| Gastric cancer(GC)is one of the most common malignant tumors in the world,which has severely threatened the health and quality of life of people.The diagnostic and treatment of GC have been greatly improved in China.However,most GC patients are in the advanced stages at the time of diagnosis,which have deprived them the opportunities of radical surgery.Moreover,radiochemotherapy can not remarkably extend their lifetime.At present,research regarding GC mechanism mostly concentrates on proliferation and invasion.Apoptosis is a major mechanism for the programmed death of mammalian cell,and inhibited apoptosis is suggested in plenty of studies to be one of the important reasons inducing tumor.The annexin A family is a calcium ion-dependent phospholipid binding protein superfamily,all members of which are involved in regulating numerous important cellular functions,such as pinocytosis,exocytosis,cytoskeleton,ion channel,cell proliferation and cell apoptosis.Annexin A7(ANXA7),which is also called synexin,belongs to the annexin A family and is abnormally expressed in multiple tumors.However,the role and molecular mechanism of ANXA7 in apoptosis of GC remain unclear.Expression of ANXA7 m RNA and protein in GC tissue and its adjacent tissue was detected in this research using real-time quantitative reverse transcriptase polymerase chain reaction(q RT-PCR)and western blot.Serum ANXA7 levels in GC patients and normal subjects were detected using enzyme linked immunosorbent assay(ELISA),so as to explore the significance of ANXA7 expression in GC serum and tissue.The relation of ANXA7 expression in GC tissue with clinicopathological factors of GC was further analyzed in this research.Meanwhile,human GC tissues and GC cell lines with various degrees of differentiation were treated as the objects of study to analyze the relation of ANXA7 expression in GC with differentiation and apoptosis using q RT-PCR,western blot,terminal-deoxynucleotidyl transferase biotin-d UTP neck end labeling(TUNEL)and flow cytometry(FCM).Effects of ANXA7 expression inhibition on apoptosis of BGC-823 and subcutaneous xenograft in nude mice were observed by cell culture and RNA interference(RNAi)technology.Furthermore,effects of ANXA7 expression inhibition on expression of apoptosis-related genes(Bcl-2,Bax,caspase-3 and caspase-9),caspase-3 and caspase-9 activities,and expression of ERK1/2,JNK1/2/3 and p38 proteins in MAPK signal transduction pathway were also examined.This could provide preliminary experimental foundation and theoretical basis for studying the role of ANXA7 in apoptosis of GC as well as molecular targeted therapy for GC with ANXA7 as the new target.This research was constituted by 5 parts.Part I Expression of ANXA7 in GC as well as its relation with clinicopathological factors of GCObjective: To examine ANXA7 expression in GC tissues,matched para-carcinoma tissues,as well as serum in GC patients and normal subjects,so as to analyze the significance of ANXA7 in GC.Methods: 105 GC patients receiving radical surgery in Third Department of Surgery of the Fourth Hospital of Hebei Medical University from January 2013 to December 2015 and were confirmed GC through postoperative pathology were collected.Their morning fasting blood,and flesh surgical specimens of GC tissues and para-carcinoma tissues were collected.Additionally,50 healthy subjects having normal physical examination results at the same period were enrolled in the control group.Expression of ANXA7 m RNA and protein in GC tissues and para-carcinoma tissues was detected using q RT-PCR and western blot.Serum ANXA7 levels in GC group and control group were detected by ELISA,so as to analyze the significance of ANXA7 in GC and to further analyze the relation of ANXA7 in GC tissue with its clinicopathological factors.Results: 1 ANXA7 m RNA and protein expression in 105 pairs of GC tissues and matched para-carcinoma tissues was detected using q RT-PCR and western blot technology.The results suggested that expression of ANXA7 m RNA and protein was higher in GC tissues than in matched para-carcinoma tissues,with the differences being of statistical significance(P<0.05).2 Concentrations of serum ANXA7 protein in GC group and normal control group detected by ELISA were(78.620±11.249)ng/L and(54.971±9.548)ng/L,respectively.Meanwhile,concentration of serum ANXA7 protein in GC group was notably higher than that in normal control group,and the difference was statistically significant(P<0.05).The area under the ROC curve in both GC group and control group was 0.941.Moreover,the sensitivity and specificity of diagnosing GC were 81.90% and 94.0%,respectively,after treating 67.21ng/L as the threshold between GC patients and normal subjects.3 Spearman correlation analysis indicated that GC tissue was positively correlated with ANXA7 protein expression in peripheral blood(r=0.607,P<0.05).4 The relation of ANXA7 m RNA and protein levels in GC tissue with clinicopathological factors in GC patients was analyzed.The results suggested that difference in expression of ANXA7 m RNA and protein in GC tissues of various degrees of differentiation was statistically significant(P<0.05).Furthermore,expression level had increased accompanied by the decrease in degree of differentiation.Among tissues with different tumor invasion depth,only differences in expression of ANXA7 m RNA and protein at T1 and T4 stages were of statistical significance(P<0.05),with T4 stage being notably higher than T1 stage.ANXA7 m RNA expression in GC tissue with lymph node metastasis was higher than that without lymph node metastasis,and the difference was statistically significant(P<0.05),but difference in ANXA7 protein expression showed no statistical significance(P>0.05).Differences in ANXA7 m RNA and protein expression in GC tissue among different gender,age,tumor size,tumor site and pathological type showed no statistical significance(P>0.05).Summary: 1 ANXA7 m RNA and protein expression in GC tissue is higher than that in para-carcinoma tissue.2 Serum ANXA7 level in GC patients is remarkably higher than that in normal control group.The sensitivity and specificity of diagnosing GC are 81.90% and 94.0%,respectively,after treating 67.21ng/L as the threshold between GC patients and normal subjects.ANXA7 may be served as a novel serum marker for GC.3 GC tissue is positively correlated with ANXA7 protein expression in peripheral blood.4 ANXA7 expression in GC tissue is related to degree of tumor differentiation,tumor invasion depth and lymph node metastasis,but it is not related to gender,age,tumor size,tumor site and pathological type.Part II Research on the correlation of ANXA7 expression with GC differentiation and apoptosisObjective: To detect ANXA7 expression and apoptosis in highly-,moderately-and poorly-differentiated GC tissues and GC cell lines,so as to analyze the relation of ANXA7 expression with tumor differentiation and apoptosis.Methods: 105 highly-,moderately-and poorly-differentiated gastric adenocarcinoma tissue specimens,as well as human highly-,moderately-and poorly-differentiated gastric adenocarcinoma cell lines MKN74,SGC7901 and BGC823 were collected.ANXA7 m RNA and protein expression was detected using q RT-PCR and western blot,apoptosis of GC tissues of various degrees of differentiation was detected by TUNEL method,apoptosis of GC cell lines of various degrees of differentiation was examined with FCM,and the relation of ANXA7 expression with GC differentiation and apoptosis was also analyzed.Results: 1 ANXA7 expression in highly-,moderately-and poorly-differentiated GC tissues was detected using q RT-PCR and western blot.The results suggested that ANXA7 was expressed in GC tissues of various degrees of differentiation,and a lower degree of differentiation was associated with higher expression,with the difference being of statistical significance(P<0.05).ANXA7 expression in highly-,moderately-and poorly-differentiated GC cell lines were examined by q RT-PCR and western blot.The results indicated that ANXA7 was expressed in GC cell lines of various degrees of differentiation,a lower degree of differentiation was associated with higher expression,and the difference was of statistical significance(P<0.05).2 Apoptotic indexes in highly-,moderately-and poorly-differentiated GC tissues detected by TUNEL were(18.12±2.40)%,(9.73±1.73)% and(4.13±0.83)%,respectively,with the difference being of statistical significance(P<0.05).3 Cell apoptotic rates in MKN74,SGC7901 and BGC823 groups detected by FCM were(10.07±1.21)%,(7.11±1.04)% and(4.25±1.02)%,respectively,and the difference was statistically significant(P<0.05).4 Spearman rank correlation analysis results had suggested that expression of ANXA7 m RNA and protein in human GC tissue was negatively correlated with degree of GC differentiation(r=-0.926,P<0.05;r=-0.950,P<0.05).GC apoptotic index was positively correlated with degree of differentiation(r=0.949,P<0.05).GC tissue apoptotic index was negatively correlated with expression of ANXA7 m RNA and protein(r=-0.978,P<0.05;r=-0.973,P<0.05).In addition,expression of ANXA7 m RNA and protein in GC cell lines showed negative correlation with degree of GC differentiation(r=-0.934,P<0.05;r=-0.938,P<0.05);apoptotic rate of GC cell lines displayed positive correlation with degree of differentiation(r=0.936,P<0.05);and apoptotic rate of GC cell lines was negatively correlated with expression of ANXA7 m RNA and protein(r=-0.917,P<0.05;r=-0.933,P<0.05).Summary: 1 Expression of ANXA7 m RNA and protein in GC tissues and cell lines is negatively correlated with degree of differentiation.To be specific,ANXA7 expression is increased accompanied by decrease in degree of GC differentiation.2 Apoptosis in GC tissues and cell lines shows positive correlation with degree of differentiation.In other words,the number of apoptotic cells is decreased accompanied by decrease in degree of GC differentiation.3 ANXA7 expression levels in GC tissues and cell lines are negatively correlated with apoptosis,which means that the apoptotic index/apoptotic rate display a decreasing trend as ANXA7 expression increases.ANXA7 can inhibit apoptosis,which may play a role as oncogene during the genesis and development of GC.Part III Effects of ANXA7 expression inhibition on GC BGC823 Cell ApoptosisObjectives: To explore the effect of ANXA7 expression inhibition on BGC823 cell apoptosis and apoptosis-related genes(Bcl-2,Bax,caspase-3 and caspase-9).Methods: Poorly-differentiated human gastric adenocarcinoma cell line BGC823 transfected with ANXA7-silencing specific si RNA sequence was constructed.Meanwhile,cells transfected with targeted ANXA7-si RNA-3 were enrolled in the experiment group(si ANXA7 group),those transfected with NS-si RNA were recruited into the negative control group(si NS group),while those in blank control group received no treatment.Changes in apoptotic rate of BGC823 cells before and after transfection were detected by FCM.Changes in m RNA and protein expression of apoptosis-related genes Bcl-2,Bax,caspases-3 and capase-9 before and after transfection were detected with q RT-PCR and western blot.Changes in caspase-3 and caspase-9 activities before and after transfection were detected.Results: 1 It was found in q RT-PCR and western blot detection after BGC823 cells were transfected with ANXA7-si RNA that,ANXA7 m RNA and protein expression in si ANXA7 group was remarkably reduced,with the difference being of statistical significance(P<0.05),but difference between si NS group and Ctrl group was not statistically significant(P>0.05).2 Cell apoptotic rates in si ANXA7 group,si NS group and Ctrl group detected by FCM were(24.87±4.80)%,(4.57±0.31)% and(3.89±0.28)%,respectively.Cell apoptotic rate in si ANXA7 group was notably higher than that in Ctrl group and si NS group(P<0.05),but difference between si NS group and Ctrl group was not statistically significant(P>0.05).3 Expression of apoptosis-related genes Bcl-2,Bax,caspases-3 and capase-9 was detected with q RT-PCR and western blot.The results showed that compared with si NS group and Ctrl group,m RNA and protein expression of Bcl-2 in si ANXA7 group was distinctly lower,while that of Bax,caspases-3 and capase-9 was evidently higher,and the differences were statistically significant(P<0.05).caspase-3 and caspase-9 activities in si ANXA7 group were 2.95 and 3.70 folds of those in Ctrl group,which were apparently higher than those before transfection,with the differences being of statistical significance(P<0.05).Differences in the above indexes between si NS group and Ctrl group were not statistically significant(P>0.05).Summary: 1 ANXA7-si RNA can effectively inhibit ANXA7 m RNA and protein expression in BGC823 cell.2 Apoptotic rate of BGC823 cell has increased after down-regulation of ANXA7 expression,indicating that ANXA7 has important biological function in the genesis and development of GC.It is an important regulatory protein of GC cell apoptosis,which can suppress GC cell apoptosis and play a role as oncogene.3 ANXA7 expression inhibition may play an important role in promoting GC cell apoptosis through improving caspase-3 and caspase-9 activities,down-regulating Bcl-2 expression,and up-regulating Bax,caspase-3 and caspase-9 expression.Part IV Effects of ANXA7 expression inhibition on MAPK signal transduction pathway of BGC823 cellObjective: To explore the effects of ANXA7 expression inhibition on expression of ERK1/2,JNK1/2/3 and p38 proteins in MAPK family,so as to analyze the influence of MAPK signal transduction pathway on GC apoptosis.Methods: Poorly-differentiated human gastric adenocarcinoma cell line BGC823 was transfected using RNA interference technology,and changes in expression and phosphorylation levels of ERK1/2,JNK1/2/3 and p38 proteins in MAPK family before and after transfection were detected by western blot.Results: 1 Western blot results suggested that differences in expression and phosphorylation levels of ERK1/2 protein among blank control group(Ctrl group),negative control group(si NS group)and experiment group(si ANXA7 group)were not statistically significant(P>0.05).2 As was indicated from western blot results,difference in JNK1/2/3 protein expression among Ctrl group,si NS group and si ANXA7 group was not statistically significant(P>0.05).Compared with Ctrl group and si NS group,JNK1/2/3 phosphorylation level in si ANXA7 group had markedly lowered,and the difference was statistically significant(P<0.05).3 Western blot results suggested that differences in expression and phosphorylation levels of p38 protein among Ctrl group,si NS group and si ANXA7 group were not statistically significant(P>0.05).Summary: 1 Inhibiting ANXA7 expression in BGC823 cell makes no significant difference to the expression of ERK1/2,JNK1/2/3 and p38 proteins as well as the phosphorylation levels of ERK1/2 and p38.However,the phosphorylation level of JNK1/2/3 protein has been notably reduced.2 JNK signaling pathway may participate in the process in which low ANXA7 expression promotes apoptosis of GC BGC823 cell.Part V Effects of ANXA7 expression inhibition on the apoptosis of human GC cell subcutaneous xenograft in nude miceObjective: To analyze the effects of ANXA7 expression inhibition on apoptosis of subcutaneous BGC823 cell xenograft in nude mice as well as the changes in apoptosis-related genes,so as to further explore the role of ANXA7 in GC apoptosis as well as its molecular mechanism.Methods: Poorly-differentiated human gastric adenocarcinoma cell line BGC823 was injected into the right axilla of nude mice subcutaneously,so as to construct the subcutaneous xenograft model.The recombinant plasmids ANXA7-sh RNA(sh ANXA7 group),NS-sh RNA(sh NS group)and saline(Ctrl group)were injected into the tumors after tumor formation.The growth of xenograft in nude mice was observed;meanwhile,tumor volume and weight were measured.Expression of ANXA7 m RNA and protein in xenograft tissues of each group was detected by q RT-PCR and western blot.Apoptotic rate in each group was measured by FCM.Expression of apoptosis-related genes(Bax,Bcl-2,caspase-3 and caspase-9)was detected by q RT-PCR and western blot,and caspase-3 and caspase-9 activities were also detected.Results: 1 Tumor growth could be seen in all the 15 experimental nude mice,with the xenograft formation rate of 100%.2 ANXA7 expression in xenograft was detected by q RT-PCR and western blot.The results suggested that compared with sh NS group and Ctrl group,ANXA7 m RNA and protein expression in sh ANXA7 group had been remarkably reduced,and the difference was statistically significant(P<0.05).But difference between sh NS group and Ctrl group was not statistically significant(P>0.05).3 Tumor volumes in sh ANXA7 group,sh NS group and Ctrl group were(614.72±121.72)mm3,(955.82±152.29)mm3 and(1072.65±238.47)mm3,respectively.Compared with Ctrl group and sh NS group,nude mouse xenograft volume in sh ANXA7 group had been notably reduced,with the difference being of statistical significance(P<0.05),but difference between sh NS group and Ctrl group was not statistically significant(P>0.05).Tumor weights in sh ANXA7 group,sh NS group and Ctrl group were(1.36±0.39)g,(2.13±0.32)g and(2.20±0.34)g,respectively.Nude mouse xenograft weight in sh ANXA7 group was markedly lower to those in Ctrl group and sh NS group,with the difference being of statistical significance(P<0.05),but difference between sh NS group and Ctrl group displayed no statistical significance (P>0.05).Meanwhile,tumor inhibiting rate in sh ANXA7 group was remarkably higher.4 Xenograft apoptotic rates in sh ANXA7 group,sh NS group and Ctrl group detected by FCM were(20.20±0.83)%,(6.10±0.21)% and(6.03±0.12)%,respectively.Apoptotic rate in sh ANXA7 group had been distinctly increased compared with Ctrl group and sh NS group,with the difference being of statistical significance(P<0.05),but difference between sh NS group and Ctrl group was not statistically significant(P>0.05).5 Expression of apoptosis-related genes Bcl-2,Bax,caspase-3 and caspase-9 in each group was detected by q RT-PCR and western blot.The results indicated that compared with Ctrl group and sh NS group,m RNA and protein expression of Bcl-2 in sh ANXA7 group had been notably reduced,while that of Bax,caspase-3 and caspase-9 had been markedly elevated,and the differences were statistically significant(P<0.05).Differences in the above indexes between sh NS group and Ctrl group were not statistically significant(P>0.05).6 Caspase-3 and caspase-9 activities in sh ANXA7 group had been distinctly elevated,which were 1.78 and 2.71 folds of those in Ctrl group,and the difference was of statistical significance(P<0.05).Differences in the above indexes between sh NS group and Ctrl group were not statistically significant(P>0.05).Summary: 1 Subcutaneous nude mouse xenograft model has been successfully constructed,with the tumor formation rate of 100%.2 Recombinant plasmid ANXA7-sh RNA can outstandingly inhibit expression of ANXA7 in nude mouse xenograft,which can effectively suppress the growth of subcutaneous xenograft in nude mice and render reduced xenograft volume and weight.3 Inhibiting ANXA7 expression can promote the apoptosis of subcutaneous xenograft in nude mice.4 Inhibiting ANXA7 expression can promote the tumor cell apoptotic rate of subcutaneous xenograft in nude mice,which may be related to elevated caspase-3 and caspase-9 activities,down-regulated Bcl-2 expression,and up-regulated Bax,caspase-3 and caspase-9 expression.Conclusion: 1 ANXA7 expression in GC tissue is higher than that in para-carcinoma tissue.2 Serum ANXA7 in GC patients is distinctly higher than that in normal control group,which may be served as a novel serum marker for GC.3 ANXA7 expression in GC tissue is related to degree of tumor differentiation,tumor invasion depth and lymph node metastasis,but it is not related to gender,age,tumor size,tumor site and pathological type.4 ANXA7 expression in GC is negatively correlated with differentiation and apoptosis.5 Inhibiting ANXA7 expression in BGC823 cell will lead to increased cell apoptotic rate,which may be related to the elevated caspase-3 and caspase-9 activities,down-regulated Bcl-2 expression,and up-regulated Bax,caspase-3 and caspase-9 expression.6 Inhibiting ANXA7 expression in BGC823 cell will give rise to lowered phosphorylation level of JNK1/2/3 protein.Moreover,the JNK signaling pathway may be involved in the process in which low ANXA7 expression promotes GC cell apoptosis.7 ANXA7 expression inhibition in nude mouse xenograft can effectively suppress the growth of subcutaneous xenograft in nude mice and elevate the apoptotic rate of subcutaneous xenograft in nude mice.It may be related to elevated caspase-3 and caspase-9 activities,down-regulated Bcl-2 expression,and up-regulated Bax,caspase-3 and caspase-9 expression.8 ANXA7 may play a role as oncogene in GC,which is an important molecular target.ANXA7 is promising to become a potential biomarker in GC diagnosis and treatment. |
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