Expression And Significance Of JNK1, P38MAPK In Arsenic Oxide Induced Development Inhibition Of Human Cholangiocarcinoma Cell Line

ObjectiveHuman cholangiocarcinoma rank second among common malignant tumors of hepatobiliary system.At present,surgeons mainly adopt surgery methods to treat the disease,and surgical radical resection rate is low.So it is an urgent need to find more effective treatment methods.In recent years,More and more evidence found that arsenic trioxide is effective on cholangiocarcinoma,its main anti-tumor mechanisms are inhibited tumor cell proliferation and induce tumor cell apoptosis.JNK1, p38MAPK are mitogen-activated protein kinase(MAPK) family members which are important on participating in apoptosis regulation,In this study,the investigation focus on expression of JNK1,p38MAPK mRNA in arsenic trioxide inhibited proliferation of cholangiocarcinoma The aim is to find feasible clues on treatment of human cholangiocarcinoma.MethodHuman cholangiocarcinoma cell line QBC939 was cultured routinely.Taking cell of logarithmic phase for the experiment,cells were randomly divided into experimental and control groups;The experimental groups were given various concentration of arsenic trioxide,while control groups without.Morphological changes were observed by invert microscope,Transmission electronic microscope was used to investigate the ultrastructure changes in experimental and control groups;CCK-8 method:The experimental group and control group of cells were cultured in 96-well plates.Five wells were set in each concentration group.Absorbance(A value)of each well of cells was detected with microplate reader so as to calculate the cell proliferation inhibition rate;The culturing process of experimental group and control group cells was terminated in 12 hours respectively,cells number of each sample had to be reached 1×10⁶.Trizol lysis method was adopted to collect cells in order to extract total RNA.Semi-quantitative RT-PCR were performed to detect expression of JNK1 mRNA,p38MAPK mRNA.ResultCell morphology1.invert contrast phase microscope:The shape of cells in experimental groups became round with cell gap increase,cell proliferation and adherent ability decreased significantly.Detached or necrosis cells gradually increased.2.TEM:For experimental cells incubated with 8μmol / L arsenic trioxide for 24 hours,such phenomenon as nucleus heterochromatin cohesion,chromatin margination, appearance of cytoplasmic vacuoles were observed;When concentration of arsenic trioxide up to 16μmol / L,the nuclear membrane dissolves occured followed by chromatin fragmentation,so apoptosis or necrosis took place.CCK-8Various concentrations of As₂O₃ had inhibitory effect on the QBC939 cell line growth,with increased concentrations of arsenic trioxide,cell growth inhibition rate increased accordingly.The differences due to various concentration of arsenic trioxide were significant(P＜0.05) except for 4μmol/L vs.8μmol/L in 72h;Cell growth inhibition rate increased as interference time prolonged,the difference was statistically significant(P＜0.05).except for 48h vs.72h in 2μmol/L.expression of JNK1,p38MAPK mRNAIn conventionally cultured QBC939 celles JNK1,p38MAPK mRNA expressed in varying degrees.JNK1 mRNA,p38MAPK mRNA expression levels are:0.53,1.98 respectively;After Arsenic trioxide intervention,JNK1 mRNA had a significantly upregulation trend,while p38MAPK mRNA had a downregulation trend.conclusion1.QBC939 cells exist JNK1 mRNA,and p38 mRNA expression;2.As₂O₃ altered JNK1 mRNA,and p38 mRNA expression level of QBC939 cells; 3.As₂O₃ inhibited QBC939 cell proliferation,induced apoptosis,one of the molecular mechanism may be through upregulating the JNK1 mRNA expression, downregulating p38 mRNA expression.