Effects of STIM1Gene Silencing by RNAi on the Biological Behavior in Human Gastrisc Cancer Cells
|Course||Pathology and Pathophysiology|
|Keywords||STIM1 Gastric Cancer Cells Biological Behavior of Tumor SOCC Ca2+|
Objective: Stromal interaction molecule1（STIM1） is a kind of type Itransmembrane protain mainly located in the endoplasmic reticulum （ER） and widelyexpressed in animal cells and tumor cells. Its main function is to detect the change ofcalcium’s concentration in ER and regulate SOCC（store-operated calcium channel）,then form CRAC (Ca2+release-activated Ca2+channel), so as to control a variety ofcells’ biological behaviour including proliferation, apoptosis, migration and so on.But reports concerning the relationship between STIM1and gastric cancer’ genesisand development are still unseen. Therefore, our project took gastric cancer cells asresearch object, silenced the gene of STIM1, observed the influence on calcium signal,proliferation, apoptosis, migration of gastric cancer cells, in order to provide a newthought and target spot for clinical prevention of gastric cancer.Methods: Our research used the lipidosome of LipofectamineTM2000, transf-ected the interference plasmid of pGPU6/shSTIM1and negative plasmid of pGPU6/shSTIM1-NC into SGC7901cells. The experiment was divided into three groups:blank control group, negative control group and STIM1silent group. After thetransfection with SGC7901cells for48h, we evaluated the transfection efficiency byfluorescence microscope; detected the silence efficiency by western blottingmeasuring the expression of STIM1protein; single cell calcium imaging system wasused to measure the change of calcium influx after STIM1was silenced; theproliferative rate of SGC7901cells was assessed by Methyl Thiazolyl Tetrazolium（MTT） assay; transwell experiment was used to test the ability of invasion andmigration; apoptosis and the changes of cell cycle were examined by flow cytometry.Results:1. pGPU6/shSTIM1and pGPU6/shSTIM1-NC plasmids were transfected intoSGC7901cells by liposome-mediated successfully, and its transfection efficiency upto80%.2. Western blotting-test shows: The grey value of blank control group, negativecontrol group and STIM1silent group were0.73±0.03，0.69±0.02，0.29±0.05, respectively. Compared with blank control group and negative control group, theexpression level of STIM1protein in STIM1silent group significantly reduced（P<0.01）, while the differences have no statistic significance （P>0.05） between thelater two groups. The results indicate that transfected specificity with STIM1-siRNAcan significantly inhibit the expression of STIM1protein.3. Single cell calcium imaging system to detect intracellular calcium: Comparedwith blank control group and negative control group, the increased extent ofintracellular calcium in STIM1slient group reduced apparently （P<0.05）, as well asthere was no significant difference between the later two groups （P>0.05）.4. MTT results showed: The proliferation of SGC7901cells was significantlyinhibited following STIM1-siRNA, and was37%,41%,27%at24h,48h,72hpost-gene-silence, respectively. Compared with negative control group, the inhibitionrate in STIM1slient group significantly increased （P<0.05）, in addition with the besttransfected time which had the best inhibited effect towards gastric cancer cells’growth was at48h.5. Flow cytometry measuring apoptosis revealed: The apoptosis rate ofSGC7901cells in the blank control group, negative control group and STIM1slientgroup were7.95±0.08,9.01±0.03,36.98±0.13, respectively. Compared with blankcontrol group and negative control group, the apoptosis rate of STIM1slient groupwas increased obviously （P<0.05）, while no statistic significance existed between thelater two groups （P>0.05）.6. Flow cytometry analysing cell cycle: After gastric cancer cell SGC7901ineach group were cultured for48h, the cell cycle distributed as follows, for blankcontrol group, the percentages of cells were35.63±1.07%in G0/G1,51.30±0.71%inS,13.14±0.81%in G2/M; for negative control group, the proportions of cells were37.46±0.67%in G0/G1,50.89±0.87%in S,11.64±0.30%in G2/M; as for the STIM1slient group, the ratios of cells were62.38±0.91%in G0/G1,29.53±0.71%in S,8.09±0.65%in G2/M.The majority of SGC7901cells in STIM1slient group werestaying at G0/G1, and the level in S and G2/M apparently decreased; moreover, itsSPF and PI value were smaller than blank control group and negative control group（P<0.05）, but there was no significant difference between the later two groups （P>0.05）.7. Transwell experiment showed: As for blank control group, negative controlgroup and STIM1slient group, the number of migrated cells were44.67±1.53,41.00±2.02,14.50±2.29, respectively. Compared with blank control group andnegative control group, the number of migrated cells in STIM1slient group decreasedobviously （P<0.05）, while the difference between the later two groups had no statisticsignificant （P>0.05）. The results suggest that lowering the expression of STIM1inhibited the ability of migration in SGC7901cells （P<0.05）.8. Experiment in vitro invasion: The number of invasive cells were26.33±1.53,21.67±2.52,7.33±1.23in blank control group, negative control group and STIM1slient group, respectively; compared with blank control group and negative controlgroup, the number of invasive cells in the STIM1slient group decreased obviously（P<0.05）, while the difference between the other two groups had no statisticsignificant （P>0.05）.The results imply that silencing STIM1can effectively inhibit theability of invasion in SGC7901cells.Conclusion: These results illustrated that silencing gene of STIM1canefficiently reduced intracellular calcium influx,inhibited cell proliferation, blockedcells cycles at G0/G1phase, triggered apoptosis, reduced the ability of cell invasionand migration. It indicates that the STIM1protein may be an important protein thatregulates gastric cancer cells’ proliferation, migration, invasion process, providing anew direction for the study of mechanism and metastasis of gastric carcinoma, andlay a solid foundation for further research.