Abstract
Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant carcinoma with an extremely high lethality. We recently reported that hypoxia-inducible factor 1 (HIF-1) targets quiescin sulfhydryl oxidase 1 to facilitate PDAC cell growth and invasion. Here, we analyzed the control of another HIF-1 target, stromal cell derived factor-1 (SDF-1), in PDAC cells. We detected significantly more CD68+ macrophages in the PDAC, compared to normal human pancreas (NT). Since macrophages are recruited to the tissue through their expression of CXCR4 in response to SDF-1, we thus examined the SDF-1 levels in the PDAC specimens. Surprisingly, the SDF-1 protein but not mRNA significantly increased in PDAC, compared to NT. Moreover, a SDF-1-targeting microRNA, miR-454, was found to decrease in PDAC. Promoter luciferase assay confirmed that bindings of miR-454 to 3′-UTR of SDF-1 mRNAs inhibited SDF-1 protein translation. Co-culture of bone marrow derived macrophages and miR-454-modified PDAC cells in a transwell migration experiment showed that macrophages migrated less towards miR-454-overexpressing PDAC cells, and migrated more towards miR-454-depleted cells. Implanted miR-454-depleted PDAC cells grew significantly faster than control, while implanted miR-454-overexpressing PDAC cells grew significantly slower than control. Together, our data suggest that miR-454 may regulate SDF-1 in the control of the growth of PDAC.
Highlights
Previous reports have shown that many miRNAs play important roles in the growth and metastasis of PANC cells
We have previously shown that HIF-1 targeted QSOX1 to facilitate pancreatic cancer cell growth and invasion[25]
QSOX1 oxidizes sulfhydryl groups to form disulfide bonds in proteins, and has been found to promote invasion of Pancreatic ductal adenocarcinoma (PDAC) cells by activating MMP-2 and MMP-9. We found that both hypoxia and hypoxia mimicking reagent up-regulated the expression of QSOX1 in human PDAC cells
Summary
All mouse experiments were approved by the Institutional Animal Care and Use Committee at Zhongshan Hospital from Fudan University (Animal Welfare Assurance). All mouse experiments were performed according to guide from the Institutional Animal Care and Use Committee at Zhongshan Hospital from Fudan University (Animal Welfare Assurance). The protein was extracted from tissue or cells or conditioned media (secreted protein into the culture media), and analyzed using SDF1 ELISA kit (R&D Systems, Los Angeles, CA, USA), according to the manufacturer’s instruction. Cultured cells or dissociated mouse cancer tissue were detached with 0.25% Trypsine solution (Invitrogen), washed three times with PBS, re-suspended, labeled with PEcy7-conjugated CD68 antibody (Becton-Dickinson Biosciences) for sorting for macrophages. Cells were collected 24 hours after transfection for assay using the dual-luciferase reporter assay system gene assay kit (Promega, Beijing, China), according to the manufacturer’s instructions. All values are depicted as mean ± standard error and are considered significant if p < 0.05
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