Journal of Traditional Chinese Medicine ›› 2024, Vol. 44 ›› Issue (4): 703-712.DOI: 10.19852/j.cnki.jtcm.20240617.002
• Original articles • Previous Articles Next Articles
High-throughput sequencing analysis of differential microRNA expression in the process of blocking the progression of chronic atrophic gastritis to gastric cancer by Xianglian Huazhuo formula (香连化浊方)
GUO Yuxi1, LI Ze1, CHENG Nan1, JIA Xuemei1, WANG Jie1, MA Hongyu2, ZHAO Runyuan3, LI Bolin1, XUE Yucong4, CAI Yanru1(
), YANG Qian1()
- 1 Department of spleen and stomach diseases, First Affiliated Hospital of Hebei University of Traditional Chinese Medicine, Shijiazhuang 050000, China
2 Department of Traditional Chinese Medicine, Hebei General Hospital, Shijiazhuang 050051, China
3 Department of Gastroenterology, the First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, China
4 College of Integrative Medicine, Hebei University of Chinese Medicine, Shijiazhuang 050011, China
-
Received:2023-03-22Accepted:2023-07-24Online:2024-08-15Published:2024-06-17 -
Contact:YANG Qian, Department of spleen and stomach diseases, First Affiliated Hospital of Hebei University of Traditional Chinese Medicine, Shijiazhuang 050000, China.yang0311qian@126.com Telephone: +86-13832355120; CAI Yanru, Department of spleen and stomach diseases, First Affiliated Hospital of Hebei University of Traditional Chinese Medicine, Shijiazhuang 050000, China.137544977@qq.com Telephone: +86-15203319182 -
Supported by:Construction Project of National Clinical Research Base of Traditional Chinese Medicine(Science Letter [2018] No. 131, State Office of Traditional Chinese Medicine);Natural Science Foundation of Hebei Province: Study on the Mechanism of Action of Traditional Chinese Medicine on Disease and Syndrome(H2023423001);Key Research Project of the Ministry of Science and Technology(2018YFC1704100);Key Research Project of the Ministry of Science and Technology: Li Diangui Famous Old Chinese Medicine of Traditional Chinese Medicine Academic View Characteristic, Diagnosis and Treatment Methods and Experience of Prevention and Control of Major Diseases(2018YFC1704102);Provincial Science and Technology Program of Hebei Province: Prevention and Treatment of Gastric Cancer by Blocking the "Inflammation-Cancer Transformation" Based on the Theory of Turbidimetric Toxicity(21377724D);Provincial Science and Technology Program of Hebei Province: to Study the Clinical Efficacy and Mechanism of Huazhuo Jiedu Formula in the Treatment of Chronic Atrophic Gastritis based on Epidermal Growth Factor Receptor/Mitogen Activated Protein Kinase/Extracellular Signal-Regulated Kinase Signaling Pathway(21377740D);Scientific Research Project of Hebei Administration of Traditional Chinese Medicine: Clinical Study of Huazhuo Jiedu Formula Blocking the Pathological Evolution of Chronic Atrophic Gastritis(2022026);Scientific Research Project of Hebei Administration of Traditional Chinese Medicine: Study on the Medication Rules of Spleen and Stomach Diseases of Famous Yanzhao Medical Doctors Based on Data Mining(2022032);Scientific Research Project of Hebei Administration of Traditional Chinese Medicine: to Explore the Mechanism of Xianglian Huazhuo Formula in the Treatment of Chronic Atrophic Gastritis based on Transcriptomics(2023022)
Cite this article
GUO Yuxi, LI Ze, CHENG Nan, JIA Xuemei, WANG Jie, MA Hongyu, ZHAO Runyuan, LI Bolin, XUE Yucong, CAI Yanru, YANG Qian. High-throughput sequencing analysis of differential microRNA expression in the process of blocking the progression of chronic atrophic gastritis to gastric cancer by Xianglian Huazhuo formula (香连化浊方)[J]. Journal of Traditional Chinese Medicine, 2024, 44(4): 703-712.
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Figure 1 Histologic changes in the gastric mucosal tissue (magnification × 200, scale bar 50 μm) A: normal group; B: model group; C: XLHZ group. normal group: drink and eat freely + the same volume of normal saline was given by gavage; model group: MNNG, 180 μg/mL + 2% sodium salicylate, 1 mL/100 g per day + hunger and satiety disorders; XLHZ: model group + XLHZ, 36 g·kg-1·d-1. HE: hematoxylin and eosin; CAG: chronic atrophic gastritis; XLHZ: Xianglian Huazhuo formula.
Figure 1 Histologic changes in the gastric mucosal tissue (magnification × 200, scale bar 50 μm) A: normal group; B: model group; C: XLHZ group. normal group: drink and eat freely + the same volume of normal saline was given by gavage; model group: MNNG, 180 μg/mL + 2% sodium salicylate, 1 mL/100 g per day + hunger and satiety disorders; XLHZ: model group + XLHZ, 36 g·kg-1·d-1. HE: hematoxylin and eosin; CAG: chronic atrophic gastritis; XLHZ: Xianglian Huazhuo formula.
Table 1 qRT-PCR validation of miRNAs expression
| Group | n | Normal | Model | XLHZ |
|---|---|---|---|---|
| miR-20a-3p | 3 | 1.08±0.49 | 6.65±1.38a | 3.31±0.76b |
| miR-320-3p | 3 | 1.04±0.31 | 0.15±0.06a | 0.44±0.15b |
| miR-34b-5p | 3 | 1.05±0.37 | 0.11±0.07a | 0.58±0.13c |
| miR-483-3p | 3 | 1.01±0.15 | 0.29±0.16a | 0.37±0.12 |
| miR-883-3p | 3 | 1.03±0.28 | 0.21±0.05a | 0.52±0.17b |
| Akap12 | 3 | 1.03±0.25 | 0.19±0.13a | 0.76±0.25b |
| DLL1 | 3 | 1.00±0.11 | 0.32±0.07a | 0.58±0.06b |
| NR4A2 | 3 | 1.06±0.40 | 3.28±0.50a | 2.25±0.49b |
| TFRC | 3 | 1.01±0.18 | 2.13±0.29a | 1.64±0.22b |
Table 1 qRT-PCR validation of miRNAs expression
| Group | n | Normal | Model | XLHZ |
|---|---|---|---|---|
| miR-20a-3p | 3 | 1.08±0.49 | 6.65±1.38a | 3.31±0.76b |
| miR-320-3p | 3 | 1.04±0.31 | 0.15±0.06a | 0.44±0.15b |
| miR-34b-5p | 3 | 1.05±0.37 | 0.11±0.07a | 0.58±0.13c |
| miR-483-3p | 3 | 1.01±0.15 | 0.29±0.16a | 0.37±0.12 |
| miR-883-3p | 3 | 1.03±0.28 | 0.21±0.05a | 0.52±0.17b |
| Akap12 | 3 | 1.03±0.25 | 0.19±0.13a | 0.76±0.25b |
| DLL1 | 3 | 1.00±0.11 | 0.32±0.07a | 0.58±0.06b |
| NR4A2 | 3 | 1.06±0.40 | 3.28±0.50a | 2.25±0.49b |
| TFRC | 3 | 1.01±0.18 | 2.13±0.29a | 1.64±0.22b |
Figure 2 Effect of XLHZ on proliferation and migration of MNNG-induced GES-1 cells A: EdU assay was used to detect cell proliferation (magnification × 200). The proliferating cells were fluorescently stained with EdU (Green). The nuclei were stained with DAPI (Blue). the higher the cell proliferation rate, the brighter the green in the plot. A1: EdU of the control group; A2: DAPI of the control group; A3: merge of the control group; A4: EdU of the model group; A5: DAPI of the model group; A6: merge of the model group; A7: EdU of the XLHZ group; A8: DAPI of the XLHZ group; A9: merge of the XLHZ group. B: Quantitative analysis of proliferating cells. C: Wound healing assay was used to detect cell migration in the normal, model and XLHZ groups (magnification × 200). C1-C5: The migration of cells in the normal group was observed at 0, 12, 24, 48, 72 h. C6-C10: The migration of cells in the model group was observed at 0, 12, 24, 48, 72 h. C11-C15: The migration of cells in the XLHZ group was observed at 0, 12, 24, 48, 72 h. D: quantitative analysis of cells migration rate at 0, 12, 24, 48, 72 h. normal group: drink and eat freely + the same volume of normal saline was given by gavage; model group: MNNG, 180 μg/mL + 2% sodium salicylate, 1 mL/100 g per day + hunger and satiety disorders; XLHZ: model group + XLHZ, 36 g·kg-1·d-1. XLHZ: Xianglian Huazhuo formula; MNNG: N-Methyl-N'-Nitro-N-Nitrosoguanidine; GES-1: human gastric mucosal epithelial cells; EdU: 5-Ethynyl-2’- deoxyuridine; DAPI: 4’,6-Diamidino-2’-phenylindole. Statistical analyses were measured using one-way analysis of variance followed by post hoc Bonferroni correction for multiple comparisons. Data were presented as mean ± standard deviation (n = 3). Compared with the normal group, aP < 0.05, bP < 0.01; Compared with the model group, cP < 0.05; dP < 0.01.
Figure 2 Effect of XLHZ on proliferation and migration of MNNG-induced GES-1 cells A: EdU assay was used to detect cell proliferation (magnification × 200). The proliferating cells were fluorescently stained with EdU (Green). The nuclei were stained with DAPI (Blue). the higher the cell proliferation rate, the brighter the green in the plot. A1: EdU of the control group; A2: DAPI of the control group; A3: merge of the control group; A4: EdU of the model group; A5: DAPI of the model group; A6: merge of the model group; A7: EdU of the XLHZ group; A8: DAPI of the XLHZ group; A9: merge of the XLHZ group. B: Quantitative analysis of proliferating cells. C: Wound healing assay was used to detect cell migration in the normal, model and XLHZ groups (magnification × 200). C1-C5: The migration of cells in the normal group was observed at 0, 12, 24, 48, 72 h. C6-C10: The migration of cells in the model group was observed at 0, 12, 24, 48, 72 h. C11-C15: The migration of cells in the XLHZ group was observed at 0, 12, 24, 48, 72 h. D: quantitative analysis of cells migration rate at 0, 12, 24, 48, 72 h. normal group: drink and eat freely + the same volume of normal saline was given by gavage; model group: MNNG, 180 μg/mL + 2% sodium salicylate, 1 mL/100 g per day + hunger and satiety disorders; XLHZ: model group + XLHZ, 36 g·kg-1·d-1. XLHZ: Xianglian Huazhuo formula; MNNG: N-Methyl-N'-Nitro-N-Nitrosoguanidine; GES-1: human gastric mucosal epithelial cells; EdU: 5-Ethynyl-2’- deoxyuridine; DAPI: 4’,6-Diamidino-2’-phenylindole. Statistical analyses were measured using one-way analysis of variance followed by post hoc Bonferroni correction for multiple comparisons. Data were presented as mean ± standard deviation (n = 3). Compared with the normal group, aP < 0.05, bP < 0.01; Compared with the model group, cP < 0.05; dP < 0.01.
Figure 3 Effect of XLHZ on apoptosis and cell cycle of MNNG-induced GES-1 cells A: flow cytometry assay was used to detect apoptosis in the normal, model and XLHZ groups. A1: normal group; A2: model group; A3: XLHZ group; A4: apoptosis rate of quantitative analysis. B: flow cytometry assay was used to detect cell cycle in the normal, model and XLHZ groups. B1: normal group; B2: model group; B3: XLHA group; B4: quantitative analysis of cell cycle distribution. normal group: drink and eat freely + the same volume of normal saline was given by gavage; model group: MNNG, 180 μg/mL + 2% sodium salicylate, 1 mL/100 g per day + hunger and satiety disorders; XLHZ: model group + XLHZ, 36 g·kg-1·d-1. XLHZ: Xianglian Huazhuo formula; MNNG: N-Methyl-N'-Nitro-N-Nitrosoguanidine; GES-1: human gastric mucosal epithelial cells. Statistical analyses were measured using one-way analysis of variance followed by post hoc Bonferroni correction for multiple comparisons. Data were presented as mean ± standard deviation (n = 3). Compared with the normal group, aP < 0.01; Compared with the model group, bP < 0.01.
Figure 3 Effect of XLHZ on apoptosis and cell cycle of MNNG-induced GES-1 cells A: flow cytometry assay was used to detect apoptosis in the normal, model and XLHZ groups. A1: normal group; A2: model group; A3: XLHZ group; A4: apoptosis rate of quantitative analysis. B: flow cytometry assay was used to detect cell cycle in the normal, model and XLHZ groups. B1: normal group; B2: model group; B3: XLHA group; B4: quantitative analysis of cell cycle distribution. normal group: drink and eat freely + the same volume of normal saline was given by gavage; model group: MNNG, 180 μg/mL + 2% sodium salicylate, 1 mL/100 g per day + hunger and satiety disorders; XLHZ: model group + XLHZ, 36 g·kg-1·d-1. XLHZ: Xianglian Huazhuo formula; MNNG: N-Methyl-N'-Nitro-N-Nitrosoguanidine; GES-1: human gastric mucosal epithelial cells. Statistical analyses were measured using one-way analysis of variance followed by post hoc Bonferroni correction for multiple comparisons. Data were presented as mean ± standard deviation (n = 3). Compared with the normal group, aP < 0.01; Compared with the model group, bP < 0.01.
Figure 4 Effects of XLHZ on EMT and proliferation of MNNG-induced GES-1 cells A: representative images of of E-cadherin, TGF-β1, vimentin and β-catenin protein expression that were exhibited from MNNG-induced GES-1 cells in the normal, model and XLHZ groups. B: quantitative analysis of E-cadherin protein; C: quantitative analysis of TGF-β1 protein; D: quantitative analysis of vimentin protein; E: quantitative analysis of β-catenin protein. normal group: drink and eat freely + the same volume of normal saline was given by gavage; model group: MNNG, 180 μg/mL + 2% sodium salicylate, 1 mL/100 g per day + hunger and satiety disorders; XLHZ: model group + XLHZ, 36 g·kg-1·d-1. XLHZ: Xianglian Huazhuo formula; EMT: epithelial mesenchymal transition; MNNG: N-Methyl-N'-Nitro-N-Nitrosoguanidine; GES-1: human gastric mucosal epithelial cells. Statistical analyses were measured using one-way analysis of variance followed by post hoc Bonferroni correction for multiple comparisons. Data were presented as mean ± standard deviation (n = 3). Compared with the normal group, aP < 0.01; Compared with the model group, bP < 0.01; cP < 0.05.
Figure 4 Effects of XLHZ on EMT and proliferation of MNNG-induced GES-1 cells A: representative images of of E-cadherin, TGF-β1, vimentin and β-catenin protein expression that were exhibited from MNNG-induced GES-1 cells in the normal, model and XLHZ groups. B: quantitative analysis of E-cadherin protein; C: quantitative analysis of TGF-β1 protein; D: quantitative analysis of vimentin protein; E: quantitative analysis of β-catenin protein. normal group: drink and eat freely + the same volume of normal saline was given by gavage; model group: MNNG, 180 μg/mL + 2% sodium salicylate, 1 mL/100 g per day + hunger and satiety disorders; XLHZ: model group + XLHZ, 36 g·kg-1·d-1. XLHZ: Xianglian Huazhuo formula; EMT: epithelial mesenchymal transition; MNNG: N-Methyl-N'-Nitro-N-Nitrosoguanidine; GES-1: human gastric mucosal epithelial cells. Statistical analyses were measured using one-way analysis of variance followed by post hoc Bonferroni correction for multiple comparisons. Data were presented as mean ± standard deviation (n = 3). Compared with the normal group, aP < 0.01; Compared with the model group, bP < 0.01; cP < 0.05.
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