| Background and objective:The incidence of breast cancer in female malignancies in China has remained high,and it has been increasing year by year in recent years.Tumor cells have the characteristics of rapid proliferation,reduced apoptosis,and strong metastasis,this is also an important reason for the rapid metastasis and development of malignant tumors.In addition,tumor cells also have different metabolic patterns than normal cells.Normal cells only use glycolysis to supply energy under conditions of insufficient oxygen,and tumor cells also preferentially provide glycolysis to supply energy under conditions of sufficient oxygen,this special energy metabolism pathway is called "aerobic sugar" "Fermentation",also known as "Warburg".The special energy metabolism pathway of tumor cells is also related to the abnormally expressed genes in tumors.lncRNA is a non-coding RNA with a length of more than 200bp,it is located in the cytoplasm and nucleus,and has no obvious open reading frame.It has been found that lncRNA is involved in RNA splicing,gene transcription,X chromosome inactivation,and the occurrence of various diseases,inhibition of miRNA expression.lncRNA is related to human tumors,and abnormal lncRNA expression has been found in tumors,these abnormally expressed lncRNAs can be used not only as potential molecular markers for early diagnosis of tumors but also as effective targets for molecular targeted therapy.lncRNA MEG3 is highly expressed in the human brain and pituitary gland,and is an imprinted gene found in humans.MEG3 expression is lost in many human tumor cell lines,such as glioma,colorectal cancer,hepatocellular carcinoma,ovarian cancer,etc.The decrease in MEG3 expression is related to the prognosis,metastasis,and clinical pathological staging of tumor patients,MEG3 also participates in the regulation During the growth,apoptosis and metastasis of tumor cells,the level of MEG3 expression is closely related to the tumor.This experiment includes the following four parts:Part one:The effect of lncRNA MEG3 on breast cancer cell proliferation,apoptosis,metastasis and Warburg;Part two:The role of lncRNA MEG3 in targeting miR-21 to influence breast cancer progression;Part three:MEG3 overexpression reverses miR-21-mediated activation of the PI3K/AKT pathway in breast cancer cells;Part four:Up-regulating MEG3 inhibits tumor growth in breast cancer cells by down-regulating miR-21.Part One The effect of lncRNA MEG3 on breast cancer cell proliferation,apoptosis,metastasis and WarburgMethods:1.qRT-PCR was used to detect changes in MEG3 expression in 40 breast cancer tissues and corresponding adjacent tissues.2.qRT-PCR was used to detect the difference of MEG3 expression between breast cancer MCF-7,MDA-MB-231,MDA-MB-453,T47D cells and normal breast epithelial MCF-10A cells.3.Transfection of pcDNA-MEG3 in breast cancer cells,qRT-PCR method to detect the effect of up-regulation,MTT method to detect cell proliferation and change,clone formation test to detect cell clone formation ability,flow cytometry to detect apoptosis change,Transwell chamber detection Changes in cell migration and invasion capabilities.Western blot detected changes in E-cadherin,N-cadherin,Vimentin,HK2,and LDHA protein expression.Kits measure cell glucose consumption,lactic acid production,and ATP levels.Hippocampal extracellular flux analyzer XF 96 cells were detected for ECAR and OCR.Results:1.The expression level of MEG3 in breast cancer tissues was lower than that of normal counterpart tissues.2.The expression levels of MEG3 in breast cancer MCF-7,MDA-MB-231,MDA-MB-453,and T47D cells were lower than those in normal breast epithelial MCF-10A cells.3.After transfection with pcDNA-MEG3,breast cancer cells have increased MEG3 expression levels,reduced cell proliferation ability,decreased cell clone formation ability,increased apoptosis,decreased cell invasion and migration ability,and N-cadherin and Vimentin proteins levels decreased,the expression level of E-cadherin protein increased,glucose consumption,glucose consumption and lactic acid production decreased,cell ATP level decreased,cell ECAR level decreased,OCR level increased,HK2 and LDHA protein expression levels in cells decreased.Part Two The role of lncRNA MEG3 in targeting miR-21 to influence breast cancer progressionMethods:1.Target gene prediction software analyzes the targeting relationship between MEG3 and miR-21,and the dual luciferase reporting system and RIP experiments verify the targeting relationship.2.Using qRT-PCR to detect miR-21 expression changes in 40 breast cancer tissues and corresponding adjacent tissues.3.qRT-PCR was used to detect the difference of miR-21 expression in breast cancer MCF-7,MDA-MB-231,MDA-MB-453,T47D cells and normal breast epithelial MCF-10A cells.4.Transfection of pcDNA-MEG3,MEG3 siRNA in breast cancer cells,qRT-PCR detection of MEG3 and miR-21 expression changes in cells.5.Co-transfection of pcDNA-MEG3 and miR-21 mimics in breast cancer cells,qRT-PCR to detect miR-21 expression changes in cells,MTT method to detect cell proliferation changes,clone formation test to detect cell clone formation ability,flow cytometry Detect changes in apoptosis,Transwell chamber to detect changes in cell migration and invasion,Western blot to detect changes in E-cadherin,N-cadherin,Vimentin,HK2,and LDHA protein expression,kit to detect cell glucose consumption,lactic acid production and ATP XF 96,a hippocampal extracellular flux analyzer,detected ECAR and OCR of cells.Results:1.MEG3 targets the regulation of miR-21 expression.2.The expression level of miR-21 in breast cancer tissues was higher than that in normal adjacent tissues.The expression levels of MEG3 and miR-21 in breast cancer tissues were negatively correlated.3.The expression levels of miR-21 in breast cancer MCF-7,MDA-MB-231,MDA-MB-453,and T47D cells were higher than those in normal breast epithelial MCF-10A cells.4.The expression level of MEG3 in breast cancer cells transfected with pcDNA-MEG3 increased and the expression level of miR-21 decreased;the expression of MEG3 in breast cancer cells transfected with MEG3 siRNA decreased and the expression level of miR-21 increased.5.Transfection of miR-21 mimics can partially reverse up-regulation of MEG3 in affect miR-21 expression,proliferation,cloning,apoptosis,invasion,migration,glucose consumption,glucose consumption,lactic acid production,ATP synthesis,ECAR and OCR levels in breast cancer cells and effects of N-cadherin,Vimentin,E-cadherin,HK2,and LDHA protein expression.Part Three MEG3 overexpression reverses miR-21-mediated activation of the PI3K/AKT pathway in breast cancer cellsMethods:1.Transfect pcDNA-MEG3 and miR-21 mimics into breast cancer cells,respectively,and given them the PI3K/AKT signaling pathway activator 740Y-P,PI3K/AKT signaling pathway inhibitor LY294002,Western blot analysis and detection protein expression of p-AKT and PI3K.2.Co-transfect miR-21 mimics and pcDNA-MEG3 in breast cancer cells.Western blot was used to detect the changes of p-AKT and PI3K protein expression in cells.Results:1.The levels of p-AKT,PI3K mRNA and protein were increased in breast cancer cells transfected with miR-21 mimics.LY294002 reduced the expression levels of p-AKT and PI3K proteins in breast cancer cells.The p-AKT and PI3K protein levels in breast cancer cells transfected with pcDNA-MEG3 decreased,and 740Y-P increased the p-AKT and PI3K protein expression levels in breast cancer cells.2.Transfection of pcDNA-MEG3 can reduce p-AKT,PI3K protein levels in breast cancer cells after miR-21 up-regulation.Part Four Up-regulating MEG3 inhibits tumor growth in breast cancer cells by down-regulating miR-21Methods:1.Infect breast cancer cells with MEG3 lentivirus,inoculate breast cancer cells under the skin of nude mice,and observe the growth volume and weight changes of nude mice.2.Infect breast cancer cells with MEG3 lentivirus and.miR-21 lentivirus,inoculate breast cancer cells under the skin of nude mice transplanted tumors,and observe the growth volume and weight changes of nude mice transplanted tumors.3.Nude mouse transplanted tumor tissues were taken and qRT-PCR was used to detect the expression levels of MEG3 and miR-21.Results:1.The growth volume and weight of nude mice transplanted with MEG3 lentivirus-infected breast cancer cells were significantly reduced.2.miR-21 lentiviral infection can partially reverse the inhibitory effect of MEG3 on breast cancer cell transplantation tumor growth volume and weight in nude mice.3.MEG3 lentivirus-infected breast cancer cells in nude mice xenograft tumor tissue MEG3 expression levels increased,miR-21 expression levels decreased;miR-21 lentivirus infection can partially reverse the up-regulation of MEG3’s inhibitory effect on miR-21 expression in breast cancer cell transplanted nude mice.Conclusion1.MEG3 was down-regulated in breast cancer tissues and cells.Upregulated MEG3 inhibited breast cancer cell proliferation,cloning,invasion,migration,EMT and Warburg,and induced apoptosis.2.miR-21 was highly expressed in breast cancer tissues and cells,and the expression of miR-21 and MEG3 in breast cancer tissues was negatively correlated.MEG3 targeted negative regulation of miR-21 expression in breast cancer cells.3.miR-21 partially reversed MEG3’s effect on breast cancer cell proliferation,cloning,invasion,migration,EMT and Warburg inhibition and apoptosis promotion.MEG3 overexpression reversed miR-21-mediated activation of the PI3K/AKT pathway in breast cancer cells:MEG3 inhibited breast cancer cells by downregulating miR-21 in nude mice.4.LncRNA MEG3 can be used as the ceRNA of miR-21 to inhibit the tumorigenic effects of breast cancer cells in vitro and in vivo by inducing PI3K/Akt conduction pathway inactivation;MEG3 and miR-21 may become potential targets for breast cancer treatment and prognosis. |
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