| Objective : Breast cancer(BC)as a common malignant tumors in the worldwide,although great progress has been made in prevention,diagnosis and individual treatment,the recurrence,metastasis and chemotherapy resistance of breast cancer still pose serious challenges to clinical treatment.Therefore,there is an urgent need to explore the molecular mechanism of the development of breast cancer,so as to provide new directions for the early diagnosis and clinical treatment of breast cancer.Long non-coding RNA(lncRNA)as a major class of non-coding RNA discovered in recent years,whose length is more than 200 nt,and it is one of the longest and most heterogeneous RNA families known.Lnc RNA is widely involved in various physiological processes of cells by regulating gene expression levels,including proliferation,differentiation and apoptosis.In addition,the abnormal expression of lnc RNA is also closely related to the development and prognosis of tumors.Long Intergenic Non-Coding RNA 00052(LINC00052)is a kind of lnc RNA.The expression of LINC00052 is abnormally increased in a variety of tumors such as lung cancer and liver cancer,which promotes the proliferation,migration and invasion of tumor cells.The expression level of LINC00052 in breast cancer tissues is higher than that in adjacent normal tissues.High expression of LINC00052 promotes the development of breast cancer,but the molecular mechanism by which LINC00052 regulates breast cancer is still unclear.Glycogen synthase 1(GYS1)is the most important rate-limiting enzyme in the last step of glycogen synthesis.Loss of GYS1 gene expression causes muscle glycogen storage disease.Tumor-related studies have shown that high levels of GYS1 expression are associated with poor prognosis.Hypoxic microenvironment can induce the increase of GYS1 expression and promote glycogen accumulation and glycolysis in tumor cells.Inhibition of GYS1 expression not only inhibited cell glycolysis,but also enhanced the activation of AMP-activated protein kinase(AMPK)and inhibited the proliferation of tumor cells.The expression levels of LINC00052 and GYS1 were positively correlated in breast cancer.By bioinformatics prediction,we found that LINC00052 and GYS1 had specific binding sites for mi R-873-3p.We hypothesized that LINC00052 could regulate the expression of GSY1 by binding to mi R-873-3p in the manner of competing endogenous RNA(ce RNA),and participate in the development of breast cancer.In this study,we used bioinformatics and molecular biology methods to screen LINC00052.Further,a series of in vivo and in vitro functional experiments showed that:LINC00052 can adsorb mi R-873-3p to regμlate the expression of GYS1 to promote the metastasis of breast cancer,and further explore the molecular mechanism of LINC00052/mi R-873-3p/GYS1 axis to regμlate the development of breast cancer,so as to provide a new strategy for the early screening and treatment of breast cancer.It further clarified that LINC00052,mi R-873-3p and GYS1 are expected to be predictive and prognostic markers of breast cancer,providing new targets for the diagnosis and treatment of breast cancer.Methods: 1.GEPIA database was used to analyze the expression difference of lnc RNA in breast cancer,and LINC00052 was identified by screening.real-time PCR was used to verify its expression in breast cancer tissues and cell lines.2.CCK-8,clonogenesis,cell scratch and Transwell tests were determined successively the effects of LINC00052 and mi R-873-3p on viability,proliferation,migration and invasion of breast cancer cells.3.The effect of LINC00052,mi R-873-3p and GYS1 on proliferation,migration,invasion and Epithelial-Mesenchymal Transition of breast cancer was confirmed by Western-Blot assay.It also confirmed that LINC00052/mi R-873-3p/GYS1 axis regulates the activation state of AMPKα/m TOR signaling pathway.4.The effect of LINC00052 on the oncogenic ability of breast cancer cells in nude mice was determined.5.The expression of mi R-873-3p in breast cancer tissues and cell lines was confirmed by real-time PCR.6.Dual luciferase reporter assay confirmed the targeting binding ability of LINC00052 and mi R-873-3p,GYS1 and mi R-873-3p respectively.7.The response experiment respectively confirmed the antagonistic effect of overexpression of mi R-873-3p on the inhibition of proliferation,migration and invasion of breast cancer cells caused by LINC00052 knockdown,the antagonistic effect of overexpression of GYS1 on the inhibition of proliferation,migration and invasion of breast cancer cells caused by overexpression of mi R-873-3p,and the antagonistic effect of overexpression of GYS1 on LINC00052 knockdown on inhibition of proliferation,migration and invasion of breast cancer cells.Results: 1.LINC00052 expression in breast cancer and its effect on proliferation,migration and invasion of breast cancer cells: 1.1 LINC00052 was highly expressed in breast cancer.In order to screen Lncrnas with differential expression in breast cancer,GEPIA database analysis was used to screen out that LINC00052 had increased expression in breast cancer,real-time PCR also confirmed similar results: the expression of LINC00052 in breast cancer tissues and cells was higher than that in adjacent normal tissues and normal breast epithelial cells.1.2 LINC00052 promotes the proliferation,migration,invasion and allogenic tumorigenesis of breast cancer cells.Phenotypic assay results showed that LINC00052 knockdown inhibited proliferation,migration and invasion of breast cancer cells.Flow cytometry showed that knockout of LINC00052 induced G0/G1 cell cycle arrest in breast cancer cells.Western-Blot results showed that LINC00052 knockdown inhibited the expression levels of G0/G1 phase related proteins CDK4,CDK6,and Cyclin D1.Western-Blot also showed that LINC00052 knockdown inhibited the expression levels of migration and invasion related proteins MMP2 and MMP9,and promoted the expression level of TIMP2.The results of tumor formation experiment in nude mice showed that LINC00052 knockdown inhibited the tumor formation ability of breast cancer cells.2.Regulation of mi R-873-3p expression by LINC00052: 2.1 mi R-873-3p was low expressed in breast cancer.Real-time PCR confirmed that the expression of mi R-873-3p in breast cancer tissues and cells was lower than that in adjacent normal tissues and normal breast epithelial cells.2.2Overexpression of mi R-873-3p inhibited proliferation,migration,and invasion of breast cancer cells.Cell phenotype experiments showed that overexpression of mi R-873-3p inhibited proliferation,migration,and invasion of breast cancer cells.Western-Blot results showed that transfection of mi R-873-3p mimics inhibited the expression levels of marker proteins NCAD and Vimentin in mesenchymal cells,and promoted the expression levels of marker proteins ECAD in epithelial cells.Transfected Scramble had no effect on the expression of the above proteins.2.3 Targeted binding interaction exists between LINC00052 and mi R-873-3p.Starbase database predicted that LINC00052 had mi R-873-3p specific interaction sequence.Dual luciferase reporter gene assay confirmed that mi R-873-3p could be used as the downstream target of LINC00052.Compared with the mimics NC transfected mi R-873-3p mimics,the relative fluorescence intensity of LINC00052 WT group was decreased.The relative fluorescence intensity of LINC00052 MUT group did not change after transfection of mimics NC and mi R-873-3p.2.4 Down-regulation of mi R-873-3p antagonized the inhibitory effect of LINC00052 knockdown on proliferation,migration and invasion of breast cancer cells.Response experiments were conducted to detect the response effect of mi R-873-3p inhibitor on knockout of LINC00052.Among them,the results of cell phenotype experiment showed that mi R-873-3p inhibitor partially restored the inhibition of proliferation,migration and invasion of breast cancer cells induced by LINC00052 knockout.Western-Blot assay showed that LINC00052 knockdown inhibited the expression of NCAD and Vimentin,and promoted the expression of ECAD.mi R-873-3p inhibitor promoted the expression of NCAD and Vimentin,but inhibited the expression of ECAD.mi R-873-3p partially restored the inhibition of EMT in breast cancer cells induced by LINC00052 knockdown,which was manifested as promoting the expression of NCAD and Vimentin.Inhibit the expression of ECAD.3.Molecular mechanism of LINC00052/mi R-873-3p/GYS1 axis regulated proliferation,migration and invasion of breast cancer cells: 3.1 GYS1 was highly expressed in breast cancer.Western-Blot confirmed that the expression of GYS1 in breast cancer tissues and cells was higher than that in para-cancer normal tissues and normal breast epithelial cells.3.2 Overexpression of mi R-873-3p inhibited proliferation,migration,and invasion of breast cancer cells.Cell phenotype experiments showed that overexpression of mi R-873-3p inhibited proliferation,migration,and invasion of breast cancer cells.Western-Blot results showed that transfection of mi R-873-3p mimics inhibited the expression levels of marker proteins NCAD and Vimentin in mesenchymal cells,and promoted the expression levels of marker proteins ECAD in epithelial cells.3.3 Targeted binding interaction between GYS1 and mi R-873-3p.It was predicted that GYS1 had mi R-873-3p specific interaction sequence using Target Scan database.Double luciferase reporter gene assay confirmed that GYS1 could be used as the downstream target of mi R-873-3p.Compared with mimics NC transfected mi R-873-3p mimics,the relative fluorescence intensity of GYS1 WT group was decreased.The relative fluorescence intensity of GYS1 MUT group did not change after transfection of mimics NC and mi R-873-3p.3.4 Overexpression of GYS1 antagonized the inhibition of proliferation,migration and invasion of breast cancer cells caused by overexpression of mi R-873-3p.The response effect of GYS1 expression on mi R-873-3p mimics was detected by the response experiment.Among them,phenotypic experiments showed that overexpression of GYS1 partially restored the inhibition of proliferation,migration,and invasion of breast cancer cells induced by transfection of mi R-873-3p mimics.Western-Blot assay showed that mi R-873-3p mimics inhibited the expression of NCAD and Vimentin,and promoted the expression of ECAD.GYS1 overexpression promoted the expression of NCAD and Vimentin,but inhibited the expression of ECAD.However,the overexpression of GYS1 partially restored the inhibition of EMT induced by mi R-873-3p mimics in breast cancer cells,which promoted the expression of NCAD and Vimentin.Inhibit the expression of ECAD.3.5Overexpression of GYS1 antagonized the inhibitory effected of LINC00052 knockdown on proliferation,migration and invasion of breast cancer cells.The response effect of overexpressed GYS1 on LINC00052 knockdown was detected in response experiment.Among them,phenotypic experiments showed that overexpression of GYS1 partially restored the inhibition of proliferation,migration,and invasion of breast cancer cells induced by LINC00052 knockdown.Western-Blot assay showed that LINC00052 inhibited the expression of NCAD and Vimentin,and promoted the expression of ECAD.GYS1 overexpression promoted the expression of NCAD and Vimentin,but inhibited the expression of ECAD.The overexpression of GYS1 partially recovered the inhibition of EMT induced by LINC00052 knockdown,which was manifested as promoting the expression of NCAD and Vimentin.Inhibit the expression of ECAD.3.6 LINC00052/mi R-873-3p/GYS1 axis regulated the activation state of AMPKα/m TOR signaling pathway.LINC00052 knockdown promoted the phosphorylation of AMPKα and inhibited the phosphorylation of m TOR.GYS1 overexpression inhibited AMPKα phosphorylation and promoted m TOR phosphorylation.GYS1 overexpression partially restored the activation inhibition of AMPKα/m TOR signaling pathway in breast cancer cells induced by LINC00052 knockdown.Conclusions: 1.LINC00052 was highly expressed in breast cancer.Knockdown of LINC00052 could inhibit the proliferation,migration,invasion ability,EMT transformation tendency and allogenic tumor formation ability of breast cancer cells.2.mi R-873-3p was significantly down-expressed in breast cancer.Overexpression of mi R-873-3p could inhibit proliferation,migration,invasion and EMT transformation of breast cancer cells.mi R-873-3p could be used as the downstream target of LINC00052,and there were targeted binding sites between the two.Transfection with mi R-873-3p inhibitor reverted to knockdown of LINC00052 induced breast cell proliferation,invasion and EMT inhibition.3.GYS1 was highly expressed in breast cancer tissues and cells.As a downstream target of LINC00052/mi R-873-3p axis,overexpression of GYS1 could restore the proliferation,invasion and EMT inhibition of breast cancer cells caused by overexpression of mi R-873-3p and knockdown of LINC00052.4.LINC00052/mi R-873-3p/GYS1 axis promoted the development of breast cancer through AMPKα/mTOR signaling pathway. |
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