| Background and objectiveIn the 1970 s,Professor Compagno proposed that "pancreatic mucinous cystadenoma will eventually become malignant over time",indicating that the invasive potential is the intrinsic property of pancreatic tumor exocrine cells rather than acquired phenotype.The 5-year survival rate of patients with invasive pancreatic mucinous cystic adenocarcinoma is poor,which is similar to pancreatic ductal adenocarcinoma.During recent years,the relationship between autophagy and tumor has attracted widespread attention.Growing evidence has found that autophagy can affect tumor metastasis.The role of autophagy in tumor metastasis is multifaceted,depending on cell type,tumor microenvironment,and genomic status.On the one hand,autophagy can inhibit tumor metastasis by blocking epithelial mesenchymal transition and fibrosis.In addition,autophagy can also promote tumor metastasis by promoting local adhesion,anti-anoikis and tumor interstitial metabolic coupling.Currently,few studies were conducted to explore the molecular mechanism underlying the development of invasive pancreatic mucinous cystadenocarcinoma.Given the poor survival associated with aggressive malignancy,we mainly studied pancreatic mucinous cystadenocarcinoma tissue and pancreatic mucinous cystadenocarcinoma cell line(MCC1)to detect the changes of autophagic activity in the state of pancreatic mucinous cystadenocarcinoma,and to reveal the effect of autophagy on the malignant migration and invasion of pancreatic mucinous cystadenocarcinoma MCC1 cells,and preliminary explored possible mechanisms.Methods1.Immunohistochemistry was employed to detect the autophagy activity of 4 pairs of paraffin-embedded human pancreatic mucinous cystadenocarcinoma tissues and adjacent tissues.Human pancreatic mucinous cystadenocarcinoma MCC1 cells and normal pancreatic ductal epithelial cells HPDE were cultured in vitro,and the expression levels of LC3-II and P62 in MCC1 cells and HPDE cells were detected by Western blot.Finally,the bright LC3 fluorescent dots(representing autophagosomes)in MCC1 cells and HPDE cells were observed under laser confocal microscopy by immunofluorescence staining.2.MCC1 cells were treated with rapamycin(100 μM)to establish an autophagic model.The MCC1 cells were divided into rapamycin-treated 4h group(4h group)and rapamycin-treated 6h group(6h group).MCC1 cells treated without rapamycin treatment was taken as control group(0h group).The expression of LC3-II and P62 in MCC1 cells of each group were detected by Western blot.Transwell assays were employed to examine the effects of autophagy on the migration and invasion of MCC1 cells.3.The expression level of miR-224-5p in MCC1 cells of 0h group,4h group and 6h group was detected by Real-time PCR.Meanwhile,the expression levels of miR-224-5p in MCC1 cells and HPDE cells were compared.Then,the stable over-expression of miR-224-5p(miR-224)and positive control group(NCm)MCC1 cells,stable low-expression of miR-224-5p(inhibitor-224)and negative control group(NCi)MCC1 cells were constructed by lentivirus infection.The expression levels of miR-224-5p in each group were detected by real-time PCR.Expression levels of LC3-II and P62 in each group were detected by Western blot.The bright LC3 fluorescent dots(representing autophagosomes)were observed under confocal laser microscopy by immunofluorescence staining.The target gene and binding site of miR-224-5p were predicted by the bioinformatics software TargetScan Human 7.2.The relationship between miR-224-5p and predicted target gene BCL2 was verified by dual luciferase reporter gene assay.The relative expression levels of miR-224-5p and BCL2 mRNA in MCC1 cells of NCm,miR-224,NCi and inhibitor-224 groups were detected by Real-time PCR.The relative expression levels of BCL2 protein in MCC1 cells of NCm,miR-224,NCi and inhibitor-224 groups were detected by Western blot.Small interfering RNA BCL2(siBCL2)and negative control(siNC)plasmids were transfected into NCi and inhibitor-224 MCC1 cells by cell transfection,respectively.They were divided into NCi-siNC group,inhibitor-224-siNC group,NCi-siBCL2 group and inhibitor-224-siBCL2 group.Finally,the expression levels of BCL2,LC3-II and P62 in MCC1 cells of each group were detected by Western blot.Results1.Compare to adjacent tissues,autophagy activity was significantly increased in human pancreatic mucinous cystadenocarcinoma.Compare with normal pancreatic ductal epithelial cell HPDE,the relative expression of LC3-II in pancreatic mucinous cystadenocarcinoma MCC1 cells was significantly increased,while the relative expression of P62 was significantly decreased.Immunofluorescence analysis showed that the amount of bright LC3 fluorescent dots in MCC1 cells was significantly more than that in HPDE cells.2.Western blot analysis showed that the relative expression levels of LC3-II in MCC1 cells of both the 4h group and the 6h group were significantly increased than that in the 0h group.Meanwhile,the expression of LC3-II in 6h group was significantly increased than that in 4h group.Compare to the 0h group,the relative expression level of P62 in MCC1 cells in 4h group and 6h group was significantly decreased,and the expression of P62 in 6h group was significantly decreased than that in 4h group.Transwell migration and invasion assays showed that the number of transmembrane cells in the 6h groups were both significantly increased than that in the 0h group MCC1 cells.3.Real-time PCR results showed that the expression of miR-224-5p in 4h and 6h group MCC1 cells was significantly enhanced than that in the 0h group,and the expression of miR-224-5p in 6h group was significantly enhanced than that in 4h group.Meanwhile,the relative expression level of miR-224-5p in MCC1 cells was significantly increased than that in HPDE cells.Compared to NCm group,the relative expression level of miR-224-5p was significantly increased in MCC1 cells of miR-224 group.The relative expression of miR-224-5p was significantly decreased in MCC1 cells of inhibitor-224 group compared with NCi group.Western blot analysis showed that compared with NCm group,the relative expression level of LC3-II in MCC1 cells of miR-224 group was significantly increased,and the relative expression level of P62 was significantly decreased.Compared to NCi group,the relative expression of LC3-II in MCC1 cells of inhibitor-224 group was significantly decreased,while the relative expression of P62 was significantly increased,and the relative expression of P62 was significantly increased.Immunofluorescence results showed that compared with the NCm group,the amount of bright LC3 fluorescent dots in MCC1 cells of the miR-224 group was significantly increased,while the amount of bright LC3 fluorescent dots in MCC1 cells of the inhibitor-224 group was significantly decreased than that in the NCI group.Bioinformatics software predicts that BCL2 as the target gene for miR-224-5p.The luciferase reporter gene analysis showed that the luciferase activity of the MCC1 cells transfected with the BCL2 3’UTR reporter gene in the miR-224 group was significantly reduced compared to the NCm group,there was no significant difference of luciferase activity between miR-224 group which transfected with the BCL2 3’UTR-Mutant reporter gene and the NCm group.Compare to NCm group,the relative expression of BCL2 mRNA and protein in MCC1 cells of miR-224 group was significantly decreased.Compared with NCi group,the relative expression of BCL2 mRNA and protein in inhibitor-224 MCC1 cells was significantly increased.Finally,Western blot analysis showed that BCL2 protein expression was significantly decreased in MCC1 cells of the NCi-siBCL2 group compared with the NCi-siNC group.Compared with inhibitor-224-siNC,the expression of BCL2 protein in MCC1 cells of inhibitor-224-siBCL2 group was significantly decreased,and there was no significant difference in the expression of BCL2 protein between MCC1 cells of NCi-siBCL2 group and inhibitor-224-siBCL2 group.Compared with the NCi-siNC group,LC3-II protein expression was significantly increased in the NCi-siBCL2 group MCC1 cells,and P62 protein expression was significantly decreased.Compared with inhibitor-224-siNC group,the expression of LC3-II protein was significantly increased in MCC1 cells of inhibitor-224-siBCL2 group,P62 protein expression was significantly decreased.The expression of LC3-II and P62 protein between NCi-siBCL2 group and inhibitor-224-siBCL2 group had no significant difference.ConclusionAutophagy is hyper-activated in human pancreatic mucinous cystadenocarcinoma tissues and MCC1 cells.High autophagy could dramatically elevate the miR-224-5p expression in MCC1 cells in a time-dependent manner,and miR-224-5p promotes autophagy in MCC1 cells by targeting BCL2.We found that a positive feedback loop between autophagy signaling and miR-224-5p,which promotes the aggressive migration and invasion of MCC1.This is the first study to evaluate the role of autophagy in pancreatic mucinous cystadenocarcinoma.Our observation provides new insight into the relationship between autophagy and tumor metastasis in pancreatic MCC. |
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