Drug monomers from Salvia miltiorrhiza pg. Enhancing tight protein expression for therapeutic effects on lung cancer

Drug monomers from Salvia miltiorrhiza pg.  Enhancing tight protein expression for therapeutic effects on lung cancer

Drugs and antibodies

Cryptotanshinone (Batch: 110852; >99% purity) and tanshinone IIA (Batch: 110766; >98.9% purity) were purchased from the National Institute of Food and Drug Supervision of China. RPMI-1640 medium fetal bovine serum (FBS) and trypsin were purchased from Gibco BRL (Grand Island, NY, USA). ZO-1 (D7D12) Rabbit mAb (8193) and GAPDH (D16H11) XP antibodies® Rabbit mAb (5174), anti-rabbit IgG horseradish peroxidase (HRP)-conjugated antibody (7074) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Occludin antibody (EPR20992) (ab216327), anti-VEGFA antibody (ab46154), anti-Ki67 antibody (37C7-12) (ab245113), and goat anti-mouse IgG H&L antibody (Alexa Fluor).® 647) (ab150115), and goat anti-rabbit IgG H&L (Alexa Fluor® 488) (ab150077) was purchased from Abcam (Cambridge, MA, USA). qRT-PCR and reverse transcription kits were purchased from TOYOBO Co., Ltd. (Osaka, Japan). PCR primers were manufactured by Wuhan Servicebio Technology Co., Ltd (Wuhan, China). WB kits were purchased from Applygen Technologies Inc. (Beijing, China).

Animal experiments and tissue harvesting

All animal experiments were previously approved by the Animal Care Committee (Ethics Committee of Guang'anmen Hospital, Chinese Academy of Medical Sciences, No. 2020-011-SQ) and were performed in accordance with the national guidelines for animal experiments. All animal experiments were performed in accordance with REACH guidelines. Male Balb/c-nu/nu nude mice of 6 to 8 weeks duration were purchased from Animal Technology at Vital River Laboratory in Beijing (Beijing, China) and housed at the Peking University Health Science Center Department of Laboratory Animal Science (SPF level, 22± 2°C), 55±5% relative humidity with 12-hour light/dark cycle). Mice were free to eat and water. 1 x 106 A549 cells were inoculated subcutaneously into mice22. The 36 model mice were randomly divided into control group (n = 6, normal saline, once daily), bevacizumab group (n = 6, 5 mg/kg/day), high-dose CPT group (n = 6, 10 mg)/kg/day), low-dose CPT group (n = 6, 5 mg/kg/day), high-dose TanIIA group (n = 6, 10 mg/kg/day), low-dose TanIIA group (n = 6 , 5 mg/kg/day). The inoculation site of mice was observed daily. The dosage of CPT and TanIIA in animals is referred to preliminary experiments and literature reports23,24,25. The longest (a) and shortest (b) diameter of the tumor was measured using a vernier caliper, and the tumor volume was calculated as V (mm3) = 1/6π ab2. Pathological changes were observed in the transplanted tumor in mice. On day 21 after modeling, explanted rat tumors were taken, fixed with paraformaldehyde, dehydrated, embedded in paraffin, and cut into slices. Changes in inflammatory cell infiltration and tumor cell morphology were observed by staining light microscopy.

Cell culture and management

A549 cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in 5% CO.2 The mammospheres were obtained from the National Cell Line Resource Infrastructure (Beijing, China) and cultured. CPT was dissolved in DMSO to prepare 10 mg/mL (34 mM) and TanIIA was dissolved in DMSO to prepare 5 mg/mL (17 mM) stock solutions for in vitro experiments. Cell viability was measured using the CCK-8 assay. A concentration of 6 μg/ml CPT (20.2 μM) and 4 μg/ml (13.6 μM) TanIIA was added to the cell for subsequent experiments.

Cell viability assay

Cell proliferation and cytotoxicity were determined using the CCK-8 assay (Dogendo Laboratories, Kumamoto, Japan). At a total density of approximately 4000 cells/well A549 cells were seeded in 96-well plates for 24 h. Cells were treated with different concentrations of CPT and TanIIA at 100 μl per well and incubated at 37°C, 5% CO .2 For 24 and 48 hours. Next, 10 μl of CCK-8 reagent was added to each well and incubated at 37°C and 5% CO.2 For 2 hours. Absorbance was measured at a wavelength of 450 nm.

Programmed cell death assay

A549 cells were plated in 6-well plates at a density of 5 × 105 Cells/well were incubated with 0, 6 µg/ml CPT and 4 µg/ml TanIIA for 24 h at 37 °C. The relative amount of Annexin V-fluorescein isothiocyanate positive/propidium iodide negative cells was detected using AnnexinV-FITC/PI Kit I (556547, BD Biosciences, Franklin Lakes, NJ, USA) and detected and analyzed using flow cytometry (BD) Biosciences, Franklin Lakes, New Jersey, USA).

Wound healing examination

A549 cells, in 2 ml of RPMI medium containing 10% FBS, were cultured in 6-well cell culture plates for 24 h. After cells formed a compact monolayer, they were scored with a pipette tip and cultured in medium containing CPT (6 μg/ml) and TanIIA (4 μg/ml) for 24 h and 48 h. Then the scratch distance was examined at the beginning of the experiment and after 24 hours and 48 hours.

If staining and analysis

Cells were seeded into 24-well plates at a density of 5 × 105 Cells per well in 500 µl of medium. After cells were treated with different methods, cells were fixed at 80% confluence on coverslips with 4% paraformaldehyde. Samples were incubated with Occludin (1:200, ab216327, abcam, Cambridge, MA, USA) and ZO1 (1:200, 8193, Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. Next, samples were washed 3 times with PBST and incubated with the secondary antibody (ab150115, ab150077, abcam, Cambridge, MA, UK) at 37°C for 60 min. Samples were stained with DAPI and maintained at 4°C. Measurements were made at 400× magnification using an optical grid.

mRNA profile detection

Total RNA extraction from A549 was performed after different treatments using TRIzol reagent. The mRNA profile of cells in different treatment groups was analyzed by RNA sequencing (RNA-Seq), and differentially expressed genes were identified based on Q value (adjusted P value) ≥ 0.05.26. The isolated RNA samples were sent to BGI Co., LTD. in Shenzhen for mRNA-seq using BGIEQ-500.

Job enrichment and path analysis

Biological Function Gene Signal Pathway (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed using the online platform “Dr. Tom 2.0” designed by BGI Tech Co., LTD. Among them, the results of GO enrichment analysis come from the three databases The following: Uniprot, gene2GO, and idmapping KEGG pathway enrichment analysis were retrieved from KEGG 93.0.

Cell transfection

Cells were distributed in a six-well plate according to a density of 5 × 105 cells/ml per well. At approximately 24 h, the miR-21-5p mimic vector was used and transfected into cells using the lipo3000 kit (Invitrogen, Shanghai, China), lipo3000 doses: miR-21-5p mimic = 3 (µl): 3 (µg). After gene transfection, cells were then cultured for 48 h and harvested for qRT-PCR and WB detection. Primers for miR-21-5p mimic oligonucleotides are listed in Table 1. Reference gene sequences were cleaned and sorted by Bowtie2, and gene expression level data were then calculated by RSEM. Finally, differentially expressed genes (DEGs) were obtained by DESeq2 analysis (Q value ≥ 0.05) (Figure 5A). GO and KEGG pathway analysis were used to understand the biological processes and pathways that affected A549 cells after CPT or TanIIA treatment. Detailed results are shown in Tables S4 to S7. Based on the Q values, we display the top 20 terms from small to large.

Full size table

Western blot analysis

Proteins are extracted and quantified according to procedures specified by the manufacturer of the reagent used. The protein extract was extracted (50 mmol/L Tris-HCL, pH 7.4; 10% SDS; 1 mmol/L PMSF, 0.25% sodium deoxycholate) and placed on ice for 30 min. Then, the supernatant was collected by centrifugation at 12,000 rpm for 10 min at 4°C and the protein concentration was determined by the BCA method. The 20 μg protein samples were separated by 5%, 10% SDS-polyacrylamide electrophoresis (PAGE) and then transferred to a polyvinylidene fluoride membrane (Millipore, USA) by electrophoresis. Using rabbit ZO1 (1:10000, 8193, CST, Danvers, MA, USA), rabbit anti-VEGFA (1:3000, ab216327, Abcam, Cambridge, MA, USA), rabbit anti-VEGFA (1:10000, ab46154, Abcam, Cambridge, MA, USA), anti-GAPDH polyclonal antibody (1:5000, 5174, CST, Danvers, MA, USA). Blots were cut before hybridization with different antibodies. The PVDF film was incubated in blocking solution at 4 °C. Finally, these membranes were incubated with an anti-rabbit IgG-HRP-conjugated antibody (1:5000,7074, abcam, Cambridge, MA, UK) at room temperature for at least 60 min and with electrochemiluminescence (ECL) reagent for approximately 30 min. second. The treated PVDF films were exposed to X-rays and imaged using a BIO-RAD ChemiDoc XRS gel imaging system. Image export and analysis using ImageProPlus 4.5 software (Media Cybernetics, Bethesda, MD, USA).

qRT-PCR analysis

A total RNA of approximately 1 × 10 was isolated6 of A549 cells according to the manufacturer's instructions. RNA samples with an OD260/OD280 ratio of 1.9-2.1 and an OD260/OD230 ratio greater than 2.0 were used for analysis. cDNA was synthesized from total RNA by reverse transcribing 1 μg of total RNA using a reverse transcription kit (FSQ-101, TOYOBO Co., Ltd., Osaka, Japan). Premier 5.0 software was used to design PCR primer sequences based on GenBank sequences, which are shown in Table 2. PCR was performed at 95°C for 15 min, followed by denaturation at 95°C for 10 s, and annealing at 60°C. for 30 s, extension at 72°C for 31 s (Foster Applied Biosystems 7300 USA), 40 cycles. Relative quantitative analysis was performed by 2− DCQ method27.

Table 2: List of primer sequences for qRT-PCR.

Statistical analyses

Statistical analysis and mapping involved in this study were performed using GraphPad Prism8 (GraphPad Software, San Diego, CA, USA). When normal distribution and variance were equal for each group, data were expressed as mean ± standard deviation (SD) and calculated using SPSS version 25.0 (SPSS Inc., Chicago, IL, USA). When data included multiple group comparisons and post hoc comparisons, one-way analysis of variance (ANOVA) and Tukey's multiple comparison test were used, respectively. We require this on both sides sThe value < 0.05, and the difference was considered statistically significant.

    (Tags for translation)Cancer

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