Berberine were purchased from Shanghai Tongtian Biotechnology Co., Ltd. (shanghai, China). Berberine were dissolved in DMSO and stored at -20°C. RPMI 1640 and Dulbeberberineo’s modified eagle medium (DMEM) for culture were purchased from Gibco (Grand Island, NY, USA), fetal bovine serum (FBS) was from HyClone (South Logan, USA) and trypsin was from Gibco (Grand Island, NY, USA). Thiazoles [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium Bromide, Methylthiazolyldiphenyl-tetrazolium bromide (MTT)] were purchased from Sigma (St. Louis, MO, USA). Propidium iodide（PI）and FITC-Annexin V were both purchased from BD Biosciences (San Jose, CA). RNA extraction kit, PrimeScript RT MasterMix kit, and SYBR Premix Ex Taq kit were provided by Takara (Dalian, China). Antibodies against POLE2 was obtained from Santa Cruz (CA, USA). Antibodies against FOXM1 and actin as well as secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
Microarray data analysis
Slides were scanned by GeneChip® Scanner 3000 (Cat#00-00212, Affymetrix, Santa Clara, CA, US) and Command Console Software 4.0 (Affymetrix, Santa Clara, CA, US) with default settings. Raw data were normalized by MAS 5.0 algorithm. Statistical significance of differential expression of probe sets between groups were detected by student t test. Probe set with p-value≦0.05 and absolute fold change≧1.5 were considered as differential expressed. One gene keeps only one most statistical significant differential expressed probe set.
Functional characterization of DEGs
The KEGG database (Release 91.0) and Gene Ontology (GO) category database were used for functional annotation of differentially expressed genes. Enrichment analysis of GO categories was performed by R clusterProfiler (v 3.14.3) package, and enrichment analysis of pathways was tested upon hypergeometric distribution by R ‘phyper’ function. Those GO categories a FDR<0.05 were considered as significant enriched. While pathways with a p-value< 0.05 were regared as enriched. Only those GO categories or pathways contains≧5 DEGs were kept for further analysis.
Reproducibility assessment using TCGA dataset
The gene expression profiles data of 526 LUAD patients and 59 adjacent cancer samples and clinical characteristics of matched patients were obtained from the Cancer Genome Atlas (TCGA) data portal18. LUAD sequencing data were downloaded. Differential expression of genes between patients and adjacent were detected by 'edgeR' (v 3.28.1) package, with a threshold of a FDR≦0.05. Then functional enrichment analysis were performed on these DEGs as described above. The clinical data of 522 patients were used for Cox regression analysis.
Cell culture and plasmid transfection
Human NSCLC cell lines, A549, H1299, and H1975 cells were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences. A549 cells were cultured with DMEM medium and H1299 and H1975 cells were grown in RPMI 1640 medium. Both DMEM medium and RPMI 1640 medium were supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin sulfate (100 μg/ml). Cells were maintained at 37°C in a humidified 5 % CO2 atmosphere. The empty plasmid EX-NEG-M02 (Genecopoeia) and human FoxM1-overexpression plasmid (Genecopoeia) were transfected into A549, H1299, and H1975 cells using Lipofectamine 3000 reagent (Invitrogen #2024201, MA, USA), according to the manufacturer’s instructions. After that, the A549, H1299, and H1975 cells transfected with the plasmid were used in subsequent experiments.
MTT colorimetric analysis for determining inhibition of cell proliferation
To assess the effect of berberine on the survival and proliferation of NSCLC cells, MTT analysis was adopted. A549, H1299 and H1975 cells in the logarithmic phase were seeded in a 96-well plate, at a density of 3000 cells/well and maintained at 37°C in a humidified 5% CO2 atmosphere for 16 h. Then the berberine at a density of 0, 30, 60, 90, 120, 150, 180, 210, 240, 270 μM were added, respectively. The total volume was 200 μl. Every concentration was set as well with 4 parallel wells in each group and kept at 37°C in a humidified 5% CO2 atmosphere for 24, 48 and 72 hours, respectively. When the time was due, 20 μl of MTT reagent (Sigma) was added to each well in the 96-well plate. Then the plate was kept at 37°C for 4 hours. After removing the supernatants, 150 μl of DMSO was supplemented into each well and then the plate was maintained at 37°C for 15 minutes. Absorbance (A) was determined with the microplate reader at 570 nm. The cell inhibition was calculated as follows:
Inhibition rate (%) = [(A of negative control group - A of test group)/ A of negative control group] ×100%
Determination of sphere formation efficiency
To clarify the effect of berberine on the tumorigenesis ability of NSCLC cells, plate clone assay was applied. A549, H1975 and H1299 cells (2000 for each type) in the logarithmic phase were seeded in a 6-well plate for 16 hours. Then berberine at a density of 0, 5, 10 and 20 μM were added to NSCLC cells, respectively. The cells were maintained at 37°C in a humidified 5% CO2 atmosphere. Ten days later, the colonies were stained with crystal violet and photographs of the stained colonies were taken by the digital camera and dissecting microscope.
Determination of the effects on transplanted tumor in C57BL/6 mice
C57BL/6 mice aged at 6-8 weeks and weighed at 18-22 g were purchased from SLAC Shanghai and fed in a standard feeding atmosphere at Jiangnan University. LLC cells (3×106) were suspended in the medium at 150 μl and then subcutaneously injected into the right axilla of C57BL/6 mice. After LLC cells were injected, berberine groups were given an intraperitoneal injection of berberine (100, 200 and 400 mg/Kg) continually for 4 weeks. The blank control group was administered intraperitoneally injected with PBS. The volume of the tumor was measured every three days with a vernier caliper. The formula for volume was as follows: V=（П/8）×a×b2. “a” represents the maximum diameter of the tumor, while “b” reflects the shorter diameter vertical to “a”. The C57BL/6 mice with the tumor over 2000 mm3 was put to death by CO2 suffocation.
HE staining of mouse tumor were observed
The tumors of mice were taken and soaked in 4% paraformaldehyde solution. The 4% paraformaldehyde solution was changed every day. One week later, the fixed specimens were routinely dehydrated with alcohol gradient, transparent xylene, embedded with paraffin, and sectioned 5 m thick. The tumor sections were stained with HE and were observed under microscope.
To demonstrate the expression of FOXM, immunohistochemistry was used to detect. The tumor sections of mice in each group were incubated with FOXM1 (Cell Signaling Technology, MA, USA) and detected, respectively, with the secondary antibodies(Cell Signaling Technology, MA, USA).
Total RNA from A549, H1299 and H1975 cells was extracted using Trizol reagent after the treatment of 0, 30, 60, 120 μM berberine for 24h. 1μg of total RNA was transcribed to cDNA using the PrimeScriptTM RT Master Mix kit (TaKaRa, China) aberberineording to the protocols. Quantitative RT-PCR was performed in a reaction volume of 20μL cDNA on ABI system (Applied Biosystems, Life Technologies), and carried out with the following parameters: 95°C for 30s, amplifications were carried out with 40 cycles at a melting temperature of 95°C for 5s and an annealing temperature of 60°C for 30s, followed by melt curve analysis. The relative expression was calculated using the 2-△△Ct. The following 4 genes were selected for analysis: RRM1, RRM2, POLE2, and LIG1. An 18S was used as an internal reference gene to normalize the expression of all genes. Primers for all genes were listed as follows: 18S forward: GTAACCCGTTGAACCCCATT,
18S reverse: CCATCCAATCGGTAGTAGCG.
POLD1 forward: ATCCAGAACTTCGACCTTCCG,
POLD1 reverse: ACGGCATTGAGCGTGTAGG.
DNA2 forward: AGAGCTGTCCTGAGTGAAACT,
DNA2 reverse: GAAACACCTCATGGAGAACCG.
POLE2 forward: TTTTGCAGAAGTCTTCACAGATG,
POLE2 reverse: GCAGAAGGTTGGTTTGAAGA.
RFC5 forward: GAAGCAGACGCCATGACTCAG,
RFC5 reverse: GACCGAACCGAAACCTCGT.
MCM2 forward: ATCTACGCCAAGGAGAGGGT,
MCM2 reverse: GTAATGGGGATGCTGCCTGT.
MCM4 forward: CTGTCCATTGCAAAGGCTG,
MCM4 reverse: GAGACTCAATGGGATTTGCTG.
MCM6 forward: CTGTGATGAGGTCCAACCT,
MCM6 reverse: GACATCAGGTGTTTCCACAC.
RFC3 forward: CCCTGAGACAGATTGGGAG,
RFC3 reverse: TCAAGGAGCCTTTGTGGAG.
FEN1 forward: GATGATTTCTTCAAGCCTTGAC,
FEN1 reverse: TCACAAACACAGACACAGC.
POLA2 forward: CTCTCCAAGTGCTACTCCC,
POLA2 reverse: ATACTCCCTGTGCTAAGCC.
PRIM1 forward: CTTAAACTTTATTACCGGAGGC,
PRIM1 reverse: GTAATTCTTTATCACTCCACCG.
PRIM2 forward: GGTTTAACTTTGGAACAGGC,
PRIM2 reverse: TTCCTCTTCCTTGTCTGGA.
Western blotting for determining protein
A549, H1299 and H1975 cells in the logarithmic phase were seeded in 10cm culture dishes. When cells occupied 80% of the dish, berberine were used for intervention. berberine at a density of 0, 30, 60, 120 μM were added to A549, H1299 and H1975 cells, respectively. 24 hours later, 100 μl RIPA was added to the dishes and cells were fully soaked. After centrifugation for 30 minutes, supernatants were collected for protein measurement. After being washed with 95°C water for 10 minutes, the proteins were separated with gel and transferred to a membrane. The membranes were incubated overnight at 4°C with the primary antibodies and then further incubated with secondary antibodies and finally visualized.
The relationship between cell cycle or DNA repair pathway related gene expression and overall survival of TCGA - LUAD patients was analyzed using COX regression. P < 0.05 was considered to have significant statistical significance. all these analysis were conducted using R (v. 3.6.0).