| Objective: Detection of the expression of mitochondrial transcription factor A(TFAM)protein in normal esophagus,low and high grade squamous intraepithelial neoplasia and esophageal squamous cell carcinoma,and its expression in esophageal squamous cell carcinoma was analyzed with the patient Relationship of pathological characteristics;To observe the effects of down-regulation of TFAM on the biological characteristics of esophageal squamous cell carcinoma cells EC109 and EC9706,such as proliferation,clone formation,migration,invasion and apoptosis,and to explore its mechanism of action.Methods: SP immunohistochemical method was used to detect the expression of TFAM protein in normal esophageal mucosal squamous epithelium,low-grade,high-grade intraepithelial neoplasia,and esophageal squamous cell carcinoma tissue samples.The relationship between the expression of TFAM and the age,gender,pathological grade,clinical stage,depth of invasion,and the presence or absence of lymphatic metastasis in esophageal cancer patients were explored.Using EC109 and EC9706 cells as models,construct a down-regulated TFAM interfering plasmid and transfect the cells in the experimental group to down-regulate TFAM expression.Real-time quantitative PCR(qRT-PCR)and protein imprinting experiments were used to detect esophageal squamous cell carcinoma cells EC109 and EC9706 in the experimental group.Transfection efficiency.MTT assay was used to determine changes in cell proliferation before and after transfection;The plate cloning experiment was used to detect the changes in the ability of each group of cells to form clones;Transwell detects the cell migration and invasion ability of each group;Flow cytometry was used to determine the apoptosis of each group before and after gene down-regulation;Western blot was used to detect the expression of caspase3 and Bax in each group.Results: Immunohistochemical results showed that the expression of TFAM protein in normal esophageal mucosal epithelial tissue,esophageal mucosal intraepithelial neoplasia,and esophageal squamous cell carcinoma tissues showed an upward trend,and the differences were statistically significant [(Z=115.44,P<0.001);(ρ=0.88,P<0.001)];The expression of TFAM protein was related to myometrial invasion,clinical stage,and lymph node metastasis in patients with esophageal squamous cell carcinoma(P<0.001,P<0.05,P<0.05).Its expression increased with the deepening of the depth of invasion of the cancer tissue(P<0.001),and increased with the increase of clinical stage(P=0.001).The expression was higher in patients with lymph node metastasis than in patients without lymph node metastasis(P<0.05);In addition,this study showed that TFAM protein expression was not related to patient age,gender,and histological grade(P>0.05).After the EC109 cells were transfected with the interference plasmid,the expression of TFAM was lower than that of the control group,and the difference was statistically significant(t=-3.30,P<0.05);The expression of TFAM in the EC9706 experimental group was lower than that in the control group,and the difference was statistically significant(t=-2.84;P<0.05);The results of MTT experiments showed that the growth rate of both experimental groups was slower than that of the control group.After EC109 cells were transfected with the interference plasmid for 12 hours,the OD value of the experimental group(0.25±0.05)was smaller than that of the control group(0.31±0.03),(t=-2.73,P<0.05);The EC9706 experimental results are consistent with EC109.At 12 hours after transfection,the OD value of the experimental group(0.26±0.01)was smaller than that of the control group(0.31± 0.01),and the difference was statistically significant(t=-6.05,P<0.001);The results of the colony formation test showed that the colony formation rate of the experimental group of EC109 cells(25.67±1.30)% was lower than that of the control group(30±1.40)%,the difference was statistically significant(t=3.93,P<0.05);The colony formation rate in the EC9706 experimental group(13.50±0.78)% was less than that in the control group(21.27±0.83)%,and the difference was statistically significant(t=11.78,P<0.001);The results of the Transwell experiment showed that the number of migrating EC109 cells in the experimental group was(34.67±4.16),which was less than that in the control group(81.33±8.39),and the difference was statistically significant(t=8.63,P<0.05);In this cell invasion experiment,the number of invasion in the experimental group(18±2.65)was significantly less than that in the control group(62.67±7.51),and the difference was statistically significant(t=9.72,P<0.05).Similarly,the number of cell migration in the EC9706 cell experimental group was(38.50±6.65)lower than that in the control group(73.75±24.17),the difference was statistically significant(t=2.81,P <0.05);The number of invasive cells in the experimental group(14.60±4.67)was lower than that in the control group(43.60±5.03),the difference was statistically significant(t=9.45,P <0.001).The results of flow cytometry experiments showed that the apoptosis rate of the EC109 experimental group was higher than that of the control group [(33.56±2.46)%vs.(6.18 ± 0.65)%;t =-18.65,P<0.001].Similarly,the apoptosis rate of the EC9706 experimental group(32.33 ± 1.84)% was higher than that of the control group(6.56±0.92)%,and the difference was statistically significant(t=-21.75,P<0.001).Western blot results showed that the expression of TFAM(0.42±0.02)in the EC109 cell experimental group was significantly lower than that in the control group(1.52±0.04);the difference was statistically significant(t=59.43,P<0.001);The gray value of TFAM in the EC9706 cell experimental group(0.65±0.01)was lower than that in the control group(1.10±0.03),and the difference was statistically significant(t=23.28,P<0.001).It was confirmed that the expression of TFAM gene was down-regulated after the experimental group was transfected with the interference plasmid.The expression of Bax(0.69 ±0.02)in the EC109 cell experimental group was higher than that in the control group(0.45± 0.02),and the difference was statistically significant(t=-16.76,P<0.001);the EC9706 cell experimental results were consistent with it [(0.32±0.02)vs.(0.87±0.04);t=-26.31,P<0.001].In addition,compared with the control group,the gray value of Cleaved Caspase3 in the EC109 cell interference group was higher [(0.75±0.02)vs.(1.08 ± 0.01);t=-27,P<0.001];The gray value of the EC9706 cell control group was smaller than the experimental group [(0.75±0.02)vs.(0.97 ±0.02);t=-17.39,P<0.001].Conclusion: The expression of TFAM in normal esophageal,esophageal squamous cell carcinoma precancerous lesions,and esophageal squamous cell carcinoma showed an increasing trend.The expression of TFAM in esophageal squamous cell carcinoma was closely related to the depth of tumor invasion,clinical stage and lymph node metastasis;TFAM can reduce the proliferation,clone formation,migration and invasion of EC109 and EC9706 esophageal squamous cell carcinoma cell lines,and may promote the apoptosis of esophageal squamous cell carcinoma by affecting the expression of caspase3 and Bax,thereby affecting the occurrence and development of esophageal squamous cell carcinoma;Down-regulating TFAM can reduce the proliferation,clone formation,migration and invasion of EC109 and EC9706 esophageal squamous carcinoma cell lines,and may promote the apoptosis of esophageal squamous cell carcinoma by affecting the expression of caspase3 and Bax,thereby affecting the occurrence and development of esophageal squamous cell carcinoma;In-depth research on TFAM can provide new information for the monitoring and prevention of esophageal squamous cell carcinoma. |
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