Cell culture
The human pancreatic cancer cells lines SW1990 and PANC-1 cells were purchased from the cell resources center of shanghai academy of life sciences (Shanghai, China). PANC-1 cells were in DMEM (KGL1206, KeyGEN Biotech, Nanjing, Jiangsu, China) with 10% fetal bovine serum (FBS). SW1990 cells were cultured in Leibovitz’s L-15 (KGM41300-500, KeyGEN Biotech, Nanjing, Jiangsu, China) with 10% FBS. All cells were incubated at 37°C in a 5% CO2 atmosphere.
Bioinformatics data mining and analysis
The differential expression of genes in the cancer genome atlas (TCGA) database was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) online website. The mRNA expression of FOSL1 and other genes in clinical cancer samples was compared with that in normal adjacent from TCGA database, and the P value was obtained by one-way ANOVA. The significant values of P-value and folding change are 0.05 and 2.0 respectively. The effects of FOSL1 and other genes on the survival of patient with pancreatic cancer were valuated using Kaplan-Meier Plotter. Samples were divided into two groups with high expression and low expression according to the median expression. The 95% confidence interval (CI), log rank risk ratio (HR) and P value of overall survival and disease-free survival were evaluated. The relationship between gene expression and prognosis was analyzed by the receiver operating characteristic (ROS) curves. The original clinical data source of 183 pancreatic cancer RNA sequencing information was obtained from the TCGA database (https://portal.gdc.com). The ROS curves and calculated area under the curve (AUC) values were analyzed by the R package pROC, and the data were visualized as ggplot2. The AUC value between 0.5 and 0.7 indicates evidence of model success, the value between 0.7 and 0.9 indicates strong evidence of model success, and the value greater than 0.9 indicates strong evidence of model success [29]. Statistical analysis and visualization were performed in R version 3.6.3. The expression correlation between the key genes of interest in the previous intersection genes and FOSL1 expression were alayzed by the GEPIA2 online tool. The expression distribution of FOSL1 mRNA in different pancreatic cancer cell lines was obtained from the CCLE dataset (https://portals.broadinstitute.org/ccle). The analysis was constructed by the R v4.0.3 software ggplot2 (V3.3.3).
RNA sequencing and data processing of DEGs
PANC-1 cells were transfected with short-hairpin RNA against FOSL1 (FOSL1 shRNA) or NC shRNA for 3 days. After treatment, cells were collected to obtain total RNA using TRIzol reagent (Vazyme, Nanjing, China) according to the manufacturer’s manual. A total of 600 ng RNA was used to prepare libraries using the NEBNext Ultra RNA Library Perp Kit for Illumina. RNA quantity and quality were assessed on an Agilent 2100 Bioanalyzer. RNA library sequencing was performed on an Illumina HiSeqTM 2500/4000 by Gene Denovo Biotechnology Co., Ltd. (Guangzhou, Guangdong, China). Differentially expressed genes (DEGs) in the NC shRNA group vs the FOSL1 shRNA group were identified based on aཛྷlog2FCཛྷ>1.0 and an adjusted P < 0.05. DEGs with a log2FC < 1.0 were considered downregulated genes, while DEGs with a log2FC > 1.0 were considered upregulated genes [30, 31].
GO, KEGG pathway and GESA enrichment analysis
The characteristic biological attributes of the DEGs were identified by gene ontology (GO) enrichment and gene set enrichment analysis (GESA) enrichment analysis. The functional attributes of the DEGs were identified by kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment and GESA enrichment analysis. GO enrichment analysis, KEGG pathway enrichment analysis and GESA enrichment analysis were performed using Omicsmart, a real-time interactive online platform for data analysis ( http://www.omicsmart.com) [29, 30].
Lentivirus Transfection
Lentiviral recombination vectors of human FOSL1 complete RNA (FOSL1 cRNA) (pGV492-FOSL1) and its scrambled control (pGV492-NC), Lentiviral recombination vectors of shRNA against FOSL1 (pGV248-shFOSL1), shRNA against HMGA1 (pGV248-shHMGA1) and the scrambled control (pGV112-shNC) were constructed and purchased from Genechem Co. Ltd. (Shanghai, China). The Lentiviral vector of GV492-FOSL1 was confirmed by PCR and sequencing. The pGV248-shFOSL1 vector and pGV248-shHMGA1 vector were confirmed by sequencing. PANC-1 cells and SW1990 cells were infected with FOSL1 cRNA, NC cRNA, FOSL1 shRNA, HMGA1 shRNA and NC shRNA Lentiviral vectors using HitransG P promoting reagent according to the manufacturer’s instructions. After infection with lentiviral vector for 3 days, culture medium containing virus was removed. Transfected cells were allowed for growth for 3–5 days, and then treated with 2.0 µg/mL puromycin for 24 h to select positive infected cells. For the rescue experiment, cells were infected with one lentiviral vector for 3 days and the culture medium containing virus was removed. The cells were then infected with another vector for 3 days, and the culture medium containing virus was removed. Cells transfected with two vectors were allowed for growth for 3–5 days, and then applied for experiments. All transfected cells were validated by Q-PCR and Western blots, and maintained in medium containing 1.0 µg/mL puromycin [30, 31]. The target sequence of FOSL1 shRNA is 5’-CTGTACCTTGTATCTCCCTTT-3’, target sequence of HMGA1 shRNA is 5’- CAACTCCAGGAAGGAAACCAA-3’, target sequence of NC shRNA is5’- CA TTCTCCGAACGTGTCACGT − 3’.
MTT assay for cell viability
Cell viability in each group was analyzed by MTT assay [30, 31]. Untreated cells served as the control. After the cells transfected with lentiviral vector were cultured at different time, the viabilities of PANC-1 cells and SW1990 cells were measured by MTT assay. The relative ratio of MTT assay was calculated by the equation: relative ratio = A Group, day/A Control, 1. Herein, A Group, day represents the absorbance value of the comparison group after culture for different days, A Control, 1 represents the absorbance value of untreated cells after culture for 1 day.
Colony formation assay
All cells were seeded in 6-well plates at a concentration of 500 cells/well. After SW1990 cells and PANC-1 cells were transfected with different lentiviral vectors, the cells were cultured in medium containing 1.0 µM puromycin for 14 days. The culture medium was changed every 3 days to allow colony formation. After treatment for 14 days, the colonies were fixed with cold methanol for 10 min, stained with 0.1% crystal violet for 10 min, and imaged using Live cell imaging system of Bio-Tek (Cytation 5, Vermont, USA). Experiments were repeated at least three times [30, 31].
Cell apoptosis assays
Cell apoptosis was analyzed by Annexin V-FITC and PI apoptosis detection kits (KeyGEN Biotech, Nanjing, Jiangsu, China). After SW1990 cells and PANC-1 cells were transfected with different lentiviral vectors, the cells were cultured in medium containing 1.0 µM puromycin for 3 days. After treatment, the cells were collected and stained with Annexin V-FITC and PI following the manufacturer’s protocol to analyze cell apoptosis [30, 31]. Cell apoptosis was detected by Agilent NovoCyte Quanteon (California, USA). The data of apoptosis were analyzed by FlowJo 7.6.
Migration and invasion assay
Transwell plate was used to perform cell migration and invasion experiments. After SW1990 cells and PANC-1 cells were transfected with different lentiviral vectors, the cells were cultured in medium containing 1.0 µM puromycin for 3 days. After treatment, the cells were collected and counted to determine cell migration and invasion. For migration, cells (1×105 /ml) were collected, suspended in 200 µl medium without FBS and seeded into the upper well of the chamber, and the lower well contained culture medium with 10% FBS. For invasion, the upper well of each group was plated with 20 µl Matrigel. After Matrigel solidifying, cells in 200 µl medium without FBS were seeded into the upper well, and the lower well contained culture medium with 10% FBS. After incubation for 48 h, the cells were fixed with 4% formaldehyde for 30 min, stained with Giemsa for 15 min. Cell migration or invasion was observed on the outside of the upper well were observed with a Nikon inverted fluorescence microscope (Ts2R-FL, Tokyo, Japan) following the manufacturer’s introduction. Five images were randomly captured per slide.
Immunofluorescence (IF) assay
After SW1990 cells and PANC-1 cells were transfected with different lentiviral vectors, the cells were cultured in medium containing 1.0 µM puromycin for 3 days. After treatment, cells were fixed with 4% formaldehyde, permeabilized with 0.2% (v/v) Triton X-100 in PBS, blocked with 1% (w/v) BSA in PBS for 1.0 h and stained with anti-CD44 antibody or anti-ABCB1 antibody overnight at 4°C, and then stained with Goat Anti-Rabbit IgG H&L labeled with Alexa Fluor 647. After staining, cellular DNA was counterstained with 4,6-diamidino-2-phenylindole (DAPI). Fluorescence signals were detected using a Carl Zeiss LSM900 laser confocal microscope (Jena, Germany). Five fields per sample were quantified.
Q-PCR analysis
After SW1990 cells and PANC-1 cells were transfected with different lentiviral vectors, the cells were cultured in medium containing 1.0 µM puromycin for 3 days. After treatment, total RNA in each group was extracted with TRIzol reagent (Vazyme, Nanjing, Jiangsu, China) according to the manufacturer’s instructions. cDNA was synthesized using HiScript II one step RT-PCR kit (R223-01, Vazyme, Nanjing, China) with 1.0 µg of total RNA in a 20 µl reaction system. The resulting cDNA was diluted 1:4 in nuclease-free water and 2.0 µl was used per Q-PCR reaction with triplicates. Q-PCR was carried out using ChamQ SYBR Q-PCR master mix (Q711-02, Vazyme, Nanjing, China) on a QuantStudio 3 real time PCR detection system (Life Tech, New York, USA) including a nontemplate negative control. GAPDH was used to normalize the level of mRNA expression. The sequences of the primers were listed in Table 1.
Table 1
Primer sequences for genes in Q-PCR.
Name | Sense (5’-3’) | Antisense (5’-3’) |
FOSL1 | ACCCATCTGCAAAATCCCGGAA | TGCAGTGCCTCAGGTTCAAGC |
CD44 | CACAAATGGCTGGTACGTCT | ATCATCAATGCCTGATCCAGA |
CD133 | TAGCTACATTATCGACCCCTT | AGTACTTAGCCAGTTTTACCG |
SOX2 | ATGGCCCAGGAGAACCCCAA | TCGCAGCCGCTTAGCCTCGTC |
BMI1 | TCTTCTTGTTTGCCTAGCC | ATTTACTGATGATTTTCGAGGT |
HMGA1 | CCAAGCAGGAAAAGGACGGCACT | GGGCTCCTTCTGACTCCCTACCA |
CDH1 | TGCCGCCATCGCTTACACC | AGGTCAGCAGCTTGAACCAC |
CDH2 | GAGTTTACTGCCATGACGTT | CTGATTCTGTACACTGCGTTC |
CTNNB1 | CCACTAATGTCCAGCGTTT | TGGTCCTCGTCATTTAGCAG |
VIM | AAATGGCTCGTCACCTTCGT | AAATCCTGCTCTCCTCGCCTT |
MMP1 | CGCACAAATCCCTTCTACCC | ATCTCTGTCGGCAAATTCGT |
ABCB1 | AGAACTCTTAGCGTATGCAA | AGAAATATTGGCTGTAATAGCTT |
ABCC1 | CCTCTATCTCTCCCGACATGACC | GCCCAGCAGACGATCCACA |
ABCC4 | TTGCACACAGATTGAACACC | ACCATCTTGTAAAATAGGCTCT |
ABCG1 | GTCGCTCCATCATTTGCACCA | GGCAGTTCAGACCCAAATCCC |
ABCG2 | TAGCTGCAAGGAAAGATCCAA | ATCTTGTACCACGTAACCTG |
SNAL | TCACCGGCTCCTTCGTCCT | CCTTTCGAGCCTGGAGATCCTT |
OCT4 | CAGATCAGCCACATCGCCCAG | AGCAGCCTCAAAATCCTCTCGTT |
ALDH1 | CAGTGTTGTATAGCCGCATC | ATATACTTCTTAGCCCGCTCA |
FN1 | TTTCCCATTATGCCGTTGGAG | AATGACCACTTCCAAAGCCTA |
ABCC2 | AAACTCGGAATGTGAATAGCC | ACAGAATTCATCACAAACGCAAG |
ABCC5 | CTTGGGCATTGAATTACCGAAC | AACATTCTCTGCCCATCGTTG |
GAPDH | GAAACTGTGGCGTGATGGC | CACCACTGACACGTTGGCAG |
Western blots analysis
After treatment, total proteins in SW1990 cells and PANC-1 cells were extracted with RIPA cell lysis buffer (Beyotime, Shanghai, China) with added protease/phosphatase inhibitor cocktail. Protein concentration was determined by the BCA assay, and equal amounts of proteins were loaded for Western blots analysis. In brief, equal amounts of total proteins were loaded for SDS-PAGE and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). Membranes with protein were blocked with 5% (w/v) skim milk, incubated with primary antibody in Supplementary Materials, and then incubated with secondary antibodies (1:2000) for detection [30, 31]. GAPDH was used to normalize the level of protein expression. Densitometric analysis was performed with ImageJ software.
Co-IP assay
Total proteins were extracted from PANC-1 cells using the lysis/washing buffer in the Protein A/G Magnetic IP/Co-IP kit (ACE Biotechnology, Nanjing, China). Protein A/G magnetic beads were incubated with anti-FOSL1 antibody, anti-HMGA1 antibody and anti-IgG antibody at 4°C overnight to obtain the anti-FOSL1-Protein A/G magnetic beads, anti-HMGA1-Protein A/G magnetic beads and the anti-IgG-Protein A/G magnetic beads, respectively. Proteins were quantified and adjusted to same concentration, then incubated with the anti- FOSL1-Protein A/G magnetic beads, the anti-HMGA1-Protein A/G magnetic beads, or the anti-IgG-Protein A/G magnetic beads at room temperature for 2 hours. After incubation, unbound proteins were washed with IP buffer. For co-IP Mass spectrometry, the resultant protein complexes were separated by SDS-PAGE gel electrophoresis. The in-gel total proteins were analyzed by Gene Denovo Biotechnology Co., Ltd. (Guangzhou, Guangdong, China). For co-IP assay, the resultant protein complexes were subjected to Western blots to determine the interaction between FOSL1 and HMGA1 [30, 31].
In vivo antitumor activity
All animal experiments were carried out in accordance with the institutional guidelines for the care and use of laboratory animals and approved by the committee on the ethics of Animal Experiments of Lanzhou University. Six- to seven-week-old athymic BALB/c-nude mice (16–18 g) were provide by the Animal House in Model Animal Institute of Nanjing University (Nanjing, Jiangsu, China). Mice were bred in an environment with a temperature of 25°C, relative humidity of 60–70%, and light and dark time of 12 h. PANC-1 cells transfected with NC shRNA and PANC-1 cells transfected with FOSL1 shRNA (1×107 cells/ml) were injected into the right flank of each mouse. After the average volume (mm3) of xenograft tumors in NC KD group reaching to 150 mm3, tumor volume and mice weight were recorded. After 32 days of feeding and observation, the mice were killed to detect the weight of tumors in each group. Tumor volume was recorded every three days. Tumor volume was measured with a vernier caliper and calculated using the formula, (ab2)/2, where a and b represent length and width of the tumor.
H&E staining
The lung, liver, heart, kidney and spleen tissue samples of xenograft model mice were fixed with 4% paraformaldehyde, dehydrated with ethanol, immersed in xylene, embedded in paraffin wax and sectioned into 4.0 µm slides. The paraffin-embedded sections were stained with H&E according to the manufacturer’s instructions (Beyotime, Shanghai, China). Each group of samples was observed with a DM6B positive fluorescence microscope (Leica, Frankfurt, Germany). Five images were randomly captured per slide.
Immunohistochemical (IHC) staining
Xenograft tumors were embedded in paraffin wax and sectioned in slides. Sections were incubated with 0.3% hydrogen peroxide for 20 min to quench endogenous peroxidase and then incubated with 1.0% BSA for 30 min to block. After blocking, the paraffin-embedded section was incubated with anti-Ki67 primary antibody at 4°C for 12 h, incubated with secondary antibody for another 1.0 h at room temperature and then counterstained for 1 min with hematoxylin. Each group was examined using the DM6B positive fluorescence microscope (Leica, Frankfurt, Germany). Five images were randomly captured per slide.
Statistical analysis
The data were analyzed using SPSS 19.0. The results are expressed as means ± SD. Differences between treatment regimens were analyzed by two-tailed Student’s t-test or one-way ANOVA. P < 0.05 was considered statistical significance.