Effect Of Pancreatic Cancer-derived Exosomes On The Production Of GIP And GLP-1 From STC-1 Cells And The Underlying Mechanism

Background:One hallmark of pancreatic cancer(PC)is high high prevalence of pancreatic cancer-related diabetes mellitus(PCDM)[15].PCDM is an important clue for early detection and diagnosis of pancreatic cancer because it often appears about 13 months before the appearance of typical PC symptoms.However,the potential mechanism of PCDM remains to be elucidated.Recently,Skrha J et al.compared the fasting incretins levels in plasma in healthy control group,PC and T2DM patients.Compared with T2DM and normal controls,the serum GIP level in patients with PC(DM-/DM+)was significantly decreased,but it rose again after tumor resection,which suggested that pancreatic cancer is the cause of GIP dcclinc[9].Although the researchers believe that a substance secreted by pancreatic cancer may inhibit GIP production,the underlying mechanism is not further explored in the literature.We intended to further explore the mechanism of GIP decrease in PC patients.K and L cells secreting GIP and GLP-1 are far away from pancreatic cancer,and exosomes are extracellular vesicles that can mediate the substances exchange between cells.So pancreatic cancer may affect the function of K and L cells by producing exosomes.Therefore,we speculated that the exosomes produced by PC act on K or L cells,thus affecting the production of GIP and GLP-1,and eventually leading to the occurrence of PCDM.Methods:In this study,STC-1 cells secreting GIP and GLP-1 were selected as target cells.Two pancreatic cancer cell lines,PANC-1 and MIA PaCa-2,and one colorectal cancer cell line SW620,were selected as mother cells.Firstly,we collected the exosomes from the supernatants of PANC-1,MIA PaCa-2 and SW620 cells,and characterized the exosomes.Then,the labeled exosomes were co-incubated with STC-1 cells to make sure that STC-1 cells could internalize these exosomes.Then,three kinds of exosomes were co-cultured with STC-1 cells,and the effects of exosomes on the proliferation of STC-1 cells were detected by MTT assay.Then,the effects of exosomes on the function of STC-1 cells were assessed by ELISA and cell immunofluorescence.Then,in order to explore the underlying mechanism,the levels of proGIP,proglucagon,PCSK1/3 and PCSK2 in STC-1 cells were detected by qRT-PCR and Western Blot,respectively.Then the differences of the expression profiles of microRNAs in different exosomes were compared by microarray,and the key microRNAs were screened and verified by qRT-PCR,transfection,ELISA and Western Blot.In other part of this study,we hypothesized that the exosomes secreted by pancreatic cancer cells were carried to K and L cells located in intestinal epithelium via pancreatic juice.We collected clinical data and fasting blood glucose records of patients with or without recurrent foci in pancreas,and tried to extract exosomes from pancreatic juice samples.In addition,animal experiments were carried out.DiR-labeled exosomes were injected into nude mice through tail vein and duodenal descending intestinal cavity,and then observed in vivo.Results:We isolated and identified these exosomes,which are designated as Exo-Pan,Exo-Mia and Exo-SW620,respectively.By co-incubation of exosomes with STC-1 cells,different exosomes can be absorbed by STC-1 cells,and they have no obvious effect on the proliferation of STC-1 cells.However,Exo-Mia can reduce the production of GIP and GLP-1 by STC-1 cells in vitro in a concentration-and time-dependent manner.The results of cellular immunofluorescence also confirm those results.The results of qRT-PCR results showed the proGip and proglucagon mRNA levels in STC-1 cells treated with Exo-Mia were increased,while the expression of PCSK1/3 responsible for modification of proGIP and proglucagon protein was decreased.This indicated that Exo-Mia inhibited the post-translational modification of proGIP and proglucagon,thereby inhibiting the production of GIP and GLP-1.Compared with Exo-Pan,Exo-Mia showed 34 up-regulated and 49 down-regulated microRNAs.After further verification by qRT-PCR,ELISA and Western Blot experiments,it was found that the expression of PCSK1/3 in Exo-Mia could be inhibited by microRNAs(miR-6796-3p,miR-6763-5p,miR-4750-3p and miR-197-3p).In addition,clinical data recorded the changes of fasting blood glucose(FBG)in two groups of patients with recurrent pancreatic cancer.There was no significant difference in FBG level between the two groups before and after operation.The FBG level decreased and reached the lowest level after operation.When the tumors recurred,the FBG level of the two groups rose again.However,the FBG levels in the group with recurrence foci in pancreas were significantly increased than another group after recurrence.We isolated a large number of exosomes from pancreatic juice.Animal experiments have shown that intestinal secretions injected through the intestinal cavity are more easily internalized by intestinal epithelial cells than those injected through the caudal vein.Conclusion:Exo-Mia secreted by MIA PaCa-2 can reduce the production of GIP and GLP-1 from STC-1 cells in vitro in a concentration-and time-dependent manner.MicroRNAs in Exo-Mia(miR-6796-3p,miR-6763-5p,miR-4750-3p and miR-197-3p)interfere with post-translational shear modification of proGIP and proglucagon by inhibiting the expression of PCSK1/3,and ultimately reduce the production of GIP and GLP-1.In addition,clinical data and animal experiments preliminarily prove that exosomes derived from pancreatic cancer may be transported to target cells(the K and L,cells in intestinal epithelium)through pancreatic juice.