MiR-133a-3p Functions as a Tumor Suppressor in Colorectal Cancer by Targeting PFKFB3

Yanjie Zhou The A liated Jiangyin Hospital of Southeast University Medical School https://orcid.org/0000-00017227-0673 Lu Jiang Wuxi Center for Disease Control and Prevention Yuanjun Cai The A liated Jiangyin Hospital of Southeast University Medical School Wendong Tang The A liated Jiangyin Hospital of Southeast University Medical School Ke Wang (  wangk@jyrmyy.com ) The A liated Jiangyin Hospital of Southeast University Medical School https://orcid.org/0000-00032684-8686


Introduction
Colorectal cancer (CRC) was the most commonly diagnosed gastrointestinal cancer, representing 1.8 million cases and 881,000 deaths globally, and constituting 1 in 10 cancer cases and deaths [1].
Advancements in pathophysiological understanding have increased the array of treatment options for local and advanced disease leading to individual treatment plans. Although these new treatment options have doubled overall survival for advanced disease to 3 years, survival is still best for those with nonmetastasised disease. As the disease only becomes symptomatic at an advanced stage, worldwide organised screening programmes are being implemented, which aim to increase early detection and reduce morbidity and mortality from colorectal cancer [2]. Additionally, although all-ages rates of colorectal cancer have largely stabilized or been in decline in high-income countries, incidence rates appear to be rising in adults younger than 50 years. Although genetic, lifestyle, obesity, and environmental factors might have some association, the exact reasons for this increase are not completely understood [3].
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFK-2/FBPase 3, PFKFB3) is a member of a homodimeric and bifunctional enzyme family (PFKFB1-4) [4], which contorls the intracellular steady-state concentration of fructose 2,6-bisphosphate(F2,6P2) [5]. F2,6BP is a potent actibator of PFK-1, and PFK-1 is essential for the rate-limiting step of glycolysis [6]. The bifunctional isoenzyme encoded by the pfkfb3 gene has the highest kinase: phosphatase activity ratio, which in turn sustains high glycolytic rates [7]. PFKFB3 expression could be upregulated in response to mitogenic, in ammatory and hypoxia stimuli and during the DNA synthesis phase of the cell cycle.18 Considering its signi cance in cancer metabolism, further explanation of the function of PFKFB3 in diverse cancers is necessary. According to previous studies, the expression of PFKFB3 gene can be regulated by different mechanisms such as signal pathways, oncogenes, noncoding RNAs, in ammatory molecules, and the PFKFB3 protein regulated both at the transcriptional and post-transcriptional levels. Although the glycolytic role of PFKFB3 in cancer progression has been the subject of numerous functional studies, some researchers have also focused on the functions of PFKFB3 beyond glycolysis. PFKFB3 expression may not only be essential for the regulation of glycolysis in the cytoplasm, but also in the control of the cell cycle in the nucleus and maintenance of an anti-apoptotic state. PFKFB3 has been suggested to play a crucial role in many types of tumor cells as well as various cells in the tumor microenvironment. miRNAs, a class of noncoding RNA (ncRNA) about 22-nucleotide RNAs in size, are highly conserved molecules regulating gene expression post-transcriptionally by binding to the 3'-untranslated region (3'-UTR) of target message RNAs (mRNAs) [8] and participating tumorigenesis, proliferation, invasion and drug resistance in cancer [9]. miR-133a-3p, which belongs to the miR-133 family, was rst experimentally characterized in mice [10]. In the human genome, miR-133a-3p is a multicopy gene, with two known copies: miR-133a-1 and miR-133a-2, located on chromosomes 18, 20 respectively. To date, multiple functional roles of miR-133a-3p have been elucidated, such as regulating myoblast proliferation and differentiation [11], inhibiting embryonic cardiomyocyte proliferation [12]and avoiding genetic cardiac hypertrophy [13].Furthermore, it has been reported that miR-133a-3p is among the most frequently downregulated miRNAs in various types of human malignancies, which suggested that miR-133a-3p may serve a critical part in tumor progression of various malignancies, including non-small cell cancer [14], ovarian cancer [15], colorectal cancer [16], bladder cancer [17], breast cancer [18] and prostate cancer [19].
In this study, we predicted PFKFB3 was a target gene of miR-133a-3p by bioinformatics analysis. qRT-PCR, western blot and luciferase assays were used to verify the relationship between PFKFB3 and miR-133a-3p, and CCK8 assay and transwell assay were applied to elucidate the effect of the miR-129-5p/PFKFB3 axis on the occurrence and development of CRC. Overall, our results demonstrated that miR-133a-3p suppresses the progression of GCs by targeting PFKFB3.
Cell culture Five CRC cell lines (LDL1, LOVO, HT29, SW480, CaCO-2) and a normal human normal colonic epithelial cell line (NCM460) were obtained from the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in DMEM medium (Thermo Fisher Scienti c, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin, and incubated at 37℃and 5% CO2.

Cell transfection
The oligonucleotides of miR-133a-3p mimics, mimics control, miR-133a-3p inhibitor, control inhibitor, PFKFB3 overexpression vector and PFKFB3 siRNA were purchased from HIPPOBIO (China) were transfected into CRC cell line SW480 by Lipo2000 (ThermoFisher, USA) kit in accordance with instructions. After 24h of transfection, transfected cells were used for subsequent experiments.

Reverse transcription-quantitative polymerase chain reaction
Total RNA was isolated from cells using TRIzol kit (Life Technologies, USA). The cDNA was synthesized using PrimeScript™ RT Master Mix Kit for qPCR (TaKaRa, Japan) and miRNA First Strand cDNA Synthesis (Sangon Biotech, China) according to relevant protocols. miR-133a-3p, PFKFB3, KI67 and MMP9 mRNA levels were quanti ed using Power SYBR™ Green PCR Master (Invitrogen, USA) with StepOne Plus Realtime PCR system. U6 and Actin were used as an internal standard control for miRNA and mRNA detection, respectively. Primer sequences used in qRT-PCR were listed in Table 1. The relative expression of miR-133a-3p and PFKFB3 mRNA was presented by 2 −ΔΔCt method. The experiment was repeated for three times. Total proteins were harvested after cells were lysed by RIPA lysis buffer, and protein concentration was assayed by BCA kit (Beyotime, China). After being denatured at a high temperature, proteins were isolated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred onto polyvinylidene fuoride membranes (PVDF; Millipore), which were then blocked with 5% skim milk for 2 h.

Cell proliferation assay
The cell proliferation ability of SW480 cells was measured using the cell counting kit-8 assay (CCK-8) (MCE, USA ). Cells were counted and seeded into the 96-well plate with 3,000 cells/well, and then incubated with 5% CO2 at 37 ℃ for 72 h. The absorbance of cultured CRC cells was measured with the micro-plate reader at 450 nm after CCK-8 solution addition for 2 h.

Transwell invasion assay
The 24-well transwell chambers (Corning, NY, USA) with or without the Matrigel (Invitrogen) were used to perform the transwell assays. 1×10 5 SW480 cells were seeded into the upper chambers in serum-free medium, while the serum-supplied medium was added to the lower chamber in 24-well plates. After being cultured for 48 h at 37 ℃, the non-invaded cells were removed, while the invaded cells were stained with 0.1% crystal violet. Numbers of stained cells in the bottom chambers were assessed from ve randomly selected elds, and the data were summarized from three individual experiments. The experiment was conducted for three times.

Statistical analysis
All experiments were executed at least three independent times. The statistical data are presented as mean ± standard deviation and analyzed using GraphPad Prism 6.0 (CA, United States). Two-tailed Student's t-tests were conducted to interpret the differences.. P value <0.05 was considered statistically signi cant.

PFKFB3 is up-regulated in CRC and signi cantly associated with poor prognosis
To explore the expression of PFKFB3 mRNA and protein in CRC cells, online databases ONCOMINE, TCGA and THPA were used. We found that PFKFB3 mRNA is up-regulated in 7 types of tumors including colorectal cancer (Fig. 1a). The TCGA-COAD data showed that PFKFB3 mRNA was prominently upregulated in CRC tissue (Fig. 1b). The THPA data showed the PFKFB3 protein is high expressed in colorectal cancer (Fig. 1c). For further con rmation, we detected the expression of PFKFB3 mRNA and protein in ve CRC cell lines (LDL1, LOVO, HT29, SW480, CaCO-2) and a normal human normal colonic epithelial cell line (NCM460). The results showed that PFKFB3 is up-regutated in all the ve CRC cell lines (Fig. 1d.e). In addition, according to the K-M survival curve plotted by THPA online tool, the upregulated PFKFB3 is signi cantly associated with poor prognosis (Fig. 1f).

PFKFB3 affects CRC cell proliferation and invasion
To evaluate weather PFKFB3 contributes to the progress of CRC, we established the PFKFB3 overexpressed and silenced CRC cell lines via SW480. We rst con rmed the expression of PFKFB3 by qRT-PCR and western blot (Fig. 2a-b). CCK8 assay and KI67 detection suggested that overexpression of PFKFB3 markedly enhanced CRC cell proliferative ability, while silencing of PFKFB3 inhibited the proliferation of CRC cells (Fig, 2d-f). Transwell assay and MMP9 detection demonstrated that overpxpress of PFKFB3 promotes the invasion of CRC cells, on the contrary silencing of PFKFB3 supressed the inbasion of CRC cells (Fig. 2g-i). Taken together, these results domonstrated that PFKFB3 enhanced CRC cell proloferation and invasion.
Low expression of miR-133a-3p has diagnostic value in CRC The online batabases Targetscan and ONCOMIR were applied to predict the regulating miRNAs of PFKFB3, we combined the prediction results of two databases. As a result, miR-26-5p and miR-133a-3p were selected as potential regulators of PFKFB3(Additional le2). To explore the expression of these microRNAs in CRC, a TCGA-COAD dataset was analysed. The results showed that miR-26a-5p and miR-26b-5p is up-regulated in colorectal cancer, while miR-133a-3p is down-regulated in colorectal cancer (Fig. 3a-c). Further more, we validated the expression level of miR-133a-3p in normal colonic epithelial cell(NCM460) and CRC cell line SW480. Additionally a ROC diagnostic curver plotted by TCGA data demonstrated that low expression of miR-133a-3p has diagnostic value in colorectal cancer miR-133a-3p directly targets PFKFB3 To further explore the regulatory efect of miR-133a-3p on PFKFB3, bioinformatics analysis was used to predict the binding sites of miR-133a-3p on PFKFB3 3'-UTR, indicating that there were binding sites between miR-133a-3p and PFKFB3 (Fig. 4a). Subsequently, dual-luciferase assay validated the targeted binding relationship between miR-133a-3p and PFKFB3, nding that overexpression of miR-133a-3p inhibited the luciferase activity of PFKFB3-WT 3'-UTR, while had no infuence on that of PFKFB3-MUT 3'-UTR (Fig. 4b). Taken together, these fndings elucidated that miR-133a-3p targeted and down-regulated PFKFB3 in CRC cells.

Disscussion
In recent years, PFKFB3 has been studied in various cancer cells. Most studies have demonstrated that the overexoression of PFKFB3 promoted cancer cell growth, proliferation, migration and metastasis [20].
In addition, inhibition of PFKFB3 expression by siRNA [21], miRNA [22] and inhibitor [23] decreases the growth, proliferation and migration of these cells. In this study we found that PFKFB3 was signi cantly up-regulated in CRC by bioimformatics analysisi and cell experiments. Besides, according to our study PFKFB3 could act as a promoter to CRC cell proliferation and invasion. These ndings provide a better understood to the regulatory mechanism of PFKFB3 in CRC.
Despite several different kinds of miRNAs, such as miR-206, miR-26b, and miR-330 have been reported to decrease the expression of PFKFB3 [23][24]. We identify a novel miRNA targeting PFKFB3 by combining the bioinformatics prediction results and TCGA expression data. Our research showed miR-133a-3p is signi cantly down-regulated in CRC and SW480 cell line, and the detection of miR-133a-3p has diagnostic value in CRC. miR-133a-3p has been reported as a supressor of several different oncogenes such as COL1A1, CORO1C and CREB1 [25][26][27], The regulatory mechanism of the miR-133a-3p/PFKFB3 axis in CRC have not been reported to date. As revealed by cell experiments, we found that PFKFB3 can be supressed by miR-133a-3p mimics. Dual-luciferase assay further validated that there was a targeted binding relationship between miR-133a-3p and PFKFB3. Additionally, rescue experiments indicated that miR-133a-3p suppressed CRC cell proliferation and invasion by targeting PFKFB3.

Conclusion
We identify miR-133a-3p as a novel biomarker in CRC, and also an suppressor of cell proliferation and invasion in CRC, wherein the targeted PFKFB3 plays a critical role. These fndings illustrate the regulatory mechanism of miR-133a-3p and PFKFB3 in CRC and provide novel potential therapeutic targets for CRC additional insight into the exploration of new biomarkers and target therapies for ccRCC.
However, the molecular mechanisms of miR-133a-3p function in CRC cell proliferation and invasion are not fully unveiled, suggesting that other targets such as lincRNAs may exit to mediate its function. Further research is needed to better exploit miR-133a-3p as a therapeutic target in CRC treatment.    miR-133a-3p directly targets PFKFB3. a The binding sites of miR-133a-3p on PFKFB3's 3'UTR were predicted by bioinformatics analysis. b The luciferase activity in different groups was detected by dualluciferase assay.