Effect of pregnancy uterine microenvironment on the expression of NKG2A,NKG2D and their ligands in decidual NK cells

AIM：To investigate the expression of NKG2A,NKG2D and their ligands in pregnancy uterine micro-environment and to probe the function of NKG2A and NKG2D imbalance expression during the immunotolerance at the fetal-maternal boundary.METHODS：Decidual lymphocytes and peripheral lymphocytes were obtained from 30 women during 6-9 weeks of pregnancy who were undergoing selective termination.FACS technology was used to detect NK cells number and NKG2A,NKG2D expression.RT-PCR was used to investigate HLA-E and MICA mRNA in trophoblast tissue.RESULTS：Natural killer cells predominate,accounting for 70% of pregnancy endometrial lymphocytes.FACS results indicated that NKG2A was significantly increased in decidual NK cells as compared with that in peripheral NK cells,accounting for 97.86%±1.75% and 33.35%±10.92%.The difference between them in NKG2A expression was significant (P<0.05).Although the levels of NKG2D in decidual NK cells were similar to that in peripheral NK cells,accounting for 93.21%±4.52% and 97.80%±1.72%,respectively,the difference between them in NKG2A expression was also significant (P<0.05).At the same time,we found HLA-E mRNA in the trophoblast tissue.There was no proof yet on the expression of NKG2D ligand-MICA on trophoblast.CONCLUSIONS：Natural killer cells are the predominant lymphocytes during the first trimester of decidual tissue.These cells have a great different antigenic phenotype with peripheral natural killer cells.Decidual natural killer cells have high expression of NKG2A and trophoblast tissue expressed HLA-E.This may be an important factor contributing to the immunotolerance at the fetal - maternal boundary during pregnancy.     

Significance and alterations of p14ARF gene of CDKN2A locus in gastric cancer

AIM:To investigate the significance and changes of p14^ARF gene in gastric cancer.METHODS:The tumors and gastric tissues neighboring carcinoma from 48 patients with gastric cancer were studied. The homozygous deletions, mutations, methylation of the CpG islands, and mRNA expression of p14^ARF gene were assessed by PCR, PCR-SSCP, PCR based methylation assay, and RT-PCR.RESULTS:①The homozygous deletion rate of p14^ARF was 31.3% (15/48), and no homozygous deletions were examined in all the gastric tissues neighboring tumor. ②There were no point mutations of p14^ARF in 33 gastric cancers without homozygous deletion and in the matched gastric tissues adjacent to tumor. ③Methylation rate of the CpG islands of p14^ARF was significantly higher in gastric cancers(47.9%, 23/48) than that in gastric tissues neighboring cancer (4.2%, 2/48)(P＜0.01).④ No expression of p14^ARF mRNA was detected in 45.8%(22/48) of gastric cancers. Moreover, the negative rate (100%, 3/3) of p14^ARF mRNA of gastric cancers with the combined methylation of exons 1β and 2 was significantly higher than that (15%, 3/20) of the sole methylation of exon2(P＜0.05). CONCLUSION:p14^ARF gene is frequently inactivated by homozygous deletion and methylation of the 5' CpG islands in gastric cancer, which may play an important role in the development of gastric cancer.     

Expression of DNMT in gastric cancer and effects of DNMT inhibitor 5-aza-2’-deoxycytidine on gastric cancer

AIM: To investigate the protein expression of DNA methyltransferases (DNMT1, DNMT2, DNMT3A, DNMT3B and DNMT3L) in gastric cancer and the effects of DNMT inhibitor (5-aza-2’-deoxycytidine) on gastric cancer. METHODS: The method of immunohistochemistry was used to detect DNMT expression in 60 pairs of gastric cancer and corresponding non-cancerous tissues. The expression of DNMT in gastric cancer cells (SGC-7901 and MKN-45) and gastric mucosa cells (GES-1) was detected by immunofluorescence and Western blotting. The proliferation, cell cycle and apoptosis in the cell lines of SGC-7901, MKN-45 and GES-1 were determined by MTT assay and flow cytometry with 5-aza-2’-deoxycytidine treatment. RESULTS: Both gastric cancer and corresponding non-cancerous tissues expressed 5 kinds of DNMT proteins, but the expression levels of DNMT1 and DNMT3A were significantly lower in gastric cancer than those in non-cancerous tissues (P<0.01). The expression levels of DNMT2 and DNMT3L were also lower in the cancer than those in non-cancerous tissues (P<0.05). No difference of DNMT3B expression level between cancer and non-cancerous tissues was observed (P>0.05). On the other hand, the cells lines of SGC-7901, MKN-45 and GES-1 also expressed DNMT proteins. Except DNMT3B, no difference of the other kinds of DNMT between gastric cancer cells and gastric mucosa cells was observed. DNMT inhibitor 5-aza-2’-deoxycytidine did not have any effect on the growth rate, cell cycle distribution or apoptotic rate in gastric cancer cells and gastric mucosa cells. CONCLUSION: Gastric cancer expresses low levels of DNMT proteins than normal gastric mucosa. 5-Aza-2’-deoxycytidine is not useful for treating gastric cancer.     

Promoting cell viability and migration of gastric cancer cells by PRRX2 via activation of Wnt/β-catenin signaling pathway

AIM: To study the effect of paired-related homeobox 2 (PRRX2) gene on the viability and migration ability of gastric cancer cells, and to analyze the underlying mechanism of regulating Wnt/β-catenin signaling pathway.METHODS: The expression of PRRX2 in gastric cancer and normal gastric tissue and the correlation between PRRX2 expression in gastric cancer tissues with the overall survival rate of gastric cancer patients were analyzed by bioinformatics. The small interfering RNA (siRNA) and over-expressed plasmids of PRRX2 were transfected into gastric cancer cells MGC-803 and SGC-7901, respectively. MTT assay and Transwell assay were used to detect the viability and migration ability of gastric cancer cells. Western blot and TOPflash/FOPflash dual-luciferase reporter gene assay were used to detect the activity of Wnt/β-catenin signaling pathway. Co-immunoprecipitation was used to detected the interaction between PRRX2 and β-catenin proteins.RESULTS: Knockdown of PRRX2 attenuated the viability and migration ability of gastric cancer cell line MGC-803 (P<0.05). Over-expression of PRRX2 enhanced the viability and migration ability of SGC-7901 cells (P<0.05), increased the protein levels of β-catenin, c-Myc and cyclin D1 (P<0.05) and the activity of TOPflash/FOPflash dual-luciferase reporter gene (P<0.05). PRRX2 interacted with β-catenin protein in gastric cancer cells.CONCLUSION: PRRX2 promotes the viability and migration ability of gastric cancer cells, which may be related to Wnt/β-catenin signaling pathway.     

Effects of rosiglitazone,a PPARγ ligand,on chemoprevention of MNNG-induced gastric cancer in rats

AIM: To examine the chemo-preventive effects of peroxisome proliferator-activated receptor γ(PPARγ) ligand rosiglitazone (RSG) on a rat model of gastric carcinogenesis induced by chemical carcinogen N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). We also attempted to identify novel anti-cancer mechanisms of rosiglitazone.METHODS: Ninety male Wistar rats were randomly allocated into six groups: group A (control group); group B (MNNG group); group C, D and E (RSG group, given different concentrations of rosiglitazone). The treatment procedures were terminated at 40th week. Stomach was harvested and gastric carcinoma was verified by histology. The gastric cancer incidence in different groups was calculated. To elucidate the mechanisms underlying the chemo-preventive effects of PPARγ ligand, we examine the gene expression profiles of MNNG induced gastric cancer and the rosiglitazone treated gastric cancer with Uniset Rat I Bioarray microarray.RESULTS: Incidence of gastric cancer in group A-E was 0% (0/10), 70% (14/20), 15% (3/20), 30% (6/20) and 30% (6/20), respectively. Gastric cancer incidence in group C, D and E was significantly lower than that in group B (P<0.01). A gene that showed prominent responses in rosiglitazone treated group was identified. The hypertension-related, calcium-regulated gene (HCaRG) was significantly upregulated in rat gastric carcinoma in rosiglitazone treated group when compared to MNNG group. The expression of HCaRG was down-regulated in human gastric cancerous tissue. CONCLUSION: PPARγ ligand rosiglitazone has a potent chemo-preventive effect against gastric cancer development in rats. Upregulation of HCaRG may be one of the mechanisms underlying the chemo-preventive effect of rosiglitazone in gastric cancer.     

Expression and prognostic significance of SLP-2 in gastric cancer

AIM: To investigate the expression of the red cell membrane integration protein SLP-2 (stomatin-like protein 2) in gastric cancer tissues and to analyze its correlation with clinicopathological manifestations and prognosis. METHODS: One hundred and ninety gastric cancer tissue samples with detailed clinical information were collected from the Department of Pathology, Sun Yat-sen University Cancer Center. The protein expression of SLP-2 in ganstric cancer was detected by the method of immunohistochemistry. The relationships between SLP-2 expression and the clinicopathological manifestations were evaluated. RESULTS: The positive rate of SLP-2 in gastric cancer tissue was 63.2% (120/190). SLP-2 expression was relevant to infiltration depth, TNM stage and lymph node metastasis (P<0.05). However, no statistical difference was observed in the SLP-2 expression associated with sex, age, differentiation, tumor size and distant metastases. Kaplan-Meier survival curves revealed that increased expression of SLP-2 was associated with poor prognosis in gastric adenocarcinoma patients (P<0.01). Based on the univariate analysis, 7 factors were found to have statistical significance of associations with overall survival, including SLP-2 expression, lymph node metastasis, histological grade, tumor size, invasive depth, distant metastases and the 7th edition of the UICC TNM classification. Only the tumor size and the 7th edition of the UICC TNM classification were independent prognostic factors for overall survival in the multivariate analysis. CONCLUSION: SLP-2 is highly expressed in gastric adenocarcinoma tissues and may play an important role in tumor progression and metastasis. Although SLP-2 is not an independent prognostic factor, it may influence the prognosis of gastric cancer. Increased expression of SLP-2 can be used for predicting unfavorable prognosis in gastric adenocarcinoma patients.     

Expression and clinical significance of Bmi-1 in gastric carcinoma

AIM: To investigate the relationship between expression of Bmi-1 (B cell-specific MLV integration site-1) in gastric cancer and its clinicopathologic significance.METHODS: 146 surgical patients with gastric carcinoma were followed up at least 2 years.Expression of Bmi-1 protein was examined by immunohistochemistry in their archival paraffin embedded tissue specimens.RESULTS: The intensive positive rate of Bmi-1 expression in gastric cancer was 67.8% (99/146).Expression of Bmi-1 was highly correlated with tumor size,clinical stage,lymph node metastasis and T classification (P<0.05),but not with sex,age,tumor differentiation,etc.(P>0.05).The survival rate in the patients with Bmi-1 expression was much lower than that in those patients without Bmi-1 expression (P<0.01).Multivariate analysis indicated that Bmi-1 expression,T classification,lymph node metastasis,distant metastasis,tumor size and postoperative chemotherapy were all significantly prognostic factors of gastric carcinoma.CONCLUSION: Overexpression of Bmi-1 in patients with gastric carcinoma enhances the possibility of invasion and metastasis,implying a poor prognosis.Bmi-1 may serve as fairly a good prognostic factor to indicate biologic behavior and prognosis in gastric carcinoma.     

Association of Pro12Ala polymorphism in the peroxisome proliferator-activated receptor-gamma2 with gastric cancers in China

AIM: To study the relationship among peroxisome proliferator-activated receptor-gamma2 (PPAR γ2) gene Pro12Ala polymorphism, Helicobacter pylori (H. pylori) infection, and gastric cancer in China.METHODS: 104 consecutive patients with gastric cancer and 104 age-and sex-matched controls from Guangdong Province of southern China were examined. PPARγ2 Pro12Ala polymorphism was analyzed by PCR-restriction fragment length polymorphism method (PCR-RFLP). H. pylori status of subjects was determined by enzyme-linked immunosorbent assay (ELISA) for anti-H. pylori IgG. RESULTS: The prevalence of H. pylori infection was significantly higher in gastric cancer patients than that in control (81.7% vs 59.6%, 2=12.27, P<0.01; OR=3.0, 95% CI=1.6-5.7). The CC, CG and GG genotype frequency of PPARγ2 gene in Chinese common people was 91.3%, 8.7%, 0 and the G allele frequency was 4.3%. The frequency of PPARγ2 G (Ala12) allele was significantly higher among patients with gastric cancer (19.2%) than that in control subjects (8.7%; P<0.05, OR=2.5, 95% CI=1.1-5.8). Moreover, the combination of PPARγ2 G allele and H. pylori infection substantially increased the risk of gastric cancer (P<0.01, OR=8.9, 95% CI=2.2-35.7). CONCLUSION: PPARγ2 G allele is associated with gastric cancer in China. The risk of developing gastric cancer is significantly increased in the PPARγ2 G allele carriers with H. pylori infection.     

Tanshinone IIA inhibits doxorubicin resistance in gastric cancer cells

AIM: To investigate the inhibitory effect and the specific mechanism of tanshinone IIA on doxorubicin (DOX)-resistant gastric cancer cells. METHODS: The sensitivity of gastric cancer cells lines to DOX was determined by MTT assay. DOX-resistant gastric cancer cell lines were established by step selection with increasing concentrations of DOX. The cell cycle arrest, apoptosis and autophagy related-markers were analyzed by flow cytometry and Western blot. The expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multi-drug resistance-associated protein 1 (MRP-1) was determined by RT-qPCR and Western blot. RESULTS: DOX-sensitive cell lines SNU-719 and SNU-601 as well as the cell lines relatively resistant to DOX including SNU-638, SNU-668, SNU-216 and SNU-620 were identified according to the IC₅₀ values of DOX for different cell lines. Two DOX-resistant cell lines SNU-719R and SNU-601R were also established. Tanshinone IIA inhibited the expression of MRP-1 in DOX-resistant cell lines. Compared with DOX treatment alone group, combined treatment of DOX and tanshinone IIA in cancer cells decreased the G₂/M phase cell number, increased the protein expression of p21, decreased the protein expressions of cyclin B1 and cyclin-dependent kinase 1 (CDK1) in the SNU-719 R cells and SNU-620 cells. In addition, compared with DOX treatment alone group, combined treatment of DOX and tanshinone IIA in the cancer cells increased the protein expressions of p53, Bax and LC3B-II, decreased the protein expression of Bcl-2 and p62 (P<0.05). CONCLUSION: Tanshinone IIA is an effective drug in the inhibition of DOX resistance in gastric cancer.     

High expression of BIRC5 enhances cell viability,inhibits apoptosis and is associated with poor prognosis in gastric cancer

AIM To investigate the expression of baculoviral inhibitor of apoptosis protein repeat-containing protein 5 （BIRC5） in gastric cancer tissue and its relationship with prognosis of gastric cancer patients, and to explore the effect of BIRC5 knock-down on the viability and apoptosis of gastric cancer cells. METHODS The expression of BIRC5 was detected by immunohistochemistry in 67 cases of gastric cancer tissues and paracancerous tissues for analyzing the relationships with clinicopathological characteristics. The mRNA and protein expression levels of BIRC5 in gastric carcinoma cell lines （AGS, MKN-1 and MGC-803） and normal gastric epithelial cell line GES-1 were detected by RT-qPCR and Western blot. The AGS cells were divided into blank group （no treatment）, Ctr-sh group （blank plasmid transfection） and BIRC5-sh group （BIRC5-shRNA plasmid transfection）. The interference efficiency of BIRC5-shRNA was evaluated by Western blot. The cell viability was measured by MTT assay, the apoptosis was analyzed by flow cytometry, and the levels of apoptosis-related proteins cleaved caspase-3, Bax and Bcl-2 were determined by Western blot. RESULTS BIRC5 was mainly expressed in cytoplasm, and the positive expression rate of BIRC5 in the gastric cancer tissues was higher than that in the adjacent tissues （P<0.01）. The positive rates of BIRC5 in the gastric cancer patients at TNM Ⅲ~Ⅳ stages and with lymph node metastasis were higher than those in the patients at TNM Ⅰ~Ⅱ stages and without lymph node metastasis, respectively （P<0.05）. The survival time of the patients with positive BIRC5 expression was shorter than that of the patients with negative BIRC5 expression （P=0.011 2）. The cell viability in BIRC5-sh group was lower than that in blank group and Ctr-sh group at time points of 48, 72 and 96 h. The apoptotic rate in BIRC5-sh group was increased compared with blank group and Ctr-sh group. The protein levels of cleaved caspase-3 and Bax in BIRC5-sh group were higher than those in blank group and Ctr-sh group, while the protein expression of Bcl-2 in BIRC5-sh group was lower than that in blank group and Ctr-sh group （P<0.05）. CONCLUSION High expression of BIRC5 in gastric cancer indicates poor prognosis. BIRC5 promotes the growth of gastric cancer cells and inhibits apoptosis.     

Clinical significance of miRNA-326 expression in gastric cancer and effect of up-regulation of its expression on viability and apoptosis of gastric cancer cells

AIM: To investigate the clinical significance of microRNA-326 (miRNA-326) expression in gastric carcinoma and the effect of up-regulation of its expression on the viability and apoptosis of gastric cancer cells. METHODS: The expression of miRNA-326 in 55 tissue samples of gastric cancer was detected by RT-qPCR, and the relationship between the expression and the clinicopathological features was analyzed. The expression of miRNA-326 in gastric cancer BGC-823 cells was detected by RT-qPCR. The BGC-823 cells were transfected by liposome method, and randomly divided into normal control group (untransfected), mimic-NC group (transfected with negative control mimic) and miRNA-326 mimic group (transfected with miRNA-326 mimic). After up-regulation of miRNA-326 expression, the cell viability was measured by CCK-8 assay, and the apoptosis of the cells was analyzed by flow cytometry. The protein levels of matrix metalloprotein 9 (MMP-9), p21, cyclin D1, Bcl-2 and cleaved caspase-3 were determined by Western blot, and the mRNA expression of cyclin D1 was detected by RT-qPCR. Whether CCND1 (the gene of cyclin D1) was the target gene of miRNA-326 was evaluated by dual-luciferase reporter assay. RESULTS: The expression of miRNA-326 in the gastric cancer tissues was significantly lower than that in the adjacent tissues (P<0.05). The miRNA-326 expression had a significant correlation with the tumor size, lymph node metastasis, differentiation, and clinical stages (P<0.05), but it had no correlation with the age and sex of the patients. Moreover, the expression of miRNA-326 was also closely related to the survival rate of the patients (P<0.05). The expression of miRNA-326 in the BGC-823 cells was significantly lower than that in the normal gastric mucosa GES-1 cells (P<0.05). Compared with normal control group, the expression of miRNA-326 in mimic-NC group did not change significantly, while that in miRNA-326 mimic group was increased significantly (P<0.05). Compared with normal control group, the cell viability in miRNA-326 mimic group was significantly decreased, and the apoptosis was increased (P<0.05). In addition, compared with normal control group, the protein levels of MMP-9, cyclin D1 and Bcl-2, and the mRNA expression of cyclin D1 in miRNA-326 mimic group were decreased, while the protein levels of p21 and cleaved caspase-3 were increased (P<0.05). However, no significant difference of above protein and mRNA levels between mimic-NC group and normal control group was observed. Compared with mimic-NC+miR-326 mimic group, the activity of luciferase in the cells transfected with pmiR-CCND1-WT plasmid was significantly decreased (P<0.05), but that in the cells transfected with pmiR-CCND1-Mut plasmid did not change significantly. CONCLUSION: The expression level of miRNA-326 in gastric cancer tissues is low, and it may promote cell viability and inhibit cell apoptosis by targeting CCND1.     

Dominant negative human epidermal growth factor receptor induces G0/G1 arrest in human gastric cancer cells

AIM：To explore the effect of dominant negative epidermal growth factor receptor (DNEGFR) on the cell cycle of human gastric cancer cells. METHODS：Two human gastric cancer cell lines were used in the study. The cells were divided into 6 groups, including untreated SGC-7901 cells (US group), SGC-7901 cells stably transfected with pEGFP-N1 (ES group), SGC-7901 cells stably transfected with pEGFPN1-DNEGFR (DS group), untreated NCI-N87 cells (UN group), NCI-N87 cells stably transfected with pEGFP-N1 (EN group), and NCI-N87 cells stably transfected with pEGFPN1-DNEGFR (DN group). The cell cycle was determined by flow cytometry. The protein levels of cyclin-dependent kinase 2 (CDK2), cyclin D1, phosphorylated glycogen synthase kinase 3 beta at Ser9 ［p-GSK-3β (Ser9)］, p21 and p27 were detected by Western blotting. RESULTS：Transfection of the human gastric cancer cells with pEGFPN1-DNEGFR led to G0/G1 arrest, and down-regulated CDK2, cyclin D1, p-GSK-3β (Ser9) and up-regulated p21 and p27 as well. CONCLUSION：DNEGFR down-regulates cyclin D1 by activating GSK-3β, down-regulates CDK2, and up-regulates p21 and p27, which induce G0/G1 arrest in human gastric cancer cells in the end.     

Effects of RARG on viability and migration ability of gastric cancer cells

AIM:To investigate the effect of retinoic acid receptor gamma (RARG) on the viability and migration ability of gastric cancer cells. METHODS:The expression of RARG in gastric cancer and normal gastric tissues and its correlation with the overall survival rate of gastric cancer patients were analyzed by bioinformatics. The expression of RARG was promoted and inhibited by over-expression plasmid transfection and RNA interference technique in gastric can-cer cells in vitro, respectively. MTT and Transwell assays were used to detect the effect of RARG on the viability and migration ability of gastric cancer cells. The effect of RARG on regulating the Wnt/β-catenin signaling pathway was evaluated by Western blot and TOP/FOP dual-luciferase reporter assay. The protein interaction of RARG and β-catenin was determined by co-immunoprecipitation and immunofluorescence co-localization assay. RESULTS:Over-expression of RARG enhanced the viability and migration ability of gastric cancer SGC7901 cells (P<0.05). Knockdown of RARG attenuated the viability and migration ability of gastric cancer MGC-803 cells (P<0.05). At the same time, RARG over-expression increased the protein expression levels of β-catenin, c-Myc, cyclin D1, Twist and Snail (P<0.05), and the activity of TOP/FOP dual-luciferase reporter gene (P<0.05). In addition, RARG interacted with β-catenin protein in the gastric cancer cells. CONCLUSION:RARG promotes the viability and migration ability of gastric cancer cells via activating the Wnt/β-catenin signaling pathway, thus playing an important role in the development of gastric cancer.     

Level and clinical significance of RNA oxidative damage in human gastric cancer and para-carcinoma tissues

AIM: To investigate the RNA oxidative damage in human gastric cancer tissue and para-carcinoma tissue for exploring the role of RNA oxidation in the occurrence of gastric cancer. METHODS: Immunohistochemical observation and LC-MS/MS analysis were performed in 61 cases of gastric carcinoma. The position and concentration of 8-oxoguanosine (8-oxoGsn) were detected, respectively. RESULTS: The results of immunohistochemical observation showed that 8-oxoGsn was obviously up-regulated in the gastric cancer. The positive staining mainly accumulated in the cytoplasm of the tumor cells. The results of mass spectrometry showed that the level of 8-oxoGsn in the gastric cancer tissues was higher than that in the para-carcinoma tissues (P<0.05). CONCLUSION: 8-oxoGsn is up-regulated in gastric cancer. RNA oxidative damage may play important roles in the occurrence of gastric cancer.     

Expression of Foxp3+ Tregs and PD1 in gastric cancer tissues and their correlation with clinicopathological factors and prognosis

AIM: To investigate the expression of Foxp3⁺ regulatory T cells (Foxp3⁺ Tregs) and programmed death receptor 1 (PD1) in gastric cancer tissues and their association with clinicopathological factors and prognosis of the patients. The correlation between the 2 molecules was also analyzed at the same time. METHODS: The tumor sections from 111 gastric cancer patients were stained for Foxp3 and PD1 by the method of immunohistochemistry. The associations of the expression levels of these 2 molecules with clinicopathological factors involved in the disease progression and prognosis were statistically analyzed. The relationship of their expression was detected. RESULTS: Foxp3⁺ Tregs and PD1 were expressed in the gastric cancer tissues, and PD1 was expressed in the tumor infiltrating lymphocytes (TILs). The expression of Foxp3 and PD1 was correlated with lymph node metastasis, clinicopathological stage and prognosis of gastric cancer patients. The expression of these 2 determinants in the patients with lymph node metastasis and an advanced clinicopathological stage was distinctly higher (P <0.05). The patients with positive expression of the 2 indexes presented a lower overall survival rate and worse prognosis (P <0.05). A significantly positive correlation between the infiltration of Foxp3⁺ Tregs and the expression of PD1⁺ TILs was also observed (P <0.01).CONCLUSION: Foxp3⁺ Tregs and PD1⁺ TILs co-infiltrate in the gastric cancer tissues, which can be used as biological markers to predict the disease progression and prognosis.     

Relationship between RUNX3 gene 364 locus C→T mutation and gastric cancer in Chinese

AIM: To study the frequency difference of RUNX3 gene 364 locus C→T mutation between normal people(controls) and gastric cancer (GC) patients, and mutation in gastric mucosa of subjects with H.pylori infection. METHODS: Genomic DNA was extracted from peripheral blood and gastric mucosal biopsy specimens of normal people and GC patients in lower or higher prevalence region. Gene mutation was analyzed by PCR-RFLP. RESULTS: The frequency of RUNX3 T/T genotype was no significant difference between controls and GC in lower (χ2=0.57, P>0.05) or higher prevalence region (χ2=0.16, P>0.05). A higher mutation rate in mucosal tissue infected with H.pylori was not discovered. CONCLUSION: RUNX3 gene C364T mutation may be not a genetic susceptibility to GC in Chinese. The mutation is impossibly involved in the pathway of H.pylori infection resulting in gastric carcinoma.     

Changes of the expression of CD27 and CD28 on cytokine-induced killer cells and natural killer cells during four week cultivation

AIM:To investigate the changes of CD27 and CD28 expression on cytokine-induced killer(CIK) cells and natural killer(NK) cells during cultivation.METHODS:The mononuclear cells were treated with interleukin-12(IL-2),IL-7,IL-15,stem cell factor and FLT3 ligand for four weeks.The CD27 and CD28 expression on CD3+CD56+ CIK cells and CD3-CD56+ NK cells were examined by flow cytometric analysis every week during four-week cultivation.RESULTS:The expression of CD27 on CIK and NK cells two weeks after cultivation reached the peak value at（44.57±4.07）% and（51.02±5.20）%, respectively.Then, their expression declined gradually to about 30% in the fourth week.The expression of CD28 on CIK and NK cells reached the peak at the third and the second week, the values were （4.40±0.66）% and （45.14±3.58）%, respectively, decreased rapidly and then were almost completely lost at the fourth week.CONCLUSION:According to the changes of expressions of CD27 and CD28 on CIK/NK cells, optimal harvest time of cultured CIK/NK cells should be at the third week of cultivation.     

Effects of safflor injection on expression of cyclooxygenase-2 mRNA during lung ischemia/reperfusion injury in rabbits

AIM: To investigate the effects of safflor injection (SI) on expression of cyclooxygenase-2 mRNA during lung ischemia/reperfusion injury (PIRI) in rabbits. METHODS: Rabbit lung model of ischemia/reperfusion injury was constructed in vivo. The rabbits were randomly divided into three groups: sham-operation group (group S), ischemia-reperfusion group (group I/R) and ischemia/reperfusion plus safflor injection group (group SI). The lung tissue sampled at the end of the experiment was assayed for wet/dry weight ratio (W/D), injured alveoli rate (IAR) and observed ultrastructure changes under electron microscope. The expressions of COX-1 and COX-2 were measured by immunohistochemistry (IHC). The expression of COX-1 mRNA and COX-2 mRNA were observed by in situ hybridization (ISH). RESULTS: The value of W/D and IAR was much higher in I/R group, but decreased in SI group. Electron microscope showed obvious ultrastructure injury brought by PIRI in I/R group, which was greatly attenuated in SI group. The IHC and ISH demonstrated that COX-2 and COX-2 expressions in pulmonary tissue of I/R group were significantly higher than those in S group (P<0.01). The difference of COX-1 and COX-1 expressions in pulmonary tissue was not observed among three groups. CONCLUSION: The lung ischemia-reperfusion insult induces the up-regulation of COX-2 in lung. Safflor injection may attenuate lung ischemia-reperfusion injury through inhibiting COX-2 expression.     

Expression of EPCAM,CD44 and CD24 in gastric cancer tissues and its clinical significance

AIM: To evaluate the relationship between epithelial cell adhesion molecule (EPCAM)/cluster of differentiation 44 (CD44)/cluster of differentiation 24 (CD24) expression and the clinicopathological characteristics/prognosis in 95 gastric cancer patients. METHODS: The expression levels of EPCAM, CD44 and CD24 were detected using the two-step method of immunohistochemistry in 95 gastric cancer patients who underwent surgical excision and were pathologically diagnosed as gastric cancer. RESULTS: There were 56 EPCAM-positive patients (58.95%), 41 CD44-positive patients (43.16%) and 56 CD24-positive patients (58.95%). Thirty patients were both EPCAM and CD44 positive (31.58%), 45 patients were both EPCAM and CD24 positive (47.37%), 32 patients were both CD44 and CD24 positive (33.68%), and 25 patients were EPCAM, CD44 and CD24 positive (26.32%). EPCAM expression was correlated with age, depth of tumor infiltration and WHO histological classification. CD44 expression was correlated with BORRMANN and WHO histological classification as well as CEA value. CD24 expression was correlated with the depth of infiltration, location of the tumor, WHO histological classification and viscera invasion. All positive expression of EPCAM, CD44 and CD24 was correlated with the depth of infiltration, location of the tumor and WHO histological classification (P<0.05). The difference of survival rate between EPCAM positive group and negative group was observed, and the CD44 positive group and negative group had the same result (P<0.05 and P<0.01, respectively). The difference of survival rate between EPCAM⁺CD44⁺CD24⁺ group and EPCAM^-CD44^-CD24^- group was statistically significant (P<0.05). The difference of survival rate between EPCAM^-CD44⁺CD24⁺ group and EPCAM^-CD44^-CD24^- group was also significant (P<0.05). CONCLUSION: The positive rates of EPCAM, CD44 and CD24 expression are high in gastric cancer tissues and these 3 proteins can be used as primary screening targets.