LncRNA AB209630 inhibits gemcitabine resistance cell proliferation by regulating PI3K/AKT signaling in pancreatic ductal adenocarcinoma

2.1 Clinical tissue samples

This study was approved by the Ethical Committee of Department of Oncology, Shanghai Medical College, Fudan University. A total of 53 pairs of PDAC tissues and adjacent normal tissue samples were obtained from January 2011 to February 2014 at Department of Oncology, Shanghai Medical College, Fudan University. All of PDAC patients underwent surgical resection and patients had no any therapy before surgery. Fresh tissues were store at - 80 ∘ C for RNA analyses.

2.2 Cell line culture

Human PDAC cell lines Panc-1 and BXPC-3 were purchased from Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM, Invitrogen, Carlsbad, CA, USA) and supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) at 37 ∘ C in a humidified atmosphere of 5% CO 2 .

2.3 Development of gemcitabine-resistant PANC-1 and BXPC-3 cells

PANC-1 and BXPC-3 cells were selected by continuous treatment of PANC-1 and BXPC-3 cells with 600 nM gemcitabine (Selleck, Houston, TX, USA), when the confluence of cells reached 50% resulting in subclones resistant to gemcitabine. Two independent GR clones of PANC-1 (PANC-1/GEM) and BXPC-3 (BXPC-3/GEM) cells were established, respectively.

2.4 Cell proliferation assay

Cell proliferation capacity was detected using CCK-8 Kit (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Transfected cells were detected at different time points (0, 24, 48, 72, and 96 h), the CCK8 solution (10 μ m) was added to each well of the plates. Then, cells were incubated at 37 ∘ C for 2 h. Cell proliferation was detected with a microplate reader (Biotek ELX-800, USA) and the absorbance at 450 nm was measured.

2.5 Colony formation assay

Transfected PANC-1/GEM and BXPC-3/GEM cells were seeded and cultured in six-well plates at a density of 300 cells/per well. Cells were cultured for 14 days at 37 ∘ C in a humidified atmosphere of 5% CO 2 . Then, colonies were fixed with 4% paraformaldehyde for 20 min and stained with a 0.1% crystal violet solution for 20 min. Finally, the numbers of cell colonies were counted under a microscope.

2.6 qPCR analysis

Total RNAs were extracted using TRIzol reagent (Invitrogen). The cDNA was transcribed by RNA using Reverse Transcriptase M-MLV kit (TAKALA, Dalian, China) according to the manufacturer’s instructions. SYBR Premix Ex Taq was used for qRT-PCR reaction on Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The mRNA expression was calculated using 2 - Δ ⁢ Δ ⁢ CT methods. LncRNA AB209630 expression was normalized to GAPDH expression. Primers for lncRNA AB209630 were as follow: lncRNA AB209630-F, 5’-GGGCTATTGTCCCTAAGTTGAT-3’, and lncRNA AB209630-R5’-TGTCTTGTAGAGCATAAGGAAACC-3’; GAPDH-F, 5’-GGGAAACTGTGGCGTGAT-3’, and GAPDH-R, 5’-GAGTGGGTGTCGCTGTTGA-3’.

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Protein were lysed using RIPA buffer and added with a protease inhibitor cocktail (Sigma). The proteins were separated on sodium dodecyl sulfate-polyacryla-mide gel electrophoresis (SDS/PAGE), and then were transferred to polyvinylidene difluoride membranes (PVDF, Millipore). The membranes were incubated with primary antibodies with PI3K (1:1000, Santa Cruz Biotechnology, CA, USA), p-PI3K (1:1000, Santa Cruz Biotechnology, CA, USA), AKT (1:1000, Santa Cruz Biotechnology, CA, USA), p-AKT (1:1000, Santa Cruz Biotechnology, CA, USA) overnight at 4 ∘ C. Following the membranes were probe with horse-radish peroxidase (HRP)-conjugated secondary antibody at room temperature, proteins blots were visualized by an enhanced chemiluminescence system.

2.8 Statistical analysis

Statistical analysis was analyzed using SPSS 18.0 software (IBM, Chicago, IL, USA). Differences between groups were compared using Student’s t test or one-way ANOVA. Values of were considered statistically significant.

3.1 Upregulation of lncRNA AB209630 expression inhibits cells proliferation in PANC-1/GEM and BXPC-3/GEM cells

In the study, we constructed the gemcitabine-resis-tant PDAC cells (PANC-1/GEM and BXPC-3/GEM) by continuous treatment of PANC-1 and BXPC-3 cells with 600 nM gemcitabine. The resistance status was determined by calculating the half maximal inhibitory concentration (IC50) of gemcitabine using MTT cytotoxicity assays. Results showed that PANC-1/GEM or BXPC-3/GEM cells had an increased IC50 value than wild PANC-1 or BXPC-3 cells (Fig. 1A–B).

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Furthermore, cell proliferation was detected by using CCK8 assays. The Results showed that cell proliferation ability was inhibited by transfecting with pcDNA3.1-AB209630 vector into PANC-1/GEM or BXPC-3/GEM cells, compared to the control groups (Fig. 2A–B). Subsequently, we performed the cell colony formation assays. Consistent with above results, PANC1/GEM or BXPC-3/GEM cells tranfected with pcDNA3.1-AB209630 vector showed reduced colonies number, compared to pcDNA3.1-vector group (Fig. 2C–D). Thus, these results indicated that upregulation of lncRNA AB209630 expression inhibited cells proliferation in gemcitabine-resistant PANC-1 and BXPC-3 cells.

PI3K/AKT signaling pathway was reported to be associated with gemcitabine resistance in some tumors [9, 10]. Western blot analysis showed that p-PI3K and p-AKT expression levels were significantly downregulated when PANC-1/GEM or BXPC-3/GEM cells were tranfected with pcDNA3.1-AB209630 vector, compared to pcDNA3.1-vector (Fig. 3A–B). However, PI3K and AKT expression levels were not significant changed, when PANC-1/GEM or BXPC-3/GEM cells were tranfected with pcDNA3.1-AB209630 vector, compared to pcDNA3.1-vecto (Fig. 3C–D). Thus, these results indicated that upregulation of lncRNA AB209630 expression inhibited PI3K/AKT signaling pathway.

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In the study, we first analyzed the expression of lncRNA AB209630 in PDAC tissues and adjacent normal tissues using qRT-PCR. The results found that lncRNA AB209630 expression was dramatically reduced in PDAC tissues compared to adjacent normal tissues (Fig. 4A). Furthermore, we analyzed the association of lncRNA AB209630 expression with prognosis of patients. Survival plots by Kaplan-Meier analysis showed that lncRNA AB209630 expression associated with poor overall survival time (Fig. 4B, log rank test, ). Thus, our results indicated that lncRNA AB209630 expression was lower in PDAC and associated with tumor prognosis.