| Objective: TF?B-related factor 2(BRF2)is a key component of the TF?B complex.The BRF2-TF?B complex is also a key factor required for Pol ? to accurately recruit to target genes and form a transcriptionally active initiation complex.In recent years,abnormal expression of BRF2 protein has been found in a variety of cancer cells and has played a role in the development of cancer,but no research has been reported in gastric cancer.The purpose of this study was to study the expression and function of BRF2 in gastric cancer and gastric precancerous diseases.Methods:1.We tested the expression of BRF2 in 806 pairs of gastric cancer tissues and adjacent tissues by immunohistochemical experiments,and verified WB,PCR experiments and database analysis of 15 pairs of fresh gastric cancers and adjacent tissues in gastric cancer and gastric cancer.Expression in adjacent tissues.2.Based on detailed clinical data,analyze the correlation between BRF2 expression level and patient pathological data,including: gender,age,tumor size,degree of differentiation,tumor invasion(p T),lymph node metastasis(p N),growth mode,etc.The survival KM curve was used to analyze the correlation between the expression of BRF2 and the survival of patients(P=0.245).Furthermore,the patients were further divided into different subgroups according to the different pathological data.In these subgroups,the expression of BRF2 and the survival of patients were analyzed.3.We extracted total RNA from 5 cell lines for PCR,including 4 gastric cancer cell lines: AGS,MGC-803,HGC-27,SGC7901,and 1 gastric mucosal cell line GES-1.BRF2 expression was studied in gastric cancer cell lines.And select two cell lines with high BRF2 gene expression: AGS and SGC7901,and use Si-RNA to knock out BRF2 gene for further cell function research,including: CCK-8 experiment for cell proliferation ability detection,cell migration ability Scratch test for detection,Transwell test for detection of cell invasion ability,and flow cytometry test.4.In order to study whether BRF2 is related to the tumorigenic stage,we selected tissue wax blocks from patients who underwent gastroscopy at the Affiliated Hospital of China Medical University in 2017 and were diagnosed with gastric precancerous diseases,including the diagnosis: gastric mucosa 20 cases of high-grade dysplasia,atrophic gastritis with intestinal metaplasia,gastric ulcer,and gastric polyp.At the same time,tissue wax blocks diagnosed as gastric cancer in the gastroscope group and normal tissues,ie,cases of superficial gastritis,were used as controls,and immunohistochemical experiments were performed again.5.Statistical analysis: The full-text statistics are calculated using SPSS 22.0.The results of immunohistochemical scoring of BRF2 expression level were analyzed by means ± standard deviation table using t-test method.The correlation between the expression level and the patient's clinicopathological data was calculated using non-parametric tests;the survival analysis was calculated using the Kaplan-Meier method.P<0.05 was used to determine whether there was a significant difference.Results:1.Our results indicate that the expression of BRF2 in gastric cancer(6.124± 2.096)is significantly higher than that in adjacent normal tissues(4.275 ± 1.886)(P<0.001).In order to further study the expression of BRF2 in tissues,we extracted RNA from 15 pairs of fresh tissues and performed PCR.The results showed that ?Ct cancer tissue was 9.818 ± 1.158,and ?Ct adjacent tissue was 11.08 ±1.319(P<0.01).WB showed that the expression of BRF2 protein was also higher in these cases(T=1.214±0.4605)than in adjacent tissues(N=0.6709±0.2261)(P<0.01).This is consistent with the results we obtained using the Oncomine database analysis.2.The statistical results showed that the expression of BRF2 had no correlation with the patient's gender,age,tumor size,degree of differentiation,tumor invasion(p T),lymph node metastasis(p N),and growth pattern.We further analyzed the survival KM curve to analyze the correlation between BRF2 expression and patient survival(P=0.245).We divided patients into different subgroups according to various pathological data.No correlation was found between the level of BRF2 expression and survival in these subgroups.Therefore,we speculate that BRF2 may not be related to tumor development,but should be changed with the stage of cancer development.3.Compared with the normal gastric mucosal cell line GES-1,BRF2 was significantly overexpressed in a variety of gastric cancer cell lines(P<0.01).Based on the results of PCR,we selected two cell lines with high BRF2 gene expression: AGS and SGC7901,and knocked out BRF2 gene with Si-RNA for further cell function research.But unfortunately,no significant difference was found between the Si-RNA group and the control NC group in the proliferation,scratch,and Transwell experiments,and the flow cytometry test.This further confirms that although BRF2 is upregulated in gastric cancer,it has no significant correlation with the stage of tumor development.4.The expression of BRF2 in the nucleus of a variety of precancerous diseases or gastric cancer(6.35±2.159)is significantly higher than superficial gastritis(4.35±1.899).These precancerous diseases include: high-grade dysplasia of the gastric mucosa(6.2±1.881)(P<0.01),atrophic gastritis with intestinal metaplasia(5.9±1.553)(P<0.01),gastric polyp tissue(6.05±2.038)(P<0.01).In gastric ulcer(4.6±1.93),the expression level was not significantly different from superficial gastritis(P=0.682).These results indicate that BRF2 expression has been clearly up-regulated in various precancerous lesions of gastric cancer,especially in highgrade gastric dysplasia and atrophic gastritis and gastric polyp tissues.Therefore,the results suggest that BRF2 has been abnormally expressed in the occurrence of gastric cancer,which provides a new idea for studying the mechanism of gastric cancer.Conclusions:1.The expression level of BRF2 in gastric cancer tissues is significantly higher than that in adjacent tissues.2.The expression of BRF2 in the lesions of gastric cancer patients had no significant correlation with the clinical pathology and prognosis of gastric cancer patients.3.Compared with GES-1,a normal gastric mucosal cell line,BRF2 is highly expressed in a variety of gastric cancer cells,such as AGS and SGC-7901,but has no significant effect on a variety of cell functions during gastric cancer progression.4.Compared with the normal gastric mucosa,BRF2 is significantly overexpressed in a variety of precancerous diseases of gastric cancer,including high-grade dysplasia of gastric mucosa,atrophic gastritis with intestinal metaplasia,and gastric polyps.This confirms that BRF2 expression has been up-regulated in the stage of gastric cancer occurrence,and its up-regulation is an early event of gastric cancer occurrence.This suggests that BRF2 may play a potential role in the occurrence of gastric cancer.BRF2 can be a potential biomarker for early diagnosis of gastric cancer and a target for early treatment. |
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