Objective To investigate influence of CDCA5 small interfering RNA(CDCA5-siRNA) on proliferation and apoptosis of human hepatocarcinoma cell line HepG2. Methods The HepG2 cells were divided into siRNA groups （A， B and C groups）， universal scrambled negative control siRNA group （NC group）and Blank group. Three CDCA5siRNAs with different sequences were transfected into HepG2 cells respectively. Western blotting method was used to measure expressions of CDCA5 protein levels at 72h after transfection. The cell proliferation was assessed by CCK-8 at 24， 48， 72， 96 and 120h after transfection. The Annexin V-FITC／PI double-labeled flow cytometry was employed to measure the apoptosis at 48h after transfection.
Results The expressions of CDCA5 protein significantly decreased in the CDCA5-siRNA groups （A，B and C groups） compared with NC group and Blank group（P＜0.05）. Group B had the lowest expression rate and highest inhibition rate（89.3％）. The relative proliferation rate of group B was significantly lower than those of NC group and Blank group since 48 hours after transfection（P＜0.05）， and the lowest proliferation rate was （66.58±2.58）％ at 72h in group B. The early and late apoptosis rates were （17.43±2.31） ％ and （22.37±2.21） ％ in B group， higher than those of other two groups（P＜0.05）. Conclusion siRNA down-regulating the expression of CDCA5 gene in human hepatocarcinoma cell line HepG2 can inhibit cell proliferation and increase cell apoptosis.

Objective To detect the expression of metastasisassociatedgene 1（MTA1） protein in tissues of small cell lung cancer（SCLC） and explore the role of MTA1 in metastasis， prognosis and chemotherapy sensitivity of SCLC. Methods The expression of MTA1 was detected in 52 non-distal metastatic SCLC tissues by immunohistochemical staining. The results were divided into low-level expression（≤6） and high-level expression（＞6） according to the immunohistochemical staining counting method. The relationships between expression of MTA1 and clinicopathological characteristics， efficacy of EP regimen（etoposide 100mg／m2 iv， d1d5；cisplatin 60mg／m2 iv d1； 3-week cycle for 4-6 cycles） and prognosis were investigated. Results Thirty-seven SCLC tissues were found of high-level MTA1 expression（71.2％）， while MTA1 was expressed at a low-level in all 18 adjacent tissues. There were significant correlations between the expression of MTA1 and tumor size， TNM stage， lymph node metastasis and vascular invasion（P＜0.05）. No significant correlation existed between the expression of MTA1 and gender， age， VALG stage and tumor sites（P＞0.05）. The low-level expression group was more sensitive to EP chemotherapy than the high-level expression group（80.0％ vs. 54.1％， P=0.020）. The median progression-free survival and overall survival of the low.level expression group were superior to those of the high-level expression group（20 months vs. 8 months, 33 months vs. 18 months） with statistical significance（P＜0.05）. Conclusion MTA1 is highly expressed in SCLC tissue， and is closely correlated with tumor metastasis， drug resistance and prognosis.

Objective To explore the influences of withaferin A （WFA） on proliferation， apoptosis and PI3K／Akt signaling pathway of non-small cell lung cancer （NSCLC） A549 cell. Methods The A549 cells were treated with different concentrations of WFA （0， 2.5， 5.0， 10.0， 20.0 μmol／L）. The MTT assay was used to measure the proliferation inhibition rates at 24， 48， 72 and 96h treated with different concentrations of WFA. The Hoechst staining and Annexin V-FITC／PI double staining were employed to detect cell apoptosis after treatment with different concentrations of WFA. The cycle distribution at 48h after treatment with WFA was detected by flow cytometry. The Western blotting was used to measure the protein levels of apoptosisrelated genes （Bcl-2， Bax and Cleaved caspase-3）， Akt and its phosphorylated form （P-Akt）. Results WFA could increase the proliferation inhibition rates in a dose- and time-dependent manner（P＜0.05）. The apoptotic indices of A549 cells after treatment with different concentrations of WFA （0， 2.5， 5.0， 10.0， 20.0 μmol／L） were 2.75±0.64， 4.6±1.36， 9.75±2.78， 12.92±3.42 and 18.68±4.31 with significant differences （P＜0.05）. In addition to 2.5μmol／L group， the early and late apoptosis rates， protein levels of apoptosis-promoting genes （Bax and Cleaved caspase-3） and the cell proportion in G0／G1phase of the remaining concentration groups were higher than those of 0μmo／L group（P＜0.05）.The p-Akt／Akt value decreased with increasing concentration and the differences were statistically significant among concentrations （P＜0.05）. Conclusion WFA can inhibit the proliferation and apoptosis of A549 cell possibly by inhibiting the activation of PI3K／Akt pathway activation.

Objective To explore the relationship between the mutation of PIK3CA， C-KIT genes and clinicopathological characteristics， prognosis of breast cancer with axillary lymph node metastasis. Methods The archival paraffinembedded tissues from 84 cases of breast cancer with axillary lymph node metastasis were collected. The Ion Torrent sequencing technique was used to detect the mutation rates and distributions of PIK3CA and C-KIT genes. The relationship between the mutations of PIK3CA， C-KIT genes and clinicopathological characteristics（age， menstruation， number of lymph node metastasis， tumor thrombus， tumor size， clinical stage， ER／PR and HER-2 expression） was investigated. The patients enrolled were divided into mutated group and nonmutated group and followed up for 3-year disease-free survival rate. Results All mutations were missense mutations with the rates of 63.1％（53／84） and 15.5％（13／84） for PIK3CA and C-KIT genes， respectively. Seven patients with PIK3CA mutation were in exon 9， 8 in exon 13， 24 in exon 20 and 14 in exons 13 and 20. All patients with C-KIT mutation were located in exon 10. Mutations in the two genes were relevant to HER-2 expression， while only the PIK3CA mutation were related to clinical stage and ER／PR expression. The 3-year disease-free survival rate was 75.5％ in PIK3CA mutated group and 48.4％ in PIK3CA non-mutated group or 76.9％ in C-KIT mutated group and 62.0％ in C-KIT non-mutated group, without significant differences（P＞0.05）. The factors（age， menstruation， number of lymph node metastasis， tumor size， clinical stage， ER／PR expression， HER-2 expression， PIK3CA mutations and C-KIT gene mutations） were not independent prognosis factors of breast cancer with lymph node metastasis. Conclusion The mutation rate of PIK3CA gene is high and relevant to clinical stage， ER／PR and HER-2 expression. Only the relevance to HER-2 expression is observed in C-KIT mutation. Mutations in both genes may play a certain role in the development of breast cancer and axillary lymph node metastasis.

Objective To investigate the frequency of different subsets of natural killer（NK） cells in tumor microenvironment and peripheral blood mononuclear cells（PBMCs） from hepatocellular carcinoma（HCC）， and discuss the relationship between NK cells subsets and HCC disease progression. Methods CD3-CD56+， CD3-CD57+ and CD3-CD161+ NK cells were tested from fresh tissue samples and PBMCs by flow cytometry and peripheral blood of 36 healthy volunteers was collected as normal control. Ten HCC specimens were tested by immunohistochemical method to detect the corresponding distribution of CD3-CD56+ NK cells， and normal tissues from 3 benign hepatic tumors were collected as control. Results Flow cytometry detection showed that frequency of CD3-CD56+ cells and CD3-CD57+ cells were（20.6±10.4）％ and（6.9±3.7）％ in 40 HCC tissues， lower than（27.9±13.5）％ and（9.6±5.1）％ in adjacent normal tissues（P＜0.05）. And there was no significant difference in CD3-CD161+ cells（P＞0.05）. The frequency of the 3 kinds of NK cells subsets in HCC PBMCs was higher than that in healthy volunteers， but without differences（P＞0.05）. Immunohistochemical staining showed that there was large amount of CD3-CD56+ NK cells infiltration in adjacent normal tissue of HCC and only a small distribution in tumor tissue， but they were less than CD3-CD56+ NK cells in normal tissues from 3 benign hepatic tumors. The proportion of NK cells in Ⅰ+Ⅱ stage HCC tissues was higher than Ⅲ+Ⅳ stage， especially CD3-CD56+ and CD3-CD161+ NK cells. No linear relationship was found between the frequency of NK cells subsets and gender， number of lesions， AFP， GOT， GUT， leukocytes， hepatic virus infection and TNM stage（P＞0.05）. Conclusion The decrease of NK cells is correlated to the natural immunity and tumor progression of HCC.

Objective To observe the effects and safety of intercalated combination of recombinant human endostatin（endostar） and cisplatin by intraperitoneal injection in mice model bearing tumor ascite. Methods The tumor ascites model were established with hepatocellular carcinoma H22 cell lines by intraperitoneal injection. One hundred and twenty ICR mice were randomly assigned into 4 groups： control group（normal saline d1-d10）， endostar group（endostar 8mg／kg，d1-d5， d7-d10； normal saline d6）， cisplatin group（cisplatin 0.04mg／kg，d6； normal saline d1-d5, d7-d10） and combined group（endostar 8mg／kg， d1-d5, d7-d10；cisplatin 0.04mg／kg，d6）. Body weight， volume of ascites， the number of tumor cells and red cells were measured. The peritoneum permeability assay， blood routine， liver function and renal function were tested in each group respectively. Implantation metastasis of tumor was observed in abdominal cavity of mice. Apoptosis of tumor cells in the ascites fluid were detected by flow cytometry. Survival time of each mouse was recorded. Results Compared with the control group， endostar group， cisplatin group and combined group could inhibit ascites accumulation， reduce the counts of tumor cells and red cells in the ascites fluid and tumor burden of abdominal cavity， as well as prolonging survival of mice（P＜0.05）. In those above items， combined group showed statistic significance compared with endostar group and cisplatin group（P＜0.05）. Combined group significantly increased apoptosis of tumor cells compared with control group and endostar group（P＜0.05）， but showed no difference with cisplatin group（P＞0.05）. Combined group obviously inhibited the peritoneum permeability compared with control group and cisplatin group（P＜0.05）， but showed no difference with endostar group（P＞0.05）. Peritoneal metastasis and weight gain were less in combined group compared with the other 3 groups. Blood routine， liver function and renal function of the each group were normal. Conclusion The intercalated combination of endostar and cisplatin by intraperitoneal injection treating H22 mice bearing tumor ascite has ideal effect and safety.

Objective To detect SNAT1 protein expression and determine its clinical significance in non-small cell lung cancer（NSCLC）. Methods Tissue microarray blocks containing 98 NSCLC tumor specimens were constructed. Expression of SNAT1 in NSCLC specimens and adjacent cancer tissues was analyzed using immunohistochemistry. A549 cells were transfected with plasmid vector which expressed sh-SNAT1. MTT assay and cloning formation assay were used to detect proliferation of A549 cells which were knocked down SNAT1 expression. Results The positive expression of SNAT1 was 53.1％ in NSCLC， while SNAT1 wasn't expressed in adjacent cancer tissues. SNAT1 expression was significantly associated with lymph node metastasis， tumor stage and differentiation. Patients with SNAT1 positive expression had significantly shorter survival time than those with SNAT1 negative expression（23.3 vs. 40.5months， P＜0.001）. Downregulating expression of SNAT1 leaded to the inhibition of cell growth. Conclusion SNAT1 expression significantly increased with the progression of NSCLC， suggesting the importance of SNAT1 as a potential biomarker in predicting the outcome of patients with NSCLC.

Objective To explore the influences of storage status （original sample or genomic DNA） and time on the mutations of EGFR gene in non-small cell lung cancer （NSCLC）. Methods Ten fresh frozen tissues and 10 formalin-fixed paraffinembedded （FFPE） tissues of NSCLC harboring EGFR mutations were collected. During the twelve months after the first detection， DNA was drawn from fresh frozen tissues （new frozen extraction group） and FFPE tissues （new FFPE extraction group） at an interval of one month. The remaining genomic DNA from fresh frozen tissues （reserved frozen group） and FFPE tissues （reserved FFPE group） were chosen as control after the first detection. The detection kits were used to qualitatively and quantitatively measure the mutations of EGFR gene in four groups， and real time PCR was performed to quantify the Ct value of reference gene to reflect the contents of genomic DNA. The mutation frequencies of EGFR gene and Ct values of reference gene were compared among 4 groups. Results The mutation of EGFR gene could be detected in 20 tissues of original samples， which were consisting with the clinical data. The concentrations of DNA drawn from fresh frozen tissues and FFPE tissues were above 50ng／μl with A260／A280 around 1.8±0.2. The Ct values were high in the reserved frozen group versus the new frozen extraction group after 10 months （P＜0.05）. No significant difference of Ct value was observed between the new FFPE extraction group and the reserved FFPE group （P＞0.05）. The mutation frequencies of EGFR gene in the new frozen extraction group after 6 months and the new FFPE extraction group after 4 months were significant higher than those in the corresponding reserved group （P＜0.05）. Conclusion Original samples are better resources than reserved genomic DNA in retrospective studies.

Objective To evaluate the association between cyclooxygenase-2 （COX-2） gene polymorphisms and digestive system tumor risk. Methods All studies published up to February 2013 on the association between765G＞C polymorphism of COX-2 and digestive system tumor susceptibility were identified by searching in PubMed， EMBASE and Web of Science databases. The correlated index was extracted for aggregate analysis in Stata／SE 12.0.
ResultsA total of 11 083 patients and 16 856 controls from 40 papers were included in the metaanalysis. Overall analysis showed that the C allele contrast model and the CG， CC／CG genotype were significantly associated with the increased digestive system tumor risk compared with the G allele contrast model and the GG genotype. Stratification analysis by ethnicity and tumor position showed that the C allele contrast model and the CG， CC／CG genotypes were significantly associated with the increased risk in Asian descendents and digestive tract tumors. No statistically significant associations were detected in Caucasian and digestive gland tumor subgroups. Stratification analysis by source of control， the C allele contrast model and the CG genotypes were significantly associated with the increased risk in the populationbased （PB） subgroup. No statistically significant associations were detected in hospitalbased （HB） subgroup.Conclusion The COX-2-765G＞C polymorphism is significantly associated with digestive system tumor susceptibility， especially among Asian populations.

Objective To investigate the expression of LRP15 gene in gastric cancer and its relationship with clinical features and prognosis. Methods The expression of LRP15 gene was detected using immunohistochemistry in tissue microarrays containing 90 specimens of gastric cancer and paired adjacent normal mucosa tissues. Survival curves were plotted using Kaplan-Meier method， and Cox regression model to evaluate the independent prognostic factors. Results The LRP15 expression was lower in the tumor tissue compared with that in the adjacent normal mucosa tissue （0.563±0.046 vs. 0.822±0.054， P＜0.001）. According to the ratio of the expression of LRP15 in cancer tissues and that in adjacent tissues were divided into two groups： high-expression group （ratio≥0.5， n=51） and low-expression group （ratio＜0.5， n=39）. LRP15 expression was statistically correlated with the tumor size （P=0.038） and lymph node metastasis （P=0.003）， but not with gender， age， tumor location， differentiation， tumor location， gross type， TNM stage and depth of invasion. The Kaplan-Meier method showed that patients with LRP15 low-expression had a significantly shorter median overall survival compared with the patients with highexpression （27.6 vs 37.3 months， P=0.040）. Cox-multivariate analysis revealed that the expression of LRP15 in cancer tissue，ratio of expression between cancer tissue and adjacent normal mucosa tissues，lymph node metastasis and depth of invasion were both independent prognostic factors.Conclusion LRP15 expression may have an important function in cell growth and is significantly associated with lymph node metastasis. LRP15 can be considered as biomarkers of prognosis and as a new target for gastric cancer therapy.