A Critical Role Of Arginine Specific Marylated GRP78/BiP Affacted By ART1 In The Progression Of KRAS Mutant Colorectal Cancer

Background:Colorectal cancer is one of the most common malignant tumors.The reports of the International Agency for Research on Cancer and The National Cancer Center of China have shown that the incidence and mortality of colorectal cancer in China have been increasing year by year.Regarding the treatment of colorectal cancer,the relevant guidelines clearly stated that KRAS(V-Ki-Ras2 Kirsten Rat Sarcoma 2 Viral Oncogene Homolog)mutations are used to exclude colorectal cancer patients from receiving specific targeted drug therapy,that is,there is currently no safe and effective targeted therapy that can be applied to KRAS mutant colorectal cancer.However,nearly 50% of colorectal cancer patients have been detected with KRAS mutation.In-depth study of the specific cellular and molecular biological changes and mechanisms of KRAS mutant colorectal cancer,seeking new drug targets,has important clinical significance for the intervention and treatment of such patients.Unfavorable microenvironmental conditions in tumors can change the protein folding ability in endoplasmic reticulum(ER)of tumor cells.By stimulating the state of “ER Stress”,it can return to “ER homeostasis”,to maintain the continuous growth of tumor cells.The accumulation of mutant proteins is one of the unfavorable microenvironments in which tumors are located.Studies have shown that the accumulation of KRAS mutant proteins can also trigger a sustained high level of endoplasmic reticulum stress,reducing unfolded protein aggregation through a long-term protective regulatory signaling pathway based on the unfolded protein response(UPR),to maintain the stability of the endoplasmic reticulum,so that KRAS mutant tumor cells continue to survive.Therefore,looking for key factors and key signaling molecules that can affect the regulation of endoplasmic reticulum homeostasis in KRAS mutant colorectal cancer will help the choice of treatment strategies for this type of tumor.Arginine specific Mono-ADP-ribosylation is one of the important modifications that can change the structure and function of the protein in vivo.ART1(Arginine-specific mono-ADP-ribosyltransferase 1)is the key enzyme reported to catalyze arginine-specific mono ADP ribosylation in human and mouse tissues.The previous research of the research group showed that the expression of ART1 in KRAS mutant colorectal cancer cells(CT26,Lo Vo cells)was significantly increased,and it could promote the proliferation and invasion of KRAS mutant colorectal cancer cells,and inhibit the apoptotic response.Other studies have shown that Glucose-regulated protein 78(GRP78/Bi P),a central protein in the processof stabilizing the endoplasmic reticulum environment,can bind to unfolded proteins in the endoplasmic reticulum on the one hand to promote the process of protein folding,on the other hand,it can regulate the UPR signaling pathway by binding or dissociating with its receptors.It has been reported that GRP78/Bi P in 293 T cells is an important target protein of ART1.When endoplasmic reticulum stress activator is applied for a short period of time,the level of ART1 and mono ADP ribosylated GRP78/Bi P increased,and the upregulation of GRP78/Bi P in the endoplasmic reticulum can be detected.However,in colorectal cancer,under the condition of KRAS mutation,the relationship between arginine specific mono-ADP-ribosylation in the regulation of endoplasmic reticulum stress and the maintenance of endoplasmic reticulum homeostasis and the specific regulatory mechanism are still unclear.Based on our previous research and existing literature reports,we speculate that,unlike wild-type colorectal cancer,under the endoplasmic reticulum stress environment of KRAS mutant colorectal cancer,ART1 may participate in the process of KRAS mutant colorectal cancer maintaining endoplasmic reticulum homeostasis by catalyzing more arginine specific mono-ADP ribosylated GRP78/Bi P to maintain survival of tumor cell.By inhibiting the arginine specific mono-ADP ribosylated GRP78/Bi P produced by the action of ART1,the progression of KRAS mutant colorectal cancer can be hindered to a certain extent.This article discusses this speculation and its molecular mechanism and specific modification sites,with the purpose of(1)clarifying the changes in the proliferation and apoptosis of KRAS mutant colorectal cancer after arginine specific mono-ADP ribosylated GRP78/Bi P affected by ART1.(2)To explore the molecular mechanism of KRAS mutant colorectal cancer proliferation and apoptosis caused by arginine specific mono-ADP ribosylated GRP78/Bi P affected by ART1.This subject provides new ideas and experimental basis for finding precise and effective targets for intervention in KRAS mutant colorectal cancer.Methods: This research is divided into three parts.1.The expression level of ART1 and endoplasmic reticulum stress markers in KRAS mutant colorectal cancer1)The expression level of ART1 in KRAS mutant colorectal cancer.Analysis in TCGA(The Cancer Genome Atlas)database: Download the whole transcriptome sequencing data and clinical information of 316 KRAS wild-type and 218 KRAS mutant colorectal adenocarcinomas in the TCGA database,draw the survival curves of KRAS wild-type group and KRAS mutant group,extract differential genes,and analyze the expression difference of ART1 in two groups in gene level.Immunohistochemical staining to detect the positive expression level of ART1 of the pathological sections of 79 colorectal cancer patients which were divided into KRAS wild type(28 cases)and KRAS(51 cases)mutant types,also,the correlation between ART1 and KRAS mutation statues was analyzed.Western Blot was used to detect the expression of ART1 in KRAS wild-type colorectal cancer cell lines SW48,Caco2 and HT-29,and KRAS mutant colorectal cancer cell lines Lo Vo(G13D/A14V),HCT-116(G13D),SW480(G12V).2)The expression level of endoplasmic reticulum stress markers in KRAS mutant colorectal cancer.Analysis in TCGA database: Using 316 KRAS wild-type and 218 KRAS mutant colorectal adenocarcinoma whole transcriptome sequencing data and clinical information in the TCGA database,to analyst the expression difference of endoplasmic reticulum stress level markers(GRP78/BiP,HSC70,CHOP)at the m RNA level,and the KEGG pathway enrichment analysis of the differential genes obtained from the transcriptome sequencing data also has been performed.immunohistochemical staining of GRP78/BiP,HSC70,CHOP was performed on 28 KRAS wild-type and 51 KRAS mutant tissue sections,the positive expression level was counted,and the correlation with KRAS mutation was analyzed.Western Blot was used to detect GRP78/BiP,HSC70,CHOP protein level in KRAS wild-type colorectal cancer cell lines SW48,Caco2 and HT-29 and KRAS mutant colorectal cancer cell lines Lo Vo(G13D/A14V),HCT-116(G13D),SW480(G12V),the endoplasmic reticulum morphological changes in the six types of cells were scanned by electron microscope.3)The correlation between the expression of ART1 and GRP78/BiP.The correlation between ART1 and GRP78/BiP at gene and protein expression level was analyzed under KRAS mutation status.2.The changes in the proliferation and apoptosis of KRAS mutant colorectal cancer after arginine specific mono-ADP ribosylated GRP78/BiP affected by ART1.1)Effect of ART1 on the level of arginine specific mono-ADP ribosylated GRP78/BiP in KRAS mutant colorectal cancer cellsIn the first part,the Western Blot method was used to detect the endoplasmic reticulum stress level markers in KRAS wild-type colorectal cancer cell lines SW48,Caco2 and HT-29 and KRAS mutant colorectal cancer cell lines Lo Vo,HCT-116,and SW480.Based on the detection of the protein expression of BiP,HSC70,CHOP,KRAS wild-type HT-29 cells were selected to construct a cell line stably expressing the KRAS mutation gene.The mutation sites were KRAS G12 D site(G12D group)and KRAS G13 D site,respectively with no load(p HBLV group)and KRAS wild type(KRAS WT group)as the control group.Western Blot was used to detect the expression changes of ART1 and endoplasmic reticulum stress markers(GRP78/BiP,HSC70,CHOP)after KRAS mutation.The GST-Pulldown experiment was performed with m Af1521 bait protein that specifically binds to the Macro region of a mono ADP ribosylation modified protein.The result of Coomassie Brilliant Blue showed a clear band at the position of 78 k Da(GRP78/BiP).Far-Westernblot showed that after incubation with m Af1521 bait protein,compared with KRAS WT group,G12 D and G13 D cells had higher levels of arginine mono-ADP ribosylation modified GRP78/BiP,p<0.01;The level of arginine mono-ADP ribosylation modified GRP78/BiP was significantly reduced after MIBG treatment in G12 D and G13 D cells,p<0.01.Confocal immunofluorescence double staining showed that the ART1 inhibitor MIBG can reduce the co-localization of GST-m Af1521 and GRP78/BiP in the KRAS mutant G12 D cells.2)The changes in the proliferation of KRAS mutant colorectal cancer after arginine specific mono-ADP ribosylated GRP78/BiP affected by ART1.In the following experiments,the level of arginine specific mono-ADP ribosylated GRP78/BiP was changed by inhibiting or knocking down ART1.The four groups of cells of p HBLV,KRAS WT,G12 D and G13 D were treated with MIBG,a specific inhibitor of ART1,through CCK8(Cell Counting Kit-8),clone formation experiment and Ed U cell proliferation experiment to observe the effects of ART1 inhibitor on the cell growth of p HBLV,KRAS WT,G12 D and G13 D cells.KRAS WT,G12 D and G13 D cells were infected lentivirus with ART1 knockdown,and KRAS WT,G12 D and G13 D cell lines with stable knockdown of ART1 were established and named KRAS WT-sh ART1,G12D-sh ART1 and G13D-sh ART1.The control groups were KRAS WT-NC,G12D-NC and G13D-NC,respectively.Nude mice were inoculated subcutaneously,the size of the transplanted tumor was measured and the growth curve of the transplanted tumor under the skin was drawn;the expression of proliferation protein Ki67 in the transplanted tumor was detected by immunohistochemistry.3)The changes in the apoptosis of KRAS mutant colorectal cancer after arginine specific mono-ADP ribosylated GRP78/BiP affected by ART1.In the following experiments,the level of arginine specific mono-ADP ribosylated GRP78/BiP was changed by inhibiting or knocking down ART1.The apoptosis status change of the p HBLV,KRAS WT,G12 D and G13 D groups after the application of ART1 inhibitors was detected by flow Annexin V/PI double staining,Hochest33258 staining and TUNEL method.Use siRNA to knock down ART1 in KRAS wild-type colorectal cancer cell lines Caco2,HT-29 and KRAS mutant colorectal cancer cell lines LOVO and HCT-116,respectively.Western Blot was used to detect the expression of Cleaved-caspase3 and Bcl2.Realtime-PCR detects the expression level of BAK,BAX,BIM,NOXA,Caspase9,Bcl2,Bclxl in Lo Vo and HCT-116.Flow cytometry was used to detect the changes in the percentage of apoptosis of Lo Vo and HCT-116 cells after ART1 knockdown.Western blot was used to detect the expression level of apoptotic proteins Cleaved-caspase3 and Caspase9 in KRAS WT-sh ART1,G12D-sh ART1,G13D-sh ART1 and each control group.The total protein was extracted from the subcutaneous transplanted tumors of nude mice in the KRAS WT-sh ART1,G12D-sh ART1 and G13D-sh ART1 groups and the control groups(KRAS WT-NC,G12D-NC and G13D-NC),and Western Blot was used to detect the expression level of apoptotic proteins Cleaved-caspase3 and PARP in each group.3.The molecular mechanism of KRAS mutant colorectal cancer proliferation and apoptosis caused by arginine specific mono-ADP ribosylated GRP78/BiP affected by ART11)ART1 affects the expression of GRP78/BiP in KRAS mutant colorectal cancerUse si RNA in KRAS wild-type colorectal cancer cell lines Caco2,HT-29 and KRAS mutant colorectal cancer cell lines Lo Vo and HCT-116,respectively to knockdown ART1 and use Western Blot to detect the expression levels of ART1 and GRP78/BiP.Western Blot detects the expression level of GRP78/BiP after the application of ART1 inhibitor MIBG in p HBLV,KRAS WT,G12 D and G13 D cells.2)ART1 affects the dissociation of GRP78 / BiP and its susceptor in KRAS mutant colorectal cancerCo-immunoprecipitation(Co-IP)experiment was used to detect the dissociation and binding status of GRP78/BiP with ATF6,IRE1α and PERK in the KRAS WT,G12 D and G13 D groups after the application of the ART1 inhibitor MIBG.3)ART1 regulates the UPR signaling pathway in KRAS mutant colorectal cancerWestern Blot was used to detect the activation status of IRE1α-XBP1-TFAF2/JNK,PERK-e IF2α-ATF4,ATF6-S1P/S2P-CHOP pathwa y in the UPR of p HBLV,KRAS WT,G12 D and G13 D groups after treatment with ART1 inhibitors.p-PERK,p-eif2α,p-IRE1α,ATF6,CHOP and the expression changes of the apoptotic protein Cleaved-C aspase3 were detected.At the same time,cells in the G12 D and G13 D groups were tra nsfected with si RNA to achieve ART1 knockdown,and Western Blot was used to confirm the activation status of the IRE1α-XBP1-TFAF2/JNK,PERK-e IF2α-ATF4,ATF6-S1P/S2P-CHOP pathway in the UP R.The expression level of p-PERK,p-eif2α,p-IRE1α,ATF6,CHOP was detected.4)The primary study of the modification sites of MARylated-GRP78/BiP in KRAS mutant colorectal cancer.According to the early stage of our research,matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS)was used to analyze the modification sites of arginine mono ADP ribosylated GRP78/BiP in human colorectal cancer cell lines SW480 and Lo Vo(KRAS mutant),combined with the modification sites of arginine mono ADP ribosylated GRP78/BiP reported in the literature,the GRP78/BiP R470+492K and R214 K site mutant plasmids were constructed and transfected into G12 D cells.Western Blot was used to detect the expression of p-PERK,p-eif2α and p-IRE1α,aim to preliminarily explore the modification site of arginine mono ADP ribosylated GRP78/BiP and its effect in KRAS mutant colorectal cancer.Results:1.The expression and correlation of ART1 and endoplasmic reticulum stress markers in KRAS mutant colorectal cancer1)ART1 is abundantly expressed in KRAS mutant colorectal cancer.In the TCGA database,there are transcriptome sequencing data of 316 cases of wild KRAS and 218 cases of mutant colorectal adenocarcinoma.Combined with follow-up information,it is found that the 10-year progression-free survival and 5-year disease-free survival of KRAS mutant colorectal cancer are shorter than those of KRAS wild-type.There were2452 differentially expressed genes between the two groups.There were1283 genes up-regulated and 1169 genes down-regulated in KRAS mutant colorectal cancer tissues.Among them,a total of 64 wild-type and 43 mutants has relative expression values of ART1 Log2(mean)data greater than 0.Statistics show that the m RNA level of ART1 in KRAS mutations is generally higher than that of wild-type(p=0.0391).Immunohistochemical staining results showed that ART1 expressed in in cell cytoplasm as fine granular form.The positive rate in KRAS mutant colorectal cancer was generally higher than that of KRAS wild-type colorectal cancer(p <0.01);and statistics show that the degree of ART1 positivity is correlated with KRAS mutation status(p<0.01).Compared with KRAS wild colorectal cancer cell lines SW48,Caco2 and HT-29,KRAS mutant colorectal cancer cell lines Lo Vo,HCT-116,SW480 have higher expression levels of ART1 protein.Among them,Lo Vo cells with double-site KRAS mutations had the most abundant expression of ART1.2)Endoplasmic reticulum stress markers are abundantly expressed in KRAS mutant colorectal cancerIn 64 wild-type and 43 mutant colorectal cancer tissues in the TCGA database,the expression level of HSPA5(GRP78/BiP)m RNA level was not significantly different between the two groups,while in the mutant group,HSPA8(HSC70)and DDIT3(CHOP)Mrna expression level were significantly higher than that of the wild type(p=0.0083,p=0.0326).In addition,the differentially expressed genes derived from the analysis of the transcriptome data of 534 cases can be enriched in the endoplasmic reticulum protein synthesis signaling pathway(map04141)in the KEGG database,with a p<0.01.Immunohistochemical staining showed that endoplasmic reticulum stress level markers(GRP78/BiP,HSC70,CHOP)were abundant in KRAS mutant colorectal cancer,p<0.05,which were related to KRAS mutation status,with P values less than 0.01.Compared with KRAS wild colorectal cancer cell lines SW48,Caco2 and HT-29,KRAS mutant colorectal cancer cell lines Lo Vo,HCT-116,SW480 have higher expression levels of GRP78/BiP,HSC70,and CHOP,which are statistically significant.In addition,the endoplasmic reticulum lumen of the KRAS mutant cell line showed a large area of swelling,suggesting that there is a higher level of endoplasmic reticulum stress to maintain its survival.3)The correlation between ART1 and endoplasmic reticulum stress markers.ART1 was positively correlated with the expression of GRP78/BiP at the m RNA level,with a correlation coefficient of 0.4963,p<0.01;ART1was positively correlated with the expression of GRP78/BiP protein,with a correlation coefficient of 0.549,p<0.05.2.The changes in the proliferation and apoptosis of KRAS mutant colorectal cancer after arginine specific mono-ADP ribosylated GRP78/BiP affected by ART1.1)Effect of ART1 on the level of arginine specific mono-ADP ribosylated GRP78/BiP in KRAS mutant colorectal cancer cellsColorectal cancer HT-29 KRAS stable mutant cell line was successfully established and named KRAS G12 D mutant group(G12D group),KRAS G13 D mutant group(G13D group),empty group(p HBLV group),KRAS wild type group(KRAS WT group)For the control group.Western Blot showed that the expression levels of ART1 and endoplasmic reticulum stress markers(GRP78/BiP,HSC70,CHOP)increased in the G12 D and G13 D groups,and both showed p<0.01 after statistics.The GST-Pulldown experiment was performed with m Af1521 bait protein that specifically binds to the Macro region of a mono ADP ribosylation modified protein.The result of Coomassie Brilliant Blue showed a clear band at the position of 78 k Da(GRP78/BiP).Far-Westernblot showed that after incubation with m Af1521 bait protein,compared with KRAS WT group,G12 D and G13 D cells had higher levels of arginine mono-ADP ribosylation modified GRP78/BiP,p<0.01;The level of arginine mono-ADP ribosylation modified GRP78/BiP was significantly reduced after MIBG treatment in G12 D and G13 D cells,p<0.01.Confocal immunofluorescence double staining showed that the ART1 inhibitor MIBG can reduce the co-localization of GST-m Af1521 and GRP78/BiP in the KRAS mutant G12 D cells.2)The changes in the proliferation of KRAS mutant colorectal cancer after arginine specific mono-ADP ribosylated GRP78/BiP affected by ART1.The plate clone formation experiment showed that after treatment with the same concentration of ART1 inhibitor,the number of cell clones in the KRAS WT,G12 D and G13 D groups were reduced compared with the control group,p<0.01,and the changes in the G12 D and G13 D groups were more obvious than those in the KRAS WT group;After the p HBLV group was treated with ART1 inhibitor,there was no significant difference in the number of cell clones compared with the DMSO group,p>0.05.Ed U cell proliferation experiments showed that after ART1 inhibitor treatment,the cell proliferation ability of p HBLV,KRAS WT,G12 D and G13 D groups were all weakened,and statistics showed p<0.01,but the changes in G12 D and G13 D groups were more obvious than p HBLV and KRAS WT.The CCK8 experiment results showed that with the increase in the application concentration of ART1 inhibitors,the cell viability of p HBLV,KRAS WT,G12 D and G13 D groups all decreased.Among them,when the MIBG application concentration was 100μmol/m L and 150μmol/m L,G12 D and G13 D The cell survival rate of the group was significantly lower than that of the KRAS WT group,p<0.01.KRAS WT,G12 D and G13 D cell lines that stably knock down ART1 were successfully established.Real-time PCR detection showed that ART1 knockdown was successful in KRAS WT-sh ART1,G12D-sh ART1 and G13D-sh ART1.28 days after nude mice were subcutaneously inoculated,the size and weight of transplanted tumors in the G12D-sh ART1 and G13D-sh ART1 groups were significantly smaller than those in the control group,p<0.01.However,the growth status of transplanted tumors in the KRAS WT-sh ART1 group was not significantly different from that in the control group,p>0.05.Immunohistochemistry showed that the positive rate of the proliferation protein Ki67 in the transplanted tumor was lower in the G12D-sh ART1 and G13D-sh ART1 groups than the control group,p<0.01;the Ki67 positive rate in the KRAS WT-sh ART1 group did not change significantly,p>0.05.Western Blot of fresh tissues showed that compared with the control group,the expression of ART1 and GRP78/BiP in the G12D-sh ART1 and G13D-sh ART1 groups decreased,p<0.01;while the expression of ART1 and GRP78/BiP in the KRAS WT-sh ART1 group was compared with the control group There is no significant difference in comparison,p>0.05.2)The changes in the apoptosis of KRAS mutant colorectal cancer after arginine specific mono-ADP ribosylated GRP78/BiP affected by ART1.Flow cytometric Annexin V/PI apoptosis experiment showed that the percentage of apoptosis of G12 D and G13 D cells in the mutant group increased after the application of ART1 inhibitor;Hochest33258 staining and TUNEL method both showed that the apoptosis of G12 D and G13 D cells in the mutant group after application of ART1 inhibitor The number of dead cells increased significantly,p<0.01;the changes of p HBLV and KRAS WT in the wild group after the application of ART1 inhibitor were not statistically significant,p>0.05.Western Blot showed that knocking down ART1 with si RNA can significantly up-regulate the expression of apoptosis molecule Cleaved-caspase3,p<0.05,and down-regulate the expression of apoptosis regulatory protein Bcl2,p<0.01.The above changes in KRAS wild-type colorectal cancer cell lines Caco2 and HT-29 were not obvious.At the same time,Realtime-PCR showed that when ART1 was knocked down,the transcription levels of key signaling molecules BAK,BAX,BIM,NOXA,and Caspase9 in the endoplasmic reticulum and mitochondrial apoptosis signaling pathways in KRAS mutant colorectal cancer cell lines Lo Vo and HCT-116 were significantly up-regulated,Bcl2 and Bclxl transcription levels were significantly down-regulated.Flow cytometry showed that Lo Vo and HCT-116 cells were knocked down by ART1 and the percentage of apoptosis increased.Western Blot showed that under the condition that both ART1 showed successful knockdown,compared with the G12D-NC and G13D-NC groups,the expression of Cleaved-caspase3 and Caspase9 in the G12D-sh ART1 and G13D-sh ART1 groups increased significantly,p<0.01;Compared with the KRAS WT-NC group,the expression changes of Cleaved-caspase3 and Caspase9 in the KRAS WT-sh ART1 group were not statistically significant,p>0.05.Western Blot showed that the expression of Cleaved-caspase3 and Caspase9 in the G12D-sh ART1 and G13D-sh ART1 groups increased significantly compared with the G12D-NC and G13D-NC groups,p<0.01;compared with the KRAS WT-NC group,the expression of Cleaved-caspase3 and Caspase9 in the KRAS WT-sh ART1 group was not statistically significant,p>0.05.Western Blot showed that the apoptotic protein Cleaved-caspase3 and PARP was significantly up-regulated in the G12D-sh ART1 and G13D-sh ART1 groups,p<0.01.There was no significant difference between the KRAS WT-sh ART1 group and the control group,p>0.05.3.The molecular mechanism of KRAS mutant colorectal cancer proliferation and apoptosis caused by arginine specific mono-ADP ribosylated GRP78/BiP affected by ART1.1)ART1 affects the expression of GRP78/BiP in KRAS mutant colorectal cancerAfter knocking down ART1 by si RNA,Western Blot showed that KRAS mutant colorectal cancer cell lines Lo Vo and HCT-116 had the most significant down-regulation trend of GRP78/BiP,while KRAS wild-type colorectal cancer cell lines Caco2 and HT-29 had less changes.After the application of ART1 inhibitor,the expression of GRP78/BiP in the cells of the G12 D and G13 D mutant groups began to decrease after24 hours.The decrease of GRP78/BiP expression in the wild group was later than that in the mutant group,occurring at 48 hours.2)ART1 affects the dissociation of GRP78/BiP and its susceptor in KRAS mutant colorectal cancer cellsCo-immunoprecipitation(Co-IP)experiments showed that when ART1 inhibitors were used,the binding of GRP78/BiP to IRE1α and PERK proteins increased in the G12 D and G13 D groups.Statistics showed that p<0.01.There was no obvious binding and dissociation change of GRP78/BiP with ATF6,p>0.05;ART1 inhibitor has no effect on the binding status of GRP78/BiP and IRE1α,PERK,ATF6 protein in KRAS WT group,p>0.05.3)ART1 regulates the UPR signaling pathway in KRAS mutant colorectal cancer cellsWestern Blot showed that p-PERK,p-eif2α,p-IRE1α,XBP1U/S,CHOP decreased significantly after treatment with the ART1 inhibitor MIBG in the G12 D and G13 D groups compared with the DMSO control groups,p<0.01.There is no significant change in the expression of ATF6,p>0.05.After p HBLV and KRAS WT were treated with ART1 inhibitor MIBG,compared with each DMSO group,the expression of p-PERK,p-eif2α,p-IRE1α,XBP1U/S,CHOP,ATF6 did not change significantly,p>0.05.At the same time,the expression of Cleaved-caspase3 in the G12 D and G13 D groups was significantly increased after ART1 inhibited MIBG treatment,and compared with each DMSO control group,p<0.01.Western Blot showed that after using si RNA to achieve ART1 knockdown,the IRE1α-XBP1-TFAF2/JNK and PERK-e IF2α-ATF4 signaling pathways were significantly inhibited in the G12 D and G13 D groups,which appeared as the expression of p-PERK,p-eif2α,p-IRE1α,CHOP decreased significantly,and statistics showed that p<0.01.The expression of ATF6 did not change significantly,p>0.05.4)Preliminary study on the single ADP ribosylation site of GRP78/BiP arginine in KRAS mutant colorectal cancer cellsAfter the GRP78/BiP R470+492K mutation and R214 K site mutation plasmid was transfected into G12 D cells,the expressions of p-PERK,p-eif2α,and p-IRE1α decreased compared with the control group,and statistics all showed p<0.01.Conclusion:1.The expression levels of ART1 and endoplasmic reticulum stress markers in KRAS mutant colorectal cancer tissues and cells increased,and under the state of KRAS mutation,the expression of ART1 and GRP78/BiP at m RNA and protein levels are positively correlated.2.The level of arginine specific mono-ADP ribosylated GRP78/BiP is higher in KRAS mutant colorectal cancer cells.By inhibiting ART1 to reduce the level of arginine specific mono-ADP ribosylated GRP78/BiP,it can effectively hinder the development of KRAS mutant colorectal cancer cells and promote the occurrence of its apoptotic response.3.By inhibiting ART1 to reduce the level of arginine specific mono-ADP ribosylated GRP78/BiP,GRP78/Bip expression can be significantly reduced,Inhibition of arginine mono-ADP ribosylated GRP78/BiP in KRAS mutant colorectal cancer cells led GRP78/BiP bind to IRE1α and PERK receptors,IRE1α-XBP1-TFAF2/JNK,PERK-e IF2α-ATF4 signaling pathways are significantly inhibited subsequently,so as damaged endoplasmic reticulum stress regulation mechanism,the cell tends to apoptosis.In this process,the modification site at 470,492,and 214 positions of GRP78/BiP may be involved to play a key role.