| Background and aim Colorectal cancer is a major public health problem in the world,with a high incidence.Although there are surgery,chemotherapy,targeted therapy and other methods,but the therapeutic effect is limited,the mortality rate of colorectal cancer is still high,so it is urgent to seek new therapeutic targets.With the progress of research,more and more attention has been paid to the epigenetics of colorectal cancer.Mono-ADP-ribosylation is a kind of epigenetics,but the modification level and target protein in colorectal cancer are not clear.The previous study of our team showed that the expression of ARTD8 and ARTD14,which catalyze mono-ADP-ribosylation,was significantly higher in colorectal cancer than that in control colorectal tissue,and both of them were expressed in nucleus of colorectal cancer,suggesting that there was abnormal mono-ADP-ribosylation in the nucleus of colorectal cancer.The MARylation of histone H3 in colorectal cancer cells was detected by LC-MS/MS.It was found that the arginine at position 117 of histone H3(H3R117)had mono-ADP-ribosylation,which could inhibit apoptosis and promote proliferation,invasion and metastasis.In transcriptome sequencing analysis,it was found that the expression of sterol regulatory element binding transcription factor 1(SREBP1)and fatty acid synthase(FASN)decreased,which was closely related to lipid metabolism.More and more evidence shows that lipid metabolism disorder is associated with colorectal cancer,which may be involved in the development of colorectal cancer in the early stage,but the specific mechanism is not clear.IGFBP1 is the molecular switch that regulates IGF-1,and the level of IGFBP1 in colorectal cancer is low.The previous research results of our team also showed that H3R117 mono-ADP-ribosylation can regulate DNA methylation and histone modification and inhibit genes expression,but whether it affects methylation of IGFBP1 is not clear.On the basis of previous studies,this study will analyze the level of mono-ADP-ribosylation in the nucleus of colorectal cancer,and explore whether H3R117 mono-ADP-ribosylation can regulate the methylation of IGFBP1 promoter and affect cell lipid metabolism,playing a role in colorectal cancer apoptosis.It provides new ideas and experimental basis for finding potential therapeutic targets of colorectal cancer.Methods This study consists of three parts: 1.Analysis of nuclear mono-ADP-ribosylation levels in human colorectal cancer Immunohistochemistry was used to investigate the level of nuclear mono-ADP-ribosylation in colorectal cancer and normal colorectal adjacent tissue from 64 CRC patients.The data of patient demographic,clinical and pathological characteristics were acquired and analyzed.2.Effect of H3R117 mono-ADP-ribosylation on lipid metabolism and apoptosis of LoVo cells(1)The effect of H3R117 mono-ADP-ribosylation on lipid metabolism in Lo V o cells The experiment was divided into four groups: Arg 117 of histone H3 was mutated into lysine(K)and alanine(A)in LoVo cells,which have been successfully constructed.Control: untreated LoVo cells;Empty vector: transfected empty vector LoVo cells;Mut-1: arginine at H3R117 site was mutated into alanine LoVo cells;Mut-2 : arginine at H3R117 site was mutated into lysine LoVo cells.1)The levels of cholesterol and fatty acid were detected by ELISA,IGF-1 was detected by the same method.2)The level of IGF-1 in cell culture supernatant was detected by ELISA.3)The expression of IGFBP1,IGF-1R and SREBP1 were detected by Western Blot.4)The expression of ACC,ACLY,FASN and HMGCR was detected by q RT-PCR.(2)The effect of H3R117 mono-ADP-ribosylation on the apoptosis of LoVo cells via IGFBP1 Small interference RNA was used to knock down IGFBP1 gene.The experiment was divided into six groups: control;empty vector;Mut-1,arginine at H3R117 site was mutated into alanine LoVo cells;Mut-2,arginine at H3R117 site was mutated into lysine LoVo cells;Mut-1+ si-IGFBP1,arginine at H3R117 site was mutated into alanine LoVo cells simultaneously knocked down IGFBP1 gene;Mut-2+si-IGFBP1,arginine at H3R117 site was mutated into lysine LoVo cells simultaneously knocked down IGFBP1 gene.1)After knocking down IGFBP1,the level of IGF-1 in the culture medium was detected by ELISA;The expressions of IGFBP1,IGF-1R and SREBP1 in LoVo cells were detected by Western blot;q RT-PCR was used to detect the expression of IGF-1R,SREBP1,ACC,FASN and HMGCR.2)The effect of knocking down IGFBP1 on cholesterol content was detected by Raman confocal microscope.3)The effect of knocking down IGFBP1 on the expression of IGF-1R in lipid raft,nucleus and cytoplasm was detected by Western Blot.4)Western Blot was used to detect the effect of knocking down IGFBP1 on Caspase3 expression,and flow cytometry was used to detect the effect of down-regulation of IGFBP1 on apoptosis rate.5)Effect of H3R117 mono-ADP-ribosylation on the expression of IGFBP1,IGF-1R,SREBP1 and Caspase3 in transplanted tumor A.Establishment of subcutaneous transplanted tumor model in nude mice.There were four groups: control;empty vector;Mut-1,arginine at H3R117 site was mutated into alanine LoVo cell s;Mut-2,arginine at H3R117 site was mutated into lysine LoVo cells.B.Calculation of subcutaneous transplanted tumor volume.The maximum diameter(A)and minimum diameter(B)of the transplanted tumor were measured,and the volume was calculated according to V= AB2/2(cm3).C.The protein of transplanted tumor was extracted and the expression of IGFBP1,IGF-1R,SREBP1 and Caspase3 was detected by Western Bolt.3.Study on the mechanism of H3R117 mono-ADP-ribosylation on lipid metabolism and apoptosis of LoVo cells via IGFBP1(1)The effect of H3R117 mono-ADP-ribosylation on the methylation of IGFBP1 promoter.The experiment was divided into four groups: control;empty vector;Mut-1,arginine at H3R117 site was mutated into alanine LoVo cells;Mut-2,arginine at H3R117 site was mutated into lysine LoVo cells.1)Disease Meth version 2.0 database was used to analyze the level of IGFBP1 promoter methylation in colorectal cancer and control colorectal tissues..2)Bisulfite genomic sequence was used to analyze the methylation of IGFBP1 promoter in LoVo cells.3)The effects of H3R117 mono-ADP-ribosylation on the H3K9me2/3 level of IGFBP1 gene promoter and the enrichment of HP1 ? and DNMT1 were detected by Ch IP.4)The effect of H3R117 mono-ADP-ribosylation on histone H3 cit expression was detected by Western Bolt.5)The effect of H3R117 mono-ADP-ribosylation on the H3 cit level of IGFBP1 gene promoter was detected by Ch IP.(2)Effect of H3R117 mono-ADP-ribosylation on IGFBP1 promoter methylation via H3 cit Histone citrullination was inhibited by inhibitor Cl-amidine.The experiment was divided into six groups: control;empty vector;Mut-1,arginine at H3R117 site was mutated into a lanine LoVo cells;Mut-2,arginine at H3R117 site was mutated into lysine LoVo cells;Mut-1+Cl-amidine,arginine at H3R117 site was mutated into alanine LoVo cells with inhibitor Cl-amidine;Mut-2+Cl-amidine,arginine at H3R117 site was mutated into lysine LoVo cells with inhibitor Cl-amidine.1)The time and concentration of inhibitor Cl-amidine were selected by CCK-8;Western Blot was used to detect the effect of inhibitor Cl-amidine on the expression of H3 cit and IGFBP1 protein.2)The co-localization of HP1 ? and DNMT1 was observed by laser confocal microscope after adding PAD inhibitor Cl-amidine.3)Methylated DNA Immunoprecipitation-q PCR was used to detect the effect of Cl-amidine on the methylation of IGFBP1 promoter.Results 1.Analysis of nuclear mono-ADP-ribosylation levels in human colorectal cancer(1)Mono-ADP-ribosylation was present in both colorectal carcinoma and control colorectal tissue.The level of mono-ADP-ribosylation in colorectal cancer was remarkably higher than that in control group(p<0.05).The expression of mono-ADP-ribosylation in nucleus was significantly higher than that in control colorectal tissue(p<0.05).(2)The clinicopathological features of colorectal carcinoma were analyzed.It was found that the level of mono-ADP-ribosylation in nucleus increased with the increase of tumor invasion depth and tumor grade(p<0.05).In addition,the level of mono-ADP-ribosylation in nucleus was positively correlated with tumor invasion and tumor grade(p<0.05).Mono-ADP-ribosylation was not related to sex,age,location,lymph node metastasis and TNM stage(p>0.05).2.Effect of H3R117 mono-ADP-ribosylation on lipid metabolism and apoptosis of LoVo cells(1)The effect of H3R117 mono-ADP-ribosylation on lipid metabolism in LoVo cells 1)The contents of cholesterol and fatty acids in cells were measured by ELISA.The results showed that compared with the control and empty vector,the content of fatty acid and cholesterol in the Mut-1 and Mut-2 were significantly reduced(p<0.05).There was no significant difference between control and empty vector,and between Mut-1 and Mut-2(p>0.05).2)The level of IGF-1 in cell culture supernatant was detected by ELISA.The results showed that compared with the control and empty vector,the level of IGF-1 in cell culture supernatant of Mut-1 and Mut-2 decreased significantly(p<0.05).There was no significant difference between control and empty vector,and between Mut-1 and Mut-2(p>0.05).3)Western Blot was used to detect the expression levels of IGFBP1,IGF-1R and SREBP1.The results showed that compared with the control and empty vector,the protein expression of IGFBP1 was increased,and IGF-1R and SREBP1 was decreased in the Mut-1 and Mut-2(p<0.05).There was no significant difference between control and empty vector,and between Mut-1 and Mut-2(p>0.05).4)The expression of ACC,ACLY,FASN and HMGCR mRNA was detected by q RT-PCR.The results showed that compared with the control and empty vector,the mRNA expressions of ACC,FASN and HMGCR were decreased(p<0.05).There was no difference in ACLY among four groups(p>0.05).There was no significant difference between control and empty vector,and between Mut-1 and Mut-2(p>0.05).(2)The effect of H3R117 mono-ADP-ribosylation on the a poptosis of LoVo cells via IGFBP1 1)The small interference RNA was used to knock down IGFBP1 gene,and si-IGFBP1-2 was selected as the optimal sequence.After knocking down IGFBP1,ELISA was used to detect the content of IGF-1 in cell culture supernatant,and the results showed that compared with Mut-1 and Mut-2,the level of IGF-1 in cell culture supernatant of Mut-1+si-IGFBP1 and Mut-2+ si-IGFBP1 was significantly higher(p<0.05).There was no statistical difference between Mut-1 and Mut-2,between control and empty vector,and between Mut-1+ si-IGFBP1 and Mut-2+ si-IGFBP1(p>0.05).2)After knockdown of IGFBP1,Western blot was used to detect the expression of IGFBP1.The results showed that the protein expression of IGFBP1 in Mut-1+si-IGFBP1 and Mut-2+ si-IGFBP1 was significantly lower than that in Mut-1 and Mut-2(p<0.05).There was no statistical difference between control and empty vector,between Mut-1 and Mut-2,and between Mut-1+ si-IGFBP1 and Mut-2+ si-IGFBP1(p>0.05).3)The mRNA expression of IGF-1R,SREBP1,ACC,FASN and HMGCR were detected by q RT-PCR after knocking down IGFBP1.The results showed that the mRNA expressions of IGF-1R,SREBP1,ACC,FASN and HMGCR in Mut-1+si-IGFBP1 and Mut-2+si-IGFBP1 were significantly increased compared with Mut-1 and Mut-2(p<0.05).There was no significant difference between Mut-1 and Mut-2,between control and empty vector,and between Mut-1+ si-IGFBP1 and Mut-2+ si-IGFBP1(p>0.05).4)The effect of knocking down IGFBP1 on cellular cholesterol was detected by Raman confocal microscope.The results showed that the characteristic peak of cholesterol standard was at 1439cm-1.Compared with Mut-1 and Mut-2,the Raman intensity and cholesterol content were significantly increased after knocking down IGFBP1(p<0.05).There was no statistical difference between control and empty vector,between Mut-1 and Mut-2,and between Mut-1+ si-IGFBP1 and Mut-2+ si-IGFBP1(p>0.05).5)The effect of knocking down IGFBP1 on the expression of IGF-1R in lipid raft,nucleus and cytoplasm was detected by Western Blot.The results showed that,after knocking down IGFBP1,compared with Mut-1 and Mut-2,the expression of IGF-1R in lipid raft,nucleus and cytoplasm of the two mutated groups increased significantly(p<0.05).There was no significant difference between control and empty vector,between Mut-1 and Mut-2,and between Mut-1+ si-IGFBP1 and Mut-2+ si-IGFBP1(p>0.05).6)After knocking down IGFBP1,Western Blot was used to analyze the expression of Caspase3.The results showed that the expression of cleaved-Caspase3 and apoptosis rate were decreased compared with Mut-1 and Mut-2(p<0.05).There was no statistical difference between control and empty vector,between Mut-1 and Mut-2,and between Mut-1+ si-IGFBP1 and Mut-2+ si-IGFBP1(p>0.05).7)The subcutaneous transplanted tumor in nude mice was successfully constructed.The results showed that the tumor volume in Mut-1 and Mut-2 was significantly lower than that in control and empty vector(p<0.05).Compared with the control and empty vector,the expression of IGFBP1 in Mut-1 and Mut-2 was increased,the expression of IGF-1R and SREBP1 protein was decreased,and the cleavage of Caspase3 was increased.There was no significant difference between control and empty vector,and between Mut-1 and Mut-2(p>0.05).3.Study on the mechanism of H3R117 mono-ADP-ribosylation on lipid metabolism and apoptosis of LoVo cells via IGFBP1(1)The effect of H3R117 mono-ADP-ribosylation on the methylation of IGFBP1 promoter.1)Bioinformatics analysis showed that the methylation of IGFBP1 promoter in colorectal cancer was significantly higher than that in control colorectal tissues(p<0.05).2)The methylation level of IGFBP1 promoter was detected by bisulfite genomic sequence.The results showed that compared with the control and empty vector,the methylation of IGFBP1 promoter in the Mut-1 and Mut-2 was significantly lower(p<0.05).Howere,there was no significant difference between Mut-1 and Mut-2,and between control and empty vector(p>0.05).3)The effects of H3R117 mono-ADP-ribosylation on the H3K9me2/3 level of IGFBP1 gene promoter and the enrichment of HP1? and DNMT1 were detected by Ch IP.The results showed that compared with the control and empty vector,the level of H3K9me2 on the IGFBP1 promoter and the enrichment of HP1 a and DNMT1 in the Mut-1 and Mut-2 were significantly lower(p<0.05);There was no significant difference in the H3K9me3 among four groups(P>0.05).There was no significant difference between control and empty vector,and between Mut-1 and Mut-2(p>0.05).4)The effect of H3R117 mono-ADP-ribosylation on the expression of histone H3 cit was detected by Western Bolt.The results showed that the expression of H3 cit in Mut-1 and Mut-2 was significantly higher than that in control and empty vector(p<0.05).There was no significant difference between Mut-1 and Mut-2,and between control and empty vector(p>0.05).5)The effect of H3R117 mono-ADP-ribosylation on the H3 cit level of IGFBP1 gene promoter was detected by Ch IP.The results showed that there was more H3 cit on IGFBP1 promoter in Mut-1 and Mut-2 than that in control and empty vector.There was no significant difference between control and empty vector,and between Mut-1 and Mut-2(p>0.05).(2)Effect of H3R117 mono-ADP-ribosylation on IGFBP1 promoter methylation via H3 cit 1)The concentration and time of inhibitor Cl-amidine was 200 ?M and 48 h.Western Blot was used to detect the effect of inhibitor Cl-amidine on the expression of H3 cit and IGFBP1.The results showed that compared with Mut-1 and Mut-2,the expression of H3 cit and IGFBP1 in the two groups decreased significantly after adding Cl-amidine(p<0.05).There was no significant difference between control and empty vector,between Mut-1 and Mut-2,and between Mut-1+Cl-amidine and mutant 2+Cl-amidine(p>0.05).2)The co-localization of HP1? and DNMT1 was observed by laser confocal microscope after the addition of Cl-amidine.The results showed that HP1? with green fluorescence and DNMT1 with red fluorescence were co-localized as yellow fluorescence.Compared with Mut-1 and Mut-2,with HP1?,the co-localization of DNMT1 increased increased after adding Cl-amidine.3)Me DIP-q PCR was used to detect the effect of Cl-amidine on the methylation of IGFBP1 promoter.The results showed that compared with Mut-1 and Mut-2,the 5m C enrichment of IGFBP1 promoter in mutant cells increased significantly after adding Cl-amidine(p<0.05).There was no difference between control and empty vector,between Mut-1 and Mut-2,and between Mut-1+Cl-amidine and Mut-2+Cl-amidine(p>0.05).Conclusion 1.The mono-ADP-ribosylation in the nucleus of colorectal cancer was positively correlated with the depth and grade of tumor.With the increase of mono-ADP-ribosylation in colorectal cancer nucleus,the deeper the invasion depth and higher the grade of tumor.It is suggested that mono-ADP-ribosylation is involved in the progression of colorectal cancer.2.H3R117 mono-ADP-ribosylation in colorectal cancer can up regulate the methylation of IGFBP1 promoter,which is related to the decrease of histone H3 citrullination,the increase of H3K9me2 and the enrichment of HP1 and DNMT1.The increased methylation of IGFBP1 promoter in colorectal cancer can increase cholesterol synthesis,inhibit cell apoptosis,and play a promoting role in the development of colorectal cancer.This study enriched the content of epigenetics of colorectal cancer,and provided a new idea and experimental basis for H3R117 mono-ADP-ribosylation as a target of colorectal cancer. |
PDF Full Text Request