2.10 Subcutaneous xenograft experiments
Four-week-old BALB/c female nude mice were purchased from Beijing HFK Bioscience Co.,LTD. The tumor masses prepared were embedded subcutaneously in nude mice, and random grouping was started when the tumor masses grew to 4 to 5mm in diameter. Forty nude mice were divided into 4 groups with 10 mice in each group, which were si-NC(HOXA11-AS) group, si-HOXA11-AS group, si-NC(Tpl2) group and si-Tpl2 group. After that, tumor volumes were measured every 2 days and HOXA11-AS and Tpl2 siRNAs were injected into the knockdown group at a dose of 10 µL of siRNA versus 10 µL of Lipofectamine TM 3000 per nude mouse. After 21 days, the nude mice were sacrificed, subcutaneous tumors were removed, images were collected and IHC staining was performed. The tumor volume was calculated with the formula Volume= (length × width2)/2.
2.11 Nuclear and cytoplasmic RNA extracted separately
After the cells were prepared, the medium was absorbed and discarded, washed twice with PBS, and the cells were scraped off with 400 µL precooled PBS. Then, transferred into 1.5ml EP tube, centrifuged at 10000rpm at 4℃ for 10s, and the supernatant was absorbed and discarded. Resuspend the cells with 400 µL PBS containing RNA enzyme inhibitor (RNA enzyme inhibitors: PBS = 1:20) and 0.1% NP-40 (NP-40: PBS = 1:1000), put it in ice for 5min, then vortexed for 5s. After centrifugation at 10000rpm at 4℃ for 20s, the supernatant was extracted from cytoplasm and transferred into a new EP tube. At this time, the supernatant was cytoplasm extract, and the precipitate was nucleus. The nuclei were resuspended by 200 µL PBS containing 0.5% NP-40 (NP-40: PBS = 1:200) and RNA enzyme inhibitor (RNA enzyme inhibitors: PBS = 1:20), and were blown for 10 times, placed on ice for 10min, and centrifuged for 20s at 13000 rpm at 4℃. The supernatant was the nuclear extract, and the nuclear extract was used for subsequent RT-qPCR experiments according to the above steps.
2.12 RNA fluorescence in situ hybridization (RNA-FISH)
The FISH kit was purchased from Shanghai GenePharma Co., Ltd. The experimental procedures were carried out in full accordance with the kit instructions.
2.13 RNA immunoprecipitation(RIP)
The Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (17–700, Merck Millipore, USA), and the experimental steps were carried out in accordance with the kit instructions. Calculate according to the following formula:
C[T] = CT = Average threshold cycle of PCR reaction
Percent Input = 10%×2(CT Input Sample − CT IP Sample)
2.14 MiRNA RT-qPCR
The miRNA RT and qPCR experiments were conducted using the miRNA kit (GenePharma, China), and the experimental procedures were conducted in accordance with the instructions of the kit.
2.15 ChIRP assay
The ChIRP experiment (BIOSENSE, China) was used to verify the binding of HOXA11-AS and miR-let7b-5p, and all the experimental steps were carried out in accordance with the kit instructions.
2.16 Single/dual luciferase reporter assay
The single and dual luciferase plasmids in this study were all purchased from Hanbio Biotechnology Co., Ltd. The luciferase plasmids were transfected with Lipofectamine 3000 (0.5µg plasmid, 1 µL P3000 reagent and 1.5 µL Lipofectamine 3000 reagent per well). At the same time, siRNA (1 µL siRNA and 1.5 µL Lipofectamine TM RNAiMAX reagent) could be co-transfected.
2.17 Western blot
The protein was extracted by RIPA lysis buffer (Solarbio, China) and the protein concentration was detected with a BCA kit (Solarbio, China). Proteins were separated by 10% SDS-PAGE gels and transferred to 0.22 µm PVDF membranes (Millipore, USA). The membranes were blocked with 5% skim milk powder and incubated with specific antibodies at 4°C overnight. The membranes were then incubated with the appropriate secondary antibodies, and an ECL detection system (Bio-Rad, USA) was used to test the protein bands. TPL2, ERK1/2, MEK1/2, p-TPL2, p-ERK1/2, p-MEK1/2 antibodies were all purchased from CST, USA. The relevant antibodies used are shown in Supplementary Table 3.
2.18 Immunofluorescence (IF)
Cell slides were made in advance, and 2×104 to 5×104 cells were planted on each 24-well cell slide. After 24h, the medium was discarded, and the cells were fixed with 4% formaldehyde for 15min. PBS was used to wash cells for three times, 3min each time. 0.5% Triton X-100 was used to incubate for 20min at room temperature, and the cells was washed with PBS for 3 times, 3min each time. PBS was absorbed and dried with absorbent paper. Then, a sufficient amount of diluted β-catenin (CST, 8480, 1:100) was added to each well. The solution was placed into a cartridge and incubated overnight at 4℃. The cells were washed with PBST for 3 times, 3min each. The diluted fluorescent secondary antibody was added and incubated for 1h in 37℃ without light. The cells were washed with PBST for 3 times, 3min each time. Each hole was dyed with 100 µL DAPI working solution for 10min, and cleaned with PBST for 4 times, 5min each time. The cover glass was dried with absorbent paper and laced upside down on the slide with anti-fluorescence attenuation sealing agent (Sigma, USA), then the fluorescence image was collected.
2.19 Chromatin Immunoprecipitation (ChIP) Assay
Chromatin immunoprecipitation assay (ChIP) was performed via the ChIP kit (56383, CST, USA), and all experimental steps were performed according to the kit instructions. Each primer corresponded to 3 samples (IgG, target protein, and INPUT), and each sample was repeated with 3 duplicate Wells. The following PCR procedures were performed: Pre-denaturation at 95℃ for 3 min; After denaturation at 95℃ for 3min, annealing extended at 60℃ for 60s, 40 cycles. The primer sequences are shown in Supplementary Table 3. Calculate according to the following formula :(IP sample was IgG and target protein sample)
C[T] = CT = Average threshold cycle of PCR reaction
Percent Input = 2%×2(CT Input Sample − CT IP Sample)
2.20 Transcriptomics and proteomics analysis
The transcriptome in this study was sequenced at Guangzhou Gene Denovo Biotechnology Co., Ltd., and the proteomic was analysed at Shanghai Zhongke New Life Biotechnology Co., Ltd. The criteria for determining differential genes in transcriptomics were |fold change|(FC) > 1.5, P < 0.05. A total of 17226 genes were detected by transcriptomics, among which 615 genes were differentially expressed. 404 genes were up-regulated and 211 genes were down-regulated. The proteomic criteria were |fold change|> 1.2, P < 0.05. A total of 5458 proteins were detected by proteomics, among which 136 were differentially expressed. 64 proteins were up-regulated and 72 proteins were down-regulated.
2.21 Actinomycin D (ActD) experiment
The cells were seeded into 6-well plates in advance, and the 6-well plates were divided into groups (0h, 2h, 4h, 6h, 8h, 12h), and 2 groups were needed for each cell line. The concentration of ActD (Sigma, USA) was 6.28 µg/µL, and the dosage was 1µg/ mL medium volume per well according to the instructions, so the amount of ActD used in each well was 2µg. After the addition of ActD, cells were collected by TRIzol every 2h, and placed at -80℃ for use at the last 4h. After all samples were collected at all time points, RNA was extracted to detect the relative expression of the target gene.
2.22 ROS sensitivity test
H2O2 was purchased from Sigma, USA, and the concentration was 29.4µmol/ µL. Firstly, we needed to detect the IC50 (half maximal inhibitory concentration) of H2O2 on U87 and LN229 cell lines, and inoculated 10000 cells per well in 96-well plates. The dosage of H2O2 was divided into 6nM, 8nM, 10nM, 12nM, 14nM, 16nM, 18nM, 20nM, 22nM, 24nM, 26nM, 28nM, 30nM, 35nM and 40nM according to the gradient. The absorbance (OD) of each well was measured by CCK-8 at 8h after dosing. The survival rate of different concentration groups was calculated by the formula, and IC50 was calculated. The formula was as follows: cell survival rate %=[(As-Ab)/(Ac-Ab)] ×100 (As was the experimental well, Ac was the control well, and AB was the blank well).
The pH-sensitive nanoparticles (NPs) we used were synthesized by Professor Wang Sheng's research group in Tianjin University, and the concentration was 1000µM. This pH-sensitive nanoparticles could exist stably and decompose into PEG-PDPA and MLH under the action of hydrogen ions after entering cells. Iron ions (forming iron pool, LIP) could react with MLH and promote the decomposition of MLH into RO· (peroxide)[22]. The concentrations of NPs were divided into 1 µM, 10 µM, 30 µM, 60 µM, 90 µM, and 120 µM. The IC50 of NPs was measured at 24h, 48h, and 72h after the addition of NPs and calculated at each time point.
2.23 In vivo glioma orthotopic model
All animal procedures were conducted in accordance with protocols approved by
the Tianjin Medical University Animal Care and Use Committee and followed guidelines for animal welfare. Four-week-old BALB/c female nude mice were purchased from Beijing HFK Bioscience Co.,LTD. Firstly, shRNA lentiviruses (or scrambled lentivirus, GenePharma, China) were used to construct U87 cell lines with HOXA11-AS(Tpl2) knockdown stably. For intracranial studies, 5×105 control or HOXA11-AS(Tpl2) knockdown U87 cells were injected into the intracranial striatum of nude mice with a stereotactic instrument and a microinfusion pump (Stoelting Co., USA). The animals were divided into 4 groups (control, control + NPs, siHOXA11-AS, siHOXA11-AS + NPs), with 10 mice in each group.
Starting on day 14 after tumor cell implantation, NPs (13.33µmol/kg) was given to the control + NPs and siHOXA11-AS + NPs groups every other day for 4 times. Other animals were treated with an equal volume of PBS alone. In order to obtain tumor growth status in live animals of different treatment groups by bioluminescent imaging, the mice were anesthetized and injected intraperitoneally with D-luciferin (150 mg/kg, beetle luciferin, potassium salt, E1605, Promega) 10 min prior to imaging with the IVIS imaging system (perkinelmer, USA) for 10–120 s. Eight weeks post implantation, the surviving nude mice in each group were sacrificed, and the brain samples were taken for hematoxylin and eosin (HE) staining and ISH. Survival analysis was determined using Kaplan–Meier survival curve
2.24 H&E staining and immumohistochemical (IHC) staining
For hematoxylin and eosin(H&E) staining, slides were stained with Mayer’s hematoxylin (Sigma-Aldrich) and 0.1% sodium bicarbonate and counterstained with Eosin Y solution (Sigma-Aldrich). For IHC, the paraffin-embedded tumor sections were deparaffinized and rehydrated. Microwave heating was performed for antigen retrieval. After incubating with the primary and secondary antibodies, the sections were incubated with diaminobenzidine and counterstained with hematoxylin (Solarbio, China). Representative images were observed using an Olympus light microscope.
2.25 Statistical analysis
The image processing software used in this study was Photoshop CS6, and the image production and statistical calculation software was GraphPad Prism 8. In this study, unpaired t-test was used for the difference comparison between the two groups involved, one-way ANOVA was used for the comparison between the multiple groups, and two-way ANOVA was used for the comparison of OD values between the multiple groups of CCK-8. The log-rank test was used for survival analysis. P < 0.05 was considered statistically significant.