| Background: Breast cancer is the highest incidence malignant tumor among women in Asia and worldwide.Although the survival rate of some patients has improved by early diagnosis,early surgery,or neoadjuvant therapy in recent years,its recurrence and metastasis still seriously affect the life and health of patients.Therefore,it is essential to deeply explore the mechanism of occurrence and progression of breast cancer and seek more meaningful diagnostic and prognostic indicators.In breast cancer,tumor invasion,recurrence,and metastasis are the main factors affecting the prognosis of patients,and these processes are related to the abnormal activation of epithelial-mesenchymal transition.Epithelial-mesenchymal transition refers to transforming epithelial cells into cells with a mesenchymal phenotype through regulating a series of factors,reducing cell junctions,disappearing cell polarity,invasive ability,and thus having malignant biological behavior.Deleting the expression of epithelial markers such as E-cadherin,ZO-1 as well as increased expression of mesenchymal markers such as vimentin and N-cadherin.Epithelial-mesenchymal transition can make cells resistant to apoptosis,leading to tumor resistance to radiotherapy and chemotherapy,and can also promote tumor recurrence and distant metastasis.In aggressive breast cancer,its markers are also increasingly associated with patient prognosis.Many studies have shown that long non-codingRNAs regulate the epithelial-mesenchymal transition of tumors.Long non-codingRNAs belong to non-codingRNAs.They are longer than 200 nt and do not directly encode proteins.T They are often involved in coding genes in various ways,such as epigenetic regulation,transcriptional level regulation,and post-transcriptional level regulation,and widely affect apoptosis,proliferation,invasion,and other related biological behaviors of tumor cells and closely related to the disease process.Therefore,they are widely regarded as essential regulators of gene expression and tumorigenesis and development,as well as potential targets for early diagnosis,adjuvant therapy,and prognosis of tumors.In breast cancer,many long non-coding chains have been confirmed to be differentially expressed and have various effects on the biological functions of cells.Some long non-codingRNAs can directly act on genes that relate to epithelial-mesenchymal transition.They act as signaling molecules or through epigenetic methods,affecting tumor cell invasion,metastasis,and epithelial-mesenchymal transition,such as TGF-β,Wnt/β-catenin,etc.In addition,some long non-codingRNAs can be indirectly regulated by competing endogenousRNA mechanisms The process of epithelial-mesenchymal transition.The competitive endogenousRNA mechanism is a mode of gene expression regulation that has attracted much attention in recent years.Its hypothesis is based on the fact that transcripts sharing microRNA binding sites can compete for the same microRNA,such as long non-codingRNAs,which can act as molecular sponges.Adsorbs microRNAs and competitively inhibits the effects of microRNAs on target genes,thereby playing a pivotal role in tumorigenesis,invasion,metastasis,and drug resistance.This study aimed to discover long non-codingRNAs which significantly differentially expressed in breast cancer and substantially impact disease progression and prognosis.Furthermore,exploring the molecular mechanism by which this lncRNA affects tumor progression will provide a new basis for diagnosing and treating breast cancer.Methods:Part 1:1.Selection of differentially expressed lncRNA: Through bioinformatics analysis,we found that several lncRNAs that were significantly highly expressed in breast cancer.A small number of samples were taken,and q RT-PCR was used to determine the expression of selected lncRNAs in breast cancer tissue and expression in cell lines to verify the results of bioinformatics analysis.2.Exploration of the essential characteristics of lncRNAs: online prediction of the subcellular localization of selected lncRNAs,and infer the possible molecular mechanisms of its effects according to the localization;online database prediction of its correlation with patient prognosis,and correlation of its expression with the collected clinicopathological data was used for correlation analysis;online database predicted its correlation with breast cancer molecular typing,and q RT-PCR was used to determine its expression in four molecularly typed breast cancers.3.The effect of lncRNA on the biological function of cells: construct a stable cell line that overexpresses and silences lncRNA,and conducts cell function experiments of overexpression and silencing of lncRNA: CCK-8 assay to determine cell proliferation activity,and scratch healing assay to determine the migration ability of cells,Transwell assay to measure the ability of cell invasion,the effect of Annexin V-FITC on apoptosis was detected,and Western Blots detected the protein expression of epithelial-mesenchymal transition markers.Part II:1.Selection of differentially expressed genes: The genes with significantly high expression in breast cancer were screened by bioinformatics analysis,and the results of bioinformatics analysis were verified by q RT-PCR.2.Expression verification of target genes in breast cancer: q RT-PCR was used to determine the expression of selected genes in breast cancer tissues and cell lines and determine their expression in four molecularly typed breast cancers.Western Blot and immunohistochemical staining were used to evaluate selected genes expression at the protein level,and fluorescence in situ hybridization was used to detect the changes of selected genes at the gene level.The clinicopathological information was used for correlation analysis.3.Selection of target miRNAs that interact with each other.Find the sequence of the selected lncRNA and the selected gene,predict the miRNAs that can interact with the two online,and take the intersection of the results;verify the results of bioinformatics analysis by q RT-PCR.Eligible miRNAs were initially selected to construct a complete ceRNA mechanism axis.4.Exploration of the basic characteristics of selected miRNAs.q RT-PCR was used to determine the expression of selected genes in breast cancer tissues and cell lines to determine their expression in four molecularly typed breast cancers.The correlation analysis was performed between the expression level and the collected clinicopathological information.5.Verification of the interaction relationship: Construct a dual-luciferase reporter gene to verify the relationship between lncRNA-miRNA and miRNA-target gene;the interaction relationship is confirmed byRNA-binding protein immunoprecipitation andRNA pull-down experiments.6.Verification of the regulatory effect of lncRNA on miRNA and its effect on cell biological function by regulating miRNA: q RT-PCR was used to measure the changes of miRNA after overexpression or silencing of lncRNA to verify the regulation of lncRNA on miRNA;design cell function experiments and functions by grouping Recovery experiments,CCK-8 experiments,scratch healing experiments,and Transwell experiments were performed to verify the regulatory effect of lncRNA on cell biological function by adsorbing miRNA.7.Verification of the regulatory effect of LncRNA/miRNA axis on target genes and the effect of regulating target genes on cell biological function.Design cell function experiments and functional recovery experiments again by grouping and conducting CCK-8 experiments,scratch healing experiments,Transwell experiments Experiments confirmed that lncRNA/miRNA plays a regulatory role in cell biological function by regulating target genes.8.Functional verification of the ceRNA axis in vivo.Inject stably transfected cells into nude mice to construct a nude mouse subcutaneous tumor model,measure the size of the tumor,and make tissue sections to detect the expression of various indicators of epithelial-mesenchymal transition by immunohistochemical staining.Results:Part 1:1.Selection of differentially expressed lncRNAs: Bioinformatics analysis based on the TCGA database showed that a total of 510 lncRNAs were significantly up-regulated in breast cancer;among the up-regulated lncRNAs,the expression abundance and differential fold were ranked from most significant to highest.Small sorting,selecting the top 10 lncRNAs and performing secondary verification on the GEPIA website.The results are consistent with the statistical conclusions of the R software.It is found that linc00504 is the most significantly up-regulated lncRNA;6 pairs of breast cancer and adjacent paired samples were selected,and the detection of the four predicted lncRNAs through q RT-PCR showed that linc00504 was most significantly up-regulated in breast cancer;therefore,linc00504 was selected as the research object.2.Exploration of the essential characteristics of Linc00504.First,it was predicted to be localized in the cytoplasm by its coding sequence,and it was speculated that it might play a role through a competitive endogenousRNA mechanism.Forty pairs of breast cancer and adjacent paired samples were further selected and confirmed by q RT-PCR.Linc00504 is highly expressed in breast cancer.GEPIA online prognostic analysis shows that linc00504 significantly impacts the prognosis of advanced/advanced breast cancer.Combined with the clinicopathological data of 40 patients,a Spearman correlation analysis was performed,and the results showed that the expression of linc00504 was closely related to the age and tissue of the patients.There was a positive correlation between the expression of ER,clinical stage,and ER,which also indicated that it was correlated with the prognosis of patients;GEPIA online correlation analysis showed that linc00504 was significantly correlated with genes related to molecular typing.Based on the similarities and differences of expression in breast cancer of various molecular types,it was found that linc00504 was most significantly up-regulated in Luminal A breast cancer.3.The effect of Linc00504 on cell biological function: The ov-linc00504 and si-linc00504 stable transfection cell lines were constructed,and after the transfection efficiency was verified,the cell function experiments were carried out in groups;CCK-8proliferation experiments showed that the si-linc00504 group The cell proliferation activity decreased,and the cell proliferation activity in the ov-linc00504 group increased;wound healing assay showed that the migration ability of the si-linc00504 group was decreased,and the migration ability of the ov-linc00504 group was increased;the Transwell experiment showed that the invasive power of the si-linc00504 group decreased,and the ov-linc00504 group increased cell invasiveness;Annexin V-FITC cell apoptosis assay showed that si-linc00504 group promoted early cell apoptosis,while ov-linc00504 group inhibited early cell apoptosis;q RT-PCR and Western Blot analysis showed that si-linc00504 group cells The expression of epithelial indexes increased and the expression of mesenchymal indexes decreased,and ov-linc00504 showed the opposite trend,indicating that linc00504 can promote cell proliferation,invasion and migration,inhibit cell apoptosis,and promote the process of epithelial-mesenchymal transition.Part II:1.Selection of differentially expressed genes: R software analysis based on the TCGA database showed that a total of 388 genes were significantly up-regulated in breast cancer;among the up-regulated genes,the expression abundance and differential fold were from large to small.Sorting,selecting the top 10 of them and verifying them twice on the GEPIA website.The results are consistent with the statistical conclusions of the R software,and it is found that MDM2 is the most significantly up-regulated gene.2.Verification of the expression of MDM2 in breast cancer: 40 pairs of breast cancer and adjacent paired samples were selected,and the high expression of MDM2 in breast cancer was confirmed by q RT-PCR;cell lines representing different molecular types were selected,the up-regulation of MDM2 in Luminal A breast cancer was the most significant was verified by q RT-PCR and Western Blot;40 tissue sections were selected for immunohistochemical staining,which proved that MDM2 was highly expressed in Luminal type A breast cancer;the paraffin sections from the same source were subjected to fluorescence in situ hybridization,which proved that MDM2 is highly expressed in Luminal type A breast cancer.Gene amplification exists in Luminal type A breast cancer;GEPIA correlation analysis showed that MDM2 was significantly correlated with breast cancer prognosis genes,and further confirmed by immunohistochemistry that its expression was significantly associated with the expression of prognostic indicators Ki-67,p53,and Bcl-2.Spearman correlation analysis was performed based on the clinicopathological data of 40 patients,and MDM2 expression was positively correlated with patient age,ER,p53,ki-67,PD-1 and PD-L1.3.Selection of target miRNAs that interact with each other: Based on data processing of the online target binding site database,nine miRNAs that can interact with linc00504 and MDM2 at the same time were found;3 pairs of breast cancer and cancer were selected Paired samples were detected by q RT-PCR on the predicted four miRNAs,which showed that among all miRNAs,miR-1244 was most significantly down-regulated in breast cancer;therefore,miR-1244 was selected as the research object.4.Exploration of the basic characteristics of miR-1244.Forty pairs of breast cancer and adjacent paired samples were selected,and the low expression of miR-1244 in breast cancer was determined by q RT-PCR;cell lines representing different molecular types were selected,q RT-PCR The results showed that the down-regulation of miR-1244 was the most significant in Luminal A breast cancer.Combined with the clinicopathological data of 40 patients,the Spearman correlation analysis displayed that miR-1244 expression was negatively correlated with the expression of ER and PD-L1;5.Verification of the interaction relationship: It was preliminarily verified that miR-1244 could interact with the mRNA of linc00504 and MDM2 by the dual-luciferase reporter gene assay;their interaction was further clarified byRNA-binding protein co-immunoprecipitation andRNA pull-down experiments.6.The effect of Linc00504 on the biological function of cells by regulating miR-1244.ov-linc00504 and si-linc00504 stable transfection cell lines were constructed.The results of q RT-PCR showed that the content of miR-1244 in the ov-linc00504 group decreased significantly;si--NC,miR-1244 inhibitor,and si-linc00504+miR-1244 inhibitor three groups were subjected to cell function and functional recovery experiments.The results of the CCK-8 experiment showed that linc00504 promoted cell proliferation by acting on miR-1244.Acting on miR-1244 promotes cell migration,and Transwell experiments show that linc00504 promotes cell invasion by acting on miR-1244.7.Linc00504/miR-1244 axis affects cell biological function by regulating MDM2.Three groups of si-NC,si-linc00504,and si-linc00504+ov-MDM2 were constructed to conduct cell function and functional recovery experiments,and CCK-8 experiment The results showed that overexpression of MDM2 after silencing linc00504 would increase the originally weakened cell proliferation activity,indicating that the linc00504/miR-1244 axis promotes cell proliferation by regulating MDM2.The results of scratch healing and Transwell experiments also confirmed that linc00504/miR-1244 Axis promotes cell migration and invasion by regulating MDM2.8.Functional verification of the Linc00504/miR-1244/MDM2 axis in vivo: We performed subcutaneous tumorigenesis experiment in BALB/C mice,and the tumor volume in the si-linc00504 group was obviously reduced,while the tumor volume in the si-linc00504+ov-MDM2 group increased,and the si-linc00504 group increased in volume.In the transplanted tumor tissue of the linc00504 group,the expression of epithelial indexes increased,and mesenchymal indexes decreased.In contrast,the expression of epithelial indexes decreased,and the expression of mesenchymal indexes increased in the si-linc00504+ov-MDM2 group,indicating that linc00504/miR-1244/MDM2 can also play a role in vivo.Regulates and promotes epithelial-mesenchymal transition.Conclusion:1.Linc00504 is highly expressed in breast cancer,and its expression has specificity in molecular typing and is related to patient prognosis;2.Linc00504 can promote the proliferation,migration,invasion,and other malignant phenotypes of breast cancer cells and facilitate epithelial-mesenchymal transition;3.Linc00504 can act as a molecular sponge to adsorb miR-1244 and competitively inhibit the binding of miR-1244 to MDM2;4.Linc00504 can promote cell proliferation,migration,invasion,and other malignant phenotypes through the linc00504/miR-1244/MDM2 axis;5.Linc00504 can promote the malignant biological behavior of cells,stimulate the epithelial-mesenchymal transition process,and promote breast cancer progression through the linc00504/miR-1244/MDM2 axis. |
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