LINC00092 Suppressed Breast Cancer Progression Via Regulating Pyruvate Carboxylase-mediated AKT/mTOR/p70S6K Pathway

BackgroundAs the most common malignant tumor in women,breast cancer seriously threatens the health and life of many people.The 5-year survival rate of patients with advanced breast cancer in my country is only about 20%.Long non-coding RNAs(lnc RNAs)are important gene expression regulators in cells.Many lnc RNAs have been found to be abnormally expressed in breast cancer cells,which participate in the occurrence and development of breast cancer.LINC00092 is a newly discovered lnc RNA.Previous studies found that the expression of LINC00092 was negatively correlated with the survival time of breast cancer patients.Whether LINC00092 is involved in the regulation of breast cancer cell proliferation,migration,invasion and glycolysis remains unclear.PurposesExplore the expression difference of LINC00092 in breast cancer tissues/cells and normal tissues/cells.Analyze the relationship between LINC00092 expression and breast cancer progression/prognosis.Study the effects of LINC00092 on breast cancer cell proliferation,migration,invasion and glycolysis,and to explore the internal related molecular mechanisms.MethodsChapter 1: Bioinformatics methods and real-time PCR analysis were used to detect the expression difference of LINC00092 in breast cancer tissues/cells and normal tissues/cells,and then explore the relationship between LINC00092 expression and breast cancer progression/prognosis.Cell transfection,Ch IP combined with PCR experiments and dual fluorescence reporter enzyme assay was utilized for analyze whether LINC00092 expression was regulated by friend leukemia virus integration 1(FLI1).After transfection of LINC00092 or sh-LINC00092,breast cancer cell viability,proliferation,migration and invasion,lactate production and glucose consumption were detected respectively.Breast cancer xenograft nude mouse model was construct to verify the regulation of LINC00092 on breast cancer growth.2-DG was added to analyze whether LINC00092 regulates cell proliferation by affecting glycolysis.Chapter 2: After overexpression or silencing of LINC00092,western blotting was used to detect the activation state of AKT/mTOR/p70S6 K pathway in breast cancer cells.Immunohistochemical experiments were used to detect the activation of AKT/mTOR/p70S6 K pathway in tumor tissues of xenografted breast cancer mice.After simultaneously transfecting with LINC00092 and treating with SC79(or shLINC00092 transfecting and MK2206 treating),breast cancer cell viability,proliferation,migration,invasion and glycolysis level were detected to further analyze the role of AKT/mTOR/p70S6 K pathway in the regulation of LINC00092 on breast cancer cell functions.Chapter 3:The proteins interacting with LINC00092 in breast cancer cells was identified by RNA pull-down experiment-combined mass spectrometry analysis.The combination between LINC00092 and pyruvate carboxylase(PC)protein in breast cancer was analyzed by fluorescence in situ hybridization experiment and RNA pulldown assay.After overexpressing or silencing LINC00092,PC m RNA and protein expression levels were detected by real-time PCR and western blotting.Immunohistochemical experiments were used to analyze the expression of PC in tumor tissues of xenograft breast cancer mice.In vitro ubiquitination assay was used to detect whether LINC00092 was involved in mediating the ubiquitination and degradation of PC protein in breast cancer cells.After simultaneously subjecting cotransfection of LINC00092 and PC(or co-transfection of sh-LINC00092 and sh-PC),breast cancer cell viability,proliferation,migration,invasion,and glycolysis,as well as the activation of AKT/mTOR/p70S6 K pathway were detected to explore whether LINC00092 regulated breast cancer cell functions and mTOR/p70S6 K pathway via affecting PC protein levels.ResultsChapter 1: LINC00092 was lowly expressed in breast cancer tissues/cells.It’s expression was correlated with breast cancer tissue size,stage and survival rate of breast cancer patients.FLI1 was also lowly expressed in breast cancer tissues.It’s expression was positively correlated with the LINC00092 expression.FLI1 could bind to the LINC00092 promoter and regulate LINC00092 expression level.Overexpression of LINC00092 inhibited breast cancer cell viability,proliferation,migration,invasion and glycolysis,as well as suppressed tumor growth in xenografted breast cancer mice.Silencing LINC00092 promoted MCF-7 and MDAMB-468 cell viability,proliferation,migration,invasion and glycolysis;2-DG treatment reduces the effect of LINC00092 silencing on MCF-7 and MDA-MB-468 cell viability and proliferation.Chapter 2: Overexpression of LINC00092 inhibited AKT/mTOR/p70S6 K pathway activity in MCF-7 and MDA-MB-468 cells;silencing of LINC00092 activated AKT/mTOR/p70S6 K pathway in MCF-7 and MDA-MB-468 cells;overexpression of LINC00092 reduced AKT/mTOR/p70S6 K pathway activity in tumor tissues of breast cancer xenografts mice;SC79 relieved the effects of LINC00092 overexpression on breast cancer cell proliferation,migration,invasion and glycolysis;MK2206 relieves the influences of LINC00092 silencing on breast cancer cell proliferation,migration,invasion and glycolysis.Chapter 3: LINC00092 could bind to PC protein in breast cancer cells.Changes of LINC00092 expression in MCF-7 and MDA-MB-468 cells had no significant effect on PC m RNA level in cells,but negatively modulated PC protein levels in cells.LINC00092 participated in regulating cellular PC protein levels through ubiquitination degradation pathway.PC overexpression reversed the effects of LINC00092 overexpression on breast cancer cell proliferation,migration,invasion,glycolysis,and AKT/ Effects of mTOR/p70S6 K pathway.PC silencing alleviated the effects of LINC00092 silencing on breast cancer cell proliferation,migration,invasion,glycolysis,and AKT/mTOR/p70S6 K pathway.Conclusion1.LINC00092 was lowly expressed in breast cancer tissues and cells,and was related to breast cancer tissue size,stage and patient survival;2.FLI1 could bind to the LINC00092 promoter and positively regulate the expression level of LINC00092 in breast cancer cells;3.LINC00092 was involved in the regulation of breast cancer cell proliferation,migration,invasion and glycolysis;4.LINC00092 regulated breast cancer cell functions by affecting the activity of AKT/mTOR/p70S6 K pathway;5.PC is the downstream target protein of LINC00092.LINC00092 affected the AKT/mTOR/p70S6 K pathway by regulating the ubiquitination and degradation of PC protein,thereby regulating breast cancer cell functions.