results of real-time PCR analysis indicated the higher levels of EGFR-AS1 in MCF-7,MDA-MB-231,MDA-MB-453,BT-549,and SK-BR-3 BC cell lines than in MCF-10A cells.Moreover,MCF-7 and MDA-MB-231 cells had the highest expression of EGFR-AS1 among the BC cell lines.Therefore,we selected the two cell lines for further analysis.FISH showed that EGFR-AS1almost located incy,short part innuclar in MCF-7.To evaluate the biological functions of EGFR-AS1 of BC,wherein si EGFR-AS1-siRNA transient transfection was used for knockdown functional analysis.The result of q PCR demonstrated the effectiveness of transfection of EGFR-AS1.As we all known,the metastasis and invasion of tumor cells is a key step in cancer progression.The process of epithelial to mesenchymal transition,that is EMT,represents a significant change in the adhesion and migration of tumor cells.As obtained from the enrichment analysis results of the KEGG database,indicated by proteins,which were associated with cell migration and invasion,the deregulation of ROCK1,Rho A,matrix metalloproteinase（MMP）2and the upregulation of Rho B,following EGFR-AS1 knockdown.In addition,the levels of EMT-related proteins such as N-cadherin,Slug,Snail L,and Vinmentin also notably decreased after EGFR-AS1 knockdown.KEGG enrichment results also show that EGFR-AS1 is related to multiple cancer regulatory pathways,such as PI3K/AKT pathway.EGFR-AS1 knockdown suppressed the expression of PI3K/AKT pathway markers,Moreover,after transfection with si EGFR-AS1,the growth curve of the MTT proliferation assay,colony formation assay and Ed U assays obtained the same results,were that the restraint of EGFR-AS1 significantly inhibited the growth of BC cells.Transwell assays showed that EGFR-AS1 knockdown significantly inhibited the migration and invasion of BC cells compared with those of the control cells.The TCGA database suggested that EGFR-AS1 expression is associated with the poor prognosis of breast cancer patients.Collectively,EGFR-AS1 promoted the EMT（epithelial-to-mesenchymal）transition,proliferation,migration,and invasion of breast cancer cells.2.By multiple database binding analysis,and mi R-149-5p was selected after pull-down assay and real-time PCR assay for regulation.The dual luciferase reporter gene demonstrated that the two can be targeted and bound negatively.Rescue experiments showed that the effects of ELP5,ROCK1,Rho A,and P21 protein expression induced by si-EGFR-AS1 were reversed by the mi R-149-5p inhibitor.Attenuation of proliferation,migration and invasion of breast cancer cells mediated by EGFR-AS1 knockdown as revealed by MTT proliferation assay,colony formation assay,Edu and transwell assays was successfully restored by mi R-149-5p inhibitor3.The dual luciferase reporter gene results showed that ELP5 interacted with mi R-149-5p.Real-time PCR and Western blot showed that the negative regulation and expression of ELP5 and mi R-149-5p at the protein and mRNA levels.Pearson correlation analysis showed that ELP5 mRNA was negatively correlated with mi R-149-5p expression in breast cancer tissues.Rescue experiments showed that the effects of si-ELP5-induced ELP5,ROCK1,Rho A,and P21 protein expression were reversed by mi R-149-5p inhibitor.MTT proliferation assay,colony formation assay,EDU and transwell assay results revealed that the attenuation of proliferation,migration and invasion of breast cancer cells mediated by ELP5 knockdown was successfully restored by mi R-149-5p inhibitor.4.We confirmed that both EGFR-AS1and ELP5 can be regulated by sponging mi R-149-5p.Thus,we established the EGFR-AS1-mi R-149-5p-ELP5 axis for the first time.Immunohistochemical analysis shows that the tissues with high expression of EGFR-AS1,ELP5 also has relatively high expression,the tissues with low expression of EGFR-AS1,ELP5 has relatively lower expression,too.As mentioned above,using real-time PCR confirmed the high expression level of ELP5 in 33 BC tissues relative to adjacent normal tissues According to the median method,an ELP5 low expression group（n=16）and an ELP5 high expression group（n=16）were divided.Then the EGFR-AS1 levels of the two groups were detected respectively,and the results showed that EGFR-AS1 in the high ELP5 group was significantly higher than that in the low ELP5 group.Pearson’s correlation analysis show the positive relationship between the ELP5 mRNA expression and the EGFR-AS1 expression in BC tissue.The result of real-time PCR and western blotting shows that EGFR-AS1 knockdown both decreased the ELP5 mRNA and protein levels.Thus,we demonstrated the positive regulation of ceRNA ELP5 by EGFR-AS1.The EMT-related markers N-cadherin and VINMENTIN are regulated by EGFR-AS1.PI3K/AKT pathway can also be activated by EGFR-AS1 and ELP5.Through rescue assays and based on the results of further experiments by western blotting,including the level protein expression of ELP5,N-cadherin,Vinmentin,PI3K,P-AKT and AKT,we determined that the ectopic ELP5 expression reversed the inhibitory effects of EGFR-AS1suppression.5.Using MTT assay to test IC50 of docetaxel in MCF-7 and MDA-MB-231 cells,different concentration gradients of docetaxel should be add into cells.The IC50s values obtained after docetaxel immersed in MCF-7 and MDA-MB-231 cells for 2days were 9.59 n M and 4.771 n M,respectively.Using real-time PCR and western blotting to detect the addition of IC50 and half of the IC50 concentration of docetaxel into BC cell lines,respectively,to evaluate the levels of EGFR-AS1,mi R-149-5p and ELP5.The results showed that the levels of EGFR-AS1 and ELP5 increased with the increase of drug concentration,and the level of mi R-149-5p decreased with the increase of drug concentration,which proved EGFR-AS1,mi R-149-5p,and ELP5 are all involved in the resistance to docetaxel.After silencing EGFR-AS1,the IC50s decreased to 5.06 n M and 2.36 n M in BC cells with docetaxel immersed,respectively.Colony formation and MTT proliferation assays were then conducted,and it was found that with the addition of the IC50 concentration of docetaxel,the cells with lower EGFR-AS1 expression had the less favorable cell viability.6.sh-EGFR-AS1 or sh-NC cultured in MCF-7 cells and injected subcutaneously into BALB/c nude mice.Subsequently,the tumor size was monitored every three days,and the tumor was removed and calculated after 21 days.In addition,compared with the negative control group,EGFR-AS1 knockdown significantly inhibited tumor growth curve,resulting in smaller tumor size and lighter weight.Using real-time PCR to analyze the tumors were harvested from mice,which revealed that the expression of EGFR-AS1 and ELP5 mRNA was decreased,the expression of mi R-149-5p was increased,a consistent ceRNA regulatory effect in vitro experiments.Using western blot in the EGFR-AS1-silenced MCF-7 cells to obtain the inhibition of ELP5,EGFR,EMT-related proteins（N-cadherin,Vinmentin,Slug,Snail）and PI3K/AKT pathway related markers.These results indicate that EGFR-AS1 promotes the growth and metastasis of BC tumors in vivo.7.TCGA,Star Base database and 33 pairs of breast cancer tissues showed that the expression level of ELP5 in breast cancer tissues was higher than that in paired adjacent tissues,both at the protein level and mRNA level in breast cancer cell lines.The expression level was also higher than that of normal mammary epithelial cells MCF-10A.Immunohistochemical results showed that ELP5 was negatively or weakly expressed in adjacent tissues,and weakly or strongly positive in cancer tissues.Western blot results showed that ELP5 activates the PI3K/AKT pathway,and can interact with and regulate EGFR at the protein level,thereby affecting the development of breast cancer.8、There are 194 bp-long base-pairing length sequence.The nucleoplasmic separation results show that EGFR-AS1 in breast cancer cells is mainly located in the cytoplasm,a small amount is located in the nucleus and thus EGFR-AS1 may involved in the post-transcriptional regulation of the target gene.Next,we detected the expression of EGFR mRNA level in the same 33 pairs of matched BC tissues by real-time PCR was upregulation,and the relationship of EGFR mRNA and EGFR-AS1 was positively correlated.The level of EGFR protein was much higher in EGFR-high samples compared with that in EGFR-low samples of the BC tissues.Furthermore,using si-EGFR-AS1 to detect the expression of EGFR mRNA and the level of EGFR protein,and EGFR level was both markedly decreased.Thereafter,actinomycin D（Act D）,a transcriptional inhibitor was exploited.The real-time PCR results demonstrated that EGFR-AS1 affects EGFR stability in BC cells.Finally,anRNA immunoprecipitation（RIP）assay result shows that there was a direct effect between EGFR-AS1 and EGFR mRNA.After interfering with EGFR-AS1,the close effect became weaker compared with the control group.To further explore the relationship between ELP5,EGFR-AS1,and EGFR,we performed co-transfection experiments with simultaneous overexpression of EGFR-AS1 and knockdown of ELP5.The expression of EGFR protein level was detected by Western blot,and the results showed that the expression of EGFR was decreased in the co-transfection group compared with the EGFR-AS1 overexpression group.It shows that ELP5 is necessary for the regulation of EGFR-AS1 on EGFR.Conclusion:1.Effect of EGFR-AS1 knockdown on inhibiting the proliferation,migration,invasion and EMT of BC cells in vitro2.Role of the EGFR-AS1-mi R-149-5p-ELP5/PI3K/AKT axis in the promotion of BC development3.Role of EGFR-AS1 silencing in the sensitivity promotion to docetaxel in BC cells4.Effect of EGFR-AS1 knockdown on inhibiting the proliferation,migration,invasion and EMT of BC cells in vivo.5.Effects of ELP5 on proliferation,metastasis,and invasion of BC Cells and regulate the stability of EGFR at the translation level.6、Regulation of EGFR by EGFR-AS1 depends on ELP5... |
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