DDI2 Regulates The Drug Resistance Of Colorectal Cancer Cells Through The MAPK Pathway

Background:As a common malignant tumor of the digestive system,the incidence and mortality of the colorectal cancer(CRC)are increasing progressively worldwide.According to the latest global data released by the International Agency for Research on Cancer(IARC)in 2021,the incidence and mortality of CRC are ranked third and second respectively,which resulted in1.93 million new cases and 940,000 deaths.We screened and determined the new high-confidence oncogene DDI2(DNA-damage inducible 1 homolog 2)of Sporadic colorectal cancer(s CRC)through the whole exome sequencing and functional experiments in vitro.In order to further explore the molecular mechanism of DDI2 involved in the occurrence and development of CRC,we analyzed the signaling pathways involved in DDI2 by transcriptome sequencing and public database,and found that DDI2 was positively correlated with mitogen activated protein kinase(MAPK)signaling pathway.MAPK is a highly conserved signal pathway in eukaryotic cells.Abnormal activation of related genes in this pathway induces tumor development and drug resistance.Therefore,we conducted in vitro functional experiments to further study the relationship between DDI2 expression and MAPK signaling pathway,and the effect of knocking down DDI2 expression on the sensitivity and anti-proliferation ability to MEK1/2 inhibitor(Trametinib)and PARP1/2inhibitor(Talazoparib),providing a theoretical basis for the precise treatment of CRC patients.Research methods:1.GEO database were used to analyze the expression of DDI2 between normal tissue samples and CRC samples,as well as the expression of DDI2 in CRC stages: stage I,stage II,stage III,and stage IV.2.Small interfering RNA(si DDI2)was transfected into SW480 cell line,total RNA was extracted,and transcriptome sequencing was performed to analyze the signaling pathways.3.QRT-PCR assay was used to analyze the effect of knocking down DDI2 expression on MAPK signaling pathway and p53 signaling pathway at the m RNA expression level.4.Western blot was used to analyze the effect of knocking down DDI2 expression on MAPK signaling pathway at the protein level.5.The Genomics of Drug Sensitivity in Cancer(GDSC)was used to analyze the effect of DDI2 on drug sensitivity and resistance.6.CCK-8 experiment,clone formation experiment and cell apoptosis experiment were used to study the effect of CRC cell lines on MEK1/2 inhibitor(Trametinib)or PARP1/2 inhibitor(Talazoparib)after knocking down DDI2,and the effect on the proliferation ability of CRC cell lines.Results:1.In clinical samples,the expression level of DDI2 in CRC samples is higher than that in normal tissue samples,and its expression is up-regulated in the early stage of CRC samples.2.Transcriptome sequencing results showed that DDI2 was significantly positively correlated with MAPK signaling pathway and negatively correlated with P53 signaling pathway.3.The q RT-RCR experiment showed that knocking down DDI2 expression in CRC cell lines down-regulates genes in the MAPK signaling pathway,and up-regulates genes in the P53 signaling pathway at the m RNA level expression.4.Western blot results showed that compared with the control group NC,knocking down DDI2 expression significantly inhibited genes in the MAPK signal pathway at the protein expression levels.5.The GDSC database showed that the low expression of DDI2 increased the sensitivity to MEK1/2(Trametinib)and PARP1/2 inhibitors(Talazoparib).MEK1 / 2 and PARP1 / 2 are the key target molecules of MAPK signaling pathway.6.Cell phenotypic experiment showed that knocking down DDI2 increased the sensitivity of CRC cell lines to Trametinib and Talazoparib,and significantly inhibited the proliferation ability of CRC cell lines.ConclusionThrough in vitro functional experiments on CRC cells,we found that the decrease of DDI2 expression can increase the drug sensitivity of CRC cells to MEK1/2 inhibitors(Trametinib)and PARP1/2 inhibitors(Talazoparib),and significantly inhibits the proliferation of CRC cells.Our research provides a theoretical basis for the precise medication of CRC patients,and a new idea for studying the resistance of anti-cancer drugs such as Trametinib and Talazoparib.