| Background Cervical cancer is one of the important causes of cancer death in women worldwide and ranks second in the incidence of gynecological malignancies.The latest data show that in 2020,there are about 600,000 new cases of cervical cancer and about 340,000 deaths worldwide.Lnc RNA CARMN(also known as MIR143HG),located at human chromosome 5q32,was initially reported to play an important role in cardiac differentiation.Numerous studies have shown that CARMN expression is downregulated in a variety of tumors including bladder,liver,laryngeal squamous cell carcinoma and breast cancer.Our group’s previous study showed that the expression of CARMN in cervical cancer tissues was lower than that in normal tissues,and a further study confirmed that high expression of CARMN could inhibit the proliferation and invasion ability of cervical cancer cells and induce cell cycle arrest and apoptosis.The exact mechanism of how CARMN affects the occurrence and development of cervical cancer cells has not been fully elucidated.In this study,we intended to overexpress CARMN in cervical cancer He La and C33 A cells,observe the effects exerted by CARMN in both cells,and explore the relationship between autophagy,cycle arrest and apoptosis induced by oxidative stress induced by CARMN and the underlying molecular mechanism.Objectives:1.To further clarify that CARMN plays a tumor suppressor gene role in cervical cancer.2.To explore the key signaling pathways and important target genes involved in CARMN exerting biological effects in cervical cancer.3.To find the potential molecular targets for precision treatment of cervical cancer,and to provide a certain theoretical reference for screening and drug treatment of people at high risk of cervical cancer.Methods and Results1.Effect of CARMN on autophagy,cycle,apoptosis,proliferation and clonogenic ability of cervical cancer cells.(1)Previous studies have shown that CARMN promotes cell cycle arrest and apoptosis in He La cells.In this study,we used western blotting(WB)to determine the protein expression levels associated with cycle and apoptosis in Hela(Hela-CAR)and C33A(C33A-CAR)cells overexpressing CARMN.We found significantly higher expression levels of the apoptosis-related proteins Caspase3,Caspase9,Bax and significantly lower expression of Bcl2 in CARMN overexpressing cells compared to their respective controls(Hela-NC,C33A-NC);Cyclin D1 expression levels were downregulated in He La-CAR cells compared with He La NC;Whereas,Cyclin B1 expression level was downregulated in C33A-CAR cells compared with C33A-NC.The above results were further verified at the molecular level that CARMN could promote the apoptosis of He La and C33 A cells and induce G1 arrest in He La cells and G2/M arrest in C33 A cells.(2)TEM showed that Hela-CAR and C33A-CAR cells had significantly higher levels of autophagosomes than their respective controls,suggesting that CARMN can activate autophagy.The expression of autophagy-related proteins was detected by WB,and we found that both LC3 B and p62 expression levels were significantly increased.The m RFP-GFP-LC3 method revealed an increased number of red puncta in Hela-CAR cells,implying a significantly increased number of lysosomes.The above results suggest that CARMN may induce autophagy-dependent cell death by blocking the fusion of autophagosomes and lysosomes and blocking the autophagic flux.(3)WB results showed that LC3 B,p62,Caspase3,Caspase9,and Bax expression levels were further increased and Bcl2 expression was further decreased by the addition of chloroquine(CQ)or bafilomycin A1(Baf-A1)to Hela-CAR and C33A-CAR cells relative to Hela-CAR or C33A-CAR alone.Cyclin D1 expression levels were further downregulated in Hela-CAR combined with CQ or Baf-A1 cells compared with Hela-CAR alone;Whereas Cyclin B1 expression levels were further downregulated in C33A-CAR combined with CQ or Baf-A1 cells compared with C33A-CAR alone.Cell phenotyping experiments revealed that CARMN in combination with CQ or Baf-A1 further aggravated cell cycle arrest,increased apoptosis,and decreased proliferation and clonogenic ability.The above results suggested that CARMN combined with CQ or Baf-A1 induced cell cycle arrest,increased apoptosis,and attenuated proliferation and clonogenic ability by further arresting autophagy.2.CARMN regulated the Akt/m TOR signaling pathway to affect cellular phenotypes(1)Sequencing and protein profiling in He La car and He La NC cells obtained 95 differentially expressed genes(DEGs)and 370 differentially expressed proteins(DEPs)associated with CARMN overexpression.DAVID website performed KEGG enrichment pathway analysis of DEGs and DEPs to obtain 54 signaling pathways associated with high expression of CARMN.The CARMN co-expressed genes in the TCGA database,as well as proteins related to CARMN gene changes(mutations,copy number changes,gene expression changes)in cervical cancer(including total and phosphorylated proteins),were accessed through c Bioportal website and KEGG analysis was performed to obtain the signaling pathways related to CARMN co-expression.Taking the intersection of KEGG results,the PI3K/Akt and m TOR signaling pathways were significantly enriched with the signaling pathways involved in CARMN.(2)WB assay identified that p-Akt and p-m TOR expression were downregulated in He La-CAR or C33A-CAR cells compared with control.However,when He La-CAR or C33A-CAR cells were treated with SC79,an Akt activator,compared with He La-CAR or C33A-CAR,the expression levels of p-Akt and p-m TOR increased,implying that this pathway was activated and LC3 B,p62,Caspase3,Caspase9,Bax,Bvl2,Cyclin D1 and Cyclin B1 were all restored to the control levels.Cell phenotyping experiments showed that CARMN induced autophagy arrest,cycle arrest,increased apoptosis and reduced clonogenic ability were alleviated after He La-CAR and C33A-CAR were treated with SC79.The above results suggested that CARMN induced autophagy arrest in cervical cancer cells by inhibiting the Akt/m TOR signaling pathway,which in turn caused cell cycle arrest,increased apoptosis and decreased clonogenic ability.3.Effect of ROS on autophagy,cycle and apoptosis in He La and C33 A cells with high CARMN expression(1)Flow cytometry showed that the level of ROS in Hela-CAR and C33A-CAR increased significantly.Treatment with antioxidant NAC can reduce the level of ROS in CARMN overexpressing cells.WB results showed that proteins p-Akt,p-m TOR,LC3 B and p62,Caspase3,Caspase9,Bax,Bcl2,Cyclin D1 and Cyclin B1 were restored to control levels after treatment with NAC compared with HeLa car and C33A-CAR alone.Cell phenotyping experiments showed that CARMN caused autophagy arrest,cycle arrest,increased apoptosis and decreased proliferation were alleviated after treatment with NAC compared with Hela-CAR and C33A-CAR alone.These results suggest that CARMN inhibits the Akt/m TOR signaling pathway by activating the level of intracellular ROS,induces the blocking of autophagy flow,and further causes cell cycle arrest,increased apoptosis and decreased proliferation.4.Role of Nrf2 in CARMN overexpressing cells(1)The protein expression of Nrf2 was down-regulated in Hela-CAR cells by WB assay,while there was no significant difference in Keap1 expression;q PCR results showed no significant differences in Nrf2 and Keap1 m RNA levels in Hela-CAR cells,whereas the m RNA levels of Nrf2 target genes GCLC and HMOX1 were significantly downregulated.Nucleocytoplasmic fractionation assays confirmed that both m RNA and protein expression of Nrf2 in the nucleus was significantly reduced in the presence of CARMN.TBHQ(Nrf2 activator)can rescue the expression of Nrf2 and its downstream antioxidant target genes and reduce ROS levels in CARMN overexpressing cells.The above results suggest that CARMN may accelerate ROS accumulation by inhibiting the nuclear translocation of Nrf2,reducing Nrf2 downstream target gene expression.(2)q PCR assay found that Nrf2 m RNA expression levels did not change after CARMN overexpression.Nrf2 protein stability experiments showed that the protein level expression of Nrf2 was lower in CARMN overexpressing cells,whereas after inhibiting the synthesis of a nascent protein with cycloheximide CHX,the Nrf2 protein appeared to have an accumulation phenomenon with the time extension,indicating that Nrf2 protein was more stable in CARMN overexpressing cells.(3)Treatment of He La car cells with MG132(a proteasome inhibitor)and Baf-A1(an autophagy inhibitor)and detection of Nrf2 and Keap1 binding by co-IP revealed that Nrf2 expression was significantly increased after MG132 treatment and downregulated after Baf-A1 treatment,suggesting that CARMN may decrease Nrf2 expression through the ubiquitin-proteasome system pathway rather than the autophagy pathway.More Keap1 bound to Nrf2 in the CARMN overexpressing cytoplasm compared to the control.These results suggested that CARMN could promote the tight association of cytosolic Keap1 and Nrf2 to some extent.Co-IP experiments revealed significantly higher levels of ubiquitinated,Nrf2 conjugated Keap1 and ubiquitinated proteins in CARMN overexpressing cells compared to control cells.The amount of Keap1 protein bound to Nrf2 was reduced after the addition of MG132,with a corresponding decrease in its ubiquitinated proteins.Taken together,CARMN overexpression accelerated Nrf2 protein degradation and reduced nuclear translocation by promoting the tight association of cytosolic Keap1 and Nrf2,leading to the downregulation of downstream antioxidant target genes and ultimately inducing an increase in intracellular ROS levels.Conclusion In conclusion,in cervical cancer cells,high expression of CARMN promoted the tight association of cytosolic Keap1 and Nrf2,accelerated Nrf2 protein ubiquitination and degradation,and then downregulated the expression of antioxidant related genes and increased intracellular ROS levels;Elevated ROS further leads to autophagy arrest,induction of cycle arrest,increased apoptosis and decreased proliferation and clonogenic capacity by inhibiting the Akt/mTOR signaling pathway. |
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