Relationship And Mechanism Between ERp29 And Chemosensitivity To Gemcitabine In Lung Cancer

Background and aimsLung cancer is one of the most common malignant tumors in the world.Endoplasmic reticulum protein 29(ERp29)is located in the endoplasmic reticulum?Abnormal expression of ERp29 was observed in several types of tumors.ERp29 was associated with pathological grade,recurrence and prognosis of cancer patients.However,a potential function of ERp29 in lung cancer is poorly understood.It has been reported that ERp29 is related with radioresistance and chemoresistance of tumor cells.The proteomic analysis suggested that ERp29 precursor was upregulated in resistant pancreatic cells when compared with gemcitabine-sensitive cells.We hypothesized that ERp29 might be involved in chemosensitivity of lung cancer to gemcitabine.The main aim of our present study was to investigate the expression pattern of ERp29 in lung cancer,and the relationship between ERp29 expression and clinicopathological features of lung cancer.We then examined effects of inhibiting ERp29 expression on the cell proliferation,migration ability and chemosensitivity to gemcitabine in lung cancer cell lines.In addition,the underlying mechanism was explored.MethodsWe detected ERp29 expression in lung tumor tissues and matched non-tumor tissues(20 pairs)by Western blot.ERp29 expression in lung adenocarcinoma tissues and matched non-tumor tissues(75 pairs)was examined by immunohistochemistry(IHC).We then investigated the correlation of ERp29 expression with the clinicopathological features of these patients,including gender,age,differentiation,TNM stage,T stage and lymph node metastasis.We employed a siRNA approach to down-regulate ERp29 expression in two lung adenocarcinoma cell lines,A549 and SPC-A1 cells.The effect of down-regulated ERp29 on cell proliferation in vitro was determined by CCK-8 assay.The wound-healing assay and transwell assay were performed to examine effects of inhibiting ERp29 expression on the cell migration.A549 and SPC-A1 cells were treated with different doses of gemcitabine.The half maximal inhibitory concentration(IC50)of gemcitabine was calculated using SPSS 13.0 software.We further examined effects of inhibiting ERp29 expression on chemosensitivity to gemcitabine of lung cancer cell lines.Both A549 and SPC-A1 cells were treated with different doses of gemcitabine,ERp29 expression were detected by Western blot.We then examined the effects of combined application with gemcitabine and ERp29 siRNA on cell apoptosis,cell cycle,and expression of HSP27.Effects of inhibiting HSP27 siRNA on chemosensitivity to gemcitabine of lung cancer cell lines were examined by CCK-8 assay.ResultsWestern blot analysis showed that ERp29 expression in lung cancer tissues was significantly higher than that adjacent tissues(P<0.05).IHC staining showed ERp29 overexpression was significantly more frequent in lung adenocarcinoma tissues(55/75,73%)than that in matched non-tumor tissues(1/75,1%),?2=83.093,P<0.01.We did not observe significant associations between ERp29 and any of the clinicopathologic characteristics,including gender,age,differentiation,TNM stage,T stage or lymph node metastasis,P>0.05.Compared with the control siRNA,ERp29 siRNA significantly reduced the expression of ERp29 protein.Inhibition of ERp29 expression did not significantly affect cell growth in either A549 cells or SPC-A1 cells.Both wound-healing assay and transwell assay showed that inhibiting ERp29 significantly impaired migrating ability of both cell lines.The IC50s of gemcitabine for A549 and SPC-Alcells were 0.51±0.06?M and 4.29±0.20?M,respectively.ERp29 downregulation significantly increased the inhibition rate of cell proliferation induced by gemcitabine,and enhanced the chemosensitivity to gemcitabine in both A549 and SPC-A1 cells(P<0.05).ERp29 expression was increased when exposed to gemcitabine.After treatment with 20?M gemcitabine,apoptotic rate of A549 cells increased from 6.43±0.55%to 20.9±2.83%,and the combined application with gemcitabine and ERp29 siRNA increased the apoptotic rate to 30.8±1.41%.After treatment with 50?M gemcitabine,apoptotic rate of SPC-A1 cells increased from 6.1±1.12%to 19.82±1.76%,and the combined application with gemcitabine and ERp29 siRNA increased the apoptotic rate to 27.53±1.11%.Gemcitabine and ERp29 siRNA synergistically increased the ratio of phosphorylated HSP27 and total HSP27.Downregulation of HSP27 significantly reduced the chemosensitivity to gemcitabine in both A549 and SPC-A1 cells.ConclusionCompared with non-tumor specimens,ERp29 levels in lung tumor tissues are significantly elevated.Downregulation of ERp29 significantly increases the inhibition rate of cell proliferation and the apoptosis rate induced by gemcitabine,and enhances the chemosensitivity to gemcitabine in both A549 and SPC-A1 cells.ERp29 may affect the chemosensitivity of lung cancer cells to gemcitabine through regulating phosphorylated HSP27.ERp29 may be a new target for the treatment of lung cancer.