| BackgroundGastric cancer is one of the most frequently reported cancers in the world, and ranks first in malignant tumors in China. Despite promising developments in multimodal treatment strategies, gastric cancer maintains a profile of low survival rates and lacks successful therapies, which may be as a result of high rates of tumor invasion and metastasis. Helicobacter pylori(H. pylori) is a gram negative bacillus. Infection with H. pylori has been well established to be a crucial event associated with the risk of gastric cancer in epidemiology, pathology and in vitro experiments. Recent studies have suggested a controversial role of H. pylori infection in gastric cancer prognosis. Micro RNAs(mi RNAs) are highly conserved, small, non-coding RNA, 18-25 nucleotides in length, involving in regulating cell growth, metabolism, proliferation, apoptosis, differentiation and other life activities. There is growing evidence that mi RNA plays a critical role in the development and progression of tumor. The mi RNA expression profiling can be used for cancer classification and subtype stratification, which provides a new idea and method for cancer diagnosis and treatment. The present study tries to investigate the impact and molecular mechanism of H. pylori status on the prognosis of patients with gastric cancer.ObjectivesThe aim of the present study is to investigate the potential impact of H. pylori status on the prognosis of patients with gastric cancer in a Chinese prospective cohort. In addition, we identified the differential mi RNA expression profiles in gastric cancer tissues and matched nontumoral tissues with and without H. pylori infection. We further investigated the effect and mechanism of differentially expressed mi R-143-3p on gastric cancer proliferation, apoptosis, migration and invasion. Thus, this project will provide solid evidence and basis for individualized therapy of gasric cancer patients and novel molecular target for developing mi RNA drug or mi RNA-based in the future. ObjectivesMethods(1) The cohort consisted of 261 consecutive patients who received curative resection for gastric cancer between 2007 and 2009. H. pylori status was defined by means of immunohistochemical(IHC) staining in gastric tumoral and matched nontumoral tissues. The association of H. pylori infection with clinicopathologic characteristics was examined using the chi-square test. Survival curves were plotted by Kaplan-Meier method. The prognostic influence of H. pylori infection was assessed by univariate and multivariate Cox proportional hazards regression models. The prognosis was measured in terms of overall survival(OS) and disease-free survival(DFS).(2) We chose 42 gastric cancer tissues and matched nontumoral tissues from the cohort. In evaluating the results of H. pylori IHC staining, 21 patients were found to be H. pylori-positive in both tumour and non-neoplastic tissues. Matched by age and gender, the other 21 patients were found to be H. pylori-negative in both tumour and nontumoral tissues. Total RNA was extracted and isolated using Recover AllTM Total Nucleic Acid Isolation Kit. We used μParaflo microfluidic microarrays to measure mi RNA expression profiles in H. pylori-positive gastric cancer tissues, H. pylori-negative gastric cancer tissues, H. pylori-positive nontumoral tissues and H. pylori-negative nontumoral tissues. The data was analyzed by t-test to identify differentially expressed mi RNA. QRT-PCR was applied to verify several mi RNAs that showed significant differences based on the microarray results.(3) We next chose the significantly upregulated mi R-143-3p for further study. The expression of mi R-143-3p in gastric cancer and adjacent nontumoral tissues and gastric cell lines was examined by q RT-PCR analysis. Cells were transfected with mi R-143-3p mimics or inhibitor using LipofectaminTM 3000 following manufacturer’s protocol. CCK-8 assay, flow cytometry, transwell migration assay and matrigel invasion assay were performed post-transfection. By using the algorithms mi Randa, Target Scan and Pic Tar, we considered Rictor and AKT2 as putative targets of mi R-143-3p. q RT-PCR and western blot analysis were generated to validate the relationship between mi R-143-3p and the putative target genes. The expression of Rictor and AKT2 was assessed by using IHC method. We further analyzed the association of mi R-143-3p with Rictor and AKT2 gene expression. The effects of Rictor or AKT2 on gastric cancer cell proliferation, apoptosis, migration and invasion were also examined.Results(1) Positivity for H. pylori infection was observed in 188 of the 261 patients(72.0%). Negativity for H. pylori infection was observed in 73 patients(28.0%). Positivity for H. pylori was significantly associated with age(78.2% in younger age versus 66.4% in older age, P=0.034). Patients who were negative for H. pylori had a significantly higher frequency of disease relapse(27.4% in negativity for H. pylori versus 16.5% in positivity for H. pylori, P=0.042) or death(26.0% in negativity for H. pylori versus 15.4% in positivity for H. pylori, P=0.043).(2) In patients positive for H. pylori, mean OS was 55.2 months(95%CI: 53.4 to 56.9 months) and mean DFS was 53.9 months(95%CI: 51.8 to 56.0); the same survivals were 45.1 months(95%CI: 42.2 to 47.9 months) and 43.7 months(95%CI: 40.4 to 47.0 months) respectively in patients negative for H. pylori. In multivariate analysis, H. pylori was an independent prognostic factor for OS(HR: 0.485; 95%CI: 0.265 to 0.889; P= 0.019).(3) Forty-three mi RNAs were significantly differentially expressed between H. pylori-positive and H. pylori-negative gastric cancer tissues, Compared with H. pylori negative gastric cancer tissues, a total of 5 mi RNAs were upregulated and 38 downregulated in H. pylori-positive gastric cancer tissues. Verified using q RT-PCR, mi R-100-5p, mi R-143-3p and mi R-145-5p expression levels were higher in H. pylori-positive gastric cancer tissues compared with H. pylori-negative gastric cancer tissues(1.24, 2.1 and 1.64 fold changes respectively). In contrast, mi R-4514 expression level was significantly higher in H. pylori-negative gastric cancer tissues compared with H. pylori-positive gastric cancer tissues(2.13 fold change).(4) Seven mi RNAs were significantly differentially expressed between H. pylori-positive and H. pylori-negative nontumoral tissues by using microarrays. Compared with H. pylori negative nontumoral tissues, a total of 2 mi RNAs were upregulated and 5 downregulated in H. pylori-positive nontumoral tissues. Verified using q RT-PCR, mi R-148a-3p, mi R-30b-5p, mi R-26b-5p expression levels were higher in H. pylori-positive nontumoral tissues compared with H. pylori-negative nontumoral tissues(1.71, 1.30 and 1.25 fold changes respectively).(5) We found that mi R-143-3p was downregulated in 73.8%(31/42) gastric cancer tissues compared with their corresponding nontumoral tissues, and the downregulated mi R-143-3p was significantly associated with tumor-node-metastasis(TNM) stage and lymph node metastasis. The expression level of mi R-143-3p was significantly reduced in all gastric cancer cell lines(SGC-7901, HGC-27, MGC-803, AGS, MKN-45 and BGC-823) compared to the normal gastric epithelial cell GES-1(P<0.001). Ectopic expression of mi R-143-3p in BGC-823 cells markedly attenuated cell proliferation, migration and invasion, and promoted apoptosis. Decreased expression of mi R-143-3p in SGC-7901 cells promoted cell proliferation, motility and invasiveness, and inhibited apoptosis.(6) Rictor and AKT2 were predicted by 3 algorithms(Miranda, Targetscan and Pic Tar) as candidate target genes. Quantitative detection of Rictor and AKT2 was performed in 42 pairs of tumour and nontumoral tissues by IHC. In evaluating the results of IHC staining, 50.2% showed Rictor positive in tumor tissue and 26.8% in adjacent nontumoral tissues. Furthermore, 71.4% showed AKT2 positive in tumor tissue and 23.8% in adjacent nontumoral tissues. Expressio of Rictor was inversely correlated with the expression of mi R-143-3p(r=-0.340,P=0.028). Expressio of AKT2 was also inversely correlated with the expression of mi R-143-3p(r=-0.422,P=0.005).(7) Overexpression of mi R-143-3p by transfecting mi R-143-3p mimics into BGC-823 cells significantly reduced the expression of Rictor and AKT2 m RNA and protein level and decreased expression of mi R-143-3p by transfecing mi R-143-3p inhibitor into SGC-7901 cells significantly upregulated the expression of Rictor and AKT2 m RNA and protein level.(8) Knockdown of Rictor and AKT2 expression both markedly attenuated cell proliferation, migration and invasion and promoted apoptosis in BGC-823 and SGC-7901 cells.Conclusions(1) The rate of H. pylori positivity was higher in nontumoral tissues than in gastric cancer tissues. Positivity for H. pylori was significantly associated with age. We identified a higher frequency of H. pylori infection in younger patients. H. pylori-positive patients showed better OS than H. pylori-negative patients. H. pylori infection as an independent beneficial prognostic factor for patients with curatively resected gastric cancer. In clinical practice, patients with curatively resected gastric cancer who are negative for H. pylori may need more careful follow-up and more aggressive anti-tumor treatment to elongate the life expectancy.(2) Forty-three mi RNAs were significantly differentially expressed between H. pylori-positive and H. pylori-negative gastric cancer tissues. The expression levels of mi R-100-5p, mi R-143-3p, and mi R-145-5p were higher in H. pylori-positive gastric cancer tissues than in H. pylori-negative gastric cancer tissues. In contrast, the expression level of mi R-4514 was lower in H. pylori-positive gastric cancer tissues than in H. pylori-negative gastric cancer tissues.(3) Seven mi RNAs were significantly differentially expressed between H. pylori-positive and H. pylori-negative nontumoral tissues. The expression levels of mi R-148a-3p, mi R-30b-5p and mi R-26b-5p were higher in H. pylori-positive nontumoral tissues than in H. pylori-negative nontumoral tissues.(4) mi R-143-3p was downregulated in gastric cancer tissues and gastric cancer cell linesand the downregulated mi R-143-3p was significantly associated withtumor-node-metastasis(TNM) stage and lymph node-metastasis. Functional assaysshowed that overexpression of mi R-143-3p suppressed gastric cancer cell proliferationmigration and invasion and induced apoptosis.(5) Rictor and AKT2 were predicted as condidate target genes of mi R-143-3p by bioinformatic analysis. Both of the two genes were significantly upregulated in gastric cancer tissues. We observed inverse correlation between mi R-143-3p and Rictor expression. The expression of mi R-143-3p was also inversely corrrelated with AKT2 expression. Mi R-143-3p could negatively regulate the expression of Rictor and AKT2 m RNA and protein levels. Knockdown of Rictor and AKT2 genes significantly inhibited gastric cancer cell proliferation, migration and invasion and induced apoptosis, resembling that of mi R-143-3p overexpression.(6) mi R-143-3p functions as a tumor suppressor in gastric cancer via regulating the expression of Rictor and AKT2,which needs to be confirmed by luciferase reporter assay. |
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