Experimental Study Of The Mitochondrial OXPHOS Regulation By STC1 In Ovarian Cancer Cells

BackgroundOvarian cancer is one of the main malignant tumors that plague female reproductive health.The incidence is significantly higher in developed regions than in less developed regions,and there is a gradual increase in the trend worldwide.Ovarian cancer is prone to recurrence and metastasis,and its mortality rate ranks first among all gynecological tumors,which seriously threatens the lives and health of women.Therefore,it is urgent to study the mechanism of ovarian cancer proliferation,metastasis and recurrence.Stanniocalcin 1,STC1,was discovered in the human genome in 1995 and has a high degree of homology with fish and other vertebrates.It is an autocrine/paracrine hormone protein whose main function is to locally regulate the transport of calcium and phosphate ions.As a secreted hormone protein,STC1 is extremely low in the serum of healthy people,but STC1 expression is significantly increased in various cancers including ovarian cancer.In the previous study,we found that STC1 can enhance the proliferation and metastasis ability of ovarian cancer cells,but the mechanism of action is unknown.The study found that STC1 is one of the hormones that act on mitochondria,which can positively regulate the mitochondrial transfer chain transmission rate,increase the respiratory efficiency of cellular mitochondria,and provide more energy,but the specific regulatory mechanism needs further study to study the regulation of STC1 on oxidative phosphorylation.The mechanism is of great significance for exploring the mechanism of STC1 promoting the development of ovarian cancer.Mitochondria are important organelles in eukaryotes.They are the main site of oxidative phosphorylation and synthesis of adenosine triphosphate(ATP)in cells,providing energy for cell activities.Although the mitochondrial gene is small(16.6 kb),the mitochondrial genome encodes 13 proteins that form an electron transport chain complex with other proteins of nuclear origin.Mitochondria are involved in important tumor characteristics,such as tumor spread and metastasis,and drug resistance.Oxidative phosphorylation is the main energy source for most malignant tumors.Studies have found that ovarian cancer cells have an increased dependence on oxidative phosphorylation metabolism and increased sensitivity to oxidative phosphorylation inhibitors.It is suggested that we can inhibit the growth of tumors by inhibiting the electron transport chain(ETC),which provides a new direction for the treatment of ovarian cancer.This study intends to explore the role of STC1 in oxidative phosphorylation in ovarian cancer,and then reveal the molecular mechanism of STC1 promoting the development of ovarian cancer,and provide a new target for the treatment of ovarian cancer.ObjectiveIn this study,nucleocytoplasmic separation and immunofluorescence in ovarian cancer cell OVCAR3 were used to verify whether STC1 was distributed in the mitochondria of OVCAR3 cells;RNA-seq technology was used to obtain the differentially expressed gene table of OVCAR3 cell lines with low and overexpression of STC1.Enrichment analysis(GSEA)analysis to find out whether STC1 is involved in oxidative phosphorylation;then in low-and over-expressing STC1 OVCAR3 cell lines,qPCR and Western blot techniques were used to detect whether STC1 had an effect on mitochondrial oxidative phosphorylation-related genes;It was verified by immunoprecipitation technique whether STC1 was combined with the electron transport chain.This study further explored the molecular mechanism of STC1 promoting the development of ovarian cancer by exploring the mechanism of STC1 regulating mitochondrial oxidative phosphorylation in ovarian cancer cells.Methods1.Determine the distribution of STC1 in the mitochondria of OVCAR3 cells using nucleoplasm separation and laser confocal immunofluorescence technology.2.Use the lentiviral system to construct OVCAR3 cell lines with low and overexpression of STC1.3.Use RNA-seq technology to obtain differential genes of OVCAR3 cell lines with low and overexpression of STC1.The differential genes are used to explore whether STC1 is involved in the process of oxidative phosphorylation by gene enrichment analysis(GSEA).4.In the OVCAR3 cell line with low and overexpression of STC1,the mitochondrial-encoded electron transport chain complex ?(ND1,ND2,ND3,ND4,ND4L,ND5,ND6),complex was detected by using Western blot and qPCR ?(CYB),Complex ?(Cox 1,Cox2,Cox3),Complex V(ATP6,ATP8)and 2 mitochondrial ribosomal RNAs(MT-12S,MT-16S)and nuclear gene-encoded nuclear respiratory factor(NRF1,NRF2),mRNA and protein expression levels of mitochondrial DNA transcription factors(TFAM,TFB2M),and mitochondrial polymerase(POLRMT).5.In the OVCAR3 cell line,verify whether STC1 interacts with the protein associated with the mitochondrial inner membrane electron transport chain complex by immunoprecipitation technology.Results1.Western blot analysis showed that STC1 protein was found in OVCAR3 whole cell lysate,mitochondrial component,nuclear component and cytoplasmic component.Laser confocal immunofluorescence detection showed that STC1 co-localized with DAPI and mitochondrial marker TOMM20,indicating that STC1 is distributed in mitochondria.2.Through GSEA analysis of RNA-seq data,the results showed that in the low-expression STC1 group,the oxidative phosphorylation genes encoded by multiple nuclear genes and mitochondrial genes were significantly down-regulated.In the overexpressing STC1 group,the oxidative phosphorylation genes encoded by multiple nuclear genes were significantly up-regulated.It is confirmed that STC1 significantly affects the oxidative phosphorylation pathway.3.In OVCAR3 cells with low expression of STC1,qPCR detection results showed that:mitochondrial encoded electron transport chain complex ?(ND2,ND3,ND4,ND4L,ND5,ND6),complex ?(CYB),complex IV(Cox3),complex ?(ATP8)and 2 mitochondrial ribosomal RNAs(MT-12S,MT-16S)and nuclear genes encoding nuclear respiration factors(NRF1,NRF2),mitochondrial DNA transcription factors(TFAM,TFB2M),mitochondria The mRNA level of polymerase(POLRMT)has been significantly reduced.Western blot detection results showed that:mitochondria encode electron transport chain complex ? protein MT-ND5 and complex ? protein MT-CYB and nuclear genes encode nuclear respiration factors(NRF1,NRF2),mitochondrial DNA transcription factors(TFB2M,etc.),The expression of mitochondrial polymerase(POLRMT)is significantly reduced.The qPCR detection results in the OVCAR3 cell line overexpressing STC1 showed that the mitochondrial encoded electron transport chain complex ?(ND3,ND4,ND6),complex ?(CYB),complex ?(Cox1,Cox2,Cox3),complex MRNA levels of mitochondrial ?(ATP6,ATP8)and mitochondrial ribosomal RNA(MT-12S)and nuclear gene-encoded mitochondrial DNA transcription factors(TFAM,TFB2M)are up-regulated;Western blot detection results show that:mitochondria encode electron transport chain related proteins The expression of MT-ND5 increased.4.The results of co-immunoprecipitation showed that STC1 in OVCAR3 cells binds to MT-ND5 protein in complex ? of mitochondrial inner membrane electron transport chain and MT-CO1 in complex ?.Conclusion1.STC1 is distributed in the nucleus,mitochondria and cytoplasm.2.STC1 can affect the expression of oxidative phosphorylation related proteins through gene regulation.3.STC1 interacts with the mitochondrial electron transport chain.