Study On Epithelial-Mesenchymal Transition Regulated By Calreticulin And The Related Mechanism In Lung Cancer Cells

Calreticulin（CRT）is a Ca²⁺binding protein in the lumen of the endoplasmic reticulum（ER）,which has many functions and is involved in a variety of cellular signaling pathways,such as calcium homeostasis,cell adhesion,protein folding and so on.Recent reports showed that CRT played an important role in a range of different tumors,such as glioblastoma multiforme,neuroblastoma,lung cancer,breast cancer,gastrointestinal cancer,and urinary tract cancers.Epithelial to mesenchymal transition（EMT）is a specific cellular process that epithelial cells translate into mesenchymal cells in specific physiological and pathological conditions.It is increasingly acknowledged that EMT makes a greater contribution to the tumor development by enhancing migratory and invasive capabilities of cancer cells.Earlier studies had found that EMT-like changes of cellular phenotype were noticed in CRT-overexpressing Madin-Darby canine kidney（MDCK）cells.Furthermore,EMT mark gene E-cadherin mRNA expression was reduced by overexpressing CRT.In gastric cancer cells,overexpressing CRT regulated EMT marker characteristics.These results suggested that there should be a direct relationship between CRT and EMT,but the internal mechanisms are yet to be fully clarified.In this study,10 ng/m L TGF-β1 was used to induce EMT models of A549 lung cancer cells.Next,we investigated the CRT mRNA and protein expression during EMT induced by TGF-β1.The recombinant expression vectors and siRNAs were used to overexpression or knockdown the CRT expression.We studied the regulation of CRT on TGF-β1 induced EMT and lung cancer A549 cells migration.Next,we studied the possible mechanism for regulation of CRT on TGF-β1induced EMT.The mainly results were as follows:（1）Using 10 ng/mL TGF-β1,we successfully made a model of EMT in A549 lung cancer cells.We demonstrated in vitro that TGF-β1-induced EMT elevated the expression of CRT in A549 lung cancer cells.（2）We confirmed that overexpression CRT had no capacity to induce A549 lung cancer cells EMT alone,but successfully enhanced TGF-β1-induced-EMT.Furthermore,knockdown of CRT in A549 lung cancer cells significantly suppressed changes of EMT marks expression induced by TGF-β1.（3）The scratch wound migration assays were used to investigate the effect of the CRT on A549 lung cancer cells migration.The results showed that overexpression of the CRT could promote migration of A549 lung cancer cells and knockdown of the CRT could inhibit migration of A549 lung cancer cells,indicating that the CRT gene positively related to the A549 lung cancer cells migration.（4）On treatment with TGF-β1,overexpression of CRT could enhance the phosphorylation of both Smad2 and Smad3.Consistently,the knockdown of CRT by siRNA-CRT could inhibit Smad signaling pathway activated by TGF-β1.The data confirm that SB-431542 is highly effective in reducing effect of CRT overexpression on TGF-β1-induced EMT.These results indicated that CRT regulates EMT induced by TGF-β1 through Smad signaling pathway.（5）TGF-β1-induced-EMT enhanced thapsigargin（TG）-induced Ca²⁺release and store-operated Ca²⁺influx in A549 cells.An increase in TG-induced Ca²⁺release responses was noticed in CRT overexpression cells,but store-operated Ca²⁺influx was no change.CRT knockdown was able to abolish the effect of TGF-β1 on TG-induced Ca²⁺release,but had failed to reduce store-operated Ca²⁺influx.（6）The alteration of intracellular Ca²⁺concentration by TG or BAPTA-AM was able to regulate TGF-β1-induced-EMT through Smad signaling pathway.Together,these data identify that CRT regulates TGF-β1-induced-EMT through modulating Smad signaling.Furthermore,TGF-β1-induced-EMT is highly calcium-dependent;CRT was partly involved in it.The targeting of CRT to regulate EMT induction may therefore offer new therapeutic approaches for combating lung cancer metastasis.