Study On The Mechanism Of Cell Cycle Arrest By C1orf109L-triggered R-loop Accumulation

C1orf109 is a gene,located in h Ch1p34.3,functions are yet remain unknown,discovered in the early studies by the our research group.NCBI database data shows that C1orf109 has multiple transcripts,which can encode multiple proteins with different length and/or sequence.The research team found that the short transcript of C1orf109 encodes a 203 amino acid protein involved in the cell cycle regulation of tumor cells and affects the migration of cells.But up to now,the functions of C1orf109 have not been reported in-depth.The main focus of this study is to reveal the biological function of the C1orf109 longest transcript translation product(C1orf109L).By revealing the molecular mechanism of C1orf109L affecting tumor cell proliferation,which led us to explore whether C1orf109L could be serve as a potential target for tumor therapy.Preliminary research determined the subcellular localization differences of the two longer transcripts translation product of C1orf109.And then confirmed the prepared C1orf109 antibody can specifically recognize multiple transcripts translation product of the gene by exogenously over-expressing C1orf109 cell line and prepared C1orf109 monoclonal antibody.On this basis,the Western blot analysis confirmed that C1orf109 expression is extremely low in a variety of tumor cells and immortalized cells(HEK-293).Moreover,Big Data analysis showed that C1orf109 has epigenetic modification in tumor cells.The cells were further treated with DNA methylase and histone deacetylase inhibitors,and the expression level of C1orf109 was detected by Western blot and real-time quantitative PCR analyses.The expression of endogenous C1orf109L in cells is up-regulated,and preliminarily confirming that the epigenetic modification of histone acetylation is involved in the regulation of C1orf109L expression.In view of the expression characteristics of C1orf109L in cells,we further analyzed the effect of C1orf109L on various cell viability.The analysis found that C1orf109L expression can inhibit the viability of various tumor cells.By constructing C1orf109L Tet-on stably transfected cell lines in He La and HEK-293 cells,the effect of C1orf109L on cell proliferation was explored.The results verified that C1orf109L can significantly inhibit cell proliferation in vitro,and the nude mouse transplantation tumor experiment further proved that C1orf109L can inhibit the growth of transplanted tumors in mice.In order to analyze the clinical value of this effect,we analyzed the impact of C1orf109 on the survival and prognosis of patients with rectal adenocarcinoma and renal clear cell carcinoma through Gene Expression Profiling Interactive Analysis.The results showed that there is a significant positive correlation between C1orf109 and prognosis of patientsTo explain the molecular basis of C1orf109L's inhibition of tumor cell proliferation,the study further analyzed the effects of C1orf109L on the cell cycle.The results showed that C1orf109L can block the cell cycle from G1 to S phase and block the cell cycle in G2 phase.Furthermore,through transcriptome data analysis,it is found that C1orf109L regulates the expression of multiple cell cycle regulation-related genes and DNA damage-related genes,especially the expression of regulatory proteins at cell cycle checkpoints.The interaction protein of C1orf109L was analyzed by LC-MS/MS,and the level of C1orf109L in the chromatin of RNAse A treated cells was analyzed by Gene Ontology annotation combined with immunoblotting.The results showed that C1orf109L may play a role in RNA processing.Further,we used bioinformatics technology to analyze the interaction network of C1orf109L interacting proteins,then study and screen C1orf109L interacting proteins involved in RNA processing,and discover the interaction of RNA helicase with DHX9 and C1orf109L,and then construct truncated DHX9 functional domains.Immunoprecipitation was utilized to confirm that C1orf109L binds to the enzymatic active region of DHX9 and the C-terminus.To explore the molecular mechanism of cell cycle arrest caused by C1orf109L,we analyzed the types of C1orf109L interacting proteins and found that more than50% of the proteins involved in R-loop regulation,and R-loop is an important factor in causing DNA damage.For this reason,R-loop was isolated and immunoprecipitated by R-loop-specific antibodies in this study,and immunoblotting technology was conducted to confirm that DHX9,PARP1 and C1orf109L proteins are located in the R-loop region.In order to determine the effect of C1orf10 L on R-loop,immunofluorescence experiments and DHX9 and PARP1 interference experiments were carried out.The results confirmed that C1orf109L can promote R-loop accumulation by competitively binding DHX9 with PARP1,and the latter is enhanced by the type I topoisomerase inhibitor camptothecin(CPT).The results of comet electrophoresis showed that the expression of C1orf109L can trigger DNA damage,and DNA damage is aggravated under the action of CPT.Further,immunoblotting analysis of DNA damage marker ?H2AX showed that?H2AX increased with the extension of CPT treatment time duration.And it was found that p21 expression was up-regulated,inactivated phosphorylated CDK1 was increased,and cell proliferation inhibition caused by C1orf109L could be restored by knocking down p21.To analyze the effect of C1orf109L on chemotherapy sensitivity of the type I topoisomerase inhibitor,the study found that C1orf109L can cause cell death after 5hours of CPT treatment through Time-Lapse experiment,and C1orf109L activates cell death signaling pathway under the action of CPT.In addition,the detection of the intracellular half-life of C1orf109L revealed that C1orf109L is degraded through the ubiquitinated proteasome pathway.Under the action of CPT,C1orf109L mainly mediates protein degradation through K48 linkage.In summary,this paper analyzes the expression of C1orf109L in a variety of cancer cells and studies its effects on cell proliferation and cell cycle,showing that it has important biological functions in regulating tumor cell cycle and inhibiting tumor cell proliferation.The fact that C1orf109L promotes R-loop accumulation,causes DNA damage and activates downstream signaling pathways,as well as enhancing CPT sensitivity,C1orf109L can play an important role in cells as a potential tumor therapy target.