| Part I.The research of endoplasmic reticulum stress affect ACC cell biology in vitroObjective: To explore the effect of endoplasmic reticulum stress on the biological behavior of human adrenocortical carcinoma cells in vitro.Methods: Using endoplasmic reticulum stress regulator Thapsigargin(TG)to intervene in ACC cell lines(SW-13 cells and NCI-H295 R cells);CCK8 method was used to detect the proliferation of the two cell lines;flow cytometry was used(FITC/PI method),Hoechst 33258 method,transmission electron microscope to detect the effect of TG on apoptosis of ACC cells;Transwell test to detect the effect of TG on the migration and invasion ability of ACC cells;cell scratch test to detect endoplasmic reticulum stress inducer TG on the migration ability of ACC cells;flow cytometry was used to detect the effect of endoplasmic reticulum stress inducer TG on ACC cell cycle.Results:(1)After the intervention of endoplasmic reticulum stress inducer TG,the proliferation capacity of ACC cell line weakened.(2)The apoptosis rate of the two groups of cells was significantly increased compared with the control group.(3)Cell invasion was significant difference in migration ability before and after TG intervention.After TG intervention,the invasion and migration ability of ACC cells decreased.(4)After TG intervention,the ratio of ACC cell cycle changed,and G0/G1 phase arrest occurred in both cell lines.Conclusion: Endoplasmic reticulum stress inducer TG can affect the proliferation,invasion,migration and apoptosis of ACC cell lines in vitro.Part II.The research of endoplasmic reticulum stress affect ACC growth and apoptosis in vivoObjective: To explore the effect of endoplasmic reticulum stress on the growth and apoptosis of adrenocortical carcinoma in vivo.Methods: The human adrenal cortical carcinoma cell line SW-13 cells were injected into nude mice to construct a model of adrenal cortical carcinoma xenograft in nude mice.After the transplanted tumor was successfully modeled,endoplasmic reticulum stress inducer TG were used for intervention,to depict the tumor growth curve and the body weight curve of nude mice,and to obtain the transplanted tumor tissue of nude mice after completing the intervention.Detect the size of the transplanted tumor and the apoptosis in the tumor tissue.Results:(1)30 nude mice were constructed with subcutaneous xenograft tumor models.The results showed that 24 nude mice formed tumors with a tumor formation rate of 80%.After staining with HE slices,they showed typical dense arrangement of tumor cells,disordered structure and large nuclei.Deep staining,irregular interstitial cells.(2)After the transplanted tumor of nude mice was formed,TG was used to intervene.Before intervention,the weight of nude mice was 19.01 ± 0.25 g in normal saline control group and 19.15 ± 0.87 g in TG group;after TG intervention,there was no significant difference in the weight between these two groups before and after intervention.(3)After the TG intervention,the size of the transplanted tumors in the TG group and the saline control group were 1.6 ± 0.85g(TG group)and 3.96 ± 0.49g(saline control group),the difference between the two groups was significant difference(P<0.05),the inhibition rate of tumor size in TG group reached 60%.(4)After TG intervention,the apoptosis rates of subcutaneously transplanted tumor cells were 5.27 ± 1.01%(saline control group)and 36.48 ± 9.77%(TG intervention group),the difference between the two groups was significant difference(P<0.05).Conclusion: Endoplasmic reticulum stress inducer TG can affect ACC proliferation and induce apoptosis in vivo.Part III The mechanism of endoplasmic reticulum stress affecting the biological behavior of ACC in vivo and in vitroObjective: To explore the mechanism of the effect of endoplasmic reticulum stress on the biological behavior of ACC in vivo and in vitro.Methods: Collect specimens before and after TG intervention in ACC in vitro and in vivo,and detect the expression of endoplasmic reticulum stress-related genes and proteins,JNK signaling pathway-related genes and proteins,apoptosis-related genes and proteins by RT-PCR and WB.Results:(1)In the vitro experiment,after intervention with different concentrations of TG,the expressions of JNK and p-JNK proteins in the two cell lines increased compared with the control group(P<0.05).(2)In vivo experiments,after TG intervention,the expression of endoplasmic reticulum stress-related pathway proteins and JNK pathway proteins JNK,p-JNK,ERK,p-ERK,IRE1?,MAPK,p-MAPK,PERK,p-PERK,GRP78 was higher than that of normal saline control group(P<0.05).(3)In the in vitro experiment,after SW-13 cells and NCI-H295 R cells were intervened with TG of different concentrations,the relative expression of JNK,ATF6,PERK,LC3 B,HSAP,bal-2 genes were higher than that of the control group(P<0.05).(4)In vivo experiments,the relative expression of related genes JNK,ATF6,PERK,LC3 B,HSAP,bal-2 in the TG group was higher than that in the control group(P <0.05).Conclusion: The endoplasmic reticulum stress mainly activates the JNK signaling pathway and further induces apoptosis to affect the biological characteristics of ACC cells both in vitro and in vivo.Part IV si RNA MAPK8(JNK1)recombinant lentivirus construction low expression of MAPK8 stable ACC cell line and its biological behaviorObjective: To construct recombinant lentiviral vectors with MAPK8 si RNA fragments,and further construct stable and low-expression MAPK8 monoclonal SW-13 cell lines to detect the biological characteristics of SW-13 cells interfered with by MAPK8 si RNA.Methods:(1)Use the selected MAPK8 si RNA as the most effective fragment to silence the MAPK8 gene.A double-stranded DNA oligo was prepared,cloned into the lentiviral expression vector GV493 using gene recombination technology,and the DNA sequencing identified the recombinant lentiviral vector.293 T cells were co-transfected with the lentiviral packaging auxiliary plasmid and the prepared recombinant lentiviral vector containing MAPK8 si RNA,the virus particles were collected and the virus titer was determined by the well dilution method.In addition,the negative sequence virus of Shanghai Jikai Company was used as a negative control.(2)Infect the adrenal cortex cancer cell line SW-13 with virus,set up 5 virus titer groups,and detect the MAPK8 inhibition rate by fluorescence quantitative PCR,and select the best inhibition rate as the next experiment.(3)MTT method was used to detect the growth curve of SW-13 cells interfered by MAPK8 si RNA,flow cytometry to detect cell apoptosis and cell cycle,Transwell to detect cell invasion ability,and cell scratch test to detect cell migration ability.Results:(1)The recombinant lentiviral RNA interference expression vector for MAPK8 was successfully constructed by sequencing;the recombinant lentiviral particles were successfully collected after packaging the recombinant lentiviral vector in 293 T cells,and the virus titer was 9.0E + 8TU/ml.Adopting 6 virus titers of recombinant lentiviral particles to infect adrenal cortical cancer cells,the infection efficiency is greater than 80%;fluorescent quantitative PCR identified the best inhibitory efficiency of adrenal cortical cancer cell lines SW-13,the optimal MAPK8 inhibition rate was 82.5%.(2)The cell proliferation rate of SW-13 cells interfered with by MAPK8 si RNA was significantly higher than that of the normal control group and negative control group(P<0.05).In the apoptosis experiment,the apoptosis rate of the MAPK si RNA group was lower than that of the normal control group and negative control group(P<0.05).In the scratch experiment,there was no significant difference in cell mobility in the three groups.In the cell cycle experiment,the G0/G1 ratio of the MAPK8 si RNA group was lower than that of the normal control group and negative control group(P<0.05).In the cell invasion experiment,the number of transmembrane cells in the MAPK8 si RNA group was higher than that in the normal control group and negative control group(P<0.05).Conclusion: The adrenocortical carcinoma cell line interfered by MAPK8 si RNA was successfully constructed.After inhibiting the expression of MAPK8,the proliferation of SW-13 cells increased,the apoptosis decreased,the cell cycle was arrested in the G0/G2 phase,and the invasion and migration ability was enhanced.It is suggested that endoplasmic reticulum stress has an effect on the biological behavior of adrenocortical cancer by mediating the JNK signaling pathway.JNK1(MAPK8)is a potentially effective target for gene therapy of adrenocortical cancer. |
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