| OBJECTIVES:By combining the small sample for high throughput gene microarray screening with the large sample of clinical tissues for verification,we aimed to find some gastric cancer related genes.We evaluated the clinical significance and prognostic value of candidate gene DDX18,which is over-expression in gastric cancer tissues.In order to provide theoretical basis for the development of new therapeutic targets for the treatment of gastric cancer,we investigated its effect on the malignant biological behavior of gastric cancer in vitro and in vivo,also discussed its mechanism.METHODS:(1)The clinicopathological characteristics and follow-up data of 585gastric cancer patients between 2005 and 2011 admitted in our hospital were collected and retrospectively analyzed.The differential profiling of m RNA expression in 5 pairs of gastric cancer and adjacent tissues was studied by Arraystar Human Lnc RNA Microarray.Combining TCGA and q RT-PCR data,we finally found that DDX18 was over-expressed in gastric cancer tissues.(2)A tissue microarray was established,based on a total of 585 human gastric cancer FFPE specimens which had been obtained from patients admitted in our hospital between 2005 and 2011.The protein expression of DDX18 was detected by immunohistochemical staining.Then the relationship between DDX18 expression and clinical pathological data and prognosis was analyzed.(3)CCK-8 assay,colony formation assay were used to investigate the effect of DDX18 on cell growth and proliferation in vitro.Transwell was also performed to examine migration and invasion of GC cells.The cell apoptosis experiment was analyzed by using FITC-Annexin V/PI double staining assay.To identify the role of DDX18 in tumorigenic ability of gastric cancer cells in vivo,we also established the gastric cancer xenografts by injecting AGS cell subcutaneously into nude mice.Co-IP,small RNAseq and western blot assays were performed to detect the mechanism of DDX18 in gastric cancer.RESULT:(1)By combining the result of gene chip and the microarray data obtained from TCGA,and further verifying through q RT-PCR in a large sample of clinical tissues,we found that 5 particular genes were up-regulated in gastric cancer tissues(P<0.05).DDX18 was overexpression in gastric cancer tissues.(2)Immunohistochemistry analysis in 585 gastric cancer cases showed that DDX18 was overexpression in 65.0%(380/585)tumor tissues,which was significantly higher than those in adjacent tissues(P<0.001).Moreover,the expression of DDX18 was closely related with tumor location(P<0.001),tumor volumn(P=0.004),Borrmann classification(P<0.001),degree of tumor differentiation(P<0.001),cancer embolus(P<0.001),lymph node metastasis(P<0.001),and TNM stage(P<0.001).The Cox's proportional-hazard model for multivariate analysis revealed that DDX18 expression,together with Lauren classification,cancer embolus,T stage,N stage,and M stage,were independently unfavorable prognostic factors for OS.(3)CCK-8 assay in vitro indicated that the proliferation of AGS after DDX18 interference was obviously inhibited.Until Day 5,the proliferation rate had already been reduced by 63.0%as compared with AGS-sh-NC group(P<0.001).In contrast,exogenous DDX18 significantly promoted the growth of SGC-7901 cells.Until Day 5,the proliferation rate had already increased by 40.7%as compared with SGC-7901-NC group(P<0.001).Colony formation assay showed that the colony formation rate of AGS cells was obviously inhibited in AGS-sh-DDX18 group(3.87±0.67%)as compared with AGS-sh-NC group(9.35±1.80%)(P<0.01).Transwell assay verified that knocking down DDX18 could reduce the migration and invasion ability of AGS cells.In the Annexin V/PI double staining assay,the apoptotic rate of SGC-7901-NC cells was42.90±5.45%after 48 hours starvation,while in the SGC-7901-OE group this rate was decreased to 11.89±2.08%(P<0.01).Meanwhile,the apoptotic rate of AGS cells in DDX18 interference group was 34.88±3.47%,which is significantly higher than17.84±1.50%in the AGS-sh-DDX18 group(P<0.01).Nude mice subcutaneous tumor experiments showed that tumor mass increased rapidly 12 days after injection.Untill Day21,the mean volume of tumors was up to 1752.99±532.69mm~3and the weight was up to1.57±0.56g.On the other hand,tumor volume curve of the interference group was relatively flat.Until Day 21,the mean volume of tumors was only 638.58±217.60mm~3and the weight was only 0.56±0.31g(P<0.01).(4)Small RNAseq analysis showed that the expression of mi RNA-21 was significantly decreased after interference with DDX18.The following q RT-PCR confirmed that DDX18 could promote the maturation of mi RNA-21.(5)CO-IP was performed to identify the direct interaction of DDX18 and Drosha,which acted as the key enzyme of maturation of mi RNAs.(6)Luciferase reporter gene indicated that mi RNA-21 could target PTEN-3'UTR and led to its degradation.(7)The results of western blot confirmed that DDX18 could promote the maturation of mi RNA-21 by interacting with Drosha,and then led the targeted gen--PTEN--to decrease,which could up-regulate the AKT signaling pathway.CONCLUSIONS:(1)DDX18 was upregulated in gastric cancer tumor tissues.(2)DDX18 protein was overexpressed in gastric tumor tissues,which was significantly higher than those in adjacent tissues.The expression of DDX18 was also closely related with tumor volumn,Borramn classification,degree of tumor differentiation,cancer embolus,lymph node metastasis,and TNM stage.DDX18 can be treated as a molecular marker to assess the prognosis of gastric cancer patients.(3)DDX18 could promote the maturation of mi RNA-21 by interacting with Drosha,leading to the decrease of PTEN expression,and up-regulating the AKT signaling pathway,finally affecting the occurrence and development of gastric cancer. |
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