| Objective:Cancer-type organic anion transporting polypeptide 1B3(Ct-OATP1B3)has been found to be highly expressed in a variety of malignant tumors,but its expression in ovarian cancer has not been reported.This study focuses on Ct-OATP1B3 expression,location,biological function and molecular mechanism in epithelial ovarian cancer(EOC)are discussed.Methods:(1)Immunohistochemical detection of the expression level of Ct-OATP1B3 in tumor tissues of EOC patients,and analysis of the relationship between the expression of Ct-OATP1B3 and the clinical stage and prognosis of EOC patients.(2)q RT-PCR analysis of the expression of Ct-OATP1B3 in EOC cell lines SKOV3,OVCAR3,A2780,HEY2 and human normal ovarian epithelial cell line HOSE.(3)Membrane and cytoplasm protein separation and cell immunofluorescence and methods were used to detect the location of Ct-OATP1B3 in EOC cell lines OVCAR3 and A2780.(4)Constructed Ct-OATP1B3 overexpression and knockdown cell models respectively,and analyzed the effects of Ct-OATP1B3 on ECO cells biological behavior using CCK-8,flow cytometry,cell scratch test,and Transwell cell invasion test.(5)Co-immunoprecipitation and mass spectrometry methods were used to detect the binding protein of Ct-OATP1B3 in EOC cell lines,search for its downstream targets,and explore the molecular mechanism of Ct-OATP1B3 promoting the invasion and migration of ovarian cancer.(6)Co-immunoprecipitation and proximity ligation assay(PLA)methods were used to verify the interaction between Ct-OATP1B3 and IGF2BP2 protein in EOC cell lines,and observe the effect of Ct-OATP1B3on IGF2BP2 m RNA and protein expression.(7)Immunohistochemical method was used to detect the expression of IGF2BP2 in EOC tissues of different clinical stages,analyze the relationship between the expression of IGF2BP2 and the survival time of EOC patients,and analyze the relationship between Ct-OATP1B3 and IGF2BP2 in EOC patients expression relevance.(8)Western blotting was used to observe the effect of Ct-OATP1B3 in EOC cell lines on the formation of homodimers of IGF2BP2 protein.(9)Used the gene sets enrichment analysis(GSEA)to analyze the molecular pathway of Ct-OATP1B3 enrichment in the TCGA database,RNA-immunoprecipitation was used to observe the effect of Ct-OATP1B3on IGF2BP2 protein and CPT1A、NDUFA2 m RNA binding in EOC cell lines.(10)q RT-PCR and Western blotting methods to observe the effect of Ct-OATP1B3 through IGF2BP2 on CPT1A and NDUFA2 transcript levels,m RNA stability and protein levels in EOC cell lines.(11)~3H-labeled isotope tracing method,NAD+/NADH detection kit,mitochondrial respiratory chain complexⅠdetection kit,ATP detection kit and Seahorse energy metabolizer methods were used to observe the FAO and OXPHOS of Ct-OATP1B3 in EOC cell lines through IGF2BP2.(12)The immunofluorescence method was used to observe the effect of Ct-OATP1B3 in EOC cell lines on the formation of invasive lamellipodia through IGF2BP2.(13)The CDX model of EOC was constructed by orthotopic injection,and the tumor growth of mouse at different time points was observed through the bioluminescence system.After the mouse were sacrificed,the number of abdominal metastases,the weight of metastases,and the volume of ascites were observed and counted.The tumor tissues were fixed and hematoxylin-eosin staining(H&E)staining was performed to observe the malignancy of tumor tissues.Immunohistochemical staining was used to observe the expression changes of Ct-OATP1B3,CPT1A and NDUFA2.Transmission electron microscopy was used to observe the number,length and width of mitochondrial cristae in tumor tissues.Results:(1)A total of 25 human normal ovarian epithelial tissues and 127 EOC tissue specimens were included in this study.Immunohistochemical results showed that Ct-OATP1B3 was not expressed in human normal ovarian epithelial tissues;while in the Federation of Gynecologists and Obstetricians(FIGO)stage I,II,III,IV EOC tissues,the expression of Ct-OATP1B3 was significantly up-regulated.And the later the stage,the higher the expression of Ct-OATP1B3.Among the 127 EOC patients,the overall survival and progression-free survival of patients with high Ct-OATP1B3 expression were significantly lower than those with low Ct-OATP1B3 expression(P<0.001).TCGA database analysis results also showed that the later the clinical stage of EOC patients,the higher the expression level of Ct-OATP1B3,and the lower the overall survival of patients(P<0.05).(2)The results of q RT-PCR experiments showed that Ct-OATP1B3expressed extremely low in normal ovarian epithelial cell HOSE,and was significantly increased in EOC cells SKOV3,OVCAR3,A2780 and HEY2.(3)The results of cell immunofluorescence and separation of cell membrane and cytoplasmic proteins showed that Ct-OATP1B3 protein is mainly distributed in the cytoplasm of OVCAR3 and A2780.(4)The results of q RT-PCR and Western blotting showed that after A2780 cells were transfected with the overexpression plasmid,Ct-OATP1B3m RNA and protein levels were 130.2 times and 3.8 times that of the empty group(P<0.01);on the contrary,OVCAR3 cells were transfected with the knockdown plasmid,Ct-OATP1B3 m RNA and protein levels were reduced by 75.0%and 37.2%respectively compared with the negative control group(P<0.01).Biological function experiments showed that Ct-OATP1B3 could significantly improve the invasion and migration ability of EOC cells:Compared with the empty group,A2780 cells overexpressed with Ct-OATP1B3 and the invasion and migration ability was enhanced(P<0.01);while OVCAR3 cells knocked down with Ct-OATP1B3,the invasion and migration ability of cells was weakened(P<0.01).In addition,Ct-OATP1B3had a weak effect on the growth and proliferation of EOC cells,but has no significant effect on the rate of apoptosis.(5)The results of co-immunoprecipitation and mass spectrometry showed that there are 33 potential binding proteins of Ct-OATP1B3 in EOC cells,of which IGF2BP2 has a higher binding fraction.(6)Co-immunoprecipitation and PLA experiments proved that there was a direct interaction between Ct-OATP1B3 and IGF2BP2 proteins in EOC cells.(7)The results of immunohistochemistry showed that IGF2BP2 was not expressed in human normal ovarian epithelial tissues;while in FIGO stage I,II,III,and IV EOC tissues,the expression level of IGF2BP2 was significantly up-regulated.Among the 127 EOC patients,the overall survival and progression-free survival of patients with high IGF2BP2 expression were significantly lower than those with low IGF2BP2 expression(P<0.001).In EOC patients,the expression of Ct-OATP1B3 and IGF2BP2 was positively correlated(P<0.001).(8)q RT-PCR and Western blotting experiments showed that Ct-OATP1B3 had no significant effect on the expression level of IGF2BP2 in EOC cells,but it could significantly promote the formation of IGF2BP2dimers.(9)GSEA analysis of EOC samples in the TCGA database found that samples with high Ct-OATP1B3 expression were enriched in the OXPHOS pathway;RNA immunoprecipitation results showed that in OVCAR3 and A2780 cells,IGF2BP2 protein and CPT1A and NDUFA2 m RNA were all binding,Ct-OATP1B3 could promote their binding.(10)In EOC cells,Ct-OATP1B3 could significantly enhance the expression of CPT1A and NDUFA2:Compared with the empty group,A2780 cells overexpression of Ct-OATP1B3 increased the m RNA stability of CPT1A and NDUFA2,and increased the protein level(P<0.01),knockdown of IGF2BP2 could reverse this effect;and knockdown of Ct-OATP1B3 in OVCAR3 cells reduced the stability of CPT1A and NDUFA2m RNA and the protein level(P<0.01).Overexpression of IGF2BP2 could reverse this effect.(11)Ct-OATP1B3 could promote FAO and OXPHOS of EOC cells through IGF2BP2:The results showed that compared with empty vector group,after overexpression of Ct-OATP1B3,FAO was significantly up-regulated(P<0.01),NAD+/NADH increased(P<0.01),complexⅠactivity and ATP production rate increased(P<0.05),and IGF2BP2 was knocked down,this effect was reversed;compared with the negative control group,after knocking down Ct-OATP1B3,cellular FAO was significantly down-regulated(P<0.01),NAD+/NADH was reduced(P<0.01),complexⅠactivity and ATP production rate were both reduced(P<0.01),this effect was reversed after overexpression of IGF2BP2.The results of Seahorse XFp detection of cell oxygen consumption rate showed that compared with the empty vector group,after overexpression of Ct-OATP1B3,the cell’s basic oxygen consumption rate,maximum oxygen consumption rate and oxygen consumption rate for ATP production all increased(P<0.05),this effect could be reversed after knockdown of IGF2BP2;compared with the negative control group,the basic oxygen consumption rate,maximum oxygen consumption rate and oxygen consumption rate of ATP production of cells after knocking down Ct-OATP1B3 were all reduced(P<0.01),and after overexpression of IGF2BP2,this effect could be saved.(12)Cellular immunofluorescence results showed that Ct-OATP1B3promoted the formation of lamellipodia in A2780 and OVCAR3 cells through IGF2BP2.(13)In vivo experiments showed that Ct-OATP1B3 promoted the metastasis of EOC:Compared with the empty vector group,the fluorescence intensity of tumor in situ increased after overexpression of Ct-OATP1B3(P<0.01),the number of metastases in the abdominal cavity,the weight of metastases,and the ascites volume increased(P<0.01),the malignant degree of tumor tissue H&E staining increased,the positive rate of Ct-OATP1B3,CPT1A and NDUFA2 immunohistochemistry all increased(P<0.01),and the number of mitochondrial cristae,length and width increased(P<0.05);compared with the negative control group,knocking down Ct-OATP1B3 had the opposite effect.Conclusion:Ct-OATP1B3 is highly expressed in EOC tissues and is related to clinical stage and prognosis.In EOC cells,Ct-OATP1B3 can directly bind to the m RNA binding protein IGF2BP2,promote the formation of its homodimers,improve its ability to bind downstream CPT1A,NDUFA2m RNA,and increase the expression levels of CPT1A and NDUFA2;After the expression of CPT1A and NDUFA2 is up-regulated,it can significantly increase the mitochondrial FAO and OXPHOS activities of cells,increase cells ATP production,promote the formation of lamellipodia at the cells edge,and ultimately lead to enhanced EOC cells invasion and migration. |
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