|Year : 2020 | Volume
| Issue : 1 | Page : 1-9
Protein disulfide isomerase A3: A potential regulatory factor of colon epithelial cells
Yang Li1, Zhenfan Huang2, Haiping Jiang3
1 Department of Clinical Nutrition, The First Affiliated Hospital of Jinan University, Guangzhou, Guangdong, China
2 Department of Gastrointestinal and Anal Surgery, The Second Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong, China
3 Department of Gastrointestinal Surgery, The First Affiliated Hospital of Jinan University, Guangzhou, Guangdong, China
|Date of Submission||10-Dec-2019|
|Date of Acceptance||24-Feb-2020|
|Date of Web Publication||25-Mar-2020|
Department of Gastrointestinal Surgery, The First Affiliated Hospital of Jinan University, Guangzhou, Guangdong
Source of Support: None, Conflict of Interest: None
Aim: This study aimed to investigate the effects of protein disulfide isomerase A3 (PDIA3) on the proliferation and apoptosis of colon epithelial cells, so as to explore its possible role in colon compensation of ultra-short bowel syndrome.
Methods: The expression of PDIA3 gene in NCM460 colonic epithelial cells was upregulated and silenced by liposome transient transfection technique. The expression of PDIA3 protein was determined by Western blotting, the proliferation rate of NCM460 cells was detected by CCK8, and the apoptosis rate of NCM460 cells was determined by flow cytometry.
Results: DNA sequencing and Western blotting results successfully verified that PDIA3 protein expression in NCM460 cells was upregulated and silenced by liposomal transfection. In the PDIA3 overexpression experiment, the proliferation rate of the experimental group was lower than that of the empty carrier group at 24 h, 48 h, and 72 h, and the apoptosis rate of the experimental group was higher than that of the empty carrier group (P < 0.05, the difference was statistically significant). In the PDIA3-silenced experiment, the proliferation rate of the experimental group was higher than that of the empty carrier group at 24 h, 48 h, and 72 h, and the apoptosis rate of the experimental group was lower than that of the empty carrier group (P < 0.05, the difference was statistically significant).
Conclusion: PDIA3 inhibits the proliferation of human colonic epithelial cells (NCM460) and promotes their apoptosis, which may not be a key regulatory protein in colon compensation of ultra-short bowel syndrome.
Keywords: Colon compensation, epithelial cells of colon mucosa, protein disulfide bond isomerase, short bowel syndrome
|How to cite this article:|
Li Y, Huang Z, Jiang H. Protein disulfide isomerase A3: A potential regulatory factor of colon epithelial cells. Cancer Transl Med 2020;6:1-9
|How to cite this URL:|
Li Y, Huang Z, Jiang H. Protein disulfide isomerase A3: A potential regulatory factor of colon epithelial cells. Cancer Transl Med [serial online] 2020 [cited 2020 May 28];6:1-9. Available from: http://www.cancertm.com/text.asp?2020/6/1/1/281364
| Introduction|| |
Short bowel syndrome (SBS) refers to malnutrition syndrome caused by physical or functional loss of large portions of small intestine, leading to too little effective absorption area in the small intestine and the remaining intestinal tract is unable to get enough nutrients to maintain the physiological and metabolic needs of the body. Ultra-SBS (USBS) can be defined when the residual bowel length is ≤35 cm. SBS is not a common disease, and the consensus in China believes that the incidence of SBS is on the rise year by year, but there is still a lack of accurate incidence nationwide. Similarly, the true incidence of SBS worldwide is not clear. Because most of the small intestinal was removed, the remaining intestinal absorption area decrease sharply, resulting in the rapid transport of intestinal contents and decreased digestive function. The pathophysiological changes of SBS patients were mainly manifested as the malabsorption of macronutrients, micronutrients and water electrolytes which can lead to diarrhea, dehydration and malnutrition. Progressive weight loss was followed by a series of symptoms and signs associated with low weight: symptoms include confusion, distraction, weakness, and lethargy, and in some cases apathy, depression, and irritability., At present, the clinical treatment of USBS mainly relies on parenteral nutrition (PN) to provide additional nutritional supplement for patients and maintain physiological and metabolic needs. The appearance of PN is of milestone significance for the treatment of SBS, which makes the long-term survival of SBS patients possible. However, while PN provided adequate nutrients for SBS patients to maintain their nutritional requirements, PN-dependent patients were still affected by various factors, resulting in low quality of life and even life-threatening. PN-related complications include catheter-related complications, metabolic-related complications, and organ function impairment., Therefore, seeking a safe and effective method to promote the compensation of residual colon function in such patients is a new idea for the treatment of USBS patients, so that they can reduce the use of PN or get rid of the dependence on PN.
In our preliminary experimental studies and domestic and foreign reports in recent years, it was found that both in the USBS animal model and in the clinical SBS patients, the residual colon had adaptive changes in structure and function.,, In our previous analysis of differential proteins in the compensatory colon mucosa of USBS rats, the high expression of protein disulfide isomerase A3 (PDIA3) protein was found. PDIA3, also known as ERp57, 1, 25 d3-marrs, and GRP58, a thiol oxidoreductase chaperone protein belonging to the PDI family, involved in many signaling transduction and regulatory links related to cell survival. In the endoplasmic reticulum, PDIA3 is often seen as a resident REDOX partner that mediates protein folding by binding to thePdomains of calreticulin and calcinetin (CRT and CNX). On the cell membrane, PDIA3 can also act as a direct binding partner of proteins, mediating a variety of cell signaling cascades. In addition to acting directly as a receptor activation signaling cascade, the binding of PDIA3 to other substrates indirectly regulates the activity of other membrane-derived signals, including activation factor 3 (STAT3), epidermal growth factor, and tumor necrosis factor-alpha (TNF-alpha). Ozaki et al. reported that PDIA3 is associated with mitochondrial μ-calpain, which can cleaved and activated apoptosis-inducing factor. In addition, in a variety of digestive system diseases such as irritable bowel syndrome and digestive tract tumors, PDIA3 has also been proved to be involved in regulation, promoting the proliferation of cancer cells,, and participating in the progression and metastasis of cancer.,,,
In combination with the regulatory roles of PDIA3 in cell proliferation and a variety of important physiological functions, we hypothesized that whether PDIA3 also plays a role in compensatory colon as a key regulatory protein. This study aims to explore the effect of PDIA3 on the proliferation and apoptosis of colonic epithelial cells by bidirectional regulation of PDIA3 expression in the human NCM460 colonic epithelial cells, so as to provide experimental basis for finding the protein regulation pathway of USBS colonic compensation.
| Methods|| |
NCM460 human colon mucosal epithelial cell lines were purchased from the ATCC cell bank, and Roswell Park Memorial Institute (RPMI)-1640 medium and Opti-MEM® were purchased from GIBCO. Lipofectamine® 2000 was purchased from Invitrogen. BCA protein quantification kit was purchased from Verde bio. Annexin V-FITC/propidium iodide (PI) and Annexin V-PE/7-ADD apoptosis detection kits were purchased from Nanjing Kaiji Biological Company. Cell Counting kit-8 was purchased from Dojindo. PDIA3 eukaryotic cell overexpression plasmid and PDIA3 eukaryotic cell silencing plasmid were constructed by Shanghai Jikaigene Company.
NCM460 cell culture
RPMI-1640 medium containing 10% calf serum was used for culture in a cell incubator at 37°C with 5% CO2.
Construction of the protein disulfide isomerase A3-overexpressed plasmid
PDIA3 ID: NM_005313 was obtained by querying GenBank, and PDIA3 primer sequence was designed by analyzing and referring to relevant literatures [Table 1]. The recombinant plasmid was constructed by polymerase chain reaction (PCR) using tool vector GV141, and the positive cloning plasmid was detected by gene sequencing.
Construction of protein disulfide isomerase A3 eukaryotic cell silencing plasmid
ShRNA targeting at three NM_005313 (PDIA3) messenger RNA target sites (876, 636, and 1769) was designed with RNAi technology [Table 2], and then PDIA3-shRNA was verified by sequencing, and finally the silencing effect was verified by preexperiment, from which the most effective silencing plasmid was selected to obtain PDIA3 eukaryotic cell silencing plasmid [Table 3].
Overexpressed and silencing protein disulfide isomerase A3 NCM460 cell models were constructed
Lipofectamine® 2000 was used to transfect PDIA3 eukaryotic cell overexpression plasmid and PDIA3 eukaryotic cell silencing plasmid into NCM460 cells by liposome transient transfection technology.
Protein disulfide isomerase A3 protein expression was detected by Western blotting
Total protein was extracted from the transfected cells by adding RIPA lysate on ice, and the protein was quantified byBCA (bicinchoninic acid) method. After denaturation at 100°C for 10min, the samples were resolved by SDS-PAGE, transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk in TBS-T for 1h, then incubated with primary antibody at 4°C for 16h and secondary antibody at room temperature for 1h. chemiluminescence imaging system was used for development and strip gray scanning, and PDIA3 protein expression level was analyzed with beta-actin as internal reference.
Cell proliferation detection
CCK8 method was applied to adjust the density of transfected cells to 3–5 × 104/ml, and then the transfected cells were put into a 96-well plate according to 100-μl/well and then cultured in an incubator. Groups of 10-μl CCK-8 were added into each well at 24 h, 48 h, and 72 h, and then cultured in an incubator for 2 h in dark. The cell proliferation was evaluated by using the absorbance value detected at the wavelength of 450 nm with the microplate reader. The cell proliferation was measured continuously for 3 days, and the growth curve was drawn.
Cell apoptosis assay
Forty-eight hours after cell transfection, the overexpressed group cells were treated with Annexin V-FITC kit and the silent group cells were treated with Annexin V-PE kit, and the apoptosis rate was determined by flow cytometry. Ex(488 nm) and Em(530 nm) were used for overexpression group. In the overexpression group, Annexin V-FITC fluorescence was green, and FL1 channel was used for detection; and Ex(488 nm) and Em(≥630 nm) were taken, and PI fluorescence was red; and FL3 channel was used for detection. In the silent group, Ex(488 nm) and Em(578 nm) were taken, Annexin V-PE fluorescence was orange red, and FL2 channel was used for detection; Ex(546 nm) and Em(647 nm) were taken, 7-aad fluorescence was red, and FL3 channel was used for detection.
Gray analysis software (Image J, National Institutes of Health, USA) was used to analyze the images of Western blotting. The remaining data were processed by SPSS 22.0 software (IBM Corp, Armonk, NY, USA). All experiments were performed in triplicate at least three times independently. Statistical analyses were performed using SPSS v.22.0. Results are presented as mean+/- S.D. Statistical analysis was performed using one-way ANOVA and SNK - q test. P < 0.05 between groups was considered significant.
| Results|| |
Construction of protein disulfide isomerase A3 overexpression plasmid (GV141-PDIA3)
Protein disulfide isomerase A3 gene results were obtained by polymerase chain reaction amplification
The electrophoresis results of PDIA3 gene showed that bright bands were visible at 1.5 kB, and the size of the products at the bright bands was determined to be 1564 bp [Figure 1]. After recovery and purification, the products could be used to construct recombinant plasmid in subsequent experiments.
|Figure 1: The result of amplification protein disulfide isomerase A3 gene|
Click here to view
Results of recombinant plasmid construction of protein disulfide isomerase A3-GV141
Eight colonies in the culture plates were selected for PCR identification. In [Figure 2], bright bands were visible between 1 kb and 1.5 kb, among which 5–12 were measured as 1209 bp.
|Figure 2: The result of plasmid identification. Note: 1: DdH2O-negative control, 2: No load self-continuous control, 3: GAPDH-positive control, 4: Marker 5–12: protein disulfide isomerase A3 group|
Click here to view
Positive clone sequencing results
Comparing the sequencing results, it was found that there were two mutant bases (GCT in the first GCC synonymy mutation and CAT in the second CAC synonymy mutation [yellow marker]), both of which were synonymy mutations. The mutation did not affect the original amino acid coding and did not change the structure and function of the translated PDIA3 protein. Overexpression plasmid (PDIA3-GV141) was constructed correctly.
Protein disulfide isomerase A3 silencing plasmid (PDIA3-shRNA) construction results
The sequencing results of three target silencing plasmids (PDIA3-RNAi) were consistent with the corresponding shRNA sequence. No mutant bases were found, indicating that the construction of the three target sites of silent plasmid (PDIA3-shRNA) was successful.
Comparing the silencing effect of three target sites in protein disulfide isomerase A3-shRNA on PDIA3 protein in NCM460 cells (Western blotting)
Experimental grouping and transfection methods are shown in methods. After 48 h, both the experimental group and the empty carrier group showed green fluorescence under a fluorescence microscope. Five field were randomly selected under 200 times fluorescence microscope vision, same as the common microscope vision, of experimental group and the empty carrier group for transfection rate calculation. The results of Western blotting showed that plasmid 63244-1 had the best silencing effect, so it was selected for the following silencing experiment [Figure 3] and [Figure 4].
|Figure 3: Detection of the expression of protein disulfide isomerase A3 in NCM460 cells|
Click here to view
|Figure 4: Relative expression of the protein disulfide isomerase A3 protein|
Click here to view
Effect of overexpressed plasmid (protein disulfide isomerase A3-GV141) on protein disulfide isomerase A3 protein expression in NCM460 cells was verified by Western blotting
Experimental grouping and transfection methods are shown in methods. It was found that with the increase of transfection amount of overexpressed plasmid (PDIA3-GV141), the expression amount of PDIA3 protein was also increased correspondingly, which presented a positive correlation trend. However, there was no significant difference in PDIA3 protein expression between the blank control group and the empty carrier group [Figure 5] and [Figure 6].
|Figure 5: Detection of the expression of protein disulfide isomerase A3 in NCM460 cells|
Click here to view
|Figure 6: Relative expression of the protein disulfide isomerase A3 protein|
Click here to view
The effect of protein disulfide isomerase A3-shRNA on protein disulfide isomerase A3 protein expression in NCM460 cells confirmed by Western blotting
Experimental grouping and transfection methods are shown in methods section. After 48 h, both the empty carrier group and the experimental group were observed under fluorescence microscope, and fluorescence could be seen in the green fluorescence channel. Five fields were randomly read under the 200-fold fluorescence field, and five fields were randomly read under the normal field [Figure 7] under the microscope mode. The average transfection rate was calculated artificially. 0.5-μg group: 30%; 1 μg: 54%; and 2 μg: 82%. It was found through many times of Western blotting detection that with the increase of transfection volume of PDIA3-shRNA, the expression of PDIA3 protein in the experimental group was significantly inhibited and negatively correlated with the transfection dose. However, there was no significant difference in PDIA3 expression between the empty vector group and the blank control group [Figure 8] and [Figure 9].
|Figure 7: Fluorescent images of NCM460 cells transfected with different doses of protein disulfide isomerase A3-shRNA after 48 h|
Click here to view
|Figure 8: Detection of the expression of protein disulfide isomerase A3 in NCM460 cells|
Click here to view
|Figure 9: Relative expression of the protein disulfide isomerase A3 protein|
Click here to view
Effect of overexpression plasmid (protein disulfide isomerase A3-GV141) and silencing plasmid (protein disulfide isomerase A3-shRNA) on the proliferation of NCM460 cells
Relative proliferation curve of transfected overexpressed plasmid (protein disulfide isomerase A3-GV141) from 0 h to 72 h
During statistical analysis, 0-h data were removed. Levene's variance homogeneity test (a = 0.10) was used to indicate that the data of each group had homogeneity of variance (statistics = 1.725, P = 0.239). Analysis of variance (a = 0.05) of completely random design data showed that the difference between each group was statistically significant (F = 88.999, P = 0.000). Snk-q test (a = 0.05) using pair-wise comparison of multiple sample means showed that there was no significant difference in relative proliferation rate between the 1-, 2-, and 4-μg groups, but there was a significant difference between the three groups and the empty carrier group. The results showed that cell proliferation was inhibited in the experimental group.
As shown in [Figure 10], after transfection of overexpressed plasmids (PDIA3-GV141) in the three experimental groups, the proliferation ability of cells was significantly inhibited, the proliferation ability was negatively correlated with the doses of transfected PDIA3-GV141, and the inhibition effect was most significant at 48 h.
Relative proliferation curve of transfected silent plasmid (protein disulfide isomerase A3-shRNA) cells at 0 h–72 h
During statistical analysis, 0-h data were removed. Levene's homogeneity of variance test (a = 0.10) indicated that the data of each group had homogeneity of variances (statistics = 2.053, P = 0.185). Analysis of variance (a = 0.05) of completely random design data showed that there was statistical significance in the difference between each group (F = 84.503, P = 0.000). Snk-q test (a = 0.05) using pair-wise comparison of multiple sample means showed that there were significant differences in relative proliferation rates between the empty carrier group, the 0.5-μg group, the 1-μg group, and the 2-μg group. The results showed that the proliferation ability of the experimental group was improved than that of the empty vector group.
|Figure 10: Relative proliferation curves of NCM460 cells between 0 and 72 h|
Click here to view
As shown in [Figure 11], after transfection of PDIA3-shRNA in three experimental groups, the 0.5-μg group and 1-μg group showed increased proliferation, whereas the 2-μg group and empty vector showed decreased proliferation. From 24 to 72 h, the 0.5-μg group and 1-μg group showed sustained proliferation, whereas the 2-μg group and empty carrier group showed sustained inhibition.
|Figure 11: Relative proliferation curves of NCM460 cells between 0 and 72 h|
Click here to view
Effect of overexpressed plasmid (protein disulfide isomerase A3-GV141) on apoptosis of NCM460 cells
Levene's variable-variance homogeneity test (a = 0.10) calculated and reported that the statistical value was 0.970, P = 0.466 > 0.10. It suggested that the variance of apoptosis rates in the five groups was homogeneous. Analysis of variance (a = 0.05) of completely randomized design data showed that F = 1336.991 and P = 0.000 < 0.05, and the difference was statistically significant. It could be considered that the mean apoptosis rate between each group was different. Finally, the pair-wise comparison of the mean numbers of several samples by snk-q test (a = 0.05) showed that the mean number of the blank control group, the empty carrier group, the 1-μg group, the 2-μg group, and the 4-μg group was all significantly different. The results showed that the apoptosis rate increased with the increase of transfection plasmid dose [Figure 12].
|Figure 12: Apoptosis rate of NCM460 cells transfected with different doses of protein disulfide isomerase A3-GV141|
Click here to view
Flow cytometry was used to detect the apoptosis rate of NCM460 cells after transfection with different concentrations of overexpressed plasmids (PDIA3-GV141). It was found that the average apoptosis rate of NCM460 cells increased with the increase of the dose of transfected overexpressed plasmids, and the apoptosis rate was positively correlated [Figure 13].
|Figure 13: The average apoptosis rate of NCM460 cells transfected with different doses of protein disulfide isomerase A3-GV141|
Click here to view
Effect of silent plasmid (protein disulfide isomerase A3-shRNA) on apoptosis of NCM460 cells
Levene's variance homogeneity test (a = 0.10) calculated that the statistical value was 1.604, P = 0.248 > 0.10, suggesting that the variance of apoptosis rates in the five groups was homogeneous. Analysis of variance (a = 0.05) of completely randomized design data showed that F = 100.479, P = 0.000 < 0.05, and the difference was statistically significant. It could be considered that the mean apoptosis rate was different between groups. Finally, the pair-wise comparison of the mean values of several samples by SNK-q test (a = 0.05) showed that the mean values of the blank control group, the empty carrier group, and the 2-μg group were significantly different. The difference between the 0.5-μg group and the 1-μg group was not significant, but the difference between them and the blank control group, the empty carrier group, and the 2-μg group could be considered significant. The results showed that the apoptosis rate of the experimental group was lower than that of the empty vector group [Figure 14].
|Figure 14: Apoptosis rate of NCM460 cells transfected with different doses of protein disulfide isomerase A3-shRNA|
Click here to view
The apoptosis rate of NCM460 cells transfected with different concentrations of PDIA3-shRNA was detected by flow cytometry. Compared with the blank control group, the average apoptosis rate of the 0.5-μg group and the 1-μg group was significantly lower, but the average apoptosis rate of the 2-μg group was higher [Figure 15].
|Figure 15: The average apoptosis rate of NCM460 cells transfected with different doses of protein disulfide isomerase A3-shRNA|
Click here to view
| Discussion|| |
Our previous experiments have verified that the colon in USBS rat model can present compensatory changes in morphology and function, and subsequent studies of proteomics differences found that PDIA3 in the compensatory colon in USBS rat model was significantly upregulated., As a resident REDOX partner of endoplamic reticulum, PDIA3 has been proved to be involved in numerous signal transduction and regulation links related to cell survival. Given its importance to cell survival, we consider that PDIA3 may be involved in the regulation of colonic compensation of USBS and may have a potential promoting effect. In this respect, our research team did the corresponding research. PDIA3 was upregulated and downregulated in colon cancer cells (SW480), and PDIA3 was found to promote proliferation and inhibit apoptosis in SW480 cells. Then, does PDIA3 have the same pro-proliferation and anti-apoptosis effect in human colon epithelial cells?
In this study, PDIA3 eukaryotic cell models with overexpressed plasmid (PDIA3-GV141) and silenced plasmid (PDIA3-shRNA) were constructed, and liposomal transient transfection was used to overexpress and silence PDIA3 in NCM460 cells. Subsequent Western blotting detection revealed that the expression of PDIA3 in NCM460 cells transfected with overexpressed plasmids increased with the transfection doses. On the contrary, the expression of PDIA3 in NCM460 cells transfected with silencing plasmid decreased with the increase of transfection doses. This indicates that the construction and transfection of plasmids were successful. In the apoptosis experiment, we found that the apoptosis rate of NCM460 cells transfected with overexpressed plasmids increased with the increase of transfection dose, and the average apoptosis rate of NCM460 cells in the transfected silent plasmid group was significantly lower than that in the empty vector group. Moreover, CCK-8 was further used to determine the effect of PDIA3 on the proliferation of NCM460 cells: overexpressed plasmids could inhibit the proliferation of NCM460 cells, whereas silenced plasmids could promote the proliferation of NCM460 cells compared with empty vectors. In conclusion, by bidirectional regulation of PDIA3 expression in NCM460 cells, we found that upregulation of PDIA3 inhibited proliferation and promoted apoptosis in NCM460 cells, whereas downregulation of PDIA3 inhibited apoptosis and promoted proliferation in NCM460 cells.
Although PDI family members are generally considered to assist in cell adaptation to unfolded protein response and promote cell survival activity, in the studies of human normal cells, most studies have proved that the overexpression of PDIA3 could promote apoptosis, while its knockdown could inhibit apoptosis process. Helton and Chen believed that although PDIA3 had the protective effect of maintaining cell homeostasis in the early stage of ER stress, with the intensification of ER stress and the effect of PDI response exceeding the threshold level, the signaling cascade effect of apoptosis would be initiated. Similar to p53, extreme levels of DNA damage will induce apoptosis, and more and more evidence have proved this finding. Xu et al. used siRNA to knock down the expression of PDIA3 in human endothelial cells and found that the decrease of its level caused human endothelial cells to be more resistant to apoptosis induced by hyperoxia or chlamycin. However, overexpression of PDIA3 aggravates apoptosis of human endothelial cells induced by hyperoxia or chlamycin, suggesting that PDIA3 participates in the regulation of apoptosis through the regulation of caspase-3 activity. Similarly, Roberson et al. found that the decrease of PDIA3 reduced the amount of influenza, a virus on bronchial epithelial cells, and weakened the subsequent activation of caspase-12 to inhibit its apoptosis. Studies by Zhao et al. have also shown that silencing the expression of PDIA3 can weaken the activity of caspase-3/7 in mouse embryonic fibroblasts (MEF), leading to decreased apoptosis of MEF cells. On the contrary, the upregulated expression of PDIA3 would intensify the activity of caspase-3/7, thus inducing the apoptosis of MEF cells. It was further verified that PDIA3 triggered mitochondrial membrane permeation (MOMP) by activating Bak, and MOMP was considered a “dead end” in the process of apoptosis signal transduction, thus leading to apoptosis. Hence, we speculate that the possible mechanism of PDIA3 promoted the apoptosis of NCM460 cells is it can activate apoptosis of mitochondrial pathway. Then bak oligomerization was promoted in outer mitochondrial membrane and trigger mitochondrial outer membrane permeabilization (MOMP), which leads to the release of apoptotic factor Apaf-1 and cytochrome c forming apoptotic body with caspase-9 in the cytoplasm. Further, the apoptotic body activating the downstream Caspase-3, 6, 7 cascade reaction and finally causing cell apoptosis. Second, this study only temporarily regulated the expression of PDIA3 in NCM460 cells through liposomal transient transfection technology and did not screen stable expression cell lines, so the effect of long-term overexpression or silencing of PDIA3 on NCM460 cells could not be observed. In addition, the study did not involve specific signaling pathways and mechanisms, and the mechanism of PDIA3 in the compensation of colon mucosal cells could not be reflected.
Comparing the results of our previous experiments on SW480 cells, the effect of PDIA3 on NCM460 cells and SW480 cells was inconsistent. But this is not contradictory, there are physiological and pathological differences between normal cells and tumor cells. Moreover, relevant studies have shown that PDIA3 does promote proliferation in tumor cells: Ramírez-Rangel et al. found that PDIA3 can promote the assembly and activation of mammalian target of rapamycin C1 (mTORC1) and participate in the proliferation and regulation of tumor cells by interacting with mTOR. Gaucci et al. found that PDIA3 silencing can lead to impaired phosphorylation of estimated glomerular filtration rate (a tyrosine kinase receptor that activates many signaling cascades, leading to cell proliferation and inhibition of apoptosis), indirectly mediating the proliferation of tumor cells. Moreover, PDIA3 was also found to be able to detach tumor-related NKG2D ligands, thereby reducing the killing effect of NK cells and T cells on tumors. These studies all proved that PDIA3 plays an important role in the regulation of tumor cell proliferation.
In the USBS rat model, proteomics analysis revealed a significant increase in PDIA3 in the compensatory colon. We hypothesize that this phenomenon may be caused by stress response. As a stress protein, ER stress occurred after the intestinal resection trauma in the USBS rat model, which promoted the large expression of PDIA3 in a short time to protect the homeostasis of the remaining colonic mucosal cells. Therefore, overexpression of PDIA3 in the compensatory colon of the USBS rat model may be only a result, rather than the occurrence of colon compensation induced by PDIA3.
| Conclusion|| |
In this study, overexpression and silencing of PDIA3 and its effect on NCM460 cells indicated that overexpression of PDIA3 could promote apoptosis of colon mucosal cells, and it may not be a key regulatory protein in the compensatory changes of USBS colon.
Further experiments are needed. Combined with the shortcomings of this experiment and the conception of the next experiment, we planned to: (1) construct lentivirus overexpression and silent vector and screen stable expression cell lines; (2) treat the stable expression cell lines with PDIA3 inhibitor to observe its effect on cells; (3) further study the relationship between overexpression or silencing of PDIA3 and cytochrome C, Bak, and caspase 3/7, and to explore its role in signal transduction; and (4) further screen the significantly different proteins in the remaining compensatory colon differential proteins for study, so as to screen out the key proteins that promote proliferation during the change of colon compensatory proteins.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Tappenden KA. Pathophysiology of short bowel syndrome: considerations of resected and residual anatomy. J Parenter Enteral Nutr
2014; 38 (Suppl 1): 14S–22.
Li YS, Cai W, Li JS, Mao Q, Chen L, Chen ZS. China consensus on diagnosis and treatment of short bowel syndrome (2016 edition). Chin Med J
2017; 97: 569–76.
Sundaram A, Koutkia P, Apovian CM. Nutritional management of short bowel syndrome in adults. J Clin Gastroenterol
2002; 34: 207–20.
van Gossum A, Cabre E, Hébuterne X, Jeppesen P, Krznaric Z, Messing B, Powell-Tuck J, Staun M, Nightingale J; ESPEN. ESPEN guidelines on parenteral nutrition: gastroenterology. Clin Nutr
2009; 28: 415–27.
Pironi L, Goulet O, Buchman A, Messing B, Gabe S, Candusso M, Bond G, Gupte G, Pertkiewicz M, Steiger E, Forbes A, van Gossum A, Pinna AD; Home Artificial Nutrition and Chronic Intestinal Failure Working Group of ESPEN. Outcome on home parenteral nutrition for benign intestinal failure: a review of the literature and benchmarking with the European prospective survey of ESPEN. Clin Nutr
2012; 31: 831–45.
Howard L, Ashley C. Management of complications in patients receiving home parenteral nutrition. Gastroenterology
2003; 124: 1651–61.
Buchman AL. Complications of long-term home total parenteral nutrition: their identification, prevention and treatment. Dig Dis Sci
2001; 46: 1–18.
Joly F, Mayeur C, Messing B, Lavergne-Slove A, Cazals-Hatem D, Noordine ML, Cherbuy C, Duée PH, Thomas M. Morphological adaptation with preserved proliferation/transporter content in the colon of patients with short bowel syndrome. Am J Physiol Gastrointest Liver Physiol
2009; 297: 116–23.
Healey KL, Bines JE, Thomas SL, Wilson G, Taylor RG, Sourial M, Pereira-Fantini PM. Morphological and functional changes in the colon after massive small bowel resection. J Pediatr Surg
2010; 45: 1581.
Jiang HP, Chen T, Yan GR, Chen D. Differential protein expression during colonic adaptation in ultra-short bowel rats. World J Gastroenterol
2011; 17: 2572–9.
Lu ZC. Study on Proteomics of Colon Compensation in Rats with Short Bowel Syndrome. Jinan University; 2012.
Wei J, Hettinghouse A, Liu C. The role of progranulin in arthritis. Ann N Y Acad Sci
2016; 1383: 5–20.
Chen J, Dosier CR, Park JH, De S, Guldberg RE, Boyan BD, Schwartz Z. Mineralization of three-dimensional osteoblast cultures is enhanced by the interaction of 1α,25-dihydroxyvitamin D3 and BMP2 via two specific vitamin D receptors. J Tissue Eng Regen Med
2016; 10: 40–51.
Guo GG, Patel K, Kumar V, Shah M, Fried VA, Etlinger JD, Sehgal PB. Association of the chaperone glucose-regulated protein 58 (GRP58/ER-60/ERp57) with Stat3 in cytosol and plasma membrane complexes. J Interferon Cytokine Res
2002; 22: 555–63.
Gaucci E, Altieri F, Turano C, Chichiarelli S. The protein ERp57 contributes to EGF receptor signaling and internalization in MDA-MB-468 breast cancer cells. J Cell Biochem
2013; 114: 2461–70.
Yang WS, Yu H, Kim JJ, Lee MJ, Park SK. Vitamin D-induced ectodomain shedding of TNF receptor 1 as a nongenomic action: D3 vs. D2 derivatives. J Steroid Biochem Mol Biol
2016; 155(Pt A): 18–25.
Ozaki T, Yamashita T, Ishiguro S. ERp57-associated mitochondrial μ-calpain truncates apoptosis-inducing factor. Biochim Et Biophys Acta Mol Cell Res
2008; 1783: 1955–63.
Zhuang ZM, Wang XT, Zhang L, Tao LY, Lv B. The effect of PDIA3 gene knockout on the mucosal immune function in IBS rats. Int J Clin Exp Med
2015; 8: 6866–77.
Zhuang Z, Zhang L, Wang X, Tao LY, Lv B. PDIA3 gene induces visceral hypersensitivity in rats with irritable bowel syndrome through the dendritic cell-mediated activation of T cells. PeerJ
2016; 4: e2644.
Zou Q, Yang ZL, Yuan Y, Li JH, Liang LF, Zeng GX, Chen SL. Clinicopathological features and CCT2 and PDIA2 expression in gallbladder squamous/adenosquamous carcinoma and gallbladder adenocarcinoma. World J Surg Oncol
2013; 11: 143.
Ménoret A, Drew D A, Miyamoto S, Nakanishi M, Vella AT, Rosenberg DW. Differential proteomics identifies PDIA3 as a novel chemoprevention target in human colon cancer cells. Mol Carcinog
2014; 53(Suppl 1): E11.
Caorsi C, Niccolai E, Capello M, Vallone R, Chattaragada MS, Alushi B, Castiglione A, Ciccone G, Mautino A, Cassoni P, De Monte L, Álvarez-Fernández SM, Amedei A, Alessio M, Novelli F. Protein disulfide isomerase A3 (PDIA3)-specific Th1 effector cells infiltrate colon cancer tissue of patients with circulating anti-PDIA3 auto-antibodies. Cardiovasc Ultrasound
2015; 11: 1–7.
Ramírez-Rangel I, Bracho-Valdés I, Vázquez-Macías A, Carretero-Ortega J, Reyes-Cruz G, Vázquez-Prado J. Regulation of mTORC1 complex assembly and signaling by GRp58/ERp57. Mol Cell Biol
2011; 31: 1657–71.
Santana-Codina N, Carretero R, Sanz-Pamplona R, Cabrera T, Guney E, Oliva B, Clezardin P, Olarte OE, Loza-Alvarez P, Méndez-Lucas A, Perales JC, Sierra A. A transcriptome- proteome integrated network identifies endoplasmic reticulum thiol oxidoreductase (ERp57) as a hub that mediates bone metastasis. Mol Cell Proteomics
2013; 12: 2111–25.
Qi YC. Bidirectional Regulation of PDIA3 on Proliferation and Apoptosis of SW480 Cells. Jinan University; 2015.
Helton ES, Chen X. p53 modulation of the DNA damage response. J Cell Biochem
2007; 100: 883–96.
Xu D, Perez RE, Rezaiekhaligh MH, Bourdi M, Truog WE. Knockdown of ERp57 increases BiP/GRP78 induction and protects against hyperoxia and tunicamycin-induced apoptosis. Am J Physiol Lung Cell Mol Physiol
2009; 297: L44–51.
Roberson EC, Tully JE, Guala AS, Reiss JN, Godburn KE, Pociask DA, Alcorn JF, Riches DW, Dienz O, Janssen-Heininger YM, Anathy V. Influenza induces endoplasmic reticulum stress, caspase-12-dependent apoptosis, and c-Jun N-terminal kinase-mediated transforming growth factor-β release in lung epithelial cells. Am J Respir Cell Mol Biol
2012; 46: 573–81.
Zhao G, Lu H, Li C. Proapoptotic activities of protein disulfide isomerase (PDI) and PDIA3 protein, a role of the Bcl-2 protein Bak. J Biol Chem
2015; 290: 8949–63.
Liu WJ, Qin HL, Ma YL. Proteomics of intestinal mucosa in patients with colorectal cancer. J Shandong Med
2008; 48: 1–3.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15]
[Table 1], [Table 2], [Table 3]