ERp29 Induces Mesenchymal-epithelial Transition Of Colorectal Cancer Cell In Endoplasmic Reticulum Stress

Background and ObjectiveColorectal cancer(CRC)is one of the high hazard malignant tumors in the worldwide.In China,the incidence of colorectal cancer is still continues to increase every year.Invasion and metastasis is an important cause of recurrence and death in patients with colorectal cancer.Therefore,it is vitual content to find out the related predictors of invasion and metastasis in CRC and investigate the molecular mechanisms of tumorigenesis and metastasis.The development of CRC is a process with multifactorial.In recent years,studies indicate that Endoplasmic reticulum stress may be involved in the development and progression of tumors.A number of stimulation in human body can cause the unbalance of homeostasis in endoplasmic reticulum,which results in endoplasmic reticulum stress(ERS).ERS is charactered by normal protein synthesis disturbed and the accumulation of unfolded and misfolded proteins in the ER.ERS can activates intracellular signaling pathways termed the unfolded protein response(UPR),which enhanced the ability of protein translation and folding,resolves the protein folding defect,attenuate protein synthesis under the translation level,and the other is the up-regulation of the genes encoding the ER chaperones in the ER.Chaperone protein namely heat shock protein or stress protein.ER molecular chaperone protein may be involved in the recognition and stability of partially folded polypeptide chain conformation,in the folding and assembled of polypeptide chain.Chaperone proteins also participate in the transportation of transmembrane protein and the repairment of thermal denaturation proteins.ERp29 protein is a novel endoplasmic reticulum protein,which expressed in a variety of human tissues.It is located on chromosome 12,and molecular weight is 29KD.Studies have shown that ERp29 is expressed in a variety of human malignancies for instance naasopharyngeal carcinoma,basal cell carcinoma,breast cancer.Our previous clinical samples studies have indicated that ERp29 is related to the poor prognosis of colorectal cancer.But whether ERp29 play a role in colorectal cancer with endoplasmic reticulum stress,as well as its mode of action is unclear.In our study,according to the endoplasmic reticulum(ER)stress induced by tunicamycin in vitro.We will investigate the relationship between ERp29 and ERS.If they does,we will explore the mechanism of ERp29 and its downstream related molecules in colorectal cancer proliferation,invasion and metastasis.And provide a new theoretical basis for clinical prevention and treatment of colorectal cancer.Method1.The endoplasmic reticulum(ER)stress of colorectal cancer cell lines was induced and the corresponding cell biological characteristic.1.1 The endoplasmic reticulum stress of CSCs induced by tunicamycin in vitro.1.2The expression level of molecular chaperone protein(GRP78,GRP94,ERp29)related to ERS detected by westernblot.1.3 The corresponding cell biological characteristic detected by CCK-8,Wound-healing assay,Transwell.2.Inhibit the formation of endoplasmic reticulum stress in colorectal cancer cell lines and the corresponding cell biological characteristic.2.1 The 4-phenylbutyric acid inhibits the endoplasmic reticulum stress of CSCs induced by tunicamycin in vitro.2.2 The expression level of molecular chaperone protein(GRP78,GRP94,ERp29)related to the inhibited of ERS detected by westernblot.2.3 The corresponding cell biological characteristic detected by CCK-8,Wound-healing assay,Transwell.3.The functional alterations of CRC cell lines with ERp29-overexpression in vitro.3.1 The expressions of ERp29 gene in seven colorectal cancer cell lines(SW620,SW480,LS174T,HT29,LoVo,DLD1 and HCT116)were detected by westernblot.3.2 Stable cell lines were established by transfected lentiviral vectors.ERp29-overexpression cell lines(SW620/ERp29,HCT116/ERp29,HT29/ERp29)and negative control groups(SW620/vector,HCT116/vector,HT29/vector).The efficiency of the overexpression was detected by quantitive real time PCR and Western blot.3.3 Assessed the functional effects of ERp29-overexpression on tumor cell proliferation,invasion and metastasis by CCK-8,Transwell,Wound-healing assay,colony formation,cell cycle,cell apoptosis experiment.4.The preliminary screening of ERp29-associated proteins4.1 The differential display of proteins between the ERp29-overexpression(SW620/ERp29)and the control cell line(SW620/vector)is determined by two-dimensional electrophoresis.4.2 The distribution of different protein is observed by confoeal laser scanning microscopy in SW620 and HCT116.4.3 The expressions of different proteins determined by two-dimensional electrophoresis in ERp29-overexpression cell lines(SW620/ERp29,HCT116/ERp29,HT29/ERp29),the control cell lines(SW620/vector,HCT116/vector,HT29/vector)and colorectal cancer cell lines(SW620,HCT116,HT29).4.4 The expression level of SSR4 in the endoplasmic reticulum stress which is detected by western blot5.The preliminary mechanism research of Endoplasmic reticulum stress in the invasion and metastasis of colorectal cancer5.1 The morphological of cell has changed which is stimulated by tunicamycin5.2The endoplasmic reticulum stress involved in the process of Mesenchymal-Epithelial Transition which isdetected by Western Blot and confoeal laser scanning microscopy5.3The overexpression of ERp29 involved in the process of Mesenchymal-Epithelial Transition which is detected by Western BlotResults1.The endoplasmic reticulum(ER)stress of colorectal cancer cells was induced and the corresponding cell biological characteristic.1.1 The optimal concentration is 20?mol/L with different concentrations of tunicamycin stimulated CRC cell line(SW620)which is detected by westemblot.1.2 The optimal concentration is 20?mol/L with different concentrations of tunicamycin stimulated CRC cell line(HCT116)which is detected by westernblot.1.3 The effect time is more than 12h with tunicamycin of 20 ?mol/L stimulated CRC cell line(SW620)different time points(0 h,12 h,24 h,36 h,48 h and 72 h)which is detected by westernblot.1.4 The effect time is more than 12h with tunicamycin of 20?mol/L stimulated CRC cell line(HCT116)different time points(0 h,12 h,24 h,36 h,48 h and 72 h)which is detected by westernblot.1.5 The increasing expression of ERp29 in CRC cell lines(S W620 and HCT116)stimulated by the optimal concentration and the effect time of tunicamycin was detected by westernblot.1.6 The results from CCK-8 assay revealed that,the growth of CRC cells lines(SW620 and HCT116)stimulated by tunicamycin of 20?mol/L decreased markedly compared with the control group(P<0.05).1.7 The results from Wound-healing assay revealed that,the scratch area changes of CRC cells lines(SW620 and HCT116)stimulated by tunicamycin of 20?mol/L decreased markedly compared with the control group(P<0.05).The Scratch area changes were observed under the inverted phase contrast microscope in different times(0h,24h,48h).1.8 The results from Transwell miagration assay revealed that,the migration abilities of CRC cells lines(SW620 and HCT116)stimulated by tunicamycin of 20?mol/L is significantly reduced compared with the control group(P<0.05).2.Inhibit the formation of endoplasmic reticulum stress in colorectal cancer cell lines and the corresponding cell biological characteristic2.1 The optimal concentration is 5mmol/L with different concentrations of 4-phenylbutyric acid stimulated CRC cell line(SW620)with endoplasmic reticulum stress which is detected by westernblot.2.2 The optimal concentration is 5mmol/L with different concentrations of 4-phenylbutyric acid stimulated CRC cell line(HCT116)with endoplasmic reticulum stress which is detected by westernblot2.3 The effect time is 48h with tunicamycin of 20?mol/L and 4-phenylbutyric acid of 5mmol/L stimulated CRC cell line(SW620)at the same time compared with the control group,single use tunicamycin or 4-phenylbutyric acid which is detected by westemblot.2.4 The effect time is 24h with tunicamycin of 20?mol/L and 4-phenylbutyric acid of 5mmol/L stimulated CRC cell line(HCT116)at the same time compared with the blank control group,single use tunicamycin or 4-phenylbutyric acid which is detected by westemblot.2.5 The decreasing expression of ERp29 in CRC cell line(SW620)stimulated by tunicamycin of 20?mol/L and 4-phenylbutyric acid of 5mmol/L for 48h which is detected by westemblot.2.6 The decreasing expression of ERp29 in CRC cell line(HCT116)stimulated by tunicamycin of 20?mol/L and 4-phenylbutyric acid of 5mmol/L for 24h which is detected by westemblot.2.7 The results from CCK-8 assay revealed that,the growth of CRC cells line(SW620)stimulated by tunicamycin of 20?mol/L and 4-phenylbutyric acid of 5mmol/L increased compared with the tunicamycin group(P<0.05);The results from Wound-healing assay revealed that,the scratch area changes of CRC cells line(SW620)stimulated by tunicamycin of 20?mol/L and 4-phenylbutyric of 5mmol/L increased compared with the tunicamycin group(P<0.05).The scratch area changes were observed under the inverted phase contrast microscope in different times(0h?12h?24h);The results from Transwell miagration assay revealed that,the migration abilities of CRC cells line(SW620)stimulated by tunicamycin of 20?mol/L and 4-phenylbutyric of 5mmol/L increased compared with the tunicamycin group(P<(0.05).2.8 The results from CCK-8 assay revealed that,the growth of CRC cells line(HCT116)stimulated by tunicamycin of 20?mol/L and 4-phenylbutyric acid of 5mmol/L increased compared with the tunicamycin group(P<0.05);The results from Wound-healing assay revealed that,the scratch area changes of CRC cells line(HCT116)stimulated by tunicamycin of 20?mol/L and 4-phenylbutyric of 5mmol/L increased compared with the tunicamycin group(P<0.05).The scratch area changes were observed under the inverted phase contrast microscope in different times(Oh?12h?24h);The results from Transwell miagration assay revealed that,the migration abilities of CRC cells line(HCT116)stimulated by tunicamycin of 20?mol/L and 4-phenylbutyric of 5mmol/L increased compared with the tunicamycin group(P<(0.05).3.The functional alterations of CRC cells lines with ERp29-overexpression in vitro3.1 We use western blot experiments to analyse the expression of ERp29 in colorectal cancer cell lines(SW620,SW480,LS174T,HT29,LoVo,DLD1,HCT116).The result show that SW620,HT29 and HCT116 have a lower expression of ERp29 protein.3.2 We use Lentiviral vectors to transfect CRC cell lines(SW620,HCT116,HT29)then subjected to FACS analysis for GFP expression.The results from westernblot and real time PCR showed that stable expression cell lines were successfully established(P<0.01).3.3 The results from CCK-8 assay revealed that compared to the control group,the growth abilities of ERp29-overexpression cells has significantly increased(P<0.01).The results from wound-healing assays revealed that compared to the control group,the scratch area changes of ERp29-overexpression cells has no significantly increased(P>0.05).The results from the cell cycle revealed analyzed by flow cytometry that compared to the control group,cells'proliferation rate of ERp29-overexpression cells has no significant decreased(P>0.05).The results from the cell apoptosis analyzed by flow cytometry,showed that compared to the control group,cells'apoptosis rate of ERp29-overexpression cell has no significant decreased(P>0.05).The results from transwell miagration assay showed that compared to the control group,the migration abilities of ERp29-overexpression cells was significantly different(P<0.01).The results from colony formation experiment showed that compared to the control group,the colony formation rates of ERp29-overexpression cells was significantly different(P<0.01).4.The preliminary screening of ERp29-associated proteins4.1 We have screen 20 kinds of differential expression protein of ERp29-overexpression(SW620/ERp29)and and the control cell line(SW620/vector).Analyse their function,then we choose four protein for the further study.They are SSR4,TBCA,CUL5,and RPA2.4.2The distribution of the four different proteins is observed by confoeal laser scanning microscopy in SW620 and HCT116.The result suggest that co-localization of ERp29 and SSR4,TBCA,CUL5,RPA2 in colorectal cancer cell lines respectively(SW620 and HCT116).4.3The expression level of TBCA,CUL5,RPA2 among the same group subjects has no significant different.But the expression level of SSR4 between the ERp29-overexpression cell lines and the control cell lines has significant different.4.4 The expression level of SSR4 in the endoplasmic reticulum stress has increased which is detected by western blot suggest that ERp29 and SSR4 participate in ERS.5.The preliminary mechanism research of Endoplasmic reticulum stress in the invasion and metastasis of colorectal cancer5.1 The morphological changes of cell which is stimulated by tunicamycin has observed by similar to the shape of the fibroblasts to "pebbles" shape.5.2 Cells with ERS exhibited weak expression levels of Vimentin,?-catenin,and N-cadherin,and highly expressed E-cadherin.5.3 ERp29-overexpressing cells exhibited weak expression levels of Vimentin,?-catenin,and N-cadherin,and highly expressed E-cadherin.Conclusion1.We have successfully build and block the model of endoplasmic reticulum stress in colorectal cancer cell lines(SW620 and HCT116).2.The growth and migration abilities of colorectal cancer cell lines have decreased in ERS.3.ERp29 has increased in the process of the ERS in colorectal cancer cells.4.The colony formation rates and migration abilities have increased in ERp29-overexpression cell lines.5.Colorectal cancer cells with endoplasmic reticulum stress is associated with the MET process.New FindingsThe aim of our study is to observe the function of ERp29 under ERS,which may contribute to the the underlying mechanism metastasis of CRC.