Study On Mettl16 Involving DNA Damage Repair And Its Role In Cisplatin Sensitivity Of Colorectal Cancer Cells

Part1 The role of METTL16 in DNA damage repair and its effect on cisplatin chemosensitivity of colorectal cancer cellsObjective: To study the function of METTL16 in DNA damage repair and to investigate its effect on sensitivity of cisplatin in colorectal cancer cells.Methods: After knocking down and overexpress the expression of METTL16 in HEK293 T,HCT116,HT29,and U2 OS cell lines respectively,constructing DR-GFP and EJ5-GFP reporter systems to detect the DNA damage repair efficiency of cells by flow cytometry;using clone formation to check the sensitivity of cisplatin in colorectal cancer cell lines;neutral comet assay and micronucleus assay was used to test the stability of cell genome;using the immunofluorescence assay to check the repair proteins recruitment in DNA damage pathway.Furthermore,verified the effect of knocking down METTL16 on cisplatin sensitivity of colorectal cancer with xenograft in nude mice.Results: Western blot showed that METTL16 knockdown and overexpression were effective in all cell lines.The results of cell flow cytometry showed that HR repair was significantly increased and NHEJ repair was basically unchanged after METTL16 knockdown;HR repair efficiency decreased after overexpression of METTL16.Clone formation showed that knockdown of METTL16 significantly reduced the sensitivity of colorectal cancer cell lines to cisplatin;overexpression increased cisplatin sensitivity.Neutral comet assay and micronucleus assay showed that knockdown of METTL16 increased genomic stability;overexpression METTL16 increased genomic instability.The results of immunofluorescence showed that r H2 AX foci decreased significantly,and Rad51,Brd U and RPA foci increased significantly;the results were opposed after overexpression of METTL16.Conclusions: METTL16 can inhibit the HR repair;knockdown METTL16 can reduce the sensitivity of colorectal cancer cell lines to cisplatin in vitro and in vivo;METTL16 can inhibit the end resection in HR repair pathway.Part 2 METTL16 interacts with MRN complex and affect cisplatin sensitivity in colorectal cancer cellsObjective: To study the interaction between METTL16 and related proteins in HR repair pathway and the molecular mechanism of METTL16 influencing cisplatin sensitivity in colorectal cancer cell line.Methods: After overexpression of mettl16 in HEK293 T cells,using immunocoprecipitation(IP)experiment to check the interaction between METTL16 and related proteins in HR repair pathway,and using p SQ/ TQ antibody to detect whether METTL16 can be phosphorylated by ATM.The protein lysates were treated with DNase or RNase respectively to observe whether they affect the interaction between METTL16 and corresponding proteins;the deletion mutant plasmids of METTL16 and its interacting protein were constructed to study the interaction domain between METTL16 and its interacting protein;the inactivated mutant plasmids of METTL16 methyltransferase were constructed to observe whether they affect the interaction between METTL16 and corresponding proteins;Using sh RNA to knock down endogenous METTL16 and transfect HEK293 T cells,colorectal cancer cell lines and U2 OS cells with its wild-type and corresponding mutant plasmids,to verify whether the transformed METTL16 plasmids can rescue HR repair efficiency,the sensitivity of colorectal cancer cell lines to cisplatin and the formation of foci related protein in HR repair process;synthesizing U6 RNA and carrying out in vitro RNA pull down experiment further confirmed that METTL16 and its interacting protein are RNA dependent.Results: IP results showed that METTL16 could interact with MRN complex(MRE11,RAD50 and NBS1)in HR repair pathway,and the interaction was significantly weakened after IR;the interaction between METTL16 and mre11 did not weaken after IR when treated with ATM inhibitor;immunofluorescence results showed that MRE11 foci increased significantly after knock down METTL16,and phosphorylation bands were found by p SQ/TQ antibody hybridization of the above IP samples.After the mutation of potential phosphorylation sites of METTL16,IP data showed that the phosphorylation of METTL16 basically disappeared when mutated the S419 site.The interaction between METTL16 and MRE11 was not weakened in the transfected S419 A mutant plasmid cells.After infecting METTL16 wild type or S419 A mutant virus in knockdown METTL16 cells,the flow cytometry results showed that HR repair efficiency was restored;clone formation showed that colorectal cancer cells could be sensitive to cisplatin again;immunofluorescence results showed that MRE11,RPA and foci of Brd U were rescued.The interaction between METTL16 and MRE11 can be completely eliminated by RNase but not by DNase.The interaction between METTL16 and MRE11 disappeared after MD5 mutation of Mre11 or MR2 mutation of METTL16.The inactivation of METTL16 methyltransferase did not affect the interaction between METTL16 and MRE11.In knockdown METTL16 cells infected with METTL16 wildtype or R82 A mutant virus,flow cytometry,clone formation and immunofluorescence showed that METTL16 both wild-type and R82 A mutation could rescue HR repair efficiency,cisplatin sensitivity or related protein foci.The results of RNA pull-down showed that the interaction between METTL16 wild type and U6 snRNA decreased after IR,but the interaction between METTL16 S419 A mutant and U6 snRNA did not change after IR.Conclusions: METTL16 S419 site can be phosphorylated by ATM after IR;METTL16 and MRE11 interact by their MR2 and MD5 domains respectively;the interaction between METTL16 and MRE11 is RNA mediated;METTL16 methyltransferase activity does not affect the interaction between METTL16 and MRE11.Part 3 Further study on the structure of METTL16Objective: To study the structure of METTL16 protein and reveal the molecular mechanism of its weaker binding with MRE11 after IR.Methods: METTL16 protein was divided into N-terminal of 1-300 amino acid and Cterminal of 301-562 amino acid from the 300 th amino acid.The wild-type and MR2 mutant plasmids of Flag-METTL16 and the wild-type or S419A/S419 D mutant plasmids of GFP-METTL16 were constructed respectively,and then detect their interaction by IP experiment.RNA pull-down experiment was used to verify whether a large number of GFP-METTL16 C-terminal proteins could compete with U6 snRNA to bind N-terminal of METTL16.Besides,we constructed GFP-METTL16-Flag wild type or S419A/S419 D mutant plasmid,and studied whether METTL16 protein could fold to N-terminal after IR by means of proximity restriction assay(PLA).Furthermore,using non-denaturing gel electrophoresis to detect whether METTL16 could form dimers or polymers before and after IR.Then we constructed the METTL16 R200E/R203E/R204E(3RE)mutant plasmid,and explored the role of these three arginine in the functional domain of METTL16 MR2 in the interaction of N-terminal and C-terminal.Results: After transfection Flag-METTL16 N-terminal plasmid and GFP-METTL16 C-terminal wild-type or S419 A mutant plasmid at the same time,IP showed that after IR,METTL16 N-terminal and C-terminal wild-type interaction increased.After transfection Flag-METTL16 N-terminal wild type or MR2 mutant plasmids and GFPMETTL16 C-terminal wild type or S419 D mutant plasmids simultaneously,IP showed that METTL16 N-terminal wild type and METTL16 C-terminal S419 D mutant plasmids could interact with each other,but the rest groups could not.The results of RNA pull-down showed that the binding of N-terminal of METTL16 and U6 snRNA decreased significantly after a large number of GFP-METTL16 C-terminal plasmids were transferred.PLA results showed that the proportion of Duo link positive cells increased significantly transfected with METTL16 wild-type plasmid cells after IR,but not in the METTL16 S419 A mutant plasmid cells.The results of non denaturing gel electrophoresis showed that METTL16 had only one band around 75 KD before and after IR.After transfection wild-type or 3R mutant plasmid of Flag-METTL16 Nterminal and wild-type plasmid of GFP-METTL16 C-terminal,IP showed that the wildtype protein of Flag-METTL16 N-terminal could interact to the wild-type protein of GFP-METTL16 C-terminal after IR,but the 3r mutant protein of Flag-METTL16 Nterminal could not.Conclusions: The N-terminal and C-terminal of METTL16 can bind to each other after IR;a large number of GFP-METTL16 C-terminal proteins can competitively bind to the N-terminal of METTL16 with U6 snRNA;METTL16 cannot form dimer or polymer in cells,and the C-terminal of METTL16 can fold to the N-terminal after IR;the conservative three arginine with positive charge in the functional domain of METTL16 MR2 play a key role in the interaction of N-terminal and C-terminal.