The Effect Of Store-operated Ca2+ Entry On The Proliferation And Epithelial-mesenchymal Transformation Of Gastric Cancer Cells Via Regulating COL1A1

Gastric carcinoma is a kind of malignancy,which ranks the fifth place of all tumors incidence rate,and the third place of all tumors death rate all over the world.Gastric carcinoma is more prevalent in China.Because of the lack of regular health examination,patient is often diagonosed as advanced gastric tumor in China.During the early stage,gastric tumor can be treated well by surgery or endoscope.While during the advanced stage,it has a worse prognosis for turmor invasion,metastasis,which need further chemotherapy combined with surgery.Moreover,the etiology of gastric tumor is associated with gene and lifestyle.Adenocarcinoma of esophagogastric junction?AEG?was first proposed by Germany surgery doctor Siewert in 1999.He classified gastric carcinoma into two groups: AEG group and cardiac gastric carcinoma group.The two groups are different obviously in etiology,clinical appearance,pathology and prognosis.In the past decades,although the incidence of gastric tumor has been decreasing all over the world,the incidence of AEG is increasing in most regions.Gastric cancer is prevalent in China.As the patients receiving gastroscopy screening in China is still fewer,most patients only go to the doctor when they have obvious clinical symptoms such as heartburn,epigastric pain,and choking on eating.At this moment,gastric cancer has developed to the advanced stage,and the operation treatment is difficult with a high recurrece.Hence,it usually requires combined chemotherapy or radiotherapy for comprehensive treatment of the advanced tumor.And it is important to study the molecular mechanisms about the proliferation,invasion and metastasis of gastric cancer and look for chemotherapy targets for gastric cancer.It will help to improve the treatment effect of gastric cancer and the prognosis of the patients.It will also reduce the pain and burden of patients.Type ? ?1 collagen?COL1A1? is a key structure protein for connection in extracellular matrix.Previous research found that COL1A1 mainly functions in the osteogenetic process and osteoporosis.Recently,some studies have reported that COL1A1 may associate with some kind of maglicant tumors and participate in the process of tumor proliferation and migration.Store-operatd Ca²⁺ entry?SOCE?is a very common mechanism to maintain intracellular Ca²⁺ homeostasis.It consists of a Ca²⁺ entry channel in cell membrane and a stromal-interacting molecule?STIM?in endoplasmic reticulum.The cell membrane channel is made of Ca²⁺-release-activated Ca²⁺ channel protein?Orai?.The STIM can detect Ca²⁺ entry and modulate Orai protein to control intracellular Ca²⁺ concentration.It is well known that Ca²⁺ is a kind of second messenger to regulate cell contraction,exocytosis,gene expression and proliferation.It is able to activate many downstream signals for various physiological functions.SOCE hase been proved in many tumor cells proliferation and migration.But its function in gastric tumor remains unclear.In most cells,STIM1 and STIM2 proteins are the key to maintain the intracellular Ca²⁺.Orai proteins usually locate on the cell membrane.They are activated to allow Ca²⁺ flows into cells when the Ca²⁺ in endoplasmic reticulum is decreased.Previous study found that Orai1 proteins and STIM1 proteins were over-expressed in many cancers,such as cervical cancer,liver cancer and glioma.And the expression level of Orai or STIM is related with survival time after operaton.Hepatocyte nuclear factor 4??HNF 4?? is a transcription factor involved in the regulation of cellular sugar,lipid metabolism,epithelial cell differentiation and inter-epithelial cell connections in the liver.Previous studies have found that it can regulate the calcium store in the cell to manipulate calcium influx by affecting the expression of STIM1 protein in the cell.In addition,hepatocyte nuclear factor 4? is also widely expressed in the epithelium of glands,kidneys,stomach and intestines.Epithelial-mesenchymal transition refers to a process in which epithelial cells undergo cytoskeleton remodeling and cell function changes after being stimulated by external signal molecules or selective expression of endogenous genes,thereby transforming them into mesenchymal cells.Epithelial-mesenchymal transition has been found to be involved in the invasion and metastasis of various malignant tumors including breast cancer,liver cancer,prostate cancer and gastric cancer.When the cell undergoes epithelialmesenchymal transition,its expression of epithelial cell characteristic proteins will decrease,such as E-cadherin.The expression of characteristic proteins of mesenchymal cells will increase,such as: N-cadherin,vinmentin and STAT3/Snai.At the same time,the ability of cells that undergo epithelial-mesenchymal transition to migrate is enhanced,and the connection between adjacent cells becomes looser,so it is easy to transfer to the distal end with blood and lymph.This study will clarify that COL1A1 affects the prognosis of gastric cancer patients and gastric cancer cell proliferation,migration,and epithelial-mesenchymal transition from the following aspects:?1?Through bioinformatics,analyze the gene chip of gastric cancer patients,and then analyze the interaction between the protein expressed by the gene,To screen out genes that may be altered in gastric cancer.Then conduct survival analysis of patients with gastric cancer to find out the genes that affect the survival time of patients;?2?Perform immunohistochemical staining on gastric cancer tissues to compare the expression of COL1A1 protein,STIM1 protein and HNF 4? protein in tumor tissue and adjacent tissues Level difference;?3?Select human gastric cancer cell line SGC7901,inhibit the expression of COL1A1 protein by si RNA transfection,and analyze the effect of COL1A1 protein on the proliferation,migration,invasion and epithelial-mesenchymal transition of SGC7901 cells by cell proliferation experiments and western blot experiments?4?Select human gastric cancer cell line SGC7901,use BTP2 or transfect Orai1 si RNA to inhibit calcium store-operated calcium influx,and analyze calcium storeoperated calcium influx on SGC7901 cell COL1A1 protein expression and epithelial interepithelialization by calcium imaging and western blotting.The impact of qualitative transformation.?5?The human gastric cancer cell line SGC7901 was selected to inhibit the expression of HNF 4? protein by si RNA transfection,and the effect of HNF 4? protein on the manipulation of calcium influx and epithelial-mesenchymal transition of SGC7901 cell calcium pool was analyzed by calcium imaging and western blotting.Through this research,we can analyze the influence of COL1A1 protein on the function of gastric cancer cells and its upstream and downstream signaling pathways,and provide new targets and ideas for non-surgical treatment of gastric cancer in the future.The research of this subject found that the genes that were significantly up-regulated in gastric cancer tissues were COL1A1 and SPP1.Among patients after gastric cancer,the survival time of the COL1A1 high expression group was significantly shorter than that of the COL1A1 low expression group,and the difference between the SPP1 high expression and low expression groups There was no significant difference in survival time after surgery.Moreover,the levels of COL1A1 protein,STIM1 protein and HNF 4? protein in gastric cancer tissues were significantly higher than those in normal tissues and adjacent tissues.Then,in the gastric cancer SGC7901 cell line,it was found that COL1A1 protein can affect cell proliferation and epithelial-mesenchymal transition.And in the gastric cancer SGC7901 cell line,the calcium store-operated calcium influx pathway can be inhibited by the calcium store-operated calcium influx inhibitor BTP2 and Orai1 si RNA,suggesting that there is a calcium store-operated calcium influx pathway in the cell.In SGC7901 cells,calcium storehouse manipulation of calcium influx can regulate the expression level of COL1A1 protein and affect the invasion and metastasis ability of SGC7901 cells.Finally,HNF 4? can regulate the storage of calcium in SGC7901 cells,the manipulation of calcium influx,the expression of STIM1 protein,and the process of epithelial-mesenchymal transition.In summary,the research of this subject has concluded that the COL1A1 gene is upregulated in gastric cancer tissues,and the expression of COL1A1 protein is elevated in gastric cancer tissues.The higher its expression level,the worse the prognosis of gastric cancer patients.Calcium storehouse manipulation of calcium influx can regulate the expression level of COL1A1 protein and the invasion and metastasis ability of SGC7901 cells,and this process is regulated by HNF 4? protein.