The Role And Mechanism Of Smac In Endoplasmic Reticulum Stress And Its Mediated Apoptosis In Human Lens Epithelial Cells

BackgroundThere are variety of theories about molecular biological mechanism of cataracts'etiology,including oxidative injury of oxygen free radicals,apoptosis by mitochondrial mediate,overload of endoplasmic reticulum,imbalance of calcium ions,apoptosis of lens epithelial cell,injury/transgenation of UVA/UVB,mutation of crystalline/regulatory gene,imbalance of local homeostasis,etc.All relevant theories rely on exact experimental basis,but most studies suggest,nevertheless,structural and functional changes of lens epithelial cells?LECs?along with lesion/oncosis/apoptosis/deputy apoptosis on cells is the real molecular basis during cataract's occurrence and development.Thus,investigating changes in LECs's physiology,biochemistry and metabolism,as well as further revealing molecular biological mechanisms both are helpful for studying the reason of cataracts'occurrence and development.Meanwhile,going a step further on intervention of LECs's lesion and apoptosis could be a new breakthrough in drug prevention and treatment of cataract.Apoptosis is a programmed cell death which is self-initiated and self-regulated.Smac is a regulatory factor associated with the promotion of mitochondrial cell apoptosis and plays an important role during the induction and regulation of apoptosis.In cells,endoplasmic reticulum?ER?is responsible for the synthesis,folding,post-translational modification and transport of most proteins,and is associated with calcium ion storage and release.When the environment out of cell changes or the body is in pathological state,such as oxidative damage,ischemia,hypoxia,abnormal glucose,calcium ion abnormalities and viral infection,will interfere with the endoplasmic reticulum homeostasis,resulting in protein folding abnormalities,making unfolded/misfolded proteins accumulate substantially in the endoplasmic reticulum.If the protein is too large,the endoplasmic reticulum can not be compensated and will activate the specific signal transduction pathway in the cell-unfolded protein response,UPR),which is called endoplasmic reticulum stress?ERS?.The early aim of UPR is to maintain and restore the homeostasis of ER,decrease the protein translation speed,ER-related false protein degradation pathway activation,express the chaperone protein to enhance protein folding ability,unfolded protein or misfolding protein production,Steady state.However,when ERS is too strong or persistent,ER can not restore homeostasis,UPR will mediate cell apoptosis.In ERS process,there are three ER stress receptors,corresponding to three classical known signal pathways:inositol Essential Enzymes?IRE1?,PKR-like endoplasmic reticulum kinase?PERK?and activating transcription factor 6?ATF6?.These three signal paths work together to complete the URP process.On the one hand,endoplasmic reticulum stress state has protective effect for cell by promoting protein folding.On the other hand,it clears cells which homeostasis is completely imbalanced via the induction of apoptosis.Therefore,maintaining the moderate stress of the endoplasmic reticulum is the key to cell survival.In order to study the relationship among age-related cataract and Smac and endoplasmic reticulum stress,we conducted the pre-experiment with the most classical inositol requirement enzyme?IRE1?,taking age-related cataract patients and normal human LECs.The expression of Smac and endoplasmic reticulum stress marker GRP78 and Caspase-12 in LECs was detected by RT-PCR with GAPDH as the internal reference.The results showed that in LECs of LECs in age-related cataract patients,the levels of Smac,GRP78 and Caspase-12 were positive in LECs of age-related cataract patients,and negative in all normal LECs.We speculate that Smac and endoplasmic reticulum stress play a very important role in the process of cataract occurrence and development.There is some unknown relationship between Smac and endoplasmic reticulum stress and apoptosis.However,at present,there are no relevant reports about the mechanism between Smac and endoplasmic reticulum stress and apoptosis.This study is divided into three parts:In the first part,the difference between the expression of Smac and ERS-related marker protein and downstream protein was compared between two groups of lens of the anterior capsule,which one is from cataract patients and another is normal.In the second part,we did artificial control on the expression of Smac in human LECs through the construction of human Smac gene overexpression and down-regulation of lentiviral vector as well as in vitro transfection of human lens epithelial cells.We observed the effect of regulation on the proliferation of human LECs.Then Screening and constructing stable expression of human Smac gene overexpression and down-regulation of human lens epithelial cell line.In the third part,the oxidized stress-induced endoplasmic reticulum stress model was established by screening the appropriate H₂O₂ concentration with stable Smac overexpression and down-regulation of human lens epithelial cell lines.We also investigated the effect of Smac overexpression and down-regulation on the proliferation of human lens epithelial cells,endoplasmic reticulum stress process and its mediated apoptosis.Then our research based on the differences in the expression of related proteins,making use of immunoprecipitation and mass spectrometry analysis to find the possible existence of bridge protein and its possible pathways and mechanisms of action.Part?Difference between expression of Smac and endoplasmic reticulum stress-related proteins in cataract and normal lens epithelial cellsObjectiveTo observe the difference of Smac and ERS-associated marker protein and downstream protein expression between the anterior capsules from normal transparent lens and cataract patients'lens.MethodsCataract lens anterior capsule and epithelial cells are obtained from the simple senile cataract patients who did Phaco+IOL implantation in our hospital ophthalmic center during January 2014 to December 2014,in which there are 33 cases?50 eyes?.All operations were completed by one experienced professor.30 cases of transparent lens anterior capsule and epithelial cells are obtained by corneal transplant donor in our hospital and the other hospital during January 2014 to December 2014.The expression difference of Smac?DIABLO?,cytochrome C,JNK,caspase-9,GRP78,GRP94,caspase-12,CHOP,XBP1,GADD34 and ATF4 were detected by immunohistochemistry semi-quantitative grading.ResultsThe results of immunohistochemistry showed that:In normal human LECs,except for Cyt-c,GRP94 expression was strongly positive to positive;caspase-9,caspase-12 expression is weak positive,the remaining protein is not positive colored;Smac,JNK,GRP78,CHOP,XBP1,GADD34,ATF4 were not expressed.In cataract patients'LECs,Smac,JNK,GRP78,CHOP,ATF4,Cyt-c,GRP94 expression from positive to strong positive;caspase-9,caspase-12 expression was positive;XBP1,GADD34 expression from weak positive to positive.From the comparison we can see that Smac,Smac,JNK,GRP78,CHOP,XBP1,GADD34 and ATF4 were expressed in LECs of cataract patients and were not expressed in normal human LECs.This result indicates that the expression of Smac and ERS-related proteins is associated with the occurrence and development of cataracts.Conclusions1.Smac,JNK,GRP78,CHOP,XBP1,GADD34,ATF4 were expressed in LECs of cataract patients and were not expressed in normal human LECs.2.Caspase-12 and Caspase-9 were highly expressed in the cytoplasm of LECs in patients with cataract and only expressed sparely in the cytoplasm of normal human LECs.3.The expression of Smac and JNK,GRP78,CHOP,XBP1,GADD34,ATF4,Caspase-12 and Caspase-9 were correlated with the occurrence and development of cataract.Part?Experimental Study of Construction of Smac gene overexpression and lentiviral vector and its transfection of HLE-B3 in vitroObjectiveConstructing human Smac gene overexpression and down-regulation of gene expression vector;packaging lentivirus;expressing transfection of HLE-B3 in vitro;observing the effect of Smac overexpression and downregulation on the proliferation of HLE-B3;screening stable expressed Smac gene overexpression and down-regulation of cell lines according to Smac expression and cell proliferation.CCK-8 was used to detect the cytotoxicity of the vector.Western blot was used to detect the expression of Smac and ERS-related proteins in the transfected cells.Purification of positive cells by puromycin screening.Methods1.Construction of Lentiviral-mediated Smac Gene Overexpression Vector.Using cDNA-Smac as template,the upstream and downstream primers containing Nhe I and NotI cleavage sites were designed and amplified by PCR.Using seamless cloning and reorganization technique combine double digested plasmid pCDH-CMV-MCS-EF1-GFP-T2A-Puro and amplified target gene.After transformation of competent bacteria,selecting the correct cloning digestion,PCR and sequencing identification.Co-transfecting HEK 293TF cells;harvesting virus stock solution after 72h;using centrifugal filtration;obtaining the virus storage solution by resuspending after ultracentrifugation;determining virus titer by gradient method.2.Construction of Lentiviral-mediated Smac Gene Interference Expression Vector.Using cDNA-Smac as template,the upstream and downstream primers containing Age1 and EcoR1 cleavage sites were designed and amplified by PCR.Using seamless cloning and reorganization technique combine double digested plasmid pLenti-hU6-Puro and amplified target gene.After transformation of competent bacteria,selecting the correct cloning digestion,PCR and sequencing identification.Co-transfecting HEK 293TF cells;harvesting virus stock solution after 72h;using centrifugal filtration;obtaining the virus storage solution by resuspending after ultracentrifugation;determining virus titer by gradient method.3.Transfected cells were screened with puromycin at concentrations of 2.0?g/ml,4.0?g/ml,6.0?g/ml,8.0?g/ml and 10.0?g/ml,respectively.4.Two kinds of lentivirus were transfected into HLE-B3 cells for 72 hours.Then CCK-8 was used to detect the survival rate of blank control group,empty vector transfection group and Smac overexpression and interfering with lentiviral vector transfected cells.5.QPCR detect Smac Mrna level of blank control group,empty vector transfection group and Smac overexpression and interference expression lentiviral vector transfection group.6.Western blot was used to detect the expression of Smac protein in blank control group,empty vector transfection group and Smac overexpression and interfering expression lentiviral vector transfection group.7.Western blot was used to detect the expression of Smac protein in the cells after passage.Results1.The sequence of Smac lentivirus expression vector was identified by digestion and sequencing.The titer of GTP-SMAC overexpressing vector virus was 4×10⁸TU/ml,PM7.1-SMAC-sh1,PM7.1-SMAC-sh2,PM7.1-SMAC-sh3 virus titers were 3×10⁸TU/ml,2×10⁸TU/ml,2×10⁸TU/ml,respectively.2.After 2 days of screening,in 8?g/ml puromycin group,all cells which with no successful transfection completely died.That is,this is the best screening concentration.3.The CCK-8 assay indicated that,the survival rate of blank control cell group HLE-B3,SMAC overexpressed no-load plasmid group GTP,SMAC overexpressed cell group GTP-SMAC,SMAC interference with no-load plasmid group PM7.1,SMAC Interference Cell Group PM7.1-SMAC-sh1,PM7.1-SMAC-sh2 and PM7.1-SMAC-sh3 are?99.56±5.12?%,?98.61±4.22?%,?93.96±2.75?%,?97.86±3.97?%,?99.12±6.34?%,?102.78±4.56?%,?104.56±5.12?%respectively,which the difference was not statistically significant?F=34.564,P=0.131?.Among them,there are no statistically significant difference between GTP and PM7.1 and HLE-B3 group pairs,?P>0.05?.The difference between GTP-SMAC group and Smac interference group was statistically significant?P<0.05?.There was no significant difference between the other groups.4.In QPCR detection,Smac amplification curve and reference curve coincidence is higher;Smac dissolution curve waveform showed a single peak curve distribution;The expression levels of Smac mRNA in each group were:HLE-B3 group?1.073±0.0746?,GTP group?1.062±0.0938?,GTP-SMAC group?4.236±0.108?,PM7.1 group?1.073±0.121?,PM7.1 SMAC-sh1 group?0.962±0.078?,PM7.1-SMAC-sh2 group?0.514±0.121?and PM7.1-SMAC-sh3 group?0.173±0.0451?;Smac mRNA was significantly up-regulated in GTP-SMAC group,Smac mRNA was significantly down-regulated in PM7.1-SMAC-sh2,PM7.1-SMAC-sh3group,and the difference was statistically significant?P<0.05?compared with HEL-B3 group;There was no significant difference in Smac mRNA between PM7.1and SMAC-sh1 group and HEL-B3 group?P>0.05?;The expression of Smac Mrna in PM7.1-SMAC-sh3 group was lower than that in PM7.1-SMAC-sh2 group,the difference was statistically significant?P<0.05?.5.Western blot results indicates that,the expression levels of Smac protein in each group were as follows:HLE-B3 group?0.521±0.059?,GTP group?0.594±0.148?,GTP-SMAC group?2.580±0.255?,PM7.1 group?0.608±0.058?,PM7.1SMAC-sh1?0.568±0.106?,PM7.1-SMAC-sh2?0.306±0.0471?and PM7.1-SMAC-sh3?0.196±0.126?.The difference among these groups was statistically significant?P<0.05?.There was no significant difference between HEL-B3 group,GTP group,PM7.1 group and PM7.1-SMAC-sh1 group?P>0.05?.The expression of Smac in GTP-SMAC group was significantly higher than that in HEL-B3group?P<0.05?.The expression of Smac in PM7.1-SMAC-sh2 group and PM7.1-SMAC-sh3 group was significantly lower than that in HEL-B3 group?P<0.05?,and the decrease of PM7.1-SMAC-sh3 group was more obvious,Compared with PM7.1-SMAC-sh2 group,the difference was statistically significant?P<0.05?.Combined with QPCR results,the expression of Smac interference was determined by PM7.1-SMAC-sh3.6.The relative expressions of Smac protein in normal control group,Smac overexpressed puromycin screened cell groups,puromycin cell passaging group,Smac interference puromycin screened cell group,puromycin cell passaging group are?0.743±0.231?,?2.580±0.896?,?2.877±0.759?,?0.216±0.556?,?0.226±0.187?respectively.Overall,the difference was statistically significant?F=3126,P=0.001?.There was no significant difference between Smac overexpression of puromycin screened cell group and puromycin screening group?P=0.975?.There was no significant difference between Smac interference puromycin screening group and puromycin screening group?P=0.854?.There was significant difference between the rest groups?P=0.001?.Conclusions1.Successful construction of Smac overexpression and lentiviral expression vector.2.Lentivirus was transfected with no cytotoxicity to HLE-B3.3.Down-regulated Smac stimulate the proliferation of HLE-B3 compared with Smac overexpression.4.Exogenous Smac protein can be stably and efficiently expressed in HLE-B3.Part?Study on Effects of Smac gene overexpression and interference on HLE-B3 endoplasmic reticulum stress and its mediated apoptosis and its mechanismObjectiveThe overexpression of Smac and the down-regulation of lens epithelial cell lines were modeled,which is for constructing the oxidative stress model by screening the appropriate H₂O₂ concentration,investigating the effect of Smac gene overexpression and interference on HLE-B3 proliferation,endoplasmic reticulum stress and its mediated apoptosis.And based on the differences in the expression of related proteins,the use of immunoprecipitation and mass spectrometry analysis may help to find the possible existence of bridge protein and its possible pathways and mechanisms of action.MethodsThe survival rate of each group was detected by CCK-8 after adding H₂O₂.According to the experimental results,200?M H₂O₂ was selected as the experimental concentration.The experiment was divided into the following 10 groups:HLE-B3group,GTP group,GTP-SMAC group,PM7.1 group,PM7.1-SMAC-sh3 group,200?M H₂O₂+HLE-B3 group,200?M H₂O₂+GTP group,200?M H₂O₂+GTP-SMAC group,200?M H₂O₂+PM7.1group,200?M H₂O₂+PM7.1-SMAC-sh3 group.MTT assay is used to detect the inhibitory effect of each group;CCK-8 to detect the proliferation of cells in each group;RT-PCR is used to detect the expression levels of Smac,ATF4,caspase-12,CHOP,GRP78 and JNK mRNA;Western blot is used to detect the expression levels of Smac,ATF4,caspase-12,CHOP,GRP78 and JNK protein.Immunoprecipitation was used to find Smac and ERS related proteins,and the results were analyzed by mass spectrometry.Results1.MTT results indicate that:different concentrations of H₂O₂ had different inhibitory effects on HLE-B3 cells in a dose-dependent manner.Half of the inhibitory concentration?IC50?is 270?M,high concentration?1000pM?H₂O₂ treatment will cause the cells almost all died after 24h.As the exogenous H₂O₂ changed the original size and morphology of the cells,and this experiment at the same time on the Smac expression and down-regulation,thus the concentration of 200?M H₂O₂ as the recommended dose of in vitro oxidative damage model.2.CCK-8 results indicate that:without H₂O₂ environment,the survival rates of HLE-B3 group,GTP group,GTP-SMAC group,PM7.1 group and PM7.1-SMAC-sh3group were?99.56±5.12?%,?98.61±4.22?%,?93.96±2.75?%,?97.86±3.97?%,?104.56±5.12?%,and the statistical results are the same as the second part;after adding 200?M H₂O₂,the survival rates of the five groups were?59.339±4.895?%,?63.943±5.125?%,?39.960±3.683?%,?61.193±33.065?%,?90.560±3.574?.The difference between the groups was statistically significant?F=57.06,P<0.001?.The survival rate of 200?M H₂O₂+GTP-SMAC group was lower than that of normal cell group?P=0.001?.The survival rate of 200?M H₂O₂+PM7.1-SMAC-sh3 group was higher than that of normal cell group and GTP-SMAC group?P=0.001?.There was no significant difference between 200?M H₂O₂+HLE-B3 group,200?M H₂O₂+GTP group and 200?M H₂O₂+PM7.1 group?P>0.05?.3.RT-PCR detection results indicate that:without H₂O₂ treatment,Smac,ATF4,GRP78,Caspase-12,CHOP mRNA were highly expressed in GTP-SMAC group.Compared with HLE-B3 group,the difference was statistically significant?P<0.05?.Smac,ATF4,GRP78,Caspase-12 and CHOP mRNA were down-regulated in PM7.1-SMAC-sh3.Compared with HLE-B3 group,the difference was statistically significant?P<0.05?.There was no significant difference in mRNA between HLE-B3group,GTP group and PM7.1 group?P>0.05?.For JNK mRNA expression,no statistically significant difference between the groups?P>0.05?.After H₂O₂ treatment,Smac,ATF4,GRP78,Caspase-12,CHOP mRNA were expressed in 200?M H₂O₂+GTP-SMAC group.Smac,ATF4 and Caspase-12 mRNA were down-regulated in200?M H₂O₂+PM7.1-SMAC-sh3.Compared with 200?M H₂O₂+HLE-B3 group,the difference was statistically significant?P<0.05?.There was no significant difference between the two groups?P>0.05?in the 200?M H₂O₂+HLE-B3 group,200?M H₂O₂+GTP group and 200?M H₂O₂+PM7.1 group.JNK mRNA expression in each group was not statistically significant?P>0.05?.4.Westen-blot detection results indicate that:without H₂O₂ treatment,Smac,ATF4,CHOP,GRP78 were highly expressed in GTP-SMAC group.Smac,ATF4,CHOP,down-regulated in the PM7.1-SMAC-sh3 group.Compared with HLE-B3group,the difference was statistically significant?P<0.05?.There was no significant difference between JNK expression group?P>0.05?and the other proteins were statistically significant?P<0.05?.There was no significant difference in the expression of HLE-B3 group,GTP group and PM7.1 group between the groups?P>0.05?.After H₂O₂ treatment,Smac,ATF4,Caspase-12,CHOP,GRP78,JNK were expressed at200?M H₂O₂+GTP-SMAC and down-regulated in 200?M H₂O₂+PM7.1-SMAC-sh3group.Compared with 200?M H₂O₂+HLE-B3 group,the difference was statistically significant?P<0.05?.The expression of each protein was not significantly different between 200?M H₂O₂+HLE-B3 group,200?M H₂O₂+GTP group and 200?M H₂O₂+PM7.1 group,the difference was not statistically significant?P>0.05?..5.Immunoprecipitation and mass spectrometry results indicate that:The obtained CO-IP pull-down proteins were:CCDC171,TSNARE1,LRRC29,cadherin-23,PDE1C,IGFBP5,MELTF,NDUFS4.Conclusions1.Smac overexpression or downregulation had no effect on normal HLE-B3proliferation.Smac gene overexpression will exacerbate decrease of oxidative stress-induced HLE-B3 survival rate.Smac gene down-regulation will play a protective role on oxidative stress-induced decreased HLE-B3 survival rate.2.Smac overexpression can up-regulate ERS,up-regulate ERS-related apoptotic protein,increase HLE-B3 apoptosis by up-regulating the expression of stress-related proteins in endoplasmic reticulum.The downregulation of Smac can inhibit the apoptosis of HLE-B3 induced by endoplasmic reticulum stress pathway,which has a protective effect on the oxidative damage of cells.This can also down-regulate the expression of stress-related proteins in endoplasmic reticulum,inhibit excessive ERS,and reduce the apoptosis of human HLE-B3.3.After HLE-B3 is oxidized,Smac overexpression can aggravate endoplasmic reticulum-mediated apoptosis by up-regulating the effector protein of endoplasmic reticulum stress pathway.Smac down-regulation can inhibit the apoptosis of endoplasmic reticulum pathway by down-regulating the apoptotic proteins of endoplasmic reticulum stress pathway,which has protective effect on oxidative stress-induced apoptosis.4.PDE1C,IGFBP5 may be the bridge proteins between Smac and ERS.