human pancreatic tumor samples.
A total of 90 cases of pancreatic cancer tissues and adjacent tissues from the pancreatic surgery department of Wuhan University People's Hospital from 2009 to 2019 were collected for RNA in situ hybridization. All patients were diagnosed with prostate cancer according to the World Health Organization's diagnostic criteria. All samples were approved by the ethics review committee and the patient's informed consent was obtained.
Two human pancreatic cancer cell lines PANC-1 and MIA-PACA2 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and both grown in high-glucose DMEM (Gibco, NY, USA). Both cell lines cultured at 37 °C in a humidified 5% CO2 incubator according to ATCC protocols.
RNA in situ hybridization
Indirect labeling method using digoxin was used to detect miR-489-3p. The RNA probe labeling method was used to construct plasmids and then synthesized by transfection. Sample processing, probe preparation, in situ hybridization, washing, blocking, and finally enzyme reaction detection. Finally, observe and take pictures under the microscope.
Cell viability analysis
Cell Counting Kit-8 (beyotime, China) was used to detect the proliferation of PC cells. According to the manufacturer's instructions, 100ul of 2x10^3 cells were seeded into a 96-well plate, and 10ul of cck8 reagent was added to each well. Absorbance values were measured every 0, 24, 48, 72 and 96 hours and recorded for statistical analysis.
Colony formation assay
Two types of pancreatic cancer cells were inoculated into six-well plates at a rate of 500 cells per well and cultured in incubators at 37°C and 5% CO2 for two weeks. The medium was then poured out, fixed with 4% paraformaldehyde for 20 minutes, and stained with 0.2% crystal violet for 30 minutes. The number of clones was observed under an optical microscope and analyzed with statistical software.
Panc-1 and mia-paca2 cells of different treatment groups were seeded into the upper chamber of Tanswell in an amount of 1x10 ^ 5 per well. 200 ul of a medium containing 10% serum was added to the upper chamber, and 700 ul of serum-free medium was added to the lower chamber. Placed in the incubator for 24 hours, discard the culture medium, washed with PBS(Hyclone, USA), and fixed with 4% paraformaldehyde(Biosharp, China) for 30 minutes. Then dyed it with 0.5% crystal violet(Solarbio,Beijing,China) solution for 30 minutes, rinse it with pure water, observe and take pictures under a microscope. Finally, the software counts and analyzes.
Quantitative real-time PCR
The cells of each group were seeded in a six-well plate, and the total RNA of each group was extracted using Trizol reagent (Invitrogen, CA, USA) when the fusion degree reached 80% -90%. CDNA was then synthesized using a PrimeScript RT reagent kit (Takara) according to the manufacturer's instructions. Real-time quantitative PCR was performed by Powerup SYBR Green PCR Master Mix (Life Technologies).
Luciferase reporter assay
Seed the cells in a good state into a six-well plate. When the six-well plate reaches 50% confluence, use lipofectamine reagent (Invitrogen) with miR-489-3p or TALD promoter-containing luciferin Enzyme reporter genes were transfected for 4-6 hours. High glucose DMEM was used to change the medium, and then cultured in an incubator for 24 hours. Then disposed according to the product instructions for the double luciferase reporter gene detection kit (Beyotime Biotechnology, China). In short, 500 microliters of reporter gene cell lysate was discarded after the medium was discarded, and the supernatant was taken for determination after sufficient lysis.
The cells of each group were seeded in a six-well plate, and when the band fusion reached 90%, the protein was obtained by lysis with RIPA lysate (beytime, China). BCA reagent (beytime, China) was used to quantify the protein, and then the protein was diluted to the same concentration, and then boiled in a refrigerator at -20 ° C until use. Electrophoresis was performed using Solarbio reagents and 10% separation gel and 5% concentrated gel were prepared according to the manufacturer's instructions. After electrophoresis and transfer, exposure is performed.
Glucose uptake andlactate production measurements
Glucose uptake was performed using the D-2-deoxyglucose method. D-2-deoxyglucose comes from Beyotime, China, and the lactic acid detection kit comes from Leagene Biological,Beijing. Analysis of glucose intake and lactic acid production was performed according to product instructions.
Cellular ATP level
Experiments were performed using an ATP detection kit (Beyotime Biotechnology, China). Add 200 μl of lysate to each well of the six-well plate according to the product instructions, and centrifuge the supernatant after full lysis. Prepare ATP working solution and add it to the detection tube to measure the luminescence value.
Extracellular acidification rate (ECAR)
Hippocampal experiments were performed with Agilent equipment: Hippocampus XFe24 Micro Edition and XFe24 cartridge. Cells of each group were incubated at 500 ° C for 7.5 × 104 cell seeds at 37 ° C for 1 hour at the hippocampal preparation station. 56 μl glucose (100 mm) (G8270σ), 62 μl oligomycin (10 μM) (σ) 75351 and 69 μl 2 dg (1 meter) (D6134, Sigma) were added to the cartridge wells. Then read the ECAR value.
In vivo assay
10 ^ 7panc-1 cells (miiR-NC group and miR-489-3p up-regulation group) were injected to 4 weeks of age female NCr nude mice were injected subcutaneously (Hua Fukang Biotechnology, Beijing). Tumor size was measured with calipers every 7 days. Over time, the size of the tumor is measured. At the end of the experiment, the mice were euthanized and subcutaneous tumors were collected. Perform other analyses.
Immunohistochemical staining was used to detect the expression of proliferation and metabolic indicators. In short, the subcutaneous tumor tissue was cut into 3um and then dewaxed. The sections were then incubated with rabbit anti-monoclonal and refrigerated at 4 ° C overnight. After washing three times with PBS, each piece was incubated with goat anti-rabbit IgG for 30 minutes and then developed with 3,3 'diaminobenzidine (DAB). All antibodies were from Proteintech, USA.
The results are calculated and analyzed using the mean ± standard deviation. GraphPad Prism 7.0 (San Diego, California, USA) was used for mapping and statistical analysis. Chi-square test was used to analyze the relationship between miR-489-3p expression level and clinicopathological characteristics in PC. The Kaplan-Meier curve method was used to analyze the overall survival rate. Student's t test was used for statistical comparison between the groups. * P <0.05, * * P <0.05.