Study On The Mechanism Of RACK1 Enhancing Lymphangiogenesis Induced By Glycolysis In Cervical Cancer

Objective:(1)To elucidate the expression of RACK1 of cervical cancer tissues,and the correlation between glycolysis and lymphangiogenesis of cervical cancer cell line;(2)To clarify the molecular mechanism of RACK1 regulating glycolysis of cervical cancer and lymph node metastasis;(3)To clarify the function and mechanism of RACK1 in regulating glycolysis of cervical cancer and lymph node metastasis.Methods:The expression of RACK1 in cervical cancer tissues and cells and normal cervical tissues and cells was detected by immunohistochemistry,and the relationship between RACK1expression and clinicopathological parameters was analyzed.The changes of metabolites in the supernatant of cervical cancer cells transfected with sh RACK1 lentivirus were detected by ~1H-NMR.Metabolic pathways related to RACK1 were determined according to metabolite changes.Glucose assay kit was used to detect the change of glucose content in supernatant,and lactic acid assay kit was used to detect the change of lactic acid content in supernatant.q RT-RCR and Western blot were used to detect the expression of key proteins.Transwll assay was used to detect the migration and invasion of cervical cancer cells after RACK1 expression was interfered.Lymphangiogenesis was detected by canalization test.In vivo lymph node metastasis was observed in nude mouse foot pad-lymph node metastasis model.Candidate transcription factors regulating RACK1expression were obtained by bioinformatics,and their binding was verified by dual luciferase reporter gene and chromatin immunoprecipitation.The online database was used to predict the possible binding of RACK1 to candidate proteins,and the binding of RACK1 to candidate proteins was observed by Co-IP and immunofluorescence techniques.KEGG database was used to further predict downstream signaling pathways,and the activation of pathways was observed by in vitro recovery experiments.Glucose uptake and lactate production capacity of cervical cancer cells were detected by glucose and lactate detection kits.Western blot was used to detect the expression of key glycolysis proteins in cervical cancer.Transwell assay was used to detect the migration and invasion of cervical cancer cells.Lymphatic vessel formation was measured by canalization assay.Results:(1)The expression of RACK1 in cervical cancer tissues was higher than that in cervicitis tissues,and the high expression of RACK1 was related to the degree of tumor differentiation(P=0.015)and lymph node metastasis(P=0.039).The expression of RACK1 in cervical cancer cell lines was higher than that in normal cervical epithelial cells(P<0.001),and the highest in MS751 and Si Ha cells.Interference with RACK1expression in these two cell lines inhibited invasion(P<0.001)and migration ability(P<0.001).Reduced lymphatic endothelial cell migration(P<0.001)and lumen formation.~1H-NMR analysis showed that RACK1 was related to glycolysis metabolism disorder(P<0.01).Further detection showed that down-regulation of RACK1 expression could reduce the expression of HK2(P<0.001),PKM2(P<0.001),GLUT1(P<0.001)and LDHA(P<0.001),leading to decreased glucose uptake(P<0.001)and lactate production(P<0.0001).(2)Bioinformatics prediction results showed that RACK1 binds to IGF1R,and AKT/m TOR pathway is the key downstream pathway.The binding of RACK1 to IGF1R was confirmed by immunoprecipitation and immunofluorescence.Bioinformatics prediction showed that POU2F2 was bound to the RACK1 promoter region,which was further confirmed by luciferin reporter gene and chromatin immunoprecipitation.sh RACK1/Si Ha and sh RACK1/MS751 cells were treated with IGF1,and AKT/m TOR pathway proteins were significantly activated(P<0.001).Increased expression of four key glycolytic proteins(P<0.001)promoted glucose uptake(P<0.001)and lactate production(P<0.001).On this basis,Rapa could significantly inhibit the expression of AKT/m TOR pathway protein in cervical cancer cells(P<0.001).Downregulation of the expression of four key glycolytic proteins(P<0.001)resulted in decreased glucose uptake(P<0.001)and lactate production(P<0.001).When POU2F2 overexpressed plasmid was transfected into sh RACK1/Si Ha and sh RACK1/MS751 cells,the expression of p-Akt(P<0.001)and p-MTOR(P<0.001)in OE POU2F2/sh RACK1group were increased.Upregulation of four key glycolytic proteins(P<0.001)resulted in increased glucose uptake(P<0.001)and lactic acid production(P<0.001).The glucose uptake(P<0.001)and lactate production(P<0.001)of OE POU2F2/sh RACK1 cervical cancer cells were decreased after 2-DG treatment.(3)The invasion ability and migration ability of sh RACK1/Si Ha and sh RACK1/MS751 cells were increased by IGF1 treatment(P<0.001).Lymphatic endothelial cell migration(P<0.001)and lumen formation were enhanced.The invasion and migration of cervical cancer cells(P<0.001)were decreased by adding Rapa on this basis.Inhibited lymphatic endothelial cell migration(P<0.001)and tube formation.The invasion(P<0.001)and migration(P<0.001)of OE POU2F2/sh RACK1group were increased by the POU2F2 overexpressed plasmid transfected into sh RACK1/Si Ha and sh RACK1/MS751 cells.Lymphatic endothelial cell migration(P<0.001)and lumen formation were enhanced.In OE POU2F2/sh RACK1group,lactate production was decreased(P<0.001),and glucose uptake was not significantly affected(P>0.05).The invasion ability(P<0.001)and migration ability(P<0.001)of cells were inhibited.Lymphatic endothelial cell migration(P<0.001)and lumen formation ability decreased.Conclusion:(1)The high expression of RACK1 in cervical cancer tissue can enhance glycolysis metabolism and promote the formation of lymphatic vessels in cervical cancer tissue microenvironment;(2)POU2F2regulates RACK1 and promotes the activation of downstream IGF1R/AKT/m TOR signaling pathway,thereby enhancing glycolysis metabolism.(3)Lactate accumulation in microenvironment can promote lymphatic endothelial cell migration and tubulogenesis.