STIM1 Knockdown Attenuates Migration And Tumorigenic Ability Of EJ Cells By Down-regulating SOCE

The ubiquitous second messenger Ca²⁺plays an important role in the regulation of cell cycle,growth,apoptosis,migration,contraction and gene expression.The regulation of intracellular Ca²⁺homeostasis is affected by extracellular Ca²⁺invoked through the calcium channel into the cell and the intracellular calcium store.Many diseases are happened with abnormal Ca²⁺motivation,including hypertension,cardiovascular disease,Alzheimer’s disease and cancer.Ca²⁺channels mainly include voltage-gated calcium channels（VGCC）,ligand gated calcium channels（LGCC）,sodium calcium exchanger（NCX）,store-operated calcium channel（SOCE）,receptor-operated calcium channels（ROCE）,and calcium channels regulated by arachidonic acid（ARC）.Store-operated calcium entry is mediated by extracellular Ca²⁺involved in its core protein located in the endoplasmic reticulum by stromal interaction molecule and located on the cell membrane Orai/TRPC protein.In Resting condiutions,STIM1 evenly distributed on the ER membrane,once the calcium release from ER,membrane STIM1 can sense within ER,the process is that once calcium store depleted,STIM1 itself could aggregate and translocated to cell membrane,coupling with the cell membrane of the four transmembraned protein Orai1 and TRPCs,the opening of Orai/TRPCs channels which induced extracellular calcium influx into the cell.Many studies have shown that the expression of STIM1 is associated with tumor process and clinical outcome.Moreover,the expression level of STIM1 and the primary tumor process and metastasis directly related to interfering the expression of STIM1 will not only lead to turbulent Ca²⁺flow in cell,and inhibiting STIM1 will arrest the proliferation of cancer cells,because of downregulating of cell cycle protein CDC25C and up regulation of anti-proliferative protein p21.The introduction of exogenous STIM1 into SiHA cells,compared with the normal control group,the overexpression of STIM1 could lead to the proliferation of tumor cells in nude mice.By comparing the expression of STIM1,we found that there was a positive correlation between STIM1 and tumor growth in 295 patients in breast cancer.Compared with the control group,the survival of patients with breast cancer had a sharp decline with low level of the STIM1 than the original high expression of mRNA.In summary,these data suggest that STIM1 is involved in the occurrence and development of malignant tumor,and play an important role.A large number of research points in breast cancer,colon cancer,cervical cancer,liver cancer,nasopharyngeal cancer,skin cancer,glioma,melanoma and so many cancers with the downregulation of STIM1/Orai1 can inhibit tumor growth and induce apoptosis.Bladder cancer is the most common malignant tumor of urinary system,and is first malignant tumors in the urinary system,with the feature of infinite proliferation and highly metastatic.It is pointed out that the cause of the currently situation is mainly focus on virus or some chemical factors acting on the body,in the end causing oncogene activation of cancer gene.Tumor formation,proliferation,migration and metastasis are accompanied with changes in ion channels,especially SOCE related proteins,and we know that Ca²⁺plays an important role in the regulation of tumorigenesis.In addition to the regulation of tumor growth and apoptosis,many studies have shown that STIM1 affects tumor metastasis,and many gene changes can affect the expression level of STIM1,which leads to abnormal growth,proliferation and metastasis in cancer patients.The process of SOCE reflects the STIM1,Orai1 and TRPCs activity,the direct effect of silencing STIM1 could down regulate the level of SOCE,so if we measure by MTT methods in cancer cells with 2-APB,SKF96365,Gd³⁺and AnCoA4 so we can verify the effect of SOCE on the proliferation actions.More and more studies in vivo and in vitro showed that STIM1 is critical in this process,so we want to explore the role of STIM1 in EJ cells.Cell clone formation experiment is an important means to detect the survival rate of inoculated cells,can shoe specific reaction treated cells after inoculation into live adherent ability and clone number,with not all adherent cells has proliferation and colony forming ability,because of the colony formation reaction is dependent and specific groups of cell proliferation.1.The expression of STIM1,Orai1 and TRPC1/3/6/7 in mRNA level in human bladder cancer cell line EJIn order to detect SOCE exist in human bladder cancer cell line EJ cells,we firstly examined the expression of the SOCE component of the certain protein in the mRNA level,human renal epithelial cells HEK293 mRNA as positive reference,with different the annealing temperature detected in HEK293 by RT-PCR to make sure the protein expression in the mRNA level.Under the same conditions,respectively using STIM1,Orai1 and TRPC1/3/4/5/6/7/GAPDH specific primers,PCR product of corresponding size,according to the principle of molecular sieve,determine the related gene expression by product size.Ultimately to determine the STIM1 EJ cells,Orai1 and TRPC1/3/5.The RT-PCR and Western blot are determining the expression level of STIM1.2.SOCE is existed in Human bladder cancer cell line EJIn order to confirm the existence of SOCE in EJ cells of human bladder cancer,this study intends to use 2 mmol/L Tg（SERCA pump irreversible inhibitor）in human bladder cancer cell line EJ,Tg is the SERCA pump irreversible inhibitor,which leads to a decrease in the calcium ion pump,breaking the ER membrane calcium ion homeostasis,so Ca²⁺can release into the cytosol,with calcium store depleted under physiological conditions,so we can detect changes in SOCE in EJ cells by calcium imaging to detect Ca²⁺influx.Experiments show that EJ cell at 2 mmol/L after Tg stimulation can produce significant influx of Ca²⁺,at the same time,with the application of SOCE inhibitors 2-APB,SKF96365,Gd³⁺and AnCoA4,Ca²⁺influx decreased significantly,indicating the existence of store-operated calcium entry namely SOCE.3.A stable STIM1 cell line was constructed by lentivirus packaging of shSTIM1We want to study whether SOCE participate in the process of human bladder cancer process,the main characteristic of cancer cells is highly reproductive,easy to metastasis.As an vital intracellular second messenger,its role is particularly important in exocytosis,cell activity,enzyme activity,calcium oscillation,muscle contraction,gene transcription,cell proliferation,inflammatory reaction,apoptosis and carcinogenesis.So we hope to observe the role STIM1 in EJ cells by silencing STIM1 to down regulation of SOCE level.As ShSTIM1 plasmid transfected into cells,firstly through the YFP fluorescence label plasmid can measure transfection efficiency.In EJ cells by immunofluorescence,and transfection efficiency is low.In order to improve the efficiency of silencing,then wo can use lentiviral packaging shSTIM1 to get better silencing effect.The protein expression level of STIM1 can determine the vector silencing efficiency.In the process of SOCE,STIM1 can translocate to cell membrane,by calcium imaging technology,compared with the mock group found significantly decreased the Ca²⁺influx.Thus selective silencing of STIM1 stable cell can verify by Western blot and imaging technology.4.Downregulation the level of SOCE induced apoptosis and arrested viability of EJ cellsApoptosis is a complex process that generally existed in physiological conditions,with the cytoplasm,mitochondria,cell volume closely,secondly,the nucleus fractured into fragments with different sizes,then there will be apoptotic bodies swallowed by macrophage phagocytosis,so we will find this process by detecting the related apoptosis index inEJ cellsafter silencing STIM1.Bcl-2 is an anti apoptotic protein that is protective protein,when cell apoptosis with low level of Bcl-2 and highly expressed of the corresponding Bax,the Bcl-2/Bax ratio can be judged by EJ cells after silencing STIM1 by JC-1 staining.In survival state and found that cells showed early mitochondrial membrane potential decreased,becasuse of JC-1 cannot be gathered in the same mitochondria.EJ cell line was detected by MTT within SKF96365,2-APB,Gd³⁺,and AnCoA4 showed low level of viability under the effect of down-regulation of SOCE by inhibitors.5.Selective silencing of STIM1 caused low level of the migration and reduced the ability of tumor formation in EJ cellsCell migration,also known as cell crawling or cell movement is an normal style after the received signal,scratch test and colony formation experiments can detect effects of a gene on the tumor cell’s physiological state,in an intuitive mode because of pictures.The clone formation experiment is testing the survival rate of cells,direct response in inoculation,this is because the adherent cells not all single cells has proliferation and colony forming ability,so under strictly conditions,colony formation reaction is dependent and showing proliferate ability.After selective silence STIM1,we can find that compared with the control group,shSTIM1 group does migration significantly inhibited.We also found that silencing STIM1 can indeed reduce the tumorigenicity of EJ cells.6.Silencing STIM1 can induce lower Phosphorylation of AKTIn theory,extracellular messenger activates GPCRs,followed by PIP₂dehydrateing IP₃ and DAG PLC hydrolysis generated,IP₃ can cause calcium store depletion,then activate SOCE in EJ cells.The development of bladder cancer is a multistep,which is a complex physiological process,involving oncogenes and tumor suppressor genes,signal transduction and cell cycle protein imbalance.Although many research focusing on AKT,but in bladder cancer,AKT and pAKT in the occurrence and development of bladder cancer is less.So it is necessary for the next study.