The Study Of The Mechanisms Of Malignant Biological Behavior Mediated By SLC7A5 In Breast Cancer

BackgroundBreast cancer always ranks first among female tumors in the world.According to the2020 global cancer burden data from International Agency for Research on Cancer(IARC),breast cancer,accounting for about 11.7%,has surpassed lung cancer to become the top one with the largest number of newly diagnosed patients in the world for the first time.With the rapid development of technologies such as high-throughput sequencing and gene chips,the research on breast cancer with typical heterogeneous characteristics has shifted from traditional pathology to the direction of molecular genetics.Exploring and discovering ideal molecular markers is of great significance for accurate diagnosis,prognostic evaluation and precise treatment of breast cancer.The solute carrier family 7 member 5(SLC7A5)is located on human chromosome 16p24.3.This gene encodes a membrane protein composed of 12 transmembrane domains.It is mainly responsible for the exchange of intracellular and extracellular nutrients,especially the large-branched chain amino acids.Amino acid transporter is an important sensor of mTORC1 signaling pathway that can accurately sense and activate amino acid signals,leading to cellular proliferation,invasion and other malignant behaviors.In recent years,a large number of studies have shown that the amino acid transporter SLC7A5 is highly expressed in a variety of tumor tissues,suggesting that it may play a role in the occurrence and progression of tumors.Micro RNAs(miRNAs)are a class of highly conserved endogenous non-coding short single-stranded RNA molecules that regulate gene expression by repressing translation or even directly degrading m RNA,and regulate target genes at the post-transcriptional level.In recent years,studies have found that miR-126-3p affect the proliferation and invasion of various tumor cells through multiple signaling pathways.ObjectivesThe purpose of this study is to comprehensively investigate the expression of SLC7A5 in breast cancer and its molecular mechanisms in tumor cell proliferation,invasion and malignant behaviors,and to evaluate the relevant clinicopathological factors,the diagnostic and prognostic value of SLC7A5 as a molecular biomarker for breast cancer.Furthermore,we also investigated the roles of AKT/mTOR signaling pathway on the malignant biological behavior of breast cancer.This work has made novel findings and characterized the roles and mechanisms of SLC7A5 in breast cancer cell proliferation,invasion and malignant behaivors,which may provide the strong evidence for precise individualized management of breast cancer patients.Methods1.RNA-sequencing data of breast cancer tissues and adjacent tissues were obtained from the TCGA official website,and 1222 samples with clinicopathological characteristics and survival data were downloaded.The correlations between SLC7A5 expression and clinical features were examined and analyzed.2.Receiver operating characteristic curve(ROC)analysis was used to predict the diagnostic value of SLC7A5 in breast cancer.3.Kaplan-Meier curves were used to analyze the SLC7A5-related survival of breast cancer.4.Cox’s proportional hazards regression model was applied to construct SLC7A5-related prediction model,and the nomogram was used for visualization.5.bc-Gen Ex Miner v4.4(Breast Cancer Gene-Expression Miner)was applied to analyze the gene chip data of METABRIC(Molecular Taxonomy of Breast Cancer International Consortium)project to verify the correlation between the expression of SLC7A5 and PAM50(Prediction Analysis of Microarray 50)classification,SBR(Scarff-BloomRichardson)classification,and NPI(Nottingham prognostic index)prognostic index.6.The SLC7A5 expression was detected and confirmed in 60 samples by immunohistochemistry method.7.Western blot and q RT-PCR were used to detect the expression of SLC7A5 in four breast cancer cell lines,including MDA-MB-231,MCF-7,T47-D and SK-BR-3,and follow-up experiments were performed.8.Lentiviral vectors and small molecular siRNA fragments were separately used to establish breast cancer cell models with SLC7A5 gene overexpression and knockdown,and further to screen and verify the changes in gene and protein expression levels.9.Cell Counting Kit 8(CCK-8)and Ed U assays were used to detect cell proliferation ability,and plate colony formation assay,transwell chamber assay,flow cytometry were carried out to detect cell clone formation ability,cell invasion and the effect on cell cycle respectively.10.The effects of SLC7A5 expression changes on the expression levels of PIK3 CA,mTOR,p-mTOR,AKT,p-AKT,S6 K,p-P70S6 K and MYC were detected by q PCR and WB.11.Based on the expression of TCGA-BRCA miRNA-seq in breast cancer,Kaplan-Meier curve was used to analyze miR-126-3p-related survival in breast cancer.12.The binding of miR-126-3p to SLC7A5 regulaory sequence was verified by dualluciferase reporter assay.13.After constructing overexpression and knockdown breast cancer cell models by transfected by miR-126-3p mimic and inhibitor,respectively,the proliferation,clone formation ability and cell cycle of MCF-7 cells were separately detected by Ed U,CCK-8,plate clone formation experiments and flow cytometry14.The roles of miR-126-3p were characterized on the expression of SLC7A5 and AKT/mTOR signaling pathway related proteins p-mTOR,p-AKT,and p-P70S6 K.15.After co-transfection of miR-126-3p inhibitor and si-SLC7A5,the effects on the proliferation of MCF-7 cells were analyzed,and the expression changes of SLC7A5,pmTOR,p-AKT and p-P70S6 K were detected.Results1.The expression of SLC7A5 in breast cancer tissues was found to be higher than that in adjacent normal tissues,and its expression levels were significantly affected by different ages,races,pathological tissue types,ER status,PR status,PAM50 molecular subtypes,TNM staging,T staging,and the outcome of overall and disease-specific survival subgroups in breast cancer(P<0.05).High expression levels of SLC7A5 were associated with invasive ductal carcinoma,ER/PR-negative status,HER2 positive status,basal-like(PAM50),luminal B(luminal subtyps),TNM stage II-IV,T2-T4 stage,and African clinical characteristics(P <0.05).2.ROC analysis results showed that SLC7A5 had good diagnostic value in the diagnosis of breast cancer,ER-positive subtypes,PAM50 molecular subtypes,and TNM staging.3.Kaplan-Meier survival analysis showed that the high expression of SLC7A5 was associated with the poor prognosis of breast cancer(P=0.002).The analysis results of different subgroups showed that the high expression of SLC7A5 was associated with advanced age(>60 years),ER positive,Luminal A,Luminal B,TNM stage II,stage III,T2,N1/N2/N3,and M0 subgroups,which were significantly related to the poor prognosis of the overall survival(P<0.005).Verification results from Kaplan-Meier Plotter database also showed that the high expression of SLC7A5 in ER-positive subgroup breast cancer was associated with poor recurrence-free survival rate(P<0.001).4.Cox univariate analysis showed that breast cancer patients with high expression of SLC7A5 had a poor prognosis(P=0.001),and multivariate analysis showed that SLC7A5 could be used as an independent risk factor for breast cancer,which affects overall survival(HR=2.469,P<0.005).The correlation between SLC7A5 expression and breast cancer risk was presented in the form of nomogram.5.The expression of SLC7A5 in breast cancer of the METABRIC dataset was significantly related to PAM50 classification,SBR grade,and NPI prognostic index(P<0.001).6.Immunohistochemical analysis showed that the expression levels of SLC7A5 in luminal and non-luminal breast cancer samples were significantly different from each other.The expression level of SLC7A5 protein in basal cell breast cancer was significantly higher than that of luminal A subtype(P<0.05).7.Western blot showed that SLC7A5 was expressed in MDA-MB-231,MCF-7,T47-D and SK-BR-3 breast cancer cell lines.8.Sequencing analyses verified that the SLC7A5 overexpression lentiviral vector was successfully constructed and transfected into MCF-7 cells.Western blot confirmed that the SLC7A5 expression significantly increased at the levels of m RNA and protein in MCF-7 cells.9.It was observed that overexpression of SLC7A5 enhanced the proliferation,clone formation,and invasion ability of MCF-7 cells(P<0.05),and the proportion of cells in S phase was increased(P<0.001).Knockdown of SLC7A5 expression inhibited the proliferation,clone formation,and invasion ability of MCF-7 cells(P<0.05),and reduced the proportion of cells in S phase(P<0.01).10.The levels of p-mTOR,p-AKT,p-P70S6 K,and MYC increased in MCF-7 cells overexpressing SLC7A5(P<0.01);While knockdown SLC7A5 expression,decreased the levels of p-mTOR,p-AKT,p-P70S6 K,and MYC(P<0.05).There was no statistical difference in the levels of total mTOR and AKT(P>0.05).11.The expression levels of miR-126-3p in breast cancer tissues and breast cancer cells were lower than that in adjacent normal tissues and breast epithelial cells(P<0.05),and low expression was associated with poor prognosis of breast cancer(P=0.001).12.The dual-luciferase reporter gene assays showed that the luciferase activity of the SLC7A5+miR-126-3p mimic co-transfection group was lower than that of the control group(P<0.01).13.After up-regulating the expression of miR-126-3p,the proliferation and clone formation of MCF-7 cells were inhibited,and the distribution ratio of cells in S phase was reduced(P<0.001).However,if the expression of miR-126-3p was knocked down,the proliferation and colony formation of MCF-7 cells were enhanced,and the number of cells in S phase was increased(P<0.05).14.The expression levels of SLC7A5,p-mTOR,p-AKT,and p-P70S6 K decreased in MCF-7cells overexpressing miR-126-3p(P<0.01);while knockdown of miR-126-3p led to increased expression levels of SLC7A5,p-mTOR,p-AKT and p-P70S6K(P<0.01).15.Knockdown of miR-126-3p and simultaneous inhibition of SLC7A5 expression were capable of reducing MCF-7 cell proliferation,clone formation,and S phase cell ratio(P<0.01).The level of p-mTOR,p-AKT and p-P70S6 K was lower as compared to miR-126-3p knocked down samples(P<0.01).Conclusions1.The expression of SLC7A5 is up-regulated in breast cancer tissues,and the differential expression level is closely related to multiple clinical features,especially the different subtypes of breast cancer.This finding has good diagnostic value.The level of SLC7A5 expression is an independent risk factor for overall survival of breast cancer patients,especially high expression level in luminal subtypes of breast cancer is associated with poor prognosis.2.The expression of miR-126-3p is down-regulated in breast cancer tissues,and low expression level is associated with poor prognosis.3.Up-regulation of SLC7A5 expression and down-regulation of miR-126-3p expression promote the proliferation activity,clone formation,and cell cycle progression of breast cancer cell line MCF-7.4.Increased SLC7A5 expression enhances the activity of the AKT/mTOR pathway by phosphorylating AKT、mTOR,and expression level of MYC,which play a key role in mediating cellular proliferation,clony formation and cell cycle progression as well as the occurrence and development of tumors and malignant behavior.5.MiR-126-3p plays role in regulating the expression of SLC7A5 in breast cancer cells,which can inhibit breast cancer cell proliferation through suppressing SLC7A5 expression and subsequent AKT/mTOR pathway.