Correlation Between Hsa-miR-520h And Its Target Gene ABCG2 In Invasion, Metastasis And Prognosis Of Pancreatic Cancer

Objective:To observe the effection of hsa-miR-520h and its target gene ABCG2in cell proliferation, cycle, apopotosis, migration, invasion and side population of pancreatic cancer PANC-1cells.Methods:We investigated ABCG2expression by qRT-PCR and western blot in four pancreatic cancer cell lines BxPC-3, CFPAC-1, PANC-1, and SW1990, and used three microRNA (miRNA) target prediction programs, and information from the existing literature to predict and identify hsa-miR-520h as a miRNA that targets ABCG2. The function of this miRNA was investigated by transient transfection of the pancreatic cancer cell line PANC-1with oligonucleotides that mimic hsa-miR-520h. The cellular growth activity was measured by MTT assay. The cell cycle, apoptosis and side population were tested by flow cytometry. The cellular migration and invasion ability were detected by transwell assay.Results:1. Pancreatic cancer cell lines expressed variable levels of ABCG2. ABCG2was overexpressed in PANC-1and SW1990cells, and underexpressed in BxPC-3and CFPAC-1cells.2. At24h post-transfection, overexpression of hsa-miR-520h resulted in a significant decrease of endogenous ABCG2protein levels and endogenous ABCG2mRNA. These observations suggested that hsa-miR-520h downregulates ABCG2expression by inhibiting translation or causing mRNA instability. Taken together, these data indicate that hsa-miR-520h targets ABCG2.3. Ectopic expression of hsa-miR-520h had no significant inhibition on cell proliferation. The cell cycle distribution of transfected cells had no significant G0/G1accumulation phenotype in PANC-1cells transfected with hsa-miR-520h (56.43%). We also examined cell apoptosis of transfected cells, and found no significant increase of apoptosis in PANC-1cells transfected with hsa-miR-520h (7.31%).4. The migration ability of hsa-miR-520h mimic-transfected cells was significantly decreased by55%, compared to controls. A Matrigel invasion assay was performed in parallel to determine the effect of transfection with hsa-miR-520h mimics. The invasion ability of hsa-miR-520h-transfected cells was significantly decreased by53%, compared to controls. Taken together, these results suggested that hsa-miR-520h is a potent suppressor of PANC-1cells migration and invasion through downregulation of ABCG2expression.5. Flow cytometry of hsa-miR-520h-transfected PANC-1cells, showed the side population was1.51%, compared to2.79%in NC-transfected, and3.06%in mock-transfected cells. These data indicated that hsa-miR-520h plays a critical role in the identification of the side population, through downregulation of ABCG2expression.Conclusions:1. ABCG2is a target gene of hsa-miR-520h in pancreatic cancer PANC-1cells.2. hsa-miR-520h plays a critical role not only in inhibiting migration and invasion, but also in decreasing the proportion of side population cells of PANC-1cells. The possible mechanism of these effects may be through downregulation of ABCG2expression. 3. Our findings provide new insight into the function of miRNA in the regulation of ABCG2expression in pancreatic cancer. Gene therapy using miRNA mimics may therefore be useful as a pancreatic cancer therapy. Objective:ABCG2has been found to be strongly expressed in a variety of human epithelial cancers, and correlates with carcinogenesis and aggressiveness. It is unclear whether ABCG2acts as a tumor metastasis promoting factor in human pancreatic cancer. This study aims to determine the prognostic value of ABCG2in patients with pancreatic adenocarcinoma.Methods:We analyzed ABCG2expression by immunohistochemistry in a retrospective cohort of110patients with resectable pancreatic ductal adenocarcinoma that had undergone surgery at the First Affiliated Hospital of Nanjing Medical University between October2006and January2010. The relationship between ABCG2expression and the clinicopathological characteristics of patients with pancreatic adenocarcinoma were evaluated. Survival data were assessed by Kaplan-Meier analyses. Parameters found to be of prognostic significance in a univariate analysis were verified in a multivariate Cox regression model.Results:ABCG2overexpression was observed in35(32%) of110pancreatic cancer patients. ABCG2overexpression correlated with histological grade (P=0.000), tumor stage (P=0.031), stage (UICC; P=0.030), lymph node metastasis (P=0.021) and perineural invasion (P=0.023). Multivariate analysis revealed that UICC stage, lymph node metastasis and perineural invasion were independent prognostic factors, but ABCG2 overexpression was not (risk ratio,1.118;95%CI,0.552-2.268, P>0.05). By Kaplan-Meier analysis, patients whose pancreatic cancer cells overexpressed ABCG2(median survival,5months) had a significantly worse prognosis than patients without ABCG2overexpression (median survival,13months; log-rank, P<0.01).Conclusions:These novel findings suggest that high expression of ABCG2by pancreatic cancer cells is significantly associated with poor prognosis by promoting tumor metastasis and invasion, which may be a useful target in the development of future targeted treatments.