The Effects And Mechanism Of ANKHD1/lncRNA MALAT1/YAP1 Feedback Loop On Radiosensitivity Of Colorectal Cancer

Part Ⅰ The Effect of ANKHD1 on Radiosensitivity of Colorectal CancerObjectives:To explore the effect of ANKHD1 on radiosensitivity and DNA damage repair in CRC.Methods:(1)The effect of ANKHD1 silencing on the radiosensitivity of CRC was detected by clone formation assay;(2)Comet assay,flow cytometry,immunofluorescence assay and WB assay were used to detect the effect of ANKHD1 silencing on DNA damage repair after IR in CRC cells.(3)The effects of ANKHD1 silencing on CRC cell cycle and apoptosis after IR were detected by flow cytometry and WB experiments.Results:(1)ANKHD1 silencing inhibited the clonal survival rate of CRC cells after IR;(2)ANKHD1 silencing promoted DNA double-strand break damage in CRC cells after IR,and inhibited MRN complex recruitment and ATM/CHK2 activation;(3)ANKHD1 silencing promotes G2/M arrest and induces apoptosis in CRC cells after IR.Conclusions:(1)ANKHD1 silencing promotes radiosensitivity of CRC cells;(2)ANKHD1 silencing promotes DNA damage and inhibits DNA damage repair after IR in CRC cells.Part Ⅱ ANKHD1 affects the radiosensitivity of colorectal cancer by regulating DNA damage repair through YAP1colorectalObjectives:(1)To explore the correlation between ANKHD1 and YAP1;(2)To explore the effect of YAP 1 on DNA damage repair after IR;(3)To reveal the role ofYAP1 in the regulation of radiosensitivity mediated by ANKHD1 in CRC.Methods:(1)Immunohistochemistry was used to detect the expression of YAP1 in CRC tissue,and the correlation between YAP1 and ANKHD1 was analyzed;(2)CoIP assay was used to detect the interaction between ANKHD1 and YAP1;(3)Flow cytometry,immunofluorescence assay and WB assay were used to detect the effect of YAP1 on DNA damage repair after IR;(4)YAP1 was overexpressed in ANKHD1-silenced CRC cells,and clone formation assay was used to detect radiosensitivity,flow cytometry,immunofluorescence experiments and WB experiments were used to detect DNA damage repair,cell cycle and apoptosis after IR.Results:(1)YAP1 is significantly highly expressed in CRC tissues,and the expression of YAP1 is positively correlated with ANKHD1;(2)ANKHD 1 and YAP1 co-precipitate with each other,and ANKHD 1 silencing inhibits the nuclear transport of YAP1 and inhibits the expression of YAP1 and its target genes;(3)YAP1 overexpression inhibits DNA damage after IR,promotes MRN complex recruitment and ATM/CHK2 signaling activation;(4)YAP1 silencing promotes DNA damage inhibits MRN complex recruitment,inhibits ATM/CHK2 signaling activation,induces cell cycle G2/M phase arrest and apoptosis after IR in CRC cells;(5)Overexpression of YAP 1 in ANKHD 1-silenced CRC cells inhibited DNA damage and promoted DNA damage repair in CRC cells after IR.Conclusions:(1)YAP1 were significantly highly expressed and positively correlated with ANKHD1 in CRC tissues;(2)ANKHD 1 interacts with YAP1 and promote the transcriptional activity of YAP 1;(3)ANKDH1 reduces the radiosensitivity of CRC cells by promoting DNA damage repair via YAP1.Part Ⅲ ANKHD1/lncRNA MALAT1/YAP1 feedback loop affects radiosensitivity of colorectal cancer via PI3K/AKT signalingObjective:(1)To explore the relationship between ANKHD1,YAP1 and MALAT1;(2)To explore the role of YAP 1 in the regulation of radiosensitivity mediated by MALAT1 in CRC;(3)To explore the role of PI3K/AKT signaling pathway in the regulation of radiosensitivity mediated by ANKHD1/MALAT1/YAP1 feedback loop in CRC.Methods:(1)RIP and RNA pull-down assays were used to detect the relationship between ANKHD1,YAP1 and MALAT1;(2)Clone formation assay was used to detect the effect of MALAT1 silencing on the radiosensitivity of CRC;(3)Flow cytometry,immunofluorescence experiments and WB experiments were used to detect the effect of MALAT1 silencing on DNA damage repair in CRC cells after IR;(4)YAP1 was overexpressed in MALAT1-silenced CRC cells,clone formation assay was used to detect the effect on radiosensitivity,flow cytometry,immunofluorescence assay and WB assay were used to detect DNA damage repair of CRC cells after IR;(5)WB assay was used to detect the effect of ANKHD1/YAP1 on the activation of PI3K/AKT signaling pathway;(6)The role of PI3K/AKT signaling pathway in ANKHD1/MALAT1/YAP1 feedback loop regulating DNA damage repair in CRC cells was detected by PI3K inhibitor.Results:(1)Both ANKHD1 and YAP1 could interact with MALAT1,and MALAT1 silencing inhibits YAP 1 nuclear transport and inhibits the expression of YAP1 and its target genes;(2)MALAT1 silencing promotes radiosensitivity of CRC cells;(3)MALAT1 silencing promotes DNA double-strand break damage after IR in CRC cells,and inhibits MRN complex recruitment and ATM/CHK2 signaling activation;(4)Overexpression of YAP 1 in MALAT1-silenced CRC cells inhibits DNA damage after IR and promotes DNA damage repair;(5)ANKHD1 silencing inhibits the activation of PI3K/AKT signaling pathway,while overexpression of YAP1 reactivates the PI3K/AKT signaling pathway;(6)PI3K inhibitors promote DNA damage after IR and inhibit YAP1-induced MRE11 expression and CHK2 activation.Conclusion:(1)ANKHD1,MALAT1 and YAP1 interact with each other and form a positive feedback loop;(2)MALAT1 reduces the radiosensitivity of CRC by promoting DNA damage repair after IR via YAP1;(3)The ANKHD1/MALAT1/YAP1 feedback loop promotes DNA damage repair after IR by activating the PI3K/AKT signaling pathway through YAP1,thereby inhibiting the radiosensitivity of CRC.