| Background and aim: In this research, we will preliminarily discuss the influence of mono-ADP-ribosylation of histone on Arginine 117 on the expression of E-cadherin and its possible mechanism in colorectal carcinoma cells.Method: This study has four parts.1. Expression and significance of ARTD8 and ARTD14 in human colorectal adenocarcinomas: Immunohistochemistry was used to investigate the expression of ARTD8 and ARTD14 in human colorectal adenocarcinoma tissues from 65 samples of colorectal adenocarcinoma.2.Detection of the difference of mono-ADP-ribosylation of histone in colorectal carcinoma cell lines with different differentiation: To check the difference of mono-ADP-ribosylation of histone in LOVO cells and SW480 cells by four-stage Tandem mass spectrometry.3.The effect of mono-ADP-ribosylation of H3 on Arginine 117 on the expression of E-cadherin and the migratory ability of LOVO cells:(1) To get amino acid mutation cell lines via infect LOVO cells with pLenti-H3 mut1-IRES-eGFP and pLenti-H3 mut2-IRES-eGFP.(2) The expression and mRNA of E-cadherin in un-transfection group, LV-control group, H3 R117 A group and H3 R117 k group were determined by western blot and QPCR, respectively.(3) Abilities of migration was determined in un-transfection group, LV-control group, H3 R117 A group and H3 R117 k group respectively by Wound healing assay and Transwell assay.4. The study of mechanism of mono-ADP-ribosylation of histone on Arginine 117 regulating the expression of E-cadherin in LOVO cells:(1) The expression of PARP1 in un-transfection group, LV-control group, H3 R117 A group and H3 R117 k group were determined by western blot(2) To observe the H3K9me2 level, the expression of H3K9me2 in each group was detected by western blot.(3) QPCR was used to detecte to mRNA level of FBP1 in each group.Result:1. Expression and significance of ARTD8 and ARTD14 in human colorectal adenocarcinomas:(1) Positive staining of ARTD8 was detected in both colorectal adenocarcinoma and normal colorectal tissue. In colorectal adenocarcinoma cells, ARTD8 located in the cytoplasm and also located in the cell nucleus in some cases, simultaneously. But, ARTD8 only expressed in the cytoplasm of normal colorectal mucosal glands. The positive staining of ARTD8 in colorectal adenocarcinoma tissues was higher than that of normal colorectal tissues. The immunostaining of ARTD8 in the cell nucleus of rectal adenocarcinoma was higher than that in normal tissues and colonic adenocarcinoma.(2) ARTD14 positivity was seen in both colorectal adenocarcinoma and normal colorectal mucosal glands. In colorectal adenocarcinoma cells, positive staining of ARTD14 located in the cytoplasm and also appeared in the cell nucleus in some cases, concurrently. In normal colorectal mucosal glands, positive staining of ARTD14 only located in the cytoplasm. The positive ratio of ARTD14 staining in colorectal adenocarcinoma was significantly higher than those of normal colorectal tissue. There was a correlation between the staining of ARTD14 and tumor differentiation. ARTD14 expressed in the cell nucleus of colonic adenocarcinoma was higher than that in normal colorectal mucosal glands.2. Detection of the difference of mono-ADP-ribosylation of histone in colorectal carcinoma cell lines with different differentiation: The result of four-stage Tandem mass spectrometry showed that there was a mono-ADP-ribosylation of histone H3 on arginine 117 in LOVO cells and the histone H3 on arginine 117 in SW480 cells did not have this modification.3. The effect of mono-ADP-ribosylation of H3 on Arginine 117 on the expression of E-cadherin and the migratory ability of LOVO cells:(1) Green fluorescence was detected in LV-control group, H3 R117 A group and H3 R117 K group and the number of fluorescent cell reached 80%.(2) Compared with un-transfection group and LV-control group, the protein and mRNA level of E-cadherin in H3 R117 A group and H3 R117 K group was enhanced. There is no significant difference between un-transfection group and LV-control group. And there is no difference between H3 R117 A group and H3 R117 K group.(3) Compared with un-transfection group and LV-control group, the abilities of migration reduced in H3 R117 A group and H3 R117 A group. There is no significant difference between un-transfection group and LV-control group. And there is no difference between H3 R117 A group and H3 R117 K group.4. The study of mechanism of mono-ADP-ribosylation of histone on Arginine 117 regulating the expression of E-cadherin in LOVO cells:(1) Compared with un-transfection group and LV-control group, the protein level of PARP1 in H3 R117 A group and H3 R117 K group was decreased. There is no significant difference between un-transfection group and LV-control group. And there is no difference between H3 R117 A group and H3 R117 K group.(2) Compared with un-transfection group and LV-control group, the H3K9me2 level in H3 R117 A group and H3 R117 K group was decreased. There is no significant difference between un-transfection group and LV-control group. And there is no difference between H3 R117 A group and H3 R117 K group.(3)The mRNA level of FBP1 in H3 R117 A group and H3 R117 K group was higher than it in un-transfection group and LV-control group. There is no significant difference between un-transfection group and LV-control group. And there is no difference between H3 R117 A group and H3 R117 K group.Conclusion:1. The positive staining of ARTD8 and ARTD14 in colorectal adenocarcinoma tissues was higher than that of normal colorectal tissues. There was a correlation between the staining of ARTD14 and tumor differentiation. These prompt that ARTD8 and ARTD14 may involve in the process of the development and progression of colorectal cancer. The immunostaining of ARTD8 in the cell nucleus of rectal adenocarcinoma was higher than that in normal tissues. ARTD14 expressed in the cell nucleus of colonic adenocarcinoma was higher than that in normal colorectal mucosal glands. These prompt that there may have mono-ADP-ribosylation in the cell nucleus of colorectal cancer cells. ARTD8 and ARTD14 may regulate colorectal carcinoma by affecting the mono-ADP-ribosylation of histone. But the site of mono-ADP-ribosylation of histone need to be further studied.2. Mono-ADP-ribosylation on arginine 117 of histone H3 can affect the expression of E-cadherin and the migratory ability of LOVO cells.3. Mono-ADP-ribosylation on arginine 117 of histone H3 may regulate H3K9me2 level by changing the expression of PARP1, resulting in changing of FBP1, which finally affect the migratory ability. |
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