as the most common malignant tumor in the gastrointestinal tract , gastric carcinoma is the second leading cancer in china , with an incidence of 400,000/per year ( approximately 42% of worldwide incidence ) . although diagnostic and treatment technology has progressed , the prognosis of gastric cancer is still unfavorable , mainly due to the occurrence of lymph node or distal metastasis by the time of first diagnosis , making radical resection impractical . moreover , the predictive power of prognosis using currently available methods is not high enough . early diagnosis and accurate prediction of prognosis is , therefore , of critical importance for improving treatment efficacy . microrna ( mir ) is a type of single - stranded non - coding rna molecule 22 nucleotides in length . it can inhibit or down - regulate the target gene translation by specific binding to the 3-untranslated region ( 3-utr ) . it is now commonly believed that mir plays a vital role in both tumor pathogenesis and tumor suppression . various mir molecules have been found to be related with gastric carcinoma , thus improving the diagnostic and treatment efficacy . studies have revealed the existence of a gene polymorphism locus at + 60 of mir-421 . this locus is within the seeding sequence and can regulate the expression of mir-421 , suggesting a close correlation between up - regulation of mir-421 and gastric cancer occurrence . the relationship between mir-421 polymorphism and gastric cancer , the present study investigated the distribution of mir-421 genotypes and its role in occurrence of gastric cancer , in an attempt to strengthen target gene prediction . a total of 96 gastric carcinoma samples were collected from surgical resection of patients from january 2010 to january 2013 in the affiliated hospital , academy of military medical sciences . no radio-/chemo- or molecular target therapy had been performed before the surgery . among the 96 patients , there were 74 males and 22 females , ages 42 to 78 years old ( average age=61.34 years ) . according to lauren type , there were 68 cases of intestine type carcinoma and 28 cases of diffused cancer . the number of cases with high , moderate , and low differentiation was 12 , 46 , and 38 , respectively . using the uicc staging system , the locations of tumors were the gastric antrum in 38 cases , the body in 16 cases , the cardia in 21 cases , and the whole stomach in 21 cases . this study was pre - approved by the ethics committee of the affiliated hospital , academy of military medical sciences , and we obtained written consent from all participants in accordance with the declaration of helsinki . pathological typing of tumor tissues was performed to evaluate the infiltration degree based on the uicc standard . post - operative follow - up was performed on all patients once per month for a maximum duration of 5.5 years ( average=3.6 years ) . total rna was extracted by use of an rna extraction kit ( sigma , usa ) from all gastric cancer tissues . the relative expression level of mir-421 was determined by taqman mirna assay kit ( santa cruz , usa ) using rnu6 as the internal reference in 2-step reactions . in brief , cdna was synthesized using stem - loop primers in an in vitro reverse transcription system using 10 ng rna as the template ( 2 ng/l ) . the gradient denaturing reaction was performed as follows : 94c for 30 s , 60c for 30 s , 72c for 60 s , ending with 72c for 7 min . in the second step , mir-421-specific primers ( forward , 5-cacag uaggc cucaa auguu uguug auga-3 ; reverse : 5-guguc guccg gguuu acaga caacu acu-3 ) were added into a 2pcr taqman universal mixture . pcr parameters were : 94c for 30 s , 60c for 30 s , and 72c for 60 s , repeated for 40 cycles , ending with 72c for 7 min . the reaction was quantified using fluorescent real - time quantitative pcr equipment ( rotor - gene , australia ) . the relative level of mir-421 was determined by 2 method ( ct = ctmir421ctrnu6 ) . pearson chi square analysis was used to determine if genotype distribution fit hardy - weinberg equilibrium . data that fit the normal distribution are presented as mean  standard deviation ( sd ) . analysis of variance ( anova ) was used to compare the differences in genotype frequencies of mir-421 , with snk correction coefficient for between - group comparison . the paired t test was used to compare the differential expression of mir-421 between tumor and normal tissues . logistic regression analysis detected if gg / gc genotypes were risk factors for gastric cancer . a kaplan - meier survival curve was plotted to analyze the postoperative survival of patients . a total of 96 gastric carcinoma samples were collected from surgical resection of patients from january 2010 to january 2013 in the affiliated hospital , academy of military medical sciences . no radio-/chemo- or molecular target therapy had been performed before the surgery . among the 96 patients , there were 74 males and 22 females , ages 42 to 78 years old ( average age=61.34 years ) . according to lauren type , there were 68 cases of intestine type carcinoma and 28 cases of diffused cancer . the number of cases with high , moderate , and low differentiation was 12 , 46 , and 38 , respectively . using the uicc staging system , the locations of tumors were the gastric antrum in 38 cases , the body in 16 cases , the cardia in 21 cases , and the whole stomach in 21 cases . this study was pre - approved by the ethics committee of the affiliated hospital , academy of military medical sciences , and we obtained written consent from all participants in accordance with the declaration of helsinki . pathological typing of tumor tissues was performed to evaluate the infiltration degree based on the uicc standard . post - operative follow - up was performed on all patients once per month for a maximum duration of 5.5 years ( average=3.6 years ) . total rna was extracted by use of an rna extraction kit ( sigma , usa ) from all gastric cancer tissues . the relative expression level of mir-421 was determined by taqman mirna assay kit ( santa cruz , usa ) using rnu6 as the internal reference in 2-step reactions . in brief , cdna was synthesized using stem - loop primers in an in vitro reverse transcription system using 10 ng rna as the template ( 2 ng/l ) . the gradient denaturing reaction was performed as follows : 94c for 30 s , 60c for 30 s , 72c for 60 s , ending with 72c for 7 min . in the second step , mir-421-specific primers ( forward , 5-cacag uaggc cucaa auguu uguug auga-3 ; reverse : 5-guguc guccg gguuu acaga caacu acu-3 ) were added into a 2pcr taqman universal mixture . pcr parameters were : 94c for 30 s , 60c for 30 s , and 72c for 60 s , repeated for 40 cycles , ending with 72c for 7 min . the reaction was quantified using fluorescent real - time quantitative pcr equipment ( rotor - gene , australia ) . the relative level of mir-421 was determined by 2 method ( ct = ctmir421ctrnu6 ) . pearson chi square analysis was used to determine if genotype distribution fit hardy - weinberg equilibrium . data that fit the normal distribution are presented as mean  standard deviation ( sd ) . analysis of variance ( anova ) was used to compare the differences in genotype frequencies of mir-421 , with snk correction coefficient for between - group comparison . the paired t test was used to compare the differential expression of mir-421 between tumor and normal tissues . logistic regression analysis detected if gg / gc genotypes were risk factors for gastric cancer . a kaplan - meier survival curve was plotted to analyze the postoperative survival of patients . demographic information , such as age and sex distribution , was not significantly different between gastric cancer and control individuals ( p>0.05 , table 1 ) . the liver and kidney functions , however , were significantly compromised in gastric cancer patients ( p<0.05 , table 1 ) . a preliminary survey of genotype ( gg and gc ) distribution showed the existence of hardy - weinberg equilibrium . in gastric cancer patients with lymph node metastasis , the frequency of gg genotype of mir-421 was higher than that in patients without lymph node metastasis , suggesting that the gg genotype might be a risk factor for lymph node metastasis . moreover , age and liver / kidney dysfunctions were shown to be major risk factors for the metastasis of gastric cancer ( table 2 ) . the frequency of gg and gc genotypes in gastric cancer patients were significantly higher than those in the control group ( 46.88% vs. 7.69% ; 29.17% vs. 12.82% ) , suggesting that both genotypes are risk factors for gastric cancer . logistic regression analysis showed that or values of gc and gg genotypes were 2.998 ( 95% ci : 1.176~7.639 ) and 2.936 ( 95% ci : 1.198~7.246 ) , respectively . the occurrence of gastric cancer was increased by 16% in gc carriers and by 28% in gg carriers . when we checked tumor metastasis , or values of gc and gg genotypes were 2.936 ( 95% ci : 0.576~1.896 ) and 1.846 ( 95% ci : 1.148~2.965 ) , respectively ( table 3 ) . the average relative expression level of mir-421 in all 96 gastric carcinoma tissues was 1.436 ( 95% ci : 0.323~0.534 ) . there were 42 cases with higher than average expression values and 54 cases had mir-421 below the average value . when comparing the prognosis of those 2 groups of patients , we found that those with high expression levels had a median survival time of 37.34 months , while mir-421 low - expression patients had a median survival time of 54.23 months . therefore , a relationship existed between disease prognosis and gg genotype frequency ( r=5.16 , p<0.05 ) but not gc genotype ( r=2.86 , p>0.05 , figure 1 ) . demographic information , such as age and sex distribution , was not significantly different between gastric cancer and control individuals ( p>0.05 , table 1 ) . the liver and kidney functions , however , were significantly compromised in gastric cancer patients ( p<0.05 , table 1 ) . a preliminary survey of genotype ( gg and gc ) distribution showed the existence of hardy - weinberg equilibrium . in gastric cancer patients with lymph node metastasis , the frequency of gg genotype of mir-421 was higher than that in patients without lymph node metastasis , suggesting that the gg genotype might be a risk factor for lymph node metastasis . moreover , age and liver / kidney dysfunctions were shown to be major risk factors for the metastasis of gastric cancer ( table 2 ) . the frequency of gg and gc genotypes in gastric cancer patients were significantly higher than those in the control group ( 46.88% vs. 7.69% ; 29.17% vs. 12.82% ) , suggesting that both genotypes are risk factors for gastric cancer . logistic regression analysis showed that or values of gc and gg genotypes were 2.998 ( 95% ci : 1.176~7.639 ) and 2.936 ( 95% ci : 1.198~7.246 ) , respectively . the occurrence of gastric cancer was increased by 16% in gc carriers and by 28% in gg carriers . when we checked tumor metastasis , or values of gc and gg genotypes were 2.936 ( 95% ci : 0.576~1.896 ) and 1.846 ( 95% ci : 1.148~2.965 ) , respectively ( table 3 ) . the average relative expression level of mir-421 in all 96 gastric carcinoma tissues was 1.436 ( 95% ci : 0.323~0.534 ) . there were 42 cases with higher than average expression values and 54 cases had mir-421 below the average value . when comparing the prognosis of those 2 groups of patients , we found that those with high expression levels had a median survival time of 37.34 months , while mir-421 low - expression patients had a median survival time of 54.23 months . therefore , a relationship existed between disease prognosis and gg genotype frequency ( r=5.16 , p<0.05 ) but not gc genotype ( r=2.86 , p>0.05 , figure 1 ) . due to the insidious onset of gastric cancer at early stage , most patients are already at the late or terminal stage by the time of first diagnosis , causing unfavorable prognosis and lower survival rate for patients with gastric cancer . mirna can exert pluripotent biological functions via modulating multiple target gene expressions , and is thus a popular research focus in oncology . with the advancement of bioinformatics , mirna has been found to be related to tumor growth , development , proliferation , invasion , and apoptosis . the expressional spectrum of mirna also determines the genotype of tumor cells and may play a vital role in subtyping of tumors and prediction of metastasis , especially for the post - operative survival time and prognosis , as suggested by previous studies showing the relationship between mirna expressional level and tumor prognosis . in various gastrointestinal tumors ( e.g. , gastric and colon cancer ) , metastatic lymph node ratio is reported to be an important prognostic factor . in recent years , high - throughput analysis has identified mir-421 as a gastric cancer - related molecule . as an important family of human irna-200 , the mir-421 coding gene is located on human chromosome 1p34.23 , where multiple tumor - related genes have been identified [ 1518 ] . a close correlation exists between up - regulation of mir-421 and gastric cancer occurrence , because single - nucleotide polymorphism ( snp ) at certain locations causes higher risk of disease . certain snp loci have been suggested to exist at the + 60 locus of the mir-421 coding sequence . this locus localizes within the seeding sequence of mir-421 , and its expression can be regulated [ 2023 ] . the relationship between mir-421 polymorphism and gastric cancer , however , has not been reported . this study thus investigated the correlation between gastric occurrence / prognosis and g / c polymorphism within the mir-421 gene , in an attempt to further illustrate the role of mir-421 in gastric cancer . we performed an analysis using sequencing technique to determine the alleles involved in snp of mir-421 from tumor samples obtained from surgical resection . multi - variant logistic regression analysis showed that both gc and gg genotypes may be risk factors for gastric cancer pathogenesis . gg genotype carriers had higher incidence of lymph node metastasis , suggesting it may be a risk factor for metastasis . age and liver / kidney dysfunctions are also identified as major risk factors for tumor metastasis . gg carriers had significantly higher incidence of gastric carcinoma occurrence and lymph node metastasis . in a further survival analysis , we found that gg carriers had shorter survival time compared to gc genotype carriers . this study demonstrated the possible involvement of mir-421 gg - genotype in the invasion and metastasis of gastric carcinoma , indicating it might be a potential drug target . we also revealed a correlation between mir-421 polymorphism and gastric cancer , suggesting the potential facilitation of gastric cancer pathogenesis by gg genotype via up - regulating mir-421 expression . however , this study has certain limitations due to the relatively small samples size and lack of long - term follow - up . we believe that more mir - related modulatory mechanisms could be identified , thus providing novel methods for diagnosis , treatment , and prognostic prediction of gastric cancer .