MicroRNA-21 Plays Multiple Oncometabolic Roles in Colitis-Associated Carcinoma and Colorectal Cancer via the PI3K/AKT, STAT3, and PDCD4/TNF-α Signaling Pathways in Zebrafish

Simple Summary The PI3K/AKT, STAT3, and PDCD4/TNF-α signaling networks, regulated by the microRNA (miR)-21, are critical for inflammatory regulation, tumor suppressor modulation, and oncogenic activation. We developed a zebrafish model (ImiR-21) with an inducible overexpression of miR-21, specifically in the intestine. The miR-21 overexpression resulted in the development of colorectal cancer (CRC) due to inflammatory bowel disease. Furthermore, the physiological, metabolic, and histological aspects of CRC were similar to those of colitis-associated cancer (CAC) induced by the intestinal carcinogens azoxymethane or dextran sodium sulfate in this model. Thus, miR-21 is critical to the pathogenesis of CRC/CAC and could serve as a novel therapeutic target to treat CRC/CAC. Abstract Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide. Patients with inflammatory bowel disease (IBD) have a high risk of developing CRC. Inflammatory cytokines are regulated by complex gene networks and regulatory RNAs, especially microRNAs. MicroRNA-21 (miR-21) is amongst the most frequently upregulated microRNAs in inflammatory responses and cancer development. miR-21 has become a target for genetic and pharmacological regulation in various diseases. However, the association between inflammation and tumorigenesis in the gut is largely unknown. Hence, in this study, we generated a zebrafish model (ImiR-21) with inducible overexpression of miR-21 in the intestine. The results demonstrate that miR-21 can induce CRC or colitis-associated cancer (CAC) in ImiR-21 through the PI3K/AKT, PDCD4/TNF-α, and IL-6/STAT3 signaling network. miR-21 activated the PI3K/AKT and NF-κB signaling pathways, leading to initial inflammation; thereafter, miR-21 and TNF-α repressed PDCD4 and its tumor suppression activity. Eventually, active STAT3 stimulated a strong inflammatory response and activated the invasion/metastasis process of tumor cells. Hence, our findings indicate that miR-21 is critical for the development of CRC/CAC via the PI3K/AKT, STAT3, and PDCD4/TNF-α signaling networks.


Introduction
Colorectal cancer (CRC), also known as colon, bowel, or rectal cancer, is a type of cancer that develops in the colon or rectum. In 2020, CRC was the second most common cancer and the second leading cause of mortality due to cancer worldwide [1]. Most CRCs develop from polyps on the inner lining of the colon or rectum. Not all polyps become malignant, but some can transform into cancer over time. Adenomatous polyps (adenoma, a precancerous condition) are an example of polyps that can transform into cancer. Hyperplastic and inflammatory polyps are more common than adenomas but are generally not precancerous. Sessile serrated polyps (SSPs) and traditional serrated adenomas (TSAs) have a higher risk of tumorigenesis of CRC [2]. Although CRC can occur at any age, it is more common in older adults. Moreover, people with a family history of CRC, adenomatous polyps, and inflammatory bowel disease (IBD) are at a high risk of developing CRC.
Clinical evidence demonstrated that microRNA (miRNA)-21 (miR-21) expression was significantly upregulated in the colon tissue of both CD and UC patients compared to that of non-IBD controls [26,27]. miR-21 promotes invasion, intravasation, and metastasis in CRC by downregulating PDCD4 [28]. Furthermore, IL-6/STAT3 signaling stimulates inflammatory pathways in UC by activating the expression of miR-21 [26]. Moreover, PDCD4 deficiency in mouse models aggravates colitis and colitis-associated CRC by promoting the IL-6/STAT3 pathway [20]. However, the molecular mechanisms mediated by miR-21 in IBD and CRC are unclear.
Zebrafish are a remarkable vertebrate model organism for biological and medical studies [29]. Over 70% of human genes have orthologs in zebrafish [30]. Compared to human cell lines or genetically engineered mouse models, zebrafish provide unique insights into the progression and pathogenesis of cancer. The features of cancer development in zebrafish are similar to those observed in humans [31,32]. Hence, in recent years, zebrafish are being increasingly used as model organisms for cancer research [33][34][35]. The cellular composition, architectural organization, and the digestive and immune functions of the intestinal tract is conserved between zebrafish and mammals [36]. However, studies examining the transgenic zebrafish as a model for GI cancer research are scarce [37][38][39].
Studies on various cancer cell lines and animal models indicate that miR-21 promotes colorectal oncogenesis and hence is referred to as an oncomiR [40,41]. In our recent study, miR-21 played multiple oncometabolic roles in nonalcoholic steatohepatitis-associated hepatocellular carcinoma (NAHCC) [42]. Hence, in this study, we aim to examine the time profiles of expression of miR-21 and its target genes during CRC genesis using an oncomiR-21 transgenic zebrafish model (ImiR-21) developed in our laboratory.

Ethics Statement
All animals were maintained in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines.

Zebrafish Lines and Maintenance
The transgenic zebrafish line ImiR-21 [Tg(fabp2a:Tet on -2A-ZsGreen, TRE:mCherry-miR-21] showed inducible and intestine-specific expression of mCherry (red fluorescent protein). Production of miR-21 was driven by the zebrafish fabp2 promoter. The fish were maintained in a controlled environment with a 14/10-h light-dark cycle at 28 • C.

Doxycycline (Dox) Treatment
The zebrafish embryos and juvenile adults were treated with Dox at a concentration of 25 µg/mL (Sigma-Aldrich, St. Louis, MO, USA) in six-well plates and 3 L tanks, respectively, and the water was changed daily.

Chemical Treatment
To screen for inflammatory markers in the intestine of zebrafish, dextran sodium sulfate (DSS, Sigma-Aldrich) was used to induce colitis in zebrafish. DSS (10 ng/4.6 nL) was injected into the zebrafish duct of Cuvier at 14 and 16 days post-fertilization (dpf). Azoxymethane (AOM, 100 ng/4.6 nL, Sigma-Aldrich) was injected into the duct of Cuvier at 14, 16, and 18 dpf.

RNA Analysis
Total RNA was extracted from intestinal tissue using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized using the RevertAid RT Kit (Thermo Fisher Scientific). Real-time quantitative reverse transcription PCR (RT-qPCR) analysis using Fast SYBR Green Master Mix (Thermo Fisher Scientific) was performed on a StepOne Real-Time PCR System (Thermo Fisher Scientific). Expression data were normalized to those of gapdh or U6 of zebrafish. The genes and their corresponding primer sequences are listed in Table S1.

Whole-Mount Alcian Blue Staining
The 4% PFA (paraformaldehyde)-PBS fixed embryos were immersed in acid ethanol (1% HCl in 70% ethanol) for 5 min at room temperature. The embryos were then transferred into alcian blue solution (0.1% alcian blue (Sigma-Aldrich), 20% acetic acid, 80% ethanol) for 16 h at 4 • C with gentle shaking. The alcian blue solution was then replaced with ethanol. Stained embryos were stored in the dark at 4 • C.

Histopathology
Freshly dissected tissues were rinsed with PBS and fixed with 1× Zinc Formal-Fixx (Thermo Fisher Scientific). Fixed tissues were then dehydrated with ethanol and embedded in paraffin (Surgipath Paraplast Plus, Leica Biosystems Division of Leica, Buffalo Grove, IL, USA). Blocks were sliced into 4 µm sections and stained with H&E (hematoxylin and eosin) and alcian blue solution (pH 2.5).

Statistical Analysis
All data are presented as mean ± standard error of the mean (SEM). Kaplan-Meier with log-rank test (build in GraphPad Prism 8.0) was used for survival analysis. The number of zebrafish larvae for survival analysis are listed in Table S2. All analyses were performed using GraphPad Prism 8.0 software (GraphPad, San Diego, CA, USA). Differences were considered statistically significant at p < 0.05.

Identification of Intestinal Inflammation Markers in Zebrafish
miR-21 has been demonstrated to play a crucial role in the inflammatory pathway [26,[44][45][46]. To determine the reactivity of inflammatory genes that are involved in CAC, WT larvae were injected with 10 ng DSS at 14 and 16 dpf to induce colitis ( Figure 2A). The seven-day post-injection survival analysis showed significantly different survival rates (p < 0.0001, n = 100 in three independent experiments) of DSS-injected larvae ( Figure 2B). Moreover, the expression levels of inflammatory genes were analyzed using RT-qPCR. The expression of some renowned inflammatory factors, such as il1b, il6, il8a, tnfa, il11b,

Effects of Intestinal miR-21 Expression on Early Onset of IBD-Like Colitis
DSS and AOM are extensively used as inflammation-related colon carcinogens in mouse models [47][48][49][50]. To investigate whether AOM could induce inflammation in the zebrafish intestine by upregulating the expression of miR-21, ImiR-21 larvae were injected with 100 ng AOM at 14, 16, and 18 dpf ( Figure 3A). The intestine became swollen in AOMinjected and ImiR-21 + Dox ( Figure 3B). The mortality rate of AOM-injected ImiR-21 was similar to that of DSS-injected WT zebrafish ( Figures 3C and 2B). In addition, the mortality rate also remarkably increased in Dox-induced (+Dox) overexpression of miR-21 in the early stage of ImiR-21. miR-21 levels in zebrafish intestines were analyzed by RT-qPCR in ImiR-21− Dox, ImiR-21 + AOM, and ImiR-21 + Dox at 21 dpf. miR-21 expression in the intestine was slightly upregulated by AOM compared to that in untreated ImiR-21 − Dox. Interestingly, the intestinal expression of miR-21 in ImiR-21 + Dox was much higher than that in AOM-treated larvae ( Figure 3D). Our previous study indicated that pdcd4b and ptenb mRNAs are direct targets of miR-21 in zebrafish [42]. This result was reflected in the RT-qPCR data, which showed that increased levels of miR-21 repressed the expression of pdcd4b and ptenb mRNAs ( Figure 3E). Furthermore, for the regulatory profiles of inflammatory genes in the intestine, ImiR-21 + Dox displayed similar features to DSS-or AOM-treated larvae (Figures 2C,D and 3F).
(dpf). (B) Survival proportions of injected WT larvae from 0 dpi (days post-injection) to 7 dpi (14 to 21 dpf, p < 0.0001, n = 100 in three independent experiments). The survival rate of DSS-injected WT larvae was approximately 50%. The survival rates were not significantly different between noninjected and PBS-injected WT larvae (>90%). (C,D) RT-qPCR analysis of inflammation-related genes in the intestines of PBS-and DSS-injected WT lines at 21 dpf. mRNA expression levels were normalized to gapdh in the intestine of PBS-injected WT controls. Statistically significant differences from the PBS-injected WT control are denoted by * (p < 0.05), ** (p < 0.01), and *** (p < 0.001) for all panels.

Effects of Intestinal miR-21 Expression on Early Onset of IBD-Like Colitis
DSS and AOM are extensively used as inflammation-related colon carcinogens in mouse models [47][48][49][50]. To investigate whether AOM could induce inflammation in the zebrafish intestine by upregulating the expression of miR-21, ImiR-21 larvae were injected with 100 ng AOM at 14, 16, and 18 dpf ( Figure 3A). The intestine became swollen in AOMinjected and ImiR-21 + Dox ( Figure 3B). The mortality rate of AOM-injected ImiR-21 was similar to that of DSS-injected WT zebrafish ( Figure 3C and Figure 2B). In addition, the mortality rate also remarkably increased in Dox-induced (+Dox) overexpression of miR-21 in the early stage of ImiR-21. miR-21 levels in zebrafish intestines were analyzed by RT-qPCR in ImiR-21− Dox, ImiR-21 + AOM, and ImiR-21 + Dox at 21 dpf. miR-21 expression in the intestine was slightly upregulated by AOM compared to that in untreated ImiR-21 − Dox. Interestingly, the intestinal expression of miR-21 in ImiR-21 + Dox was much higher than that in AOM-treated larvae ( Figure 3D). Our previous study indicated that pdcd4b and ptenb mRNAs are direct targets of miR-21 in zebrafish [42]. This result was reflected in the RT-qPCR data, which showed that increased levels of miR-21 repressed the expression of pdcd4b and ptenb mRNAs ( Figure 3E). Furthermore, for the regulatory profiles of inflammatory genes in the intestine, ImiR-21 + Dox displayed similar features to DSS-or AOM-treated larvae ( Figures 2C,D and 3F).

Overexpression of miR-21 Causes Intestinal Epithelial Barrier Impairment
Upregulation of miR-21 has been observed in various tissue inflammatory conditions [44,45] in human patients and animal models of IBD [26,46]. Overexpressed miR-21 leads to intestinal epithelial barrier impairment [51]; thus, we examined the early onset of intestinal inflammation in ImiR-21 fish. WT and ImiR-21 larvae were treated with Dox or AOM ( Figure 3A). Whole-mount alcian blue staining displayed significant signals in the posterior intestine of ImiR-21 + AOM and ImiR-21 + Dox at 21 dpf ( Figure 4A). Alcian blue stains acid mucins, which are secreted by various connective and epithelial tissue tumors. The presence of goblet cells in the injured villi indicates an inflammation in the posterior intestine of ImiR-21 + AOM and ImiR-21 + Dox. For histological analysis, paraffinembedded zebrafish intestines at 21 dpf were sliced along the transverse plane into 4 µm sections ( Figure 4B). Histopathological examination revealed goblet cell hyperplasia (GCH, indicated by arrows) in the villi of ImiR-21 + AOM and ImiR-21 + Dox ( Figure 4C-F).

Overexpression of miR-21 Causes Intestinal Epithelial Barrier Impairment
Upregulation of miR-21 has been observed in various tissue inflammatory conditions [44,45] in human patients and animal models of IBD [26,46]. Overexpressed miR-21 leads to intestinal epithelial barrier impairment [51]; thus, we examined the early onset of intestinal inflammation in ImiR-21 fish. WT and ImiR-21 larvae were treated with Dox or AOM ( Figure 3A). Whole-mount alcian blue staining displayed significant signals in the posterior intestine of ImiR-21 + AOM and ImiR-21 + Dox at 21 dpf ( Figure 4A). Alcian blue stains acid mucins, which are secreted by various connective and epithelial tissue tumors. The presence of goblet cells in the injured villi indicates an inflammation in the posterior intestine of ImiR-21 + AOM and ImiR-21 + Dox. For histological analysis, paraffin-embedded zebrafish intestines at 21 dpf were sliced along the transverse plane into 4 μm sections ( Figure 4B). Histopathological examination revealed goblet cell hyperplasia (GCH, indicated by arrows) in the villi of ImiR-21 + AOM and ImiR-21 + Dox ( Figure 4C-F).

Chronic Effects of Intestinal miR-21 Expression on Colitis
As ImiR-21 + Dox fish showed a similar gene regulatory feature to the intestinal carcinogen-treated fish, we analyzed whether the ImiR-21 intestine was predisposed to CAC development from the beginning. Because of the high mortality rate in the early stages of development of ImiR-21 with upregulated miR-21 expression, miR-21 was induced by Dox at 5 months post-fertilization (mpf) in the adult ImiR-21 intestine ( Figure 5A). After one month of miR-21 induction, oncogenic p-Akt and p-Stat3 expression increased significantly, and the tumor-suppressive genes pten and pdcd4 were substantially repressed in the intestine of ImiR-21 + Dox ( Figures 5B,C and S1). Alcian blue-stained sections indicate GCH ( Figure 5D, black arrow) and reduction of the mucus layer (blue arrow) in ImiR-21 + Dox. These results indicated that the intestinal epithelial cells suffered from miR-21mediated inflammation.

Chronic Effects of Intestinal miR-21 Expression on Colitis
As ImiR-21 + Dox fish showed a similar gene regulatory feature to the intestinal carcinogen-treated fish, we analyzed whether the ImiR-21 intestine was predisposed to CAC development from the beginning. Because of the high mortality rate in the early stages of development of ImiR-21 with upregulated miR-21 expression, miR-21 was induced by Dox at 5 months post-fertilization (mpf) in the adult ImiR-21 intestine ( Figure 5A). After one month of miR-21 induction, oncogenic p-Akt and p-Stat3 expression increased significantly, and the tumor-suppressive genes pten and pdcd4 were substantially repressed in the intestine of ImiR-21 + Dox ( Figures 5B,C and S1). Alcian blue-stained sections indicate GCH ( Figure 5D, black arrow) and reduction of the mucus layer (blue arrow) in ImiR-21 + Dox. These results indicated that the intestinal epithelial cells suffered from miR-21-mediated inflammation.

miR21 Promotes CAC Development by Activating PI3K/AKT, IL-6/JAK/STAT3, and PDCD4/NF-κB/TNF-α (PSP) Signaling Networks
miR-21 is a well-known oncogenic miRNA that is upregulated in several human cancers. In this study, we compared the miR-21-mediated gene networks to DSS-or AOMinduced colitis in the zebrafish intestine and found that they have similar gene regulatory profiles during CAC development. By repressing Pten, miR-21 activates the Pi3k/Akt pathway and eventually the Nf-κB pathway. Activated Nf-κB promotes the release of the inflammatory cytokines Il-1β, Il-6, and Tnf-α. Il-6 activates Stat3 through the Jak signaling 3.7. miR21 Promotes CAC Development by Activating PI3K/AKT, IL-6/JAK/STAT3, and PDCD4/NF-κB/TNF-α (PSP) Signaling Networks miR-21 is a well-known oncogenic miRNA that is upregulated in several human cancers. In this study, we compared the miR-21-mediated gene networks to DSS-or AOM-induced colitis in the zebrafish intestine and found that they have similar gene regulatory profiles during CAC development. By repressing Pten, miR-21 activates the Pi3k/Akt pathway and eventually the Nf-κB pathway. Activated Nf-κB promotes the release of the inflammatory cytokines Il-1β, Il-6, and Tnf-α. Il-6 activates Stat3 through the Jak signaling pathway; thus, p-Stat3 (active) binds to the promoter region of miR-21 and creates a positive feedback loop for miR-21. Tnf-α antagonizes Pdcd4. miR-21 targets tumor suppressor genes such as pdcd4, btg2, and tpm1 to enhance cell proliferation, release more inflammatory cytokines, and activate oncogenes. Hence, the dysregulated gene networks and chronic inflammation mediated by miR-21 eventually lead to tumorigenesis during CAC development (Figure 7). pathway; thus, p-Stat3 (active) binds to the promoter region of miR-21 and creates a positive feedback loop for miR-21. Tnf-α antagonizes Pdcd4. miR-21 targets tumor suppressor genes such as pdcd4, btg2, and tpm1 to enhance cell proliferation, release more inflammatory cytokines, and activate oncogenes. Hence, the dysregulated gene networks and chronic inflammation mediated by miR-21 eventually lead to tumorigenesis during CAC development (Figure 7).
We demonstrated that miR-21 regulates at least three molecular and pathophysiological pathways during CAC development: Akt/Nf-κb, Pdcd4/Tnf-α, and Il-6/Stat3. First, miR-21 represses pten and activates Akt/Nf-κb pathways. Activated Aky/Nf-κb promotes the release of inflammatory cytokines, such as Il-6 and Tnf-α. Tnf-α not only promotes colon cancer cell migration and invasion [61] but also represses Pdcd4, and Pdcd4 deficiency may aggravate inflammatory response. Stat3 is activated by Il-6 through the Jak pathway. Activated Stat3 promotes the transcription of miR-21 and forms a positive feedback loop, which explains why miR-21 is upregulated in the AOM-treated intestine ( Figure 3). Moreover, miR-21 directly targets several tumor suppressors, such as pdcd4 and btg2, to enhance cell proliferation, release more inflammatory cytokines, and activate oncogenes. All the above events accelerate the early onset of tumorigenesis and metastasis in the ImiR-21 + Dox intestine (Figures 3 and 5, Figures 6 and 7). In our previous study, we showed that miR-21 targeted smad7 to increase the phosphorylation of Smad3 in the zebrafish liver [42]. Another study showed that IL-6/JAK/STAT3 and TGF-β/SMAD pathways are required for the epithelial-mesenchymal transition in the early stages of cancer [62]. These studies further support our data wherein we observed a large invasive tumor in ImiR-21 + Dox within 3 months of miR-21 induction ( Figure 6E).
The intestinal length of ImiR-21 + Dox was significantly shorter than that of the WT + Dox control at 2 mpt. Moreover, hyperplastic tissues and tumors were observed in the intestine of ImiR-21 + Dox (Figures 6 and S2). Similar symptoms were observed in AOM/DSS-treated mouse models [63,64]. Hence, miR-21 has strong oncogenic effects and is critical for CAC and CRC development. Furthermore, since dysregulated miR-21 is easily detected in blood, serum miR-21 can be used as a promising biomarker for the early detection and prognosis of CRC [65,66], thereby providing an effective target for the development of novel therapeutic strategies to treat CRC.

Conclusions
In conclusion, our study emphasized the role of PSP signaling networks in inflammatory cytokine regulation, tumor suppressor modulation, and oncogenic activation. Our zebrafish model could successfully recreate the development of IBD to CAC/CRC with respect to physiological, metabolic, and histological aspects. Chronic inflammation is a critical risk factor for the development of tumors. Our findings, in addition to those of previous studies, contribute toward understanding the critical role of miR-21 in tumorigenesis. This knowledge would further help in developing novel therapeutics to target miR-21 to attenuate CRC/CAC progression.
Institutional Review Board Statement: No human materials were used in this study. This study was conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines, and the protocol was approved by National Yang Ming Chiao Tung University (IACUC number: 1070709 and 31 July 2018).

Data Availability Statement:
The data presented in this study are available in Supplementary Materials here.