The two human pancreatic cancer cell lines SW1990 and PANC-1 used in this study were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). Both cell lines were incubated under suitable conditions with 5% CO2 (37°C). For hypoxia induction, cells were cultured with 1% O2 balanced with nitrogen and 5% CO2.
Quantitative real-time PCR (qRT-PCR) analysis
QRT-PCR analysis was performed according to the manufacturer’s protocol as described previously . GAPDH was used as the reference gene. Primer sequences (5’ to 3’): GLUT1-F: CTGTGCTCCTGGTTCTGTTCT; GLUT1-R: CAGCTCCTCGGGTGTCTTGT; HK2-F: GATGGCGTGAACGATGCT; HK2-R: GACGTTGGACGAGATGAGGT; LDHA-F: CAGCCCGATTCCGTTACCTA; LDHA-R: TCAGAGAGACACCAGCAACA; MALAT1-F: GAATTGCGTCATTTAAAGCCTAG; MALAT1-R: GTTTCATCCTACCACTCCCAATT; HIF-1α-F: ACTAGTGCCACATCATCACC; HIF-1α-R: ACAGATAACACGTTAGGGCTTC; GAPDH-F: GACGCTGGGGCTGGCATTG; GAPDH-R: GCTGGTGGTCCAGGGGTC.
Western blot analysis
Western blot analysis was conducted as described previously . In brief, proteins in the cell lysates were electrophoretically separated on sodium dodecyl sulfate polyacrylamide gels and then transferred onto PVDF membranes (Millipore, Burlington, MA, USA). After they were blocked with 5% skim milk, the blots were incubated with primary antibodies at 4°C and secondary antibodies (Aspen, Wuhan, China) at room temperature, followed by visualization with an ECL substrate (Thermo Fisher, Waltham, MA, USA). Primary antibodies against HIF-1α, HK2, GLUT1, LDHA, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA).
Glucose uptake and lactate production assays
SW1990 and PANC-1 cells were seeded into 6-well plates and incubated with different treatments. Glucose uptake and lactate release were detected according to the protocols of a glucose uptake cell-based assay kit (Cayman Chemical, Ann Arbor, MI, USA) and lactic acid assay kit (Nanjing Jiancheng Bio. Nanjing, China), respectively. The production of lactate in each sample was normalized to their respective control groups as indicated.
The chemosensitivity of pancreatic cancer cells to gemcitabine was measured by the MTT assay, which was described previously . In brief, cells (7×103/well) were seeded into 96-well plates. After different treatments, 20 μL of MTT (5 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) as incubated per well for 4 h and subsequently replaced with dimethyl sulfoxide (Sigma-Aldrich). The absorbance values were recorded by an ELISA reader at 490 nm. Each concentration was set up with five replicates.
Apoptosis was evaluated with Annexin V/propidium iodide (PI) staining. Briefly, harvested cells were washed in ice-cold PBS and resuspended in binding buffer at a density of 100×104 cells. Then, cells were mixed with Annexin V-FITC and PI according to the manufacturer’s protocols (KeyGEN Biotech). The fluorescence values for Annexin V-FITC and PI were detected on a flow cytometer (BD Biosciences).
SiRNAs targeting MALAT1 and HIF-1α and negative control siRNA were synthesized by RiboBio Co. (Guangzhou, China). SiRNA transfection was carried out with a mixture of LipofectamineTM 2000 (Invitrogen) as described previously . The lentiviral vectors with the sequence for Si-MALAT1 (LV-Si-MALAT1) and negative control (LV-Si-NC) were purchased from GeneChem (Shanghai, China). Transfection of the lentiviral vectors into SW1990 cells was conducted according to the manufacturer’s protocols. The siRNA sequences (5’ to 3’): MALAT1 siRNA: GCAGCCCGAGACUUCUGUA; HIF-1α siRNA: GGAUGGAUUCAUAUUUCUU; NC siRNA: GGUGUUUCUUUUCUCCCUU.
Chromatin immunoprecipitation (ChIP) assay
HIF-1α binding to the promoter of MALAT1 was tested using a ChIP assay as described previously . All procedures were carried out according to the protocol of the EZ-ChIPTM Chromatin Immunoprecipitation Kit (Millipore). The Primer sequences (5’ to 3’): Target 1-F: AGTGCAGTGACAGCGCAGA; Target 1-R: AACCGGCTCTAGCCGGTC; Target 2-F: CGCAGTTGGAGAGACTG; Target 2-R: CGCAAATGGGGATTTGG.
The effect of MALAT1 inhibition on gemcitabine chemosensitivity in vivo was examined in a tumor xenograft model. SW1990 cells transfected with LV-Si-MALAT1 were harvested and resuspended in phosphate-buffered saline (PBS). Approximately 5×106 cells were subcutaneously injected into the right flanks of male nude mice (n=5; HFK Bioscience Co., Beijing, China). After approximately 5 days, the nude mice were divided into Control, LV-Si-MALAT1, GEM, GEM+LV-Si-MALAT1 groups. For the gemcitabine-treated group, 20 mg/kg gemcitabine (Selleck.cn, Shanghai, China) was intraperitoneally injected every 3 days. An equal amount of PBS was injected as control. The tumor size was periodically measured and calculated by the following formula: 1/2 × length × width2. After 5 weeks, the mice were euthanized with 2% sodium pentobarbital (50 mg/kg), followed by cervical dislocation. Then, the complete xenograft tumors were excised, fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm sections. The proliferative activity of tumor cells was determined by Ki-67 immunohistochemical staining, while the apoptosis level was measured by TUNEL immunofluorescent staining.
The data are presented as the means ± SD. Comparisons between 2 groups were conducted with Student’s t-test. SPSS version 18.0 was used for data analysis. P<0.05 was considered to be the threshold of significance.