Exosomal microRNA-let-7b-5p Derived From Pancreatic Cancer Cells Possibly Promotes Insulin Resistance in C2C12 Myotube Cells by Targeting SLC6A15

Background Cancers trigger systemic metabolic disorder usually associated glucose intolerance, which as initial apparent phenomenon. One of the features of pancreatic cancer (PC) metabolic reprogramming is the crosstalk between PC and peripheral tissues (skeletal muscle and adipose tissues), emphasized by insulin resistance (IR). In our previous study (Sci Rep. 2017;7(1):5384), we reported that mice pancreatic cancer derived exosomes could induce skeletal muscle cells(C2C12) IR and exosomal microRNAs (miRNAs) may exert an important effect. This work was carried out to investigate whether there exist a direct functional relationship between PC exosomal miRNAs and C2C12 cell genes, in the pathological process of IR. Methods The expression proles of exosomal miRNAs were evaluated using the Agilent Mouse miRNA V21.0 chip (GSE95741). The differentially expressed genes were screened through Agilent SurePrint G3 Mouse GE V2.0 chip (GSE174058). TargetScan and miRbase databases were used for target genes prediction and the prediction results were veried by dual-luciferase reporter gene assay. Results The biochips (GSE95741 and GSE174058) revealed that exosomes derived from mouse pancreatic cancer cells had higher levels of miRNA-let-7b-5p (Log FC 8.6); SLC6A15 gene expression was down-regulated in C2C12 cells (Log FC -4.7). Related mice and human studies has showed that SLC6A15 is associated with IR of metabolic disorder. In this work, luciferase assays conrmed that a direct interaction between miRNA-let-7b-5p and the SLC6A15 3΄-untranslated region (3΄-UTR) was established, as predicted by the TargetScan and miRbase. Our data suggest that exosomal miRNA-let-7b-5p may promote IR in C2C12 myotube cells targeting SLC6A15. Lipofectamine 2000 (Invitrogen), HEK-293T cells were contransfected with the reporter constructs, miRNA-let-7b-5p mimics and negative control. Luciferase activity was determined after 48h using the Dual-Glo substrate system (Promega) and a Beckman Coulter LD400 luminometer. Data are presented as the ratio of experimental (Renilla) luciferase to control (Firey) luciferase.


Abstract
Background Cancers trigger systemic metabolic disorder usually associated glucose intolerance, which as initial apparent phenomenon. One of the features of pancreatic cancer (PC) metabolic reprogramming is the crosstalk between PC and peripheral tissues (skeletal muscle and adipose tissues), emphasized by insulin resistance (IR). In our previous study (Sci Rep. 2017;7(1):5384), we reported that mice pancreatic cancer derived exosomes could induce skeletal muscle cells(C2C12) IR and exosomal microRNAs (miRNAs) may exert an important effect. This work was carried out to investigate whether there exist a direct functional relationship between PC exosomal miRNAs and C2C12 cell genes, in the pathological process of IR.
Methods The expression pro les of exosomal miRNAs were evaluated using the Agilent Mouse miRNA V21.0 chip (GSE95741). The differentially expressed genes were screened through Agilent SurePrint G3 Mouse GE V2.0 chip (GSE174058). TargetScan and miRbase databases were used for target genes prediction and the prediction results were veri ed by dual-luciferase reporter gene assay.

Results
The biochips (GSE95741 and GSE174058) revealed that exosomes derived from mouse pancreatic cancer cells had higher levels of miRNA-let-7b-5p (Log FC 8.6); SLC6A15 gene expression was down-regulated in C2C12 cells (Log FC -4.7). Related mice and human studies has showed that SLC6A15 is associated with IR of metabolic disorder. In this work, luciferase assays con rmed that a direct interaction between miRNA-let-7b-5p and the SLC6A15 3΄-untranslated region (3΄-UTR) was established, as predicted by the TargetScan and miRbase.

Background
Pancreatic cancer(PC) is a morbid disease and there are currently no reliable forms of early detection, which leads to a 5-year survival rate for PC patients that is under 9% [1]. Therefore, the main goals of PC research lie in looking for markers of the early stage, understanding the biological behaviors of the tumor and developing novel therapeutic methods and targets.
Multiple clinical studies have shown that pancreatic cancer-associated new-onset diabetes mellitus (PC-DM) may be one of the early indicators of PC [2][3][4][5]. The fundamental cause is "metabolic reprogramming" and "metabolic crosstalk"[6-8], characterized by PC. In other words, PC affects peripheral tissue (skeletal muscle and adipose tissues) through certain mechanisms, and results in insulin resistance (IR) in these tissues, which is the main pathological component of PC-DM and type II diabetes mellitus (T2DM) [9].
How does PC exert its in uence on peripheral tissues from afar? In our previous study (Sci Rep. 2017;7(1):5384), we reported that mice pancreatic cancer derived exosomes could induce skeletal muscle cells(C2C12) IR and exosomal microRNAs (miRNAs) may exert an important effect. But the mechanisms between speci c exosomal miRNAs and target genes are still waiting to be elucidated.
This work aims to nd the candidate exosomal miRNAs and target genes, through microarray technology and bioinformatics analysis, and then check the functional relationship by dual-luciferase reporter gene assay. This may help clarify PC biological behaviors and develop potential therapeutic targets for PC.

Microarray assay
The miRNA microarray analysis was performed by OE Biotech.Co.,Ltd.

Statistical analysis
Results are expressed as the means ± standard error. Analyses between different groups were performed using the two-tailed Student t-test or one-way analysis of variance. Statistical signi cance was set as follows: a P value of 0.05 was signi cant, and a value of 0.01 was highly signi cant.

Discussion
PC is one of the most invasive carcinomas worldwide,which causes distinct symptoms of malnutrition and altered glucose homeostasis. The only curative therapeutic treatments rely on surgical resection, yet the e ciency of this approach is limited by the highly aggressive behaviors and lack of inceptive diagnosis. About 90% of PC patients who lost the chance of surgery eventually show cachexia --a severe complication involving pathological weight loss due to the wasting of skeletal muscle and adipose tissue [10,11]---The clinical syndrome also indicates high metastasis rate, dangerous condition, poor prognosis and low survival rate. [12].
During the disease progression, the severity of IR is closely related to the development of cancer cachexia [13,14]. In the state of IR, the normal signal pathway is inhibited, and the proteolysis system is activated, leading to the degradation of muscle protein [15], which is the primary characteristic of cancer cachexia.
How does PC exert its in uence on peripheral tissues IR from afar? As previously reported(Sci Rep. 2017;7(1):5384), mice pancreatic cancer derived exosomes could induce skeletal muscle cells(C2C12) IR and exosomal microRNAs (miRNAs) may exert an important effect. But the mechanisms in this led are still waiting to be clari ed.
In this work,the biochips (GSE95741 and GSE174058) revealed that exosomes derived from mouse pancreatic cancer cells had higher levels of miRNA-let-7b-5p (Log FC 8.6); SLC6A15 gene expression was down-regulated in C2C12 cells (Log FC -4.7). Related mice and human studies has showed that SLC6A15 is associated with IR of metabolic disorder [16,17], and luciferase assays con rmed that a direct interaction between miRNA-let-7b-5p and the SLC6A15 3΄-untranslated region (3΄-UTR) was established, as predicted by the TargetScan, and miRbase.

Conclusion
This work suggest that exosomal miRNA-let-7b-5p may promote IR in C2C12 myotube cells by targeting SLC6A15. Our data paves the way towards discovering potential targets for correcting metabolic disorders and improving the diagnosis/treatment of PC.

Con ict of interest
The author declare that there is no con ict of interest. Figure 1 miRNA-let-7b-5p directly regulates SLC6A15 expression in HEK-293T cells. (A). The complementary sequences of miR-let-7b-5p were discovered in 3'UTR of SLC6A15 mRNA using TargetScan and miRBase. The mutagenesis was performed in the complementary sites for the seed region of miR-let-7b-5p (wt, wild type; mut, mutant type.) (B). miR-let-7b-5p inversely modulated the luciferase activity of plasmids that carried wt 3'UTR of SLC6A15 rather than mut 3'UTR of SLC6A15. n=four independent experiments. Values are the MEAN ± SD. *P < 0.05 vs Control. **P < 0.01 vs Control.