LncRNA MIR17HG promotes colorectal cancer liver metastasis by mediating a glycolysis-associated positive feedback circuit

Glycolysis plays a crucial role in reprogramming the metastatic tumor microenvironment. A series of lncRNAs have been identified to function as oncogenic molecules by regulating glycolysis. However, the roles of glycolysis-related lncRNAs in regulating colorectal cancer liver metastasis (CRLM) remain poorly understood. In the present study, the expression of the glycolysis-related lncRNA MIR17HG gradually increased from adjacent normal to CRC to the paired liver metastatic tissues, and high MIR17HG expression predicted poor survival, especially in patients with liver metastasis. Functionally, MIR17HG promoted glycolysis in CRC cells and enhanced their invasion and liver metastasis in vitro and in vivo. Mechanistically, MIR17HG functioned as a ceRNA to regulate HK1 expression by sponging miR-138-5p, resulting in glycolysis in CRC cells and leading to their invasion and liver metastasis. More interestingly, lactate accumulated via glycolysis activated the p38/Elk-1 signaling pathway to promote the transcriptional expression of MIR17HG in CRC cells, forming a positive feedback loop, which eventually resulted in persistent glycolysis and the invasion and liver metastasis of CRC cells. In conclusion, the present study indicates that the lactate-responsive lncRNA MIR17HG, acting as a ceRNA, promotes CRLM through a glycolysis-mediated positive feedback circuit and might be a novel biomarker and therapeutic target for CRLM.


Cell culture and vector construction
Eight CRC cell lines (Caco2, HT29, SW480, SW620, RKO, HCT116, HCT8, and DLD-1) were obtained from the Cell Bank of the Chinese Academy of Sciences, and one normal colon mucosal epithelial cell line (FHC) was obtained from ATCC. The cell lines were characterized using short tandem repeat markers and tested for mycoplasma contamination. Patients who died from non-cancerous causes (such as car accidents and cardiovascular disease) were excluded. The entire patient cohort was divided into two groups according to the expression level of MIR17HG or miR-138-5p (high: fold change of equal to or greater than the mean; low: fold change of less than the mean). For metabolic analysis, a total of 50 patients who underwent 18 F-FDG PET/CT examination at FUSCC between 2019 and 2020 were enrolled. Imaging diagnoses were processed independently by two radiologists. The sample size difference between each group was less than 3 times to ensure adequate power to detect a pre-specified effect. The present study was approved by the Ethics Committee of Fudan University Shanghai Cancer Center (FUSCC, ID: 050432-4-1911D), and informed consent was obtained from all patients before enrollment in this study.

Chromatin immunoprecipitation (ChIP) assay
We performed a ChIP assay to investigate the direct interaction between Elk-1 and the MIR17HG promoter using a Plus Enzymatic ChIP Kit (CST, Boston, MA, USA) according to the kit's reference guide. Briefly, we added 37% formaldehyde to a final concentration of 1% and incubated cells for 10 minutes at room temperature before glycine was added to terminate cross-linking. After mixing with the micrococcal nuclease provided in the kit, cells were incubated for 30 minutes at 37°C for DNA digestion, which was stopped by incubation with EDTA on ice for 2 minutes. For each immunoprecipitation reaction, an anti-Elk-1 antibody (2 μg) or a protocol-recommended internal control antibody was added to each microcentrifuge tube and incubated overnight at 4°C prior to incubation with the provided magnetic beads for 2 hours at 4°C with rotation. Then, elution of chromatin, reversal of cross-links, and DNA purification were sequentially performed as recommended. The enrichment degree was determined by quantitative real-time PCR. Each experiment was repeated five times.

Cell migration and invasion assay
To perform the wound healing assay, we marked the back of 6-well plates with horizontal lines spaced 0.5~1 cm apart before seeding of cells in logarithmic phase, which made it easier to observe the same regions at different time points. We used a 200 μL pipette tip to scratch the cells in the wells, and the initial scratch area (0 h area) was assessed after washing 3 times with sterile PBS. Cells were incubated in a 5% CO2 37℃ atmosphere as usual, and we then measured and recorded the wound area after 24 hours. The migration ability was calculated with the following equation

Immunohistochemical (IHC) assay
Following dewaxing and hydration, sections were incubated with 3% hydrogen peroxide in a wet box for 10 minutes. After washing with PBS three times, antigen retrieval was performed in 10 mM citrate buffer (pH=6.0). Blocking of unspecific antigen binding sites using 10% normal goat serum was performed at room temperature for 1 hour. Subsequently, sections were incubated with a primary antibody purchased from Abcam (ab150423, 1:100) overnight at 4°C. Then, sections were incubated with an HRP-conjugated secondary antibody at room temperature for 1 hour and washed two times with PBS. Then, slides were stained with diaminobenzidine at room temperature for 10 minutes and counterstained with 20 hematoxylin.

Dual luciferase assay
To confirm the interaction between miR-138-5p and MIR17HG or HK1, wild-type or mutated fragments of the MIR17HG gene and the HK1 3'UTR with or without the putative miR-138-5p complementary sequences were synthesized by GenePharma (Shanghai, China) and inserted downstream of the firefly luciferase coding sequence in the pmirGLO vector. The biotinylated miR-138-5p mimics and mutants were synthesized by RiboBio (Guangzhou, China).

RNA immunoprecipitation (RIP) assay
MiR-138-5p mimics and the miR-138-5p mutant were transfected into cells using Glucose uptake, lactate production and ATP production assays CRC cells were seeded into a 96-well plate and were then analyzed using a colorimetric glucose uptake assay kit (AAT Bioquest, Sunnyvale, CA, USA) and a colorimetric L-lactate assay kit (AAT Bioquest, Sunnyvale, CA, USA) according to the manufacturer's protocols.
For the ATP assay, cells cultured in 6-well plates were lysed in 200 μL/well lysis buffer on ice and centrifuged at 4°C. The ATP content in the supernatant was determined using an enhanced ATP assay kit (Beyotime Biotechnology, Shanghai, China) in adherence to the recommended protocol. The content was normalized to the cell number. Each experiment was repeated five 22 times.

Extracellular acidification rate assay
The extracellular acidification rate (ECAR) was measured using a Seahorse XFe24 extracellular flux analyzer (Seahorse Bioscience, Billerica, MA, USA) according to the manufacturer's guidelines. Briefly, on the day before the ECAR assay, the sensors were submerged in the calibrant and placed in a non-CO2 37℃ incubator overnight for improved hydration. Cells were seeded into a 24-well plate, and medium was added to a final volume of 250 μL/well after 5 hours. After overnight incubation, assay medium containing 2 mM glutamine was used to rinse cells two times and was added to a final volume of 500 μL/well.
Then, cells were incubated in a non-CO2 37°C atmosphere for 1 hour prior to the assay.
Glucose (10 mM), oligomycin (1 μM), and 2-DG (100 mM) were injected sequentially to measure the ECAR with 3 repeated tests after each injection. Each point represents the average of three independent samples.

In vivo experiment
Spleen injection, with the highest success rate is the most effective method for the construction of CRC liver metastasis causing little damage to mice [1]. To establish liver metastasis models, CRC cells (1×10 7 cells/200 μL) were injected into the spleens of female BALB/c-nude mice (six weeks of age, 15 mice/group). Then, five randomly selected mice from each group were sacrificed, and the livers were collected for hematoxylin and eosin (HE) staining at week 6. The liver metastatic colonies were counted under a light microscope. The rest of the mice in each group were observed until week 12 for survival analysis. Mice that had died from other causes (such as fighting and infection) were excluded. Our experiments 23 were approved by the Animal Experiment Ethics Committee of FUSCC.

Statistical analysis
Quantitative data for two or more than two groups were analyzed using two-sided t-test or one-way ANOVA, respectively. The variance was similar between the groups that were statistically compared using t-test. Qualitative data were analyzed by the chi-square test or Fisher's exact test. The Kaplan-Meier method with the log-rank test was used for survival analysis. Correlations were analyzed by Pearson's or Spearman's test. R program 3.0.1 was used for comprehensive WGCNA. SPSS v25 (IBM Inc., Armonk, NY, USA) was used for all analyses, and Prism v8 (GraphPad Software Inc., San Diego, CA, USA) was employed for plotting. All experiments were repeated at least three times, and the data are shown as the mean ± standard deviation (SD) values. P<0.05 was considered to indicate statistical significance.