The Functions And Mechanisms Of UTP11 And BRIX1 In Cancer

UTP11 regulates cancer progression via nucleolar stress and ferroptosisBackground:As a major public health problem,cancer is a serious threat to human health.At present,most cancer treatments are still traditional surgical therapy,radiotherapy and chemotherapy,which have many limitations,such as inevitable side effects.Therefore,it is important to know more molecular targets for cancer treatment.At present,a lot of studies have shown that the biosynthesis of the ribosome is closely related to tumors.Therefore,the related proteins in the biosynthesis of ribosomes are expected to be promising therapeutic targets.UTP11 is an important regulator of the small subunit of the ribosome and its deficiency affects the synthesis of 18 S r RNA in yeast cells.Besides,the level of UTP11 is correlated with the prognosis of tumor patients.However,the functions and mechanisms of UTP11 in cancers have not been explored.Consequently,the aim of this study is to find more targets for tumor treatment by exploring the effects and mechanisms of UTP11 in tumors.Materials and methods:1.The expression of UTP11 in tumor tissues and its relationship with tumor prognosis were analyzed by mining public databases and performing Western blot,real-time quantitative PCR and immunohistochemistry.2.The role of UTP11 in tumor cells was investigated by CCK8,clone formation,cell cycle and transwell assay;the effect of UTP11 in vivo was verified by subcutaneous tumor formation in nude mice.3.The potential mechanisms of UTP11 in cancer development were analyzed by RNA-seq.4.The level of p53 and its target genes,such as p21,BTG2 and MDM2 in UTP11-depleted tumor cells was detected by Western blot and RT-q PCR.The function of UTP11 in 18 S r RNA synthesis was confirmed by semi-quantitative PCR,fluorescent real-time quantitative PCR,immunofluorescence and Co-IP experiments.The CHX half-life experiment was used to verify the stability of p53 protein.The Co-IP experiments were used to test the interaction between MDM2 and RPL5/RPL11 in cancer cells after treated with UTP11 si RNA.The effect of MDM2 on the ubiquitination level of p53 protein in UTP11 depleted-tumor cell was verified by ubiquitination experiments.5.We employed the isogenic colorectal cancer cell lines,HCT116 p53+/+ and HCT116 p53-/-to examine whether the tumor-regressive outcomes of depleting UTP11 were p53-dependent.6.SLC7A11,as a top candidate ferroptosis-associated genes,was detected by Western blot and RT-q PCR in UTP11-depleted tumor cells.The level of GSH synthesis was detected by GSH detection kit.NRF2 is the transcription factor of SLC7A11,the binding of UTP11 and NRF2 m RNA was investigated by RIP experiment.The effect of UTP11 deficiency on the stability of NRF2 m RNA was verified by m RNA stability experiment.The Ch IP experiment was conducted to investigate the effect of NRF2 on the transcription level of SLC7A11 when UTP11 level was downregulated.Results:1.UTP11 was overexpressed in breast cancer and colon cancer,and its overexpression was associated with poor prognosis of tumor patients.2.UTP11 overexpression promoted proliferation,clone formation and migration ability of breast and colon cancer cells,knockdown of UTP11 inhibited proliferation,clone formation and migration ability of breast cancer cells.3.Ablation of UTP11 activated several tumor suppressive pathways,including the p53 pathway and ferroptosis.4.The Western blot and real-time quantitative PCR results verified that the depletion of UTP11 activated p53 and its target genes.Besides,Semi-quantitative PCR, fluorescent real-time quantitative PCR,immunofluorescence and Co-IP experiments confirmed that UTP11 colocalized and interacted with MPP10 and UTP11 downregulation inhibited the biosynthesis of 18 S r RNA and triggered nucleolar stress.Then,Co-IP and ubiquitination experiments revealed that UTP11 depletion promoted the interaction between MDM2 and ribosomal proteins RPL5 and RPL11,and inhibited the ubiquitin-mediated degradation of p53 by MDM2.We also found that UTP11 si RNA could increase the protein stability of p53 through the CHX protein stability experiment.5.UTP11 depletion inhibited proliferation,clone formation and migration of colon cancer cells in a p53 partly dependent manner.Furthermore,UTP11 deficiency suppressed the growth of HCT116 p53+/+ cell-derived xenograft tumors more markable than that of HCT116 p53-/-cell-derived tumors.These results suggested that a p53-independent mechanism might contribute to the effect of depleting UTP11 in the cells.6.Considering that UTP11 depletion was associated with alterations of some ferroptosis-associated genes,and SLC7A11 was a top candidate gene,we desired to explore whether UTP11 depletion would trigger ferroptosis through SLC7A11.The results of IB and RT-q PCR assays revealed that UTP11 knockdown significantly reduced m RNA and protein levels of SLC7A11.In addition,GSH detection and cell proliferation experiments revealed that UTP11 depletion inhibited GSH synthesis and the inhibition of cell proliferation caused by UTP11 depletion could be partially rescued by Ferrostatin-1,which is a ferroptosis inhibitor.7.NRF2 was the master transcription factor that regulated the expression of a myriad of antioxidant genes in response to oxidative stress,such as SLC7A11.Thus,we tested if UTP11 might regulate SLC7A11 expression through NRF2.The IB and RT-q PCR assays showed that UTP11 knockdown significantly reduced m RNA and protein levels of NRF2.Through RIP experiment and m RNA stability experiment,we found that UTP11 protein could bind to NRF2 m RNA,and its m RNA stability would be impaired by UTP11 deficiency.In addition,Ch IP experiment revealed that the reduction of UTP11 inhibited the transcriptional regulation level of NRF2 on SLC7A11,thereby reducing the level of SLC7A11.ConclusionUTP11 is overexpressed in human cancers and associated with poor prognosis.Overexpression of UTP11 promotes cancer cell growth,interestingly,depletion of UTP11 inhibits cancer cell growth in vitro and in vivo through p53-depedednt and-independent mechanisms.On the one hand,ablation of UTP11 triggers nucleolar stress by impeding 18 S r RNA biosynthesis,thereby preventing MDM2-mediated p53 ubiquitination and degradation through ribosomal proteins,RPL5 and RPL11.On the other hand,UTP11 deficiency represses the expression of SLC7A11 by promoting the decay of NRF2 m RNA,resulting in reduced levels of glutathione(GSH)and enhanced ferroptosis.BRIX1 regulates cancer development via nucleolar stressBackground:Ribosome synthesis is a multi-step assembly process and is vital for maintaining the active translation machine required for protein production in rapidly growing cancer cells.Ribosomal or nucleolar stress often occurs when any step of the ribosomal biogenesis is disrupted by genetic alterations,nutrient depletions,or therapeutic agents,consequently leading to cell cycle arrest and apoptosis.BRIX1 is an important regulator of the large subunit of ribosomes.Some studies have shown that Brix is involved in the processing of pre-RNA in African Xenopus frogs and yeast,and cooperates with Ebp2 to impinge on the synthesis of the 60 S large subunit of ribosome.In addition,studies have shown that BRIX1 is involved in tumor development,and can be used as a potential therapeutic target for liver cancer,gastric cancer and colorectal cancer.However,there are few studies on the functions and mechanisms of BRIX1 in tumors.Therefore,we will study the functions and mechanisms of BRIX1 in tumors to provide more targets for tumor treatment.Materials and methods:1.The expression of BRIX1 in tumor tissues and its relationship with tumor prognosis were analyzed by mining public databases and performing Western blotting,realtime quantitative PCR and immunohistochemistry.2.The role of BRIX1 in tumor cells was investigated by CCK8,clone formation,cell cycle,apoptosis and transwell assay.3.The potential mechanisms of BRIX1 in cancer development were analyzed by RNA-seq.4.The downstream target genes of p53 in BRIX1-depleted tumor cells were detected by Western blot and RT-q PCR.The function of BRIX1 in 28 S r RNA synthesis was confirmed by semi-quantitative PCR,fluorescent real-time quantitative PCR,immunofluorescence and Co-IP experiments.The CHX half-life experiment was used to verify the stability of wild-type p53 protein.The Co-IP experiments were used to test the interaction between MDM2 and RPL5/RPL11 after treating cancer cells with BRIX1 si RNA.The effect of MDM2 on the ubiquitination level of p53 protein in BRIX1-depleted tumor cell was verified by ubiquitination experiments.5.We employed the isogenic colorectal cancer cell lines,HCT116 p53+/+ and HCT116 p53-/-to examine whether the tumor-regressive outcomes of depleting BRIX1 were p53-dependent.Results:1.BRIX1 was overexpressed in cancers,and its overexpression was associated with poor prognosis of tumor patients.2.BRIX1 overexpression promoted proliferation,clone formation,cell cycle and migration ability of breast cancer.3.By analyzing the results of RNA-seq,we found that the genes affected by the decreased BRIX1 were enriched in the p53 pathway.Next,the protein immunoblotting and real-time quantitative PCR results verified that the depletion of BRIX1 activated p53 and its target genes,such as p21,BTG2 and MDM2.Besides,semi-quantitative PCR,fluorescent real-time quantitative PCR,immunofluorescence and Co-IP experiments confirmed that BRIX1 interacted with BOP1 and PES1,which are important components of the Pe Bo W complex.In addition,the downregulation of BRIX1 could inhibit the biosynthesis of 28 S r RNA and cause nucleolar stress.Then,Co-IP,ubiquitination experiments and CHX protein stability experiment revealed that BRIX1 deficiency promoted the interaction between MDM2 and ribosomal proteins RPL5 and RPL11,thus inhibited the ubiquitination degradation of p53 by MDM2.4.BRIX1 depletion inhibited proliferation,clone formation,cell cycle,migration ability and triggered apoptosis of colon cancer in a p53 largely dependent manner.ConclusionBRIX1 is overexpressed in human cancers and associated with poor prognosis. Overexpression of BRIX1 promotes cancer cell growth.Depletion of BRIX1 inhibits cancer cell growth in vitro and in vivo in a p53 largely dependent manner.Mechanically,ablation of BRIX1 blocks 28 S r RNA biosynthesis by affecting Pe Bow complex to trigger nucleolar stress,thereby preventing MDM2-mediated p53 ubiquitination and degradation via ribosomal proteins,RPL5 and RPL11.