RUNX3 regulates trastuzumab resistance of gastric cancer cells: a metabolomic analysis based on UPLC-Q Exactive Focus Orbitrap mass spectrometry / 南方医科大学学报

OBJECTIVE@#To explore the role of Runt-related transcription factor 3 (RUNX3) in metabolic regulation of trastuzumab-resistant gastric cancer cells and investigate the mechanism of RUNX3 knockdown-mediated reversal of trastuzumab resistance.@*METHODS@#We performed a metabolomic analysis of trastuzumab-resistant gastric cancer cells (NCI N87R) and RUNX3 knockdown cells (NCI N87R/RUNX3) using ultra performance liquid chromatography (UPLC) coupled with Q Exactive Focus Orbitrap mass spectrometry (MS). Multivariate combined with univariate analyses and MS/MS ion spectrums were used to screen the differential variables. MetaboAnalyst 5.0 database was employed for pathway enrichment analysis. Differential metabolites-genes regulatory relationships were constructed based on OmicsNet database. The changes in GSH/GSSG and NADPH/NADP ratios in NCI N87R/RUNX3 cells were measured using detection kits.@*RESULTS@#The metabolic profile of NCI N87R cells was significantly altered after RUNX3 knockdown, with 81 differential metabolites identified to contribute significantly to the classification, among which 43 metabolites were increased and 38 were decreased (P < 0.01). In NCI N87R cells, RUNX3 knockdown resulted in noticeable alterations in 8 pathways involving glutamine metabolism, glycolysis, glycerophospholipid, nicotinate-nicotinamide and glutathione metabolism, causing also significant reduction of intracellular GSH/GSSG and NADPH/NADP ratios (P < 0.01). The differential metabolites-genes network revealed a regulatory relationship between the metabolic molecules and genes.@*CONCLUSION@#RUNX3 reverses trastuzumab resistance in gastric cancer cells by regulating energy metabolism and oxidation-reduction homeostasis and may serve as a potential therapeutic target for trastuzumab-resistant gastric cancer.

Ziyin Huatan Recipe, a Chinese herbal compound, inhibits migration and invasion of gastric cancer by upregulating RUNX3 expression / 中西医结合学报

OBJECTIVES@#Ziyin Huatan Recipe (ZYHT), a traditional Chinese medicine comprised of Lilii Bulbus, Pinelliae Rhizoma, and Hedyotis Diffusa, has shown promise in treating gastric cancer (GC). However, its potential mechanism has not yet been clearly addressed. This study aimed to predict targets and molecular mechanisms of ZYHT in treating GC by network pharmacology analysis and to explore the role of ZYHT in GC both in vitro and in vivo.@*METHODS@#Targets and molecular mechanisms of ZYHT were predicted via network pharmacology analysis. The effects of ZYHT on the expression of metastasis-associated targets were further validated by Western blot and quantitative real-time polymerase chain reaction. To explore the specific molecular mechanisms of the effects of ZYHT on migration and invasion, the runt-related transcription factor 3 (RUNX3) gene was knocked out by clustered regularly interspaced short palindromic repeats/Cas9, and lentiviral vectors were transfected into SGC-7901 cells. Then lung metastasis model of GC in nude mice was established to explore the anti-metastasis effect of ZYHT. Western blot and immunohistochemistry were used to explore the impact of ZYHT on the expression of metastasis-related proteins with or without RUNX3 gene.@*RESULTS@#The network pharmacology analysis showed that ZYHT might inhibit focal adhesion, migration, invasion and metastasis of GC. ZYHT inhibited the proliferation, migration and invasion of GC cells in vitro via regulating the expression of metastasis-associated targets. Knocking out RUNX3 almost completely reversed the cell phenotypes (migration and invasion) and protein expression levels elicited by ZYHT. In vivo studies showed that ZYHT inhibited the metastasis of GC cells to the lung and prolonged the survival time of the nude mice. Knocking out RUNX3 partly reversed the metastasis of GC cells to the lung and the protein expression levels elicited by ZYHT.@*CONCLUSION@#ZYHT can effectively inhibit the invasion and migration of GC in vitro and in vivo, and its molecular mechanism may relate to the upregulation of RUNX3 expression.

Label-free quantitative proteomic study of RUNX3 regulating Herceptin resistance in gastric cancer cells / 药学学报

Resistance of tumor cells is a complex biological process involving multiple mechanisms and factors, in which anti-apoptosis is the most important cause of drug resistance. Previous studies have shown that the DNA binding activity of Runt related transcription factor 3 (RUNX3) increased prominently in Herceptin resistant gastric cancer cells (NCI N87R) while the relevance of which to drug resistance has not yet been confirmed. In this study, we employed CRISPR/Cas9 to establish RUNX3 knock-out cell line (△RUNX3/NCI N87R) to investigate the functions of RUNX3 in Herceptin resistance of NCI N87R cells and its potential mechanisms. We investigated proteomics profiling of △RUNX3/NCI N87R cells based on label free quantitative proteomics. Differentially expressed proteins were screened out according to fold change and significance level between △RUNX3/NCI N87R and NCI N87R cells. Pathway enrichment analysis was done using GeneAnalytics database, and gene ontology analysis was conducted by DAVID Bioinformatics Resources database. Protein-protein interaction networks were constructed based on STRING database. The results showed that △RUNX3/NCI N87R cells increased the sensitivity to Herceptin. Proteomic data demonstrated that the expression of 577 genes changed significantly in △RUNX3/NCI N87R cells, among which 191 genes were up-regulated while 386 ones down-regulated comparing with NCI N87R cells. Pathway analysis showed that autophagy, cell cycle, apoptosis, mitochondrial fatty acid β oxidation, neurogenic locus notch homolog protein 1 (NOTCH1), mammalian target of rapamycin (mTOR), Hedgehog and DNA damage response pathways exhibited notable changes based on pathway enrichment ratio and significance level (P < 0.05). These results indicated that RUNX3 knock-out altered multiple signaling pathways of NCI N87R cells. Western blotting manifested that the expression of autophagy regulatory molecules autophagy-related protein (ATG) 13, 7 and BECN1 increased remarkably while cell cycle molecules serine/threonine-protein kinase Chk2 (CHEK2) and apoptosis regulator Bcl-2 (BCL2) decreased prominently in △RUNX3/NCI N87R cells. The p-AKT expression decreased significantly in △RUNX3/NCI N87R cells compared with NCI N87R cells (P < 0.01) and was suppressed by Herceptin. These results indicated that RUNX3 knock-out altered cell cycle, increased inhibition to p-AKT by Herceptin, promoted autophagy and induced cell apoptosis of NCI N87R cells. These results suggested that RUNX3 may be a potential therapeutic target for reversing or reducing Herceptin resistance in gastric cancer cells.

Expression of Runt-related Transcription Factor 3 in Human Colon Cancer Cell Line HCT-116 Resistant to 5-Fluorouracil and the Mechanism of Drug Resistance / 中国医学科学院学报

Objective To establish a human colon cancer cell line HCT-116/5-FU resistant to 5-fluorouracil(5-FU)and explore the relationship between runt-related transcription factor 3(RUNX3)and drug resistance of colorectal cancer.Methods The human colon cancer cell line HCT-116/5-FU with resistance to 5-FU was established by low concentration gradient increment combined with high-dose intermittent shock.CCK-8 method was used to determine the half maximal inhibitory concentration(IC

Relationship between Expression of Runt-related Transcription Factor 3 and Enhancer of zeste Homolog 2 Proteins and Sensitivity to Neoadjuvant Chemotherapy in Locally Advanced Rectal Cancer / 中国医学科学院学报

Objective To investigate the expression and correlation of Runt-related transcription factor 3(RUNX3)and enhancer of zeste homolog 2(EZH2)in rectal cancer,and to reveal the relationship between the expression of RUNX3 and EZH2 and the sensitivity of XELOX regimen to neoadjuvant chemotherapy in locally advanced rectal cancer patients. Methods The carcinoma and paracancerous tissues of 31 patients with rectal adenocarcinoma and no preoperative antitumor therapy were selected as cancer group and paracancer group,respectively.The relative mRNA levels of RUNX3 and EZH2 in the two groups were measured by real-time quantitative reverse transcription-polymerase chain reaction,and the protein levels were determined by immunohistochemical assay.The expression of RUNX3 and EZH2 was compared between cancer tissue and paracancerous tissue.The pre-treatment wax blocks of 26 patients with locally advanced rectal cancer who received 3 cycles of XELOX regimen as neoadjuvant chemotherapy before surgery were selected as the pre-neoadjuvant therapy group,and the postoperative pathological wax blocks were selected as the post-neoadjuvant treatment group.Tumor regression grade(TRG)was determined to evaluate the efficacy of neoadjuvant therapy.Immunohistochemical assay was used to detect the protein levels of RUNX3 and EZH2 in the two groups,and then the relationship between the expression patterns of the two proteins and the efficacy of neoadjuvant chemotherapy was analyzed. Results Compared with paracancerous tissue,the cancer tissue showed down-regulated mRNA level and reduced positive protein expression rate of RUNX3,while up-regulated mRNA level(

Regulatory effects of miR-138 and RUNX3 on Th1/Th2 balance in peripheral blood of children with cough variant asthma / 中国当代儿科杂志

OBJECTIVES@#To study the expression levels of microRNA-138 (miR-138) and Runt-related transcription factor 3 (RUNX3) in peripheral blood of children with cough variant asthma (CVA) and their regulatory effects on Th1/Th2 balance.@*METHODS@#Sixty-five children with CVA (CVA group) and 30 healthy children (control group) were enrolled. Peripheral venous blood samples were collected for both groups, and CD4@*RESULTS@#Compared with the control group, the CVA group showed significantly decreased levels of IFN-γ and IL-2 from CD4@*CONCLUSIONS@#MiR-138 regulates Th1/Th2 balance by targeting RUNX3 in children with CVA, providing a new direction for the treatment of CVA.

MiRNA-106a Induces Multidrug Resistance of Gastric Cancer Cells by Targeting RUNX 3 / 华中科技大学学报(医学版)

Objective To explore the effect of miRNA‐106a(miR‐106a) expression on multidrug resistance(MDR)of gastric cancer(GC)cells and the involvement of runt‐related transcription factor 3 RUNX3.Methods The expression of miR‐106a was detected in two human gastric adenocarcinoma cell lines with MDR by immunoblotting and apoptosis assay. The sensitivity of GC cells to anticancer drugs was observed by detecting the expression of miR‐106a by using immunoblotting and PCR ,and the relationship between miR‐106a and RUNX3 was determined by luciferase activity assay.Results miR‐106a was significantly in‐creased in GC cells with MDR ,and it suppressed the sensitivity of GC cells to anticancer drugs. It could modulate MDR by tar‐geting RUNX3.Conclusion miR‐106a can induce the MDR by targeting RUNX3 in GC.

Relationship between the rs760805 polymorphism of the RUNX3 gene and susceptibility to gastric cancer / 中国癌症杂志

Background and purpose:Runt-related transcription factor 3 (RUNX3) has a signiifcant relation with gastric cancer. Many researches have confirmed that rs760805 AA can increase the risk of gastric cancer. The purpose of the study was to investigate the relationship between the rs760805 polymorphism of the RUNX3 gene and gastric cancer. Methods: The rs760805 genotypes were determined by PCR-based DNA sequence measuring analysis and direct DNA sequencing in 310 incident cases with gastric cancer and 327 controls recruited in Shandong. Results:The frequency of TT genotype was 15.16%in gastric cancer patients and 20.49%in normal controls, and the corresponding percentages for AT and AA genotypes were 48.39%and 36.45%, and 52.60%and 26.91%, respectively. Compared to TT genotype, AT genotypes were associated with an increased risk of gastric cancer (OR=1.24, 95%CI`:0.81-1.92;OR=1.83, 95%CI:1.15-2.92). Conclusion:The rs760805 polymorphism of the RUNX3 gene is associated with increased susceptibility to gastric cancer.

Role of MSH2 and RUNX3 gene methylation in DNA prepared from nasopharyngeal swabs in early diagnosis and outcome prediction of nasopharyngeal carcinoma / 解放军医学杂志

Objective To explore the role of methylation of Human MutS homolog 2 (MSH2) and Human runt-related transcription factor 3 genes (RUNX3) in DNA prepared from nasopharyngeal swabs in early diagnosis and prognosis prediction of nasopharyngeal carcinoma. Methods The methylation-specific PCR was used to detect hypermethylation of MSH2 and RUNX3 genes in DNA prepared from nasopharyngeal swabs from 54 nasopharyngeal carcinoma patients, 18 chronic nasopharyngitis patients and 20 healthy volunteers. The specificity and sensitivity in early diagnosis of nasopharyngeal carcinoma by detecting MSH2 and/or RUNX3 gene methylation were evaluated. The relationship between methylation of MSH2 or RUNX3 gene and the biological behavior of nasopharyngeal carcinoma was analyzed. Results Hypermethylation of MSH2 and RUNX3 gene was respectively detected in 38 out of 54 (70.37%) and in 28 out of 54 (51.85%) nasopharyngeal swabs obtained from nasopharyngeal carcinoma patients, while there was no methylation in nasopharyngeal swabs from 18 chronic nasopharyngitis patients and 20 healthy volunteers. The differences were statistically significant (P0.05). Conclusions Parallel combined testing of MSH2 and RUNX3 gene methylation in DNA prepared from nasopharyngeal swabs determined by methylation-specific PCR could increase the specificity and sensitivity in early diagnosis of nasopharyngeal carcinoma, and is of important clinical significance. However it may not serve as an index in evaluating the clinical prognosis of nasopharyngeal carcinoma at present.

Role of MSH2 and RUNX3 gene methylation in DNA prepared from nasopharyngeal swabs in early diagnosis and outcome prediction of nasopharyngeal carcinoma / 解放军医学杂志

Objective To explore the role of methylation of Human MutS homolog 2 (MSH2) and Human runt-related transcription factor 3 genes (RUNX3) in DNA prepared from nasopharyngeal swabs in early diagnosis and prognosis prediction of nasopharyngeal carcinoma. Methods The methylation-specific PCR was used to detect hypermethylation of MSH2 and RUNX3 genes in DNA prepared from nasopharyngeal swabs from 54 nasopharyngeal carcinoma patients, 18 chronic nasopharyngitis patients and 20 healthy volunteers. The specificity and sensitivity in early diagnosis of nasopharyngeal carcinoma by detecting MSH2 and/or RUNX3 gene methylation were evaluated. The relationship between methylation of MSH2 or RUNX3 gene and the biological behavior of nasopharyngeal carcinoma was analyzed. Results Hypermethylation of MSH2 and RUNX3 gene was respectively detected in 38 out of 54 (70.37%) and in 28 out of 54 (51.85%) nasopharyngeal swabs obtained from nasopharyngeal carcinoma patients, while there was no methylation in nasopharyngeal swabs from 18 chronic nasopharyngitis patients and 20 healthy volunteers. The differences were statistically significant (P0.05). Conclusions Parallel combined testing of MSH2 and RUNX3 gene methylation in DNA prepared from nasopharyngeal swabs determined by methylation-specific PCR could increase the specificity and sensitivity in early diagnosis of nasopharyngeal carcinoma, and is of important clinical significance. However it may not serve as an index in evaluating the clinical prognosis of nasopharyngeal carcinoma at present.

Expression of Suppressor Gene Runt-Related Transcription Factor 3 in Gastric Carcinoma and Its Relationship with Clinical Pathological Parameters / 中国普外基础与临床杂志

Objective To investigate the expression of suppressor gene Runt-related transcription factor 3(Runx3)in gastric carcinoma and its relationship with clinicopathologic parameters.Methods RT-PCR and Western blot were used to determine the mRNA expression and protein expression of Runx3 gene in primary tumor and corresponding normal tissues respectively in 52 patients with gastric carcinoma.The relationship between Runx3 expression and clinicopathologic parameters was analyzed.Results RT-PCR and Western blot analysis in 52 patients with gastric carcinoma showed down-regulation of Runx3 mRNA and Runx3 protein in 59.6%(31/52)and 48.1%(25/52)of the primary tumors tested,and in none of the normal tissues(P

Effect of tumor suppressor factor RUNX3 on the expression of apoptosis-related genes in gastric carcinoma cells / 中国病理生理杂志

AIM:To investigate the effect of tumor-suppressor RUNX3 on the transcription of apoptosis-related genes bcl-2,bax,caspase-3,caspase-8,caspase-9 in human gastric carcinoma cells BGC823,and to reveal the apoptosis molecular mechanism promoted by RUNX3. METHODS:The eukaryotic expression vector of human Runx3 gene pcDNA3.1-Runx3 was constructed. pcDNA3.1-Runx3 and blank vector pcDNA3.1 were transfected into BGC823 cells,respectively. After 48 h,the total mRNA and protein were acquired and the expression level of Runx3 was determined by RT-PCR and Western blotting. Then,the mRNA and protein expression of bcl-2,bax,caspase-3,caspase-8 and caspase-9 was determined by RT-PCR and Western blotting. ?-actin was used as a control. RESULTS:The eukaryotic expression vector pcDNA3.1-Runx3 was constructed successfully and transfected into BGC823 cells. RT-PCR and Western blotting confirmed that RUNX3 level was higher in pcDNA3.1-Runx3 transfected BGC823 cells than that in blank vector-transfected cells (P