| Gastric cancer?GC?is a major public health issue.It is the fourth most common cancer and the second leading cause of cancer-related death in the world.At diagnosis,about half of GC patients have advanced disease,with a 5-year survival rate lower than 30%.A full understanding of GC aggressiveness and why this disease frequently presents at such an advanced stage are urgently required.Epithelial-mesenchymal transition?EMT?is an essential early step and a critical process in tumor metastasis.During EMT,tumor cells lose their epithelial characteristics,including E-cadherin expression,cell-cell adhesion,and polarity,and obtain mesenchymal traits,including motility and invasiveness.It has been demonstrated that EMT correlates with poor tumor staging,an increased risk of cancer recurrence,and a decreased survival rate for several types of cancers,such as breast cancer,colorectal cancer,bladder cancer,lung cancer,and GC.MicroRNAs?miRNAs?,a class of endogenous,highly conserved non-coding short RNAs of 18-22 nucleotides in size,were reported to have major roles in regulating gene expression through mRNA cleavage,decay or translational repression.MiRNAs play an important role in tumor and development.MicroRNAs,as players and signals in the metastatic cascade,have implications for the development of novel anti-metastatic therapies.In this study,culture gastric cancer cell lines and clinicial samples of GC tissue were selected as the research objects.The experimental methods including quantitative real-time PCR?qRT-PCR?,western blot,pathology,immunohistochemistry,Fluo-4/AM based Ca2+ measuremen,Cell Counting Kit-8,flow cytometry and Transwell were used.The relationship between the expression of miR-185-5p in GC and clinicopathologic characteristics of GC patients was investigated.The expression of miR-185-5p in gastric cancer cell lines was examined by qRT-PCR.The effect of miR-185-5p on GC cell biological behavior was detected by adopting the method of transient transfection to transfer miR-185-5p mimics,miR-185-5p inhibitor or negative control into the gastric cancer cell lines.Then,the relationship of miR-185-5p and Stromal interaction molecule 1 in GC and GC cells lines was analyzed by qRT-PCR and Western blot.After transfering miR-185-5p mimics,miR-185-5p inhibitor or negative control into the gastric cancer cell lines,we analyzed the effect of miR-185-5p on Store-operated Ca2+ entry of GC cell lines.In this study,we also detected the expression of STIM1 in GC and GC cell lines by qRT-PCR and immunohistochemistry,and investigated the relationship between the expression of STIM1 in GC and clinicopathologic characteristics of GC patients.In order to understand the effect of STIM1 on gastric cancer cell biological behavior,we explored the proliferation of GC cells by Cell Counting Kit-8,detected cell apoptosis using flow cytometry instrument,and studied the abilities of migration and invasion by Transwell after knockdown of STIM1.All the three parts of studies were aimed to investigate the roles of miR-185-5p,STIM1,SOCE in epithelial–mesenchymal transition in gasric cancer,and explore the effects of miR-185-5p,STIM1,SOCE on the generation,migration and invasion of GC,and provide evidence as well as target for diagnosis and treatment of GC.These methods and results of our study are as follows: Part one Effect of miR-185-5p-STIM1-SOCE pathway on epithelial-mesenchymal transition in gastric cancerObjective: To verify the gastric cancer cells with miR-185-5p negative regulation of STIM1,to investigate the effect of miR-185-5p on storeoperated Ca2+ influx and to find the effects of miR-185-5p and SOCE inhibitor SKF96365 on the proliferation,migration and invasion of gastric cancer cells and the relationship with EMT of gastric cancer cell lines.Methods:1 Using the qRT-PCR tested miR-185-5p expression of human gastric mucosal epithelial cell line GES-1 and gastric cancer cells.The SGC-7901 cells were transfected with miR-185-5p mimics and the BGC-823 cells were transfected with miR-185-5p inhibitor.The correlation of miR-185-5p and STIM1 expression was detected by qRT-PCR and Western-blot.2 Using the qRT-PCR and immunohistochemistry tested the correlation of miR-185-5p and STIM1 expression in gastric carcinoma.3 After SGC-7901 cells were transfected with miR-185-5p mimics and BGC-823 cells were transfected with miR-185-5p inhibitor,the effect of miR-185-5p on store operated Ca2+ entry was investigated by confocal microscopy.4 These effects of miR-185-5p and SOCE inhibitor SKF96365 on SGC-7901 and BGC-823 cells were assayed for proliferation by CCK8 method.Meanwhile,the cells migration and invasion were detected by Transwell Chambers.5 Using the qRT-PCR and Western blot tested these effects of miR-185-5p and SOCE inhibitor SKF96365 on the expressions of the markers of EMT E-cadherin,N-cadherin and viminten in both SGC-7901 and BGC-823 cells.Results:1 Expression of miR-185-5p in different gastric cancer cell lines.The expression of miR-185-5p in gastric cancer cells SGC-7901,MKN-28,MGC-803,BGC-823 and AGS was low,when GES-1 as control.The expression of miR-185-5p in BGC-823 cells was relatively higher,and the SGC-7901 was relatively lower.Transfected with miR-185-5p inhibitor,miR-185-5p expression in BGC-823 was knocked down and miR-185-5p mimics was used to up-regulate miR-185-5p expression in SGC-7901.Then,these cells were used in subsequent tests.2 miR-185-5p negatively regulates the expression of STIM1 in gastric cancer cells.Compared with the negative control group,after miR-185-5p overexpression in SGC-7901 cells,the expression of STIM1 mRNA and protein levels were decreased,P<0.01.Knockdown of miR-185-5p in BGC-823 cells,compared with the negative control group,the expression of STIM1 mRNA and protein levels were increased,P <0.01.3 Evaluated the correlation between miR-185-5p and STIM1 expression in GC.The expression of miR-185-5p in GC was significantly lower than that in adjacent normal gastric tissue,which was correlated with lymph node metastasis and TNM high stage.The expression of miR-185-5p in STIM1 immunohistochemical staining positive specimen was significantly lower than that in STIM1 immunohistochemical staining negative specimen,P=0.004.4 Effects of miR-185-5p on store operated Ca2+ entry.Overexpression of miR-185-5p in SGC-7901 cells and these changes of SOCE after knocking down miR-185-5p in BGC-823 cells were detected by Fluo-4/ AM fluorescent probe method.Compared with the negative control group,these results showed that TG activated Ca2+ amplitude in SGC-7901 cells which miR-185-5p was overexpressed had no significant change?P>0.05?,and added SOCE activated by calcium was significantly reduced?P<0.01?.After Knocking down miR-185-5p in BGC-823 cells,compared with the negative control group,TG activated Ca2+ amplitude had no significant change,and added SOCE activated by calcium was significantly increased,P <0.01.5 Effects of miR-185-5p and SOCE inhibitor SKF96365 on proliferation of SGC-7901 and BGC-823 cells.The proliferation of gastric cancer cells BGC-823 and SGC-7901 were detected by CCK8 method.Compared with the negative control group,the ability of proliferation in miR-185-5p group was decreased in SGC-7901 cells.At the time of 24 h,OD values of NC group and miR-185-5p mimics group were 0.736±0.0743 and 0.596±0.012,respectively?P=0.000?.At the time of 48 h,OD values of NC group and miR-185-5p mimics group were 0.826±0.0912 and 0.663± 0.0393,respectively?P=0.002?.At the time of 72 h,OD values of NC group and miR-185-5p mimics group were 0.816±0.159 and 0.648±0.047,respectively?P=0.015?.However,compared with the negative control group,the ability of proliferation in miR-185-5p group was increased in BGC-823 cells.At the time of 24 h,OD values of NC group and miR-185-5p mimics group were 1.538±0.087 and 1.673±0.123?P=0.031?,respectively.At the time of 48 h,OD values of NC group and miR-185-5p mimics group were 1.564±0.067 and 1.994±0.397?P=0.007?,respectively.At the time of 72 h,OD values of NC group and miR-185-5p mimics group were 1.582±0.293 and 2.02±0.185?P=0.003?.Added the SOCE inhibitor SKF96365 with the final concentration of 1.5uM in the medium,the effect of miR-185-5p on the proliferation of gastric cancer cells SGC-7901 and BGC-823 disappeared,P>0.05.6 Effects of miR-185-5p and SOCE inhibitor SKF96365 on migration of SGC-7901 and BGC-823 cells.After transfection of miR-185-5p mimics or miR-185-5p inhibitor 48 h,the migration of gastric cancer cells BGC-823 and SGC-7901 was detected by Transwell,negative group as control.SGC-7901 miR-185-5p mimics group was significantly lower than the negative control group;the migration ability of miR-185-5p inhibitor group in BGC-823 was significantly higher than that in negative control group,the difference was statistically significant?P <0.01?.After treated with SKF 96365 at the same time,the difference was not obvious P >0.05.7 Effects of miR-185-5p and SOCE inhibitor SKF96365 on invasion of SGC-7901 and BGC-823 cells.After transfection of miR-185-5p mimics or miR-185-5p inhibitor 48 h,the invasion of gastric cancer cells BGC-823 and SGC-7901 was detected by Transwell,negative group as control.SGC-7901 miR-185-5p mimics group was significantly lower than the negative control group;the invasion ability of miR-185-5p inhibitor group in BGC-823 was significantly higher than that in negative control group,the difference was statistically significant?P<0.01?.After treated with SKF 96365 at the same time,the difference was not obvious?P>0.05?.8 Effects of miR-185-5p and SOCE inhibitor SKF96365 on the expression of E-cadherin,N-cadherin and Viminten in SGC-7901 and BGC-823 cells.After 24 hours of transfection of miR-185-5p mimics or miR-185-5p inhibitor,these expressions of mRNA in STIM1,E-cadherin,N-cadherin and Viminten of gastric cancer cells BGC-823 and SGC-7901 were detected by qRT-PCR,negative group as control.These expressions of STIM1,E-cadherin,N-cadherin and Viminten protein in SGC-7901 and BGC-823 cells were detected by Western blot after 48 h of transfection.The mRNA and protein levels of STIM1,N-cadherin and Viminten were significantly decreased after miR-185-5p mimics increased the expression of miR-185-5p in SGC-7901 cells,while the mRNA and protein levels of E-cadherin was significantly increased?P<0.01?.The expression of miR-185-5p in BGC-823 cells was inhibited by miR-185-5p inhibitor,STIM1,N-cadherin and Viminten mRNA and protein levels were significantly increased,while E-cadherin mRNA and protein levels were significantly decreased?P<0.01?.After administration of the SOCE inhibitor SKF96365,the mRNA and protein expression of STIM1 were negatively correlated with miR-185-5p expression in miR-185-5p overexpressing SGC-7901 cells and miR-185-5p knockdown BGC-823 cells.However,compared with the negative control group,there was no significant difference in the expression of E-cadherin,N-cadherin and Viminten mRNA and protein in these cells,P>0.05.Conclusions:1 The low expression of miR-185-5p was in both gastric cancer cell lines and gastric carcinoma.2 miR-185-5p mimics was able to transfect SGC-7901 and up-regulate miR-185-5p expression.miR-185-5p inhibitor was able to transfect BGC-823 and down-regulated miR-185-5p expression.3 miR-185-5p has negative regulations of STIM1 expression and SOCE amplitude in SGC-7901 and BGC-823 cells.4 Overexpression of miR-185-5p can inhibit the proliferation of SGC-7901 cells,and knockout miR-185-5p can promote the proliferation of BGC-823 cells.SOCE inhibitor SKF96365 can eliminate the effect of miR-185-5p on the proliferation of SGC-7901 and BGC-823 cells.5 Overexpression of miR-185-5p can inhibit the migration and invasion of SGC-7901 cells,and knockout miR-185-5p can promote the migration and invasion of BGC-823 cells.SOCE inhibitor SKF96365 can eliminate the effect of miR-185-5p on the migration and invasion of SGC-7901 and BGC-823 cells.6 Overexpression of miR-185-5p can promote the epithelial – mesenchymal transition in SGC-7901 cells.Knockout miR-185-5p can promote the epithelial–mesenchymal transition in BGC-823 cells.SOCE inhibitor SKF96365 can eliminate the effect of miR-185-5p on the epithelial-mesenchymal transition of SGC-7901 and BGC-823 cells.Part two Effect of STIM1 on the proliferation,apoptosis,migration and invasion of gastric cancer cells and role of STIM1 in epithelial interstitial transformation in gastric cancer cells.Objective: To study the effect of STIM1 on the biological behavior of gastric cancer cells in the aspect of proliferation,apoptosis,migration and invasion,to verify the correlation between STIM1 and epithelial – mesenchymal transition in gastric cancer cells.Methods:1 Using the qRT-PCR and Western blot tested STIM1 expression of human gastric mucosal epithelial cell line GES-1 and gastric cancer cells.2 Gastric cancer cell lines BGC-823 with the highest expression of STIM1 and SGC-7901 with relatively lower STIM1 expression were chosen for subsequent experiment.These cells were transfected with STIM1-siRNA or negative control using Lipofectaminetm2000 and then assayed for proliferation by CCK8 method,apoptosis with flow cytometry instrument and migration and invasion abilities with Transwell Chambers.3 The effect on the expression of the Epithelial-mesenchymal transition?EMT?-associated marker E-cadherin,N-cadherin and viminten by knockdown of STIM1 in SGC-7901 and BGC-823 cells was analyzed by qRT-PCR and Western blot methods.Results:1 Expression of STIM1 was qualified using real-time quantitative polymerase chain reaction?qRT-PCR?and Western blot analysis in five gastric cancer cell lines?SGC-7901,MKN-28,MGC-803,BGC-823,AGS?and immortalized human gastric mucosal epithelial cell line GES-1.The expression of STIM1 was significantly higher in these gastric cancer cell lines than in GES-1.Of them,BGC-823 had the highest expression and STIM1 expression in SGC-7901 was relatively lower.2 STIM1 expressions in BGC-823 and SGC-7901 cells transfected with STIM1-siRNA.BGC-823 and SGC-7901 cells were transfected with STIM1-siRNA using liposome transfection techniques,then detected the expression of STIM1 by qRT-PCR and Western blot.These results showed that the expression of STIM1 in BGC-823 and SGC-7901 cells transfected with STIM1-siRNA was significantly lower than that in negative control group and blank control group?P<0.01?,which indicated that the transfection was successful and feasible.3 Effects of knockout STIM1 on the proliferation of gastric cancer cells SGC-7901 and BGC-823.After transfection of STIM1-siRNA 24 h,48h and 72 h,the proliferation of gastric cancer cells BGC-823 and SGC-7901 was detected by CCK8 method,negative group as control.OD values of NC group and siRNA group in SGC-7901 cells at 24 h were 0.809 ± 0.073 and 0.801 ± 0.0145,respectively.There was no statistical significance,P=0.938.OD values of NC group and siRNA group at 48 h were 0.96±0.34 and 0.65±0.184?P <0.05?,respectively.OD values of NC group and siRNA group at 72 h were 0.938±0.259 and 0.874±0.215?P <0.01?respectively.In BGC-823 cells,OD values of NC group and siRNA group at 24 h were 1.563±0.195 and 1.332±0.0654?P <0.05?,respectively.OD values of NC group and siRNA group at 48 h were 1.699±0.142 and 1.425±0.0389?P<0.01?,respectively.OD values of NC group and siRNA group at 72 h were 1.911 ±0.110 and 1.611±0.138?P<0.05?,respectively.4 Effects of knockout STIM1 on the apoptosis of gastric cancer cells SGC-7901 and BGC-823.The apoptosis of gastric cancer cells BGC-823 and SGC-7901 with transfection of STIM1-siRNA after 48 h was detected by flow cytometry,negative group as control.SGC-7901 cells NC group,siSTIM1 group values were 4.753±0.15%,6.533 ±0.153%,P<0.01.BGC-823 cells NC group,siSTIM1 group values were 2.733±0.208%,6.763 ±0.764%,P <0.01.5 Effects of knockout STIM1 on the migration of gastric cancer cells SGC-7901 and BGC-823.The migration of gastric cancer cells BGC-823 and SGC-7901 with transfection of STIM1-siRNA after 48 h was detected by Transwell,negative group as control.SGC-7901 cells NC group,siSTIM1 group number of migrated cells were 443.33±21.825,133±15.716,P=0.000.BGC-823 cells NC group,siSTIM1 group number of migrated cells were 642.67±31.342,254.33±17.502,P=0.000.6 Effects of knockout STIM1 on invasion of gastric cancer cells SGC-7901 and BGC-823.After transfection of STIM1-siRNA for 48 hours,the invasion of gastric cancer cells BGC-823 and SGC-7901 was detected by Transwell,negative group as control.SGC-7901 cells NC group,siSTIM1 group number of invaded cells were 431±19,94±11,P=0.000.BGC-823 cells NC group,siSTIM1 group number of invaded cells were 259±18,99±10,P=0.000.7 Effects of knockout STIM1 on the expression of E-cadherin,N-cadherin and Viminten in gastric cancer cells SGC-7901 and BGC-823.After transfection of STIM1-siRNA for 24 hours,the expression of mRNA in STIM1,E-cadherin,N-cadherin and Viminten of gastric cancer cells BGC-823 and SGC-7901 was detected by qRT-PCR,negative group as control.The results showed that N-cadherin and Viminten in siSTIM1 group were significantly lower than in the NC group?P<0.01?,but the E-cadherin was significantly increased?P<0.05?.After transfection of STIM1-siRNA for 48 hours,the protein expression of STIM1,E-cadherin,N-cadherin and vimentin in SGC-7901 and BGC-823 were detected by Western-blot,negative group as control.These results showed that N-cadherin and Viminten in siSTIM1 group were significantly lower than in the NC group?P <0.01?,but the E-cadherin was significantly increased?P <0.01?.Summary:1 STIM1 was overexpressed in SGC-7901,MKN-28,MGC-803,BGC-823,AGS,consistent with the result of gastric carcinoma.2 STIM1-siRNA was able to transfect SGC-7901 and BGC-823 cells and down-regulate the expression of STIM1 in SGC-7901 and BGC-823 cells.3 Down-regulation of STIM1 expression inhibited the proliferation of SGC-7901 and BGC-823 cells.4 Down-regulation of STIM1 expression can promote the apoptosis of SGC-7901 and BGC-823 cells.5 Down-regulation of STIM1 expression can inhibit the migration and invasion of SGC-7901 and BGC-823 cells.6 Down-regulation of STIM1 expression,SGC-7901 and BGC-823 cells occurred EMT reversal,that is,interstitial epithelial transformation.Part three Stromal interaction molecule 1 overexpression promotes epithelial-mesenchymal transition and is associated with poor survival in gastric cancer patientsObjective: The aim of this study was to investigate stromal interaction molecule 1?STIM1?expression in gastric cancer?GC?and explore the relationship between STIM1 and epithelial–mesenchymal transition.Methods:1 GC and adjacent normal tissue samples were obtained from 170 GC patients with histologically confirmed gastric adenocarcinoma that was initially resected between June 2009 and October 2011 in The Fourth Hospital of Hebei Medical University.All patients underwent surgical resection of the stomach with lymph node clearance,with no chemotherapy or radiotherapy before surgery;no other cancers were diagnosed.Immunohistochemical staining was performed to detect STIM1,E-cadherin,and ?-cadherin in 170 GC and 35 adjacent normal gastric tissue samples.2 All data were processed using SPSS 19.0 statistical software.Statistical significance was defined as a P value of < 0.05.Cell count data are expressed as n?%?.The chi-square test was used to analyze the relationship between STIM1 expression and clinical features.A binary logistical regression model Summary: was used to identify factors associated with STIM1 positive expression.Cohen's kappa statistic was used for determining the association between STIM1 expression and abnormal E-cadherin and ?-catenin expression.The Kaplan-Meier method was used to calculate patient survival rate,and the Cox proportional hazards models were used to identify factors related to patient survival.Results:1 STIM1 expression in GC tissues was predominantly cytoplasmic.The STIM1 expression rate in GC tissues was 43.5%?74/170?,which was significantly higher than in tumor adjacent tissues?8.60%,3/35,?2 = 15.12,P< 0.001?.2 The STIM1 expression rate in GC patients with LNM was significantly higher than in patients without LNM?P < 0.001?.STIM1 expression in stage I–II GC tissues was 33.5%?17/57?,which was significantly lower than in stage III–IV tumors?66.5%,57/113,P = 0.01?.However,STIM1 expression in GC tissues did not correlate with sex,age,the degree of histologic differentiation and location of the tumor,and tumor size?P> 0.05?.Cox risk regression analysis indicated that lymphatic metastasis is the only independent risk factor for STIM1 expression in GC patients.3 Using Kaplan–Meier analysis,we founded that the overall patient survival rate was significantly lower for those with STIM1-expressing GC tumors than in those with GC tumors without STM1 expression?P = 0.043?Factors that significantly correlated with patient survival rate,including STIM1 expression,LNM,and a high TNM stage,were identified by univariate analysis.Cox risk regression analysis indicated that STIM1 expression and LNM are independent prognostic factors for GC patients.4 E-cadherin and ?-catenin were abnormally expressed in the cytoplasm or nucleus of GC cells.Abnormal E-cadherin and ?-catenin expression were seen in 61.8%?105/170?and 52.4%?89/170?,respectively,of GC tissue samples.However,abnormal E-cadherin and ?-catenin expression were seen in 11.4%?4/35?and 20.0%?7/35?,respectively,of adjacent normal gastric tissues.Differences in the rates of abnormal E-cadherin and ?-catenin expression between GC tissues and adjacent normal gastric tissues were significant?P < 0.001 for both?.In addition,abnormal E-cadherin expression was positively associated with lymph node metastasis and a higher clinical stage?P<0.001?.In this study,the ?-catenin expression correlated significantly with tumor size,LNM,and the clinical stage of GC tissues?P< 0.001?,however,there was no correlation between ?-catenin expression and other clinicopathological parameters?P > 0.05?.5 Potential associations between STIM1,E-cadherin,and ?-catenin expression patterns in GC were evaluated.We observed that 78.4%?58/74?of STIM1-positive tumors are E-cadherin positive,and that 90.5%?67/74?of STIM1-positive tumors are positive for ?-catenin.Chi-square tests showed that STIM1 expression correlated significantly with abnormal E-cadherin expression??2 = 34.555,P < 0.001,? = 0.447?and with abnormal ?-cadherin expression??2 = 45.947,P < 0.001,? = 0.486?.Moreover,79.8%?71/89?of E-cadherin-positive tumors were also positive for ?-cadherin,and this relationship was statistically significant?P< 0.001?.Summary:1 STIM1 expression in GC tissues were significantly higher than in adjacent normal tissues.2 STIM1 expression was significantly associated with lymph node metastasis and tumor–node–metastasis stage in GC tissues.3 The overall survival rate was significantly lower in STIM1-positive patients than in STIM1-negative patients.Positive STIM1 expression and lymph node metastasis were independent prognostic factors for GC.4 The rate of abnormal E-cadherin and ?-catenin expression in GC tissues were significantly higher than in adjacent normal tissues.5 STIM1 expression in GC tissues was significantly associated with abnormal E-cadherin and ?-cadherin expression.Conclusion:1 miR-185-5p expression in gastric cancer cell lines and gastric cancer tissues was low,but STIM1 expression in gastric cancer cell lines and gastric cancer tissues was significantly higher.miR-185-5p expression and STIM1 expression in gastric cancer tissues was significantly associated with lymph node metastasis and TNM stages.The overall patient survival rate was significantly lower for those with STIM1-expressing GC tumors than in those with GC tumors without STIM1 expression.STIM1 expression and lymph node metastasis were independent prognostic factors for gastric cancer patients.miR-185-5p expression was negatively correlated to STIM1 expression in gastric cancer.miR-185-5p and STIM1 can be forecast factors of gastric cancer metastasis.2 miR-185-5p has negative regulation of STIM1 expression and SOCE amplitude in gastric cancer cell lines.3 miR-185-5p can inhibit the proliferation,migration and invasion of gastric cancer cell lines,but SOCE inhibitor SKF96365 can eliminate the effect of miR-185-5p.4 Knockout STIM1 can inhibit the proliferation,migration and invasion of gastric cancer cell lines.5 STIM1 promotes epithelial-mesenchymal transition in gastric cancer.STIM1 expression correlated significantly with abnormal E-cadherin expression and ?-catenin expression patterns in GC.Down-regulation of STIM1 expression in GC cells occurred EMT reversal,that was mesenchymal-epithelial transition.6 miR-185-5p-STIM1-SOCE pathway regulates epithelial-mesenchymal transition in gastric cancer.7 miR-185-5p,STIM1 and SOCE may become the new targets for anti invasion and metastasis of gastric cancer therapy. |
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