Downregulation of Circ-PITHD1 Suppressed Colorectal Cancer via Glycolysis Inhibition through miR-590-5p/HK2 Axis

Colorectal cancer (CRC) is a frequent malignancy around the globe. Circular RNAs (circRNAs) are implicated in CRC development. Nevertheless, the regulatory mechanisms and biological functions regarding circRNAs in CRC progression are largely unclear. The present investigation employed next-generation sequencing (NGS) to study the abnormal circRNA expression in CRC tissues. The regulatory mechanism and targets were then analyzed by bioinformatics, luciferase reporter analysis, CCK8, colony formation, and Transwell migration. In vivo metastasis and tumorigenesis assays were conducted to elucidate circ-PITHD1 roles regarding CRC. The data showed that circ-PITHD1 expression increased in a CRC cell line and tissues, which indicated that circ-PITHD1 functioned in CRC progression. circ-PITHD1 downregulation inhibited CRC invasion and proliferation in the experiments. Luciferase reporter results confirmed that both miR-590-5p and hexokinase 2 (HK2) were circ-PITHD1 downstream targets. HK2 overexpression or miR-590-5p suppression reversed CRC cell proliferation and invasion after silencing of circ-PITHD1 by regulation of glycolysis. Taken together, this investigation discovered that circ-PITHD1 downregulation suppressed CRC progression by inhibiting glycolysis via the miR-590-5p/HK2 axis.


Introduction
Colorectal cancer (CRC) is the 3rd most general tumor and the 4th most deadly cancer on the globe. ∼10% of all new cancer patients are CRC [1,2]. ough neoadjuvant chemoradiotherapy, postoperative chemoradiotherapy, surgery, and immunotherapy are broadly utilized and are rapidly gaining acceptance among CRC patients, the prognosis of patients with advanced CRC remains poor [3]. e specific mechanism of CRC occurrence and development is still unclear, which is a major reason for the poor prognosis of CRC. us, it is urgent to identify CRC progression pathogenesis and develop special diagnostic biomarkers along with precise therapy targets.
Circular RNA (circRNA) is initiated due to pre-mRNAback-splicing [4]. Exonic circRNAs are the most common circRNAs, which are primarily localized in the cytoplasm to act with microRNAs (miRNAs) or RNAbinding proteins [5,6]. An accumulation study found that circRNAs have important functions in various cancers. As a miRNA sponge, circRNAs regulate mRNA expression and ultimately improve their functions in cancer cell proliferation and metastasis [7,8]. Previous investigations discovered that circ-0087862 promoted CRC progression by upregulating BACH1 expression via miR-142-3p sponging [9]. e expression of circ-0000212 promotes CRC cell proliferation by modulating FOXP4 expression via sponging miR-491 [10]. e role of circRNA in CRC progression is still unknown. e present investigation sought to determine circRNA expression in CRC and to identify underlying mechanisms. e data showed that circ-PITHD1 is highly expressed in CRC cell lines, which increased CRC cell invasion and proliferation. Additionally, we found that circ-PITHD1 knockdown repressed CRC progression by inhibiting hexokinase 2 (HK2)-mediated glycolysis through sponging miR-590-5p. ese data demonstrate that circ-PITHD1 may represent a novel CRC diagnosis and treatment target.

Patients.
In total, we obtained six paired CRC samples and adjacent normal tissues from the Second Affiliated Hospital of Soochow University. e Ethics Committee in the Second Affiliated Hospital of Soochow University approved our study after receiving written consent from the patients. Tissues were stored at −80°C.

RNA Sequencing, Quantification, and Identifications.
Total RNA was obtained from the freshly frozen CRC and adjacent tissue pairs. We used an Agilent 2200 system (Agilent Technologies, USA) to confirm the quality of the RNA. A RiboMinus eukaryote kit (QIAGEN, Valencia, CA, USA) was used to eliminate ribosomal RNA. We performed NGS with the Illumina HiSeq 3000 (Illumina, San Diego, CA, USA) and aligned reads. We collected unmapped reads to characterize circRNAs. We counted reads that aligned to circRNA junctions having an overhang of ≥6 nt for each candidate.

Cell Proliferations.
e technician seeded cells into 96well plates with 2 × 10 3 cells/well density. At established time points, each sample absorbance was determined at 450 nm by applying the CCK-8 assay (Yeasen Biotech Co., Ltd, Shanghai, China). A cell viability curve was then plotted.

Transwell Migration Assay.
A cell suspension of 2.0 × 10 5 /mL was added to each well (200 μL/well) in a Transwell chamber (Millipore, Billerica, MA, USA) on the upper side. e 500 μL medium, including 10% FBS, was put on the lower side. After 1 d incubation, migrated cells were fixed to the bottom side by 4% paraformaldehyde for 15 min before staining with 0.1% crystal violet for 5 min. We observed and calculated the number of migrated cells using a microscope. Six fields of view were randomly selected for each sample.

Lactate Production and Glucose Uptake Assay.
Lovo cells together with SW480 cells were cultured in a glucose-free DMEM medium for 16 h. e medium was changed by highglucose DMEM medium and the CRC cells were cultivated for another day. Lactate production and glucose uptake were determined by utilizing a lactate oxidase-based colorimetric assay and a fluorescence-based glucose assay kit (BioVision, Milpitas, CA, USA), respectively.
2.11. In Vivo Experiments. CRC nude mouse models were prepared by injecting SW480 cells with sh-NC or sh-circ-PITHD1 into the mice flank. Tumor weight and volume were measured.
e Animal Ethics Committee of the Second Affiliated Hospital of Soochow University approved experiments. e present research followed the Guide for Care and Use of Laboratory Animals.
For tumor metastasis analysis, stably transfected luminescence-labeled SW480 cells with sh-circ-PITHD1 or sh-NC, which we suspended in sterile PBS, were injected into every 4-week-old male nude mouse tail vein. After 4 w, the in vivo bioluminescence imaging system was used for lung metastasis evaluation. Metastatic foci numbers within lung tissues were measured following HE staining.

Statistical
Analyses. Data are denoted by means ± SD. Statistics analysis was made utilizing GraphPad Prism (La Jolla, USA) to compare significance between groups. P value ≤0.05 was considered as statistically significance. Two-tailed Student's t-tests were utilized to calculate significant differences between groups. One-way ANOVA with post hoc Bonferroni tests was applied to compare significant differences between the groups.

Circ-PITHD1 Functions Importantly for CRC Progression.
More and more studies have reported that circRNA has important functions in CRC progression [11], while the regulatory mechanism is unknown. e present investigation employed NGS and found that circRNA was abnormally expressed in CRC tissues compared with adjacent normal tissues (Figure 1(a)). RT-qPCR data revealed that five highly expressed circRNAs were observed according to NGS results. e data showcased that only circ-PITHD1 expression was upregulated significantly in CRC tissues (Figure 1(b)). e RT-qPCR data verified that circ-PITHD1 expression was increased in CRC cells, including SW480, SW620, and Lovo compared with normal human colonic epithelial NCM460 cells. Additionally, Lovo and SW480 cells had higher circ-PITHD1 expression (Figure 1(c)). FISH detection showcased that circ-PITHD1 expression increased in CRC tumor tissues compared to adjacent normal tissues. e outcomes confirmed that circ-PITHD1 was mainly distributed in the cytoplasm (Figure 1(d)). e circ-PITHD1 originated from cyclizing six exons of PITHD1, which are located at chr1:24104875-24114722. PITHD1 is 9847 bp and spliced mature circRNA is 1622 bp (Figure 1(e)). So our team termed hsa_circ_0010889 as circ-PITHD1.
e outcome data suggested that circ-PITHD1 downregulations inhibited CRC proliferation and tumor growth.
e results showed that HK2 was the miR-590-5p downstream target. To obtain correlations between miR-590-5p and HK2, we used WT or MUT 3′-UTR-HK2 sequences such as the miR-590-5p binding sequence in the luciferase reporter vector (Figure 4(d)). e technician transfected the luciferase reporter vector into HEK293 cells combined with or without the miR-590-5p mimic.

HK2 Overexpression or miR-590-5p Inhibition Reversed CRC Cell Aerobic Glycolysis after Circ-PITHD1 Silencing.
e circ-PITHD1 silencing effects on the Warburg effect were analyzed. e data demonstrated that lactate production and glucose uptake decreased in the circ-PITHD1 silenced groups compared with the si-NC group. HK2 was found to be a dynamic metabolic enzyme in glycolysis, which can enhance glucose uptake. us, HK2 overexpression or miR-590-5p suppression reversed glucose uptake and lactate production after circ-PITHD1 silencing in Lovo and SW480 cells (Figures 6(a)-6(d)).
is study verified that HK2 had an important function in glycolytic metabolism regulation [20]. HK2 is a critical glycolytic enzyme catalyzing glucose conversion to glucose-6-phosphate, which is required for glycolysis. HK2 upregulation promoted cell invasion and migration by increasing glycolysis [21]. Aerobic glycolysis is also known as the Warburg Effect, which is a cancer hallmark. It is identified by changing its energy and glycolysis [22]. e current investigation also discovered that circ-PITHD1 silencing inhibited lactate production and glucose uptake, but HK2 overexpression restored inhibitory effects regarding si-circ-PITHD1 upon aerobic glycolysis. It is suggested that circ-PITHD1 silencing suppressed CRC progression via promoting miR-590-5p and inhibiting HK2 expression.
In conclusion, the current research provided proof that circ-PITHD1 downregulation decreased the invasion and proliferation ability of CRC by regulating miR-590-5p/ HK2 signaling-mediated aerobic glycolysis. ese findings show that circ-PITHD1 is a promising biomarker in CRC diagnostics, which will extend drug applications targeting circ-PITHD1 and indicates a role for circ-PITHD1 in CRC treatment.

Data Availability
All data in this study are available from the corresponding author.

Disclosure
Shiguang Yang and Kui Zhao are the co-first authors.
Evidence-Based Complementary and Alternative Medicine 9