Multi-Omics Reveal the Immunological Role and the Theragnostic Value of miR-216a/GDF15 Axis in Human Colon Adenocarcinoma

Colon adenocarcinoma (COAD) is the most common type of gastrointestinal cancer and is still the third leading cause of cancer-related mortality worldwide. Accurate screening tools for early diagnosis and prediction of prognosis and precision treatment strategies are urgently required to accommodate the unmet medical needs of COAD management. We herein aimed to explore the significance of the microRNA (miR)-216a/growth differentiation factor 15 (GDF15) axis in terms of clinical value, tumor immunity, and potential mechanisms in COAD by using multi-omic analysis. The gene expression levels of miR-216a and GDF15 showed an increase in the COAD group compared to those of the normal group. The expression of miR-216a presented a negative correlation with GDF15 in COAD tumor tissue. The use of an in vitro luciferase reporter assay and bioinformatic prediction revealed that miR-216a-3p acted toward translational inhibition on GDF15 by targeting its 3′untranslated region (UTR) site. High miR-216a expression was associated with decreased overall survival (OS), while the high expression of GDF15 was associated with increased OS. Enriched type 1 T-helper (Th1), enriched regulatory T (Treg), enriched eosinophils, and decreased nature killer T-cells (NKTs) in COAD tumor tissue may play counteracting factors on the tumor-regulatory effects of miR-216a and GDF15. In addition, high GDF15 expression had associations with suppressed immunoinhibitory genes and negative correlations with the infiltration of macrophages and endothelial cells. The enrichment analysis revealed that GDF15 and its co-expression network may be implicated in mitochondrial organization, apoptosis signaling, and endoplasmic reticulum (ER) stress response. The Genomics of Drug Sensitivity in Cancer (GDSC) and Cancer Therapeutics Response Portal (CTRP) analysis identified that Gemcitabine acted as a precision treatment for COAD when GDF15 expression was low. This study supports the miR-216a/GDF15 axis as a diagnostic/prognostic panel for COAD, identifies Th1, Treg, eosinophils, and NKTs as counteracting factors, indicates potential relationships underlying immunomodulation, mitochondrial organization, apoptotic signaling, and ER stress and unveil Gemcitabine as a potential drug for the development of treatment strategy when combined with targeting GDF15.


Introduction
Colon adenocarcinoma (COAD) is the most frequently diagnosed histological subtype of colorectal cancer (CRC), which is the third most common type of malignant tumor, and in 2020, it ranked second in terms of cancer-related deaths globally [1]. In recent decades, considerable progress has been made regarding early diagnosis and multidisciplinary management strategies; however, the invasion, migration, metastasis, and recurrence of Int. J. Mol. Sci. 2021, 22, 13636 2 of 20 COAD have been challenges for improving long-term survival rates, preventing the 5-year survival rate from exceeding 30% [2,3]. Therefore, the development of highly accurate screening tools for the early diagnosis and prediction of prognosis, as well as precision treatment strategies, is urgently required to accommodate the unmet medical needs of COAD management.
Growth differentiation factor 15 (GDF15) is a well-known endocrine-acting mitokine, which is induced and secreted in response to mitochondrial stress [4]. GDF15 belongs to the transforming growth factor β (TGF-β) superfamily and thus presents the characteristic structure of the TGF-β superfamily [5,6]. The precursor protein of GDF15 contains 308 amino acids. After furin protease cutting, it becomes a mature protein and is subsequently secreted from the cell. GDF15 exerts autocrine and paracrine functions both on the cell, and it is secreted from the surrounding cells [5,6]. In a normally functioning human body, the expression of GDF15 exhibits tissue specificity, primarily distributed in the prostate, kidney, and pancreas [7], although its expression is increased at the kidney and liver lesions of several cancers [7,8]. However, the role of GDF15 in COAD remains controversial. For example, elevated GDF15 levels as a poor prognostic marker and tumor promoter have been noted [9][10][11], whereas its tumor-suppressive role has also been revealed [12]. microRNAs (miRs) are small non-coding RNAs with approximately 22 nucleotides, which play a regulatory role in the transcriptional control mechanisms for the maintenance of metabolic homeostasis. miRs are transcribed by RNA polymerase II or III in the nucleus to generate primary miRs (pri-miRs), which are then processed to become precursor miRNAs (pre-miRs) [13]. The miRs subsequently bind to the 3 untranslated region (3 UTR) of target mRNA(s), which can affect mRNA degradation or translational repression [14]. Despite displaying such repressive activity, it has been noted that some miRs act to enhance gene expression [15]. In addition, mechanisms including miRNAs binding to 5 UTR or the coding sequence of mRNA [16], toll-like receptors [17,18], or mitochondrial transcripts [19] have recently been reported. While the tumor-suppressive role of miR-216a in COAD has been shown [20,21], a more recent study reported miR-216a presents a pro-COAD effect [22]. Thus, more studies are warranted to clarify this debatable issue.
The purpose of this study was to explore the significance of the miR-216a/GDF15 axis in terms of clinical value, tumor immunity, and potential mechanisms in COAD. We first explored the gene expression profiles and the association of miR-216a and GDF15 in human samples of COAD and confirmed the miR-216a/GDF15 interaction at the transcriptional level. Second, we investigated the prognostic value of miR-216a and GDF15 and found the counteracting factors based on immune cell content. Third, we analyzed the correlation between GDF15 and immunoinhibitory markers and immune infiltration. Next, we employed a gene enrichment approach to reveal the molecular mechanisms and biological pathways of GDF15 in COAD. Finally, we combined Genomics of Drug Sensitivity in Cancer (GDSC) and Cancer Therapeutics Response Portal (CTRP) analyses to identify that Gemcitabine acts to simulate the GDF15 gene profile and is a potential candidate for the treatment of COAD. Our results may support the use of miR-216a and GDF15 as diagnostic/prognostic biomarkers for COAD, further clarify the molecular basis underlying immunomodulation, mitochondrial organization, and apoptotic signaling and reveal Gemcitabine as a potential precision treatment for COAD.

Multi-Omic Analysis
The coding gene expression profiles across various cancer types were accessed from UALCAN (http://ualcan.path.uab.edu/; accessed on 25 October 2021) [23] and GEPIA2 (http://gepia2.cancer-pku.cn/#index; accessed on 25 October 2021) [24]. The non-coding gene profiles were obtained from The Encyclopedia of RNA Interactomes (ENCORI; star-Base v3.0) web server, an open-source platform for pan-cancer analysis on the miRNA-ncRNA, miRNA-mRNA, ncRNA-RNA, RNA-RNA, RBP-ncRNA, and RBP-mRNA interac-tions from CLIP-seq, degradome-seq, and RNA-RNA interactome data [25]. We utilized the Human Protein Atlas (http://www.proteinatlas.org; accessed on 25 October 2021), a Swedish-based program initiated in 2003 to map human proteins in cells, tissues and organs, to determine the immunohistochemical (IHC) staining signal of GDF15 expression [26][27][28]. The validation test data for the differential expression of miR-216a were assessed with the OncoMir Cancer Database (OMCD, a repository enabling systemic comparative genome analysis of miR expression sequencing data derived from over 10,000 cancer patients with associated clinical information and organ-specific controls present in TCGA (https://www.oncomir.umn.edu/omcd/; accessed on 25 October 2021) [29] and GDS2609 of the Gene Expression Omnibus (GEO, a database repository of high throughput gene expression data and hybridization arrays, chips, and microarrays). The prediction of the direct binding between miR-216a and GDF15 3 UTR was conducted by TargetScanHuman v8.0 (http://www.targetscan.org/vert_80/; accessed on 25 October 2021). The mutation landscape was retrieved in cBioPortal (https://www.cbioportal.org/; accessed on 25 October 2021), which is a web platform of gene-based data exploration. The Kaplan-Meier analysis based on gene expression levels was conducted in the Kaplan-Meier plotter web server (available online: https://kmplot.com/analysis/; accessed on 25 October 2021), which includes data sources from the GEO, the European Genome-phenome Archive (EGA), and TCGA to enable the assessment of the effect of 54,000 genes, including mRNA, miR, and protein on survival in 21 cancer types [30]. In addition, the OncoLnc web server permits users to link TCGA survival data to mRNA and miRNA expression levels (http://www.oncolnc.org/; accessed on 25 October 2021). The construction of proteinprotein interaction (PPI) and functional enrichment analysis were conducted by using GeneMANIA v3.6.0 (https://genemania.org/; accessed on 25 October 2021) and STRING v11.5 (https://string-db.org/; accessed on 25 October 2021). The enrichment analysis integrating gene ontology sources including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome gene sets, and canonical pathways was conducted by Metascape algorithm [31] (https://metascape.org/gp/index.html#/main/step1; accessed on 25 October 2021). The drug sensitivity profiling based on GDF15 expression was examined by the CRISPR-screen data repository of GDSC algorithm in Q-omics v0.95 (accessed on 1 November 2021) [32] and by CTRP-based algorithm in GSCALite (accessed on 1 November 2021) [33]. The expression of each gene in the gene set based on GSCALite was performed by Spearman correlation analysis with the CTRP drug sensitivity (half maximal inhibitory concentration, IC 50 ) data. The positive correlation means that the low expression of the gene is sensitive to the drug, and vice versa. The filtering criteria for multi-omic analysis were stated in Supplementary Materials.

In Vitro Cell Experiment
HEK293, HEK293T, and HCT116 cell lines were cultured at 5% CO 2 and 37 • C in DMEM (Thermo Scientific, San Diego, CA, USA) supplemented 10% FBS (Thermo Scientific), penicillin (100 U/mL; Thermo Scientific), and streptomycin (100 µg/mL; Thermo Scientific). The GDF15 3 UTR or GDF15 3 UTR mutant sequences were cloned into the multiple cloning site of pMIR-REPORTERTM plasmid after cytomegalovirus (CMV) promoterdriven luciferase (Figure 1i). To observe luciferase signals, the reporter plasmid was subsequently introduced into the HEK-293 cells, which were then treated with miR negative control sequence (HMC0003; Sigma-Aldrich ® , St. Louis, MO, USA), miR-216a-3p mimic (HMI1979; Sigma-Aldrich ® ), or no treatment (NT). The HEK-293 cells were seeded at 1.5 × 10 6 cells in a 100 mm dish for 18 h. Five micrograms of reporter plasmids with GDF15 3 UTR or GDF15 3 UTR mutant were then individually transfected into the HEK-293 cells using Lipofectamine™ 2000 Transfection Reagent (11668030; Thermo FisherScientific, Rockford, IL, USA) for 16 h. The cells were then trypsinized and seeded at 7.5 × 10 5 cells in a 60 mm dish with a fresh growth medium. Subsequently, 20 nM miR-216a-3p mimic, miRs negative control, or NT were transfected into cells with RNAiMAX transfection reagent (13778-150; Invitrogen, Carlsbad, CA, USA) for 24 h. The cells were then lysed to detect luciferase signals with the Neolite Reporter Gene Assay System (PerkinElmer, Waltham, MA, USA). The procedure of detecting luciferase signals was followed according to the standard protocol of the manufacturer of the Neolite Reporter Gene Assay System. For Western blotting, 30 µg proteins were separated in SDS-PAGE gels and were transferred onto 0.45-µm PVDF membranes (Millipore, Burlington, MA, USA) in a Trans-Blot ® SD Semi-Dry Transfer Cell (Bio-Rad). The membrane was blocked in 5% non-fat milk powder/PBST (1X PBS, 0.1% Tween-20 (Sigma-Aldrich, St. Louis, MO, USA) and incubated overnight at 4 • C with a blocking buffer containing primary antibodies. The membrane was washed and then incubated for 1 h with 5% non-fat milk powder and a PBS-T-containing secondary antibody. The signals were developed using the Ultra ECL-HRP Substrate (#TU-ECL02, TOOLS) using X-ray films. Primary antibodies were purchased from Santa Cruz Biotechnology (anti-GDF15 (sc-377195) and anti-β-actin (sc-8432)), and the secondary antibody was purchased from Jackson ImmunoResearch (111-035-003).

Immunological Databases
The correlation between GDF15 and the immunoinhibitory genes in COAD was analyzed by TISIDB, which is an integrated repository web portal for tumor-immune system interactions [34]. The association between GDF15 expression and immune cell infiltration in COAD was determined using The Tumor Immunology Estimation Resource version 2.0 (TIMER 2.0), which is an online tool for systematically assessing the expression of gene sets associated with infiltrating immune cells in TCGA datasets.

Construction of PPI Network and Enriched Analysis
The significantly co-expressed genes of GDF15 were determined by the correlation repository of UALCAN. The genes displaying prognostic significance in patients with COAD were selected for PPI analysis using GeneMANIA and STRING. GeneMANIA is an open website for building PPI networks and demonstrating gene function [35,36]. By applying bioinformatics methodologies, this website analyzes genes or gene lists, which includes data regarding physical interaction, co-expression, and co-location, as well as enrichment and predictive analyses. To determine PPI, we utilized STRING, which is an online data-mining platform and includes data regarding physical and functional interplays [37].

Statistical Analysis
All values are expressed as mean ± standard error (SE). Quantitative data were analyzed using unpaired t-test for two-group comparison or one-way analysis of variance (ANOVA) for three-or more-group comparison when appropriate. Bonferroni test was used for post-hoc analysis. Two-sided p-values less than 0.05 were considered to be statistically significant.  (h) Target scan human-based bioinformatics prediction of the complementary relationship between miR-216a-3p and GDF15 3 untranslated region (UTR). (i) Graphic illustration of the luciferase reporter system for the detection of miR-216a-3p/GDF15 3 UTR interaction. (j) miR-216a-3p-mediated suppression of luciferase signal in HCT116 cells. Cells were treated with miR negative control (NC) or miR-216a-3p (mimic) for 24 h. Six independent experiments for each group were conducted. (k) Representative Western blotting image of the expression levels of GDF15 and β-actin. (l) Quantification results of GDF15 normalized to β-actin. (m) The illustration of the inhibitory activity of miR-216a on GDF15 by direct binding to its mRNA 3 UTR. * p < 0.05, *** p < 0.001 between groups.

Differential Expression Profile and the Molecular Interaction of miR-216a and GDF15
The gene expression levels of miR-216a and GDF15 showed an increase in the COAD group compared to in the normal group (Figure 1a,b). The protein expression levels of GDF15 presented a similar profile (Figure 1c). In addition, the IHC-determined GDF15 protein expression presented a higher quantity of GDF15 staining signals in COAD than in normal tissue (Figure 1d), in line with the aforementioned findings. The miR-216a expression levels retrieved from the OncoMir Cancer Database (OMCD) (Figure 1e) and the GDF15 accessed from GDS2609 of GEO ( Figure 1f) database confirmed the findings. We noted that the expression of miR-216a presented a negative correlation with GDF15 in COAD tumor tissue ( Figure 1g). We thus investigated whether miR-216a acts to suppress GDF15 at the transcriptional level. The TargetScanHuman survey predicted that miR-216a-3p has a complementary relationship with GDF15 at its 3 UTR site ( Figure 1h). To verify this, we employed a luciferase reporter assay, in which a CMV-driven plasmid was inserted with a fusion sequence of luciferase with GDF15 3 UTR or GDF15 3 UTR mutant ( Figure 1i). Then, the luciferase reporter plasmid was transfected into the HCT116 colorectal carcinoma cell line. The luciferase activity was decreased by the presence of miR-216a-3p mimic, while unaltered in cells harboring the GDF15 3 UTR mutant (Figure 1j), suggesting the direct binding of miR-216a to GDF15 3 UTR. Similar results were seen in HEK-293 and HEK-293T cells (Supplementary Figure S1). The Western blotting results confirmed that miR-216a-3p mimic suppressed the protein expression level of GDF15 (Figure 1k,l). Collectively, these results indicated that miR-216a and GDF15 featured the diagnostic value for COAD and that miR-216a targeted 3 UTR of GDF15 to suppress its expression.

Prognostic Value of miR-216a and GDF15
The correlation between the expression of miR-216a and GDF15 and the corresponding clinical follow-up information was analyzed by Kaplan-Meier curves and the logrank test. High miR-216a expression was found to be associated with decreased overall survival (OS; Figure 2a), while the high expression of GDF15 was associated with increased OS (Figure 2b). Specifically, miR-216a and GDF15 served as independent prognostic factors in COAD patients with stage 3, but not in stages 1, 2, and 4 ( Figure 2c-j). These results indicated the prognostic significance of the miR-216a/GDF15 axis, especially at the late stage.

Prognostic Value of miR-216a and GDF15 in the Context of Different Immune Cell Contents
We further examined the Kaplan-Meier results in the context of different immune cell contents. High miR-216a-associated poor OS was reversed in the context of decreased type 1 T-helper (Th1) cells, enriched regulatory T (Treg) cells, and enriched eosinophils (Figure 3a-g). In addition, high GDF15-associated favorable OS was reversed in the presence of decreased nature killer T-cells (NKTs; Figure 3h-j). In light of the findings that the overall predictive value was reversed in the presence of certain immune cell content, we suggested that enriched Th1, enriched Treg, enriched eosinophils, and decreased NKTs may be counteracting factors on the tumor-regulatory role of miR-216a and GDF15.
reporter system for the detection of miR-216a-3p/GDF15 3′UTR interaction. (j) miR-216a-3p-mediated suppression of luciferase signal in HCT116 cells. Cells were treated with miR negative control (NC) or miR-216a-3p (mimic) for 24 h. Six independent experiments for each group were conducted. (k) Representative Western blotting image of the expression levels of GDF15 and β-actin. (l) Quantification results of GDF15 normalized to β-actin. (m) The illustration of the inhibitory activity of miR-216a on GDF15 by direct binding to its mRNA 3′UTR. * p < 0.05, *** p < 0.001 between groups.

Mutation Landscape of miR-216a and GDF15
We investigated the mutation landscape of miR-216a and GDF15 in 10,953 patients. As shown in Figure 4a, the mutation spectra/counts of miR-216a and GDF15 did not synchronize with the corresponding tumor mutational burden (TMB). The genetic alteration of miR-216a stood at 0.6%, mainly composed of amplification and deep deletion. The genetic alteration of GDF15 was 1.4%, which primarily included amplification, deep deletion, missense mutation, truncated mutation, and structural variant. Interestingly, we noted that the TMB synchronized with the missense mutation and truncated mutation of GDF15. Nevertheless, no genetic alteration of miR-216a was noted in COAD, while that of GDF15 stood at 1.85% of 594 cases (Figure 4b). The number and mutation distribution across GDF15 is demonstrated in Figure 4c. Two missense mutations T90M (TCGA-D5-6391-01) and R217 (TCGA-CM-6674-01) were identified in COAD samples of TCGA PanCancer Atlas (Figure 4c).

Generalization Value of the miR-216a/GDF15 Axis in Pan-Cancer
To investigate whether miR-216a and GDF15 have a broad value, we performed a series of studies on miR-216a and GDF15 across all cancer types. We used ENCORI and TIMER analysis and revealed that miR-216a and GDF15 expression status varied in different cancers (Figure 5a,b). We noted that stomach adenocarcinoma (STAD) and uterine corpus endometrial carcinoma (UCEC) presented similar patterns to COAD, wherein tumors expressed high miR-216a expression levels along with high GDF15 expression compared to normal tissue (Figure 5a,b). The Kaplan-Meier analysis showed that high miR-216a expression had significant associations with short OS, while high GDF15 expression had significant associations with long OS in bladder carcinoma (BLCA), cervical squamous cell carcinoma (CSCA), stomach adenocarcinoma (STAD), and uterine corpus endometrial carcinoma (UCEC) (Figure 5c-j).

Generalization Value of the miR-216a/GDF15 Axis in Pan-Cancer
To investigate whether miR-216a and GDF15 have a broad value, we performed a series of studies on miR-216a and GDF15 across all cancer types. We used ENCORI and TIMER analysis and revealed that miR-216a and GDF15 expression status varied in different cancers (Figure 5a,b). We noted that stomach adenocarcinoma (STAD) and uterine corpus endometrial carcinoma (UCEC) presented similar patterns to COAD, wherein tumors expressed high miR-216a expression levels along with high GDF15 expression compared to normal tissue (Figure 5a,b). The Kaplan-Meier analysis showed that high miR-216a expression had significant associations with short OS, while high GDF15 expression had significant associations with long OS in bladder carcinoma (BLCA), cervical squamous cell carcinoma (CSCA), stomach adenocarcinoma (STAD), and uterine corpus endometrial carcinoma (UCEC) (Figure 5c-j).

GDF15 Negatively Correlated with Immunoinhibitory Genes and Immune Infiltration
The results of TISIDB demonstrated that GDF15 negatively correlated with a majority of the immunoinhibitory genes in COAD tumor tissues (Figure 6a). In this regard, immunoinhibitors including cytotoxic T-lymphocyte-associated protein 4 (CTLA4), programmed death-ligand 1 (PD-L1, also known as CD274), CD96, and T cell immunoreceptor with Ig and ITIM domains (TIGIT) exhibited negative correlations with GDF15 levels (Figure 6b). The TIMER2.0 analysis demonstrated that GDF15 had significant negative correlations with total macrophage, macrophage M1, macrophage M2, cancer-associated fibroblast, and endothelial cells (Figure 6c).

Co-Expression Network and Biological Functions of GDF15
To gain molecular insights into the role of GDF15 in COAD, we further used UALCAN analysis to acquire 22 genes positively correlated with GDF15 in COAD tumor tissue (Figure 7a). Among these genes, high expressions of early growth response 1 (EGR1), BCL2-associated X protein (BAX), immediate early response 3 (IER3), and JunD Proto-Oncogene (JUND) were identified to have associations with longer OS (Figure 7b-e). The PPI analysis revealed that the co-expression networks mainly participate in mitochondrial organization, apoptosis process, transcriptional activity, and endoplasmic reticulum (ER) stress (Figure 7f,g). After constructing this network, we applied the Metascape algorithm that processes enrichment analysis integrating gene ontology sources, including the GO biological process, the KEGG pathway, Reactome gene sets, and canonical pathways. As shown in Figure 7h, the top 20 clusters of the enriched sets were identified. As such, these genes were enriched in the molecular categories implicated in mitochondrial organization, apoptosis signaling, unfolded protein response, pyroptosis (a type of immunogenic cell death), and ER stress response.

Gemcitabine as a Therapeutic Option in the Context of Low GDF15
To explore potential pharmaceutical approaches that can effectively target COAD, we utilized the GDSC repository in Q-omics analysis to identify potent drugs that may act to activate GDF15. We conducted cross-associations between drug response and single guide RNA (sgRNA)-mediated GDF15 knockdown based on the CRISPR approach in large intestine cells. As shown in Figure 8a, four out of 478 drugs were identified, including Gemcitabine, NU7441, IMD-0354, and obatoclax mesylate. Moreover, large intestine cells with high sgRNA-GDF15 efficiency exhibited higher log(IC 50 Figure 8e). Among the four drugs, Gemcitabine showed the highest predictivity and descriptibility, as well as the highest fold change in the scenario of high sgGDF15 efficiency, indicating that Gemcitabine exerted the most significant suppressive effect in the context of GDF15 deficiency. In addition, we examined the relationship between GDF15 expression and drug sensitivity using the CTRP IC 50 drug data repository. As shown in Figure 8f, Gemcitabine showed a positive correlation with GDF15 expression, indicating that low GDF15 expression was sensitive to Gemcitabine. Collectively, these results suggested that Gemcitabine can serve as a therapeutic option when GDF15 levels are low.     We then employed CCLE analysis to further verify the effect of GDF15 expression levels on Gemcitabine sensitivity in 14 COAD cell lines. As shown in Figure 9, the Gemcitabine sensitivity featured a positive correlation with GDF15 expression (Pearson r = 0.636, p-value = 0.01), supporting the notion that Gemcitabine serves as a therapeutic option in the context of low GDF15 expression.

Discussion
The biological role and clinical implications of miR-216a and GDF15 in COAD remain unclear. In this study, we employed a variety of databases to reveal elevated miR-216a and GDF15 levels as diagnostic biomarkers and as independent risk predictors for favorable and unfavorable outcomes, respectively. We demonstrated that the miR-216a/GDF15 axis may account for the underlying molecular basis of COAD progression, offering an opportunity for the development of anti-miR-216a and GDF15-mimic approaches as therapeutic strategies. In addition, we revealed that tumor immune cell contents, such as enriched Th1, enriched Treg, enriched eosinophils, and decreased NKT, may play counteracting roles in the tumor-regulatory activity of the miR-216a/GDF15 axis. Furthermore, we found that the diagnostic and prognostic value of miR-216a and GDF15 in COAD can be extrapolated to other cancer types, including BLCA, CSCA, STAD, and UCEC. The favorable survival time associated with high GDF15 expression levels may be due to the suppression of immunoinhibitory genes and reduced immune infiltration of macrophage, cancer-associated fibroblast and endothelial cells, as well as perturbed mitochondrial organization, apoptosis signaling, ER stress, and immunomodulation. Importantly, our investigation indicated that Gemcitabine can act as a therapeutic option when present with low GDF15 expression levels, offering a promising precision treatment strategy. A proposed model is summarized in Figure 10.

Discussion
The biological role and clinical implications of miR-216a and GDF15 in COAD remain unclear. In this study, we employed a variety of databases to reveal elevated miR-216a and GDF15 levels as diagnostic biomarkers and as independent risk predictors for favorable and unfavorable outcomes, respectively. We demonstrated that the miR-216a/GDF15 axis may account for the underlying molecular basis of COAD progression, offering an opportunity for the development of anti-miR-216a and GDF15-mimic approaches as therapeutic strategies. In addition, we revealed that tumor immune cell contents, such as enriched Th1, enriched Treg, enriched eosinophils, and decreased NKT, may play counteracting roles in the tumor-regulatory activity of the miR-216a/GDF15 axis. Furthermore, we found that the diagnostic and prognostic value of miR-216a and GDF15 in COAD can be extrapolated to other cancer types, including BLCA, CSCA, STAD, and UCEC. The favorable survival time associated with high GDF15 expression levels may be due to the suppression of immunoinhibitory genes and reduced immune infiltration of macrophage, cancer-associated fibroblast and endothelial cells, as well as perturbed mitochondrial organization, apoptosis signaling, ER stress, and immunomodulation. Importantly, our investigation indicated that Gemcitabine can act as a therapeutic option when present with low GDF15 expression levels, offering a promising precision treatment strategy. A proposed model is summarized in Figure 10. Figure 10. Proposed model depicting the significance of the miR-216a/GDF15 axis in diagnosis, prognosis, tumor immunity, and precision treatment with Gemcitabine in COAD and its correlation for mitochondrial organization, apoptotic signaling, endoplasmic reticulum (ER) stress, and immunomodulation.
As an independent predictor, GDF15 was reported to be overexpressed in many tumors, including colon, prostate, and breast [38][39][40][41]. In addition, Danta et al. demonstrated that baseline GDF15 serum levels increase in subjects with polyps compared to those with no polyps and that the levels decreased in post-polypectomy subjects compared to in prepolypectomy ones, indicating the significance of GDF15 as a predictive factor in colonic neoplasia [42]. Our study revealed that the high expression of GDF15 had associations with COAD tissue, supporting its value as a diagnostic/prognostic marker. As a mitokine in response to mitochondrial stress, the role of GDF15 in mediating stress resistance to promote cellular longevity through modulating mitochondrial homeostasis is gradually emerging [43]. Kang et al. reported that the upregulation of GDF15 levels is induced by mitochondrial dysfunction. The knockdown of GDF15 impedes metastatic behaviors, indicating the tumor-promoting effect of GDF15 in thyroid cancer [44]. Furthermore, GDF15′s tumor promoting role in COAD has been noted in several reports [9][10][11]. For instance, secreted GDF15 of fibroblasts was identified to be involved in fostering a senescence-associated tumor microenvironment that promotes proliferation and invasion of COAD cells [45]. On the other hand, some studies demonstrated the role of GDF15 in mediating the anti-tumoral activity of pharmacological treatments. Shin et al., in this regard, reported that GDF15 is required for 2'-hydroxyflavanone-induced apoptosis in the HCT116 colon cancer cell line [46]. Guo et al. have indicated that GDF15 acts as a tumor suppressor of COAD in the context of traditional Chinese medicine treatment [12]. In this study, we revealed that an elevated GDF15 expression level is an independent predictor Figure 10. Proposed model depicting the significance of the miR-216a/GDF15 axis in diagnosis, prognosis, tumor immunity, and precision treatment with Gemcitabine in COAD and its correlation for mitochondrial organization, apoptotic signaling, endoplasmic reticulum (ER) stress, and immunomodulation.
As an independent predictor, GDF15 was reported to be overexpressed in many tumors, including colon, prostate, and breast [38][39][40][41]. In addition, Danta et al. demonstrated that baseline GDF15 serum levels increase in subjects with polyps compared to those with no polyps and that the levels decreased in post-polypectomy subjects compared to in pre-polypectomy ones, indicating the significance of GDF15 as a predictive factor in colonic neoplasia [42]. Our study revealed that the high expression of GDF15 had associations with COAD tissue, supporting its value as a diagnostic/prognostic marker. As a mitokine in response to mitochondrial stress, the role of GDF15 in mediating stress resistance to promote cellular longevity through modulating mitochondrial homeostasis is gradually emerging [43]. Kang et al. reported that the upregulation of GDF15 levels is induced by mitochondrial dysfunction. The knockdown of GDF15 impedes metastatic behaviors, indicating the tumor-promoting effect of GDF15 in thyroid cancer [44]. Furthermore, GDF15 s tumor promoting role in COAD has been noted in several reports [9][10][11]. For instance, secreted GDF15 of fibroblasts was identified to be involved in fostering a senescenceassociated tumor microenvironment that promotes proliferation and invasion of COAD cells [45]. On the other hand, some studies demonstrated the role of GDF15 in mediating the anti-tumoral activity of pharmacological treatments. Shin et al., in this regard, reported that GDF15 is required for 2'-hydroxyflavanone-induced apoptosis in the HCT116 colon cancer cell line [46]. Guo et al. have indicated that GDF15 acts as a tumor suppressor of COAD in the context of traditional Chinese medicine treatment [12]. In this study, we revealed that an elevated GDF15 expression level is an independent predictor for longer survival time. However, the question of whether GDF15 exerts anti-COAD effects requires further in-depth study.
Wei et al. demonstrated that COAD tumor presents higher levels of miR-216a than normal issue and that the high expression of miR-216a serves as one prognostic factor for shorter OS time [47]. This is in tandem with the results of our study. Nevertheless, one limitation of our result with respect to miR-216a expression levels was the small sample size, which may lead to statistical Type II error. Further study with a larger sample size is warranted. Whether miR-216a acts to promote or suppress COAD remains a debatable issue. In this study, the TCGA dataset shows that elevated miR-216a levels is an independent risk factor for COAD patients, suggesting that miR-216a may be a positive factor for COAD progression. This notion is in line with a study reported by Zeng et al., which revealed that downregulated miR-216a acts toward COAD suppression [22]. In addition, the fact that miR-216a binds to GDF15 3 UTR and that there exists a reverse prognostic outcome between the two markers suggests that the activity of the miR-216a/GDF15 axis may have associations in controlling COAD progression. However, a major caveat of this study lies in the majority use of database analysis that is in need of further molecular verification through an investigation to clarify the exact mechanisms in a COAD experimental model. While further study in this regard is warranted, our study reveals novel insights into the possible role of the miR-216a/GDF15 axis in COAD.
One critical factor impacting on CRC development is gut microbiota, which can act to promote oncogenic transformation and alter tumor-associated inflammation by perturbing redox status and the activity of immune cells [48]. Wang et al. demonstrated that Enterococcus faecalis infection in macrophage promotes superoxide production, leading to DNA damage in epithelial cells via a bystander effect [49,50]. Mangerich et al. showed that Helicobacter hepaticus plays a role in the accumulation of macrophage and neutrophil in the colon and acts to promote nitric oxide (NO) production. These effects have been observed to occur concomitantly with gut inflammation and colon cancer development [51].
In this study, we demonstrated that GDF15 expression inversely correlated with tumorassociated macrophage. That raises a possibility that macrophage may be a common target of GDF15 and gut microbiota in the context of colonic tumorigenesis and that GDF15 may interplay with gut microbiota to regulate immune response. Nevertheless, more investigations are needed to clarify the exact mechanisms.
Gemcitabine (brand name Gemzar) is an FDA-approved drug for pancreatic, lung, and metastatic breast cancer, reaching phase 2 clinical trials (NCT00220155 and NCT00007943). Zheng et al. reported on the anti-tumoral effect of Gemcitabine in a mouse CT26 COAD model [52]. Several clinical studies have revealed that Gemcitabine is clinically active and well-tolerated when combined with the chemotherapeutic agent fluoropyrimidine [53]. Saif et al. have also suggested that the combination of Gemcitabine and capecitabine is an effective option for the treatment of CRC patients in advanced stages [54]. However, studies into Gemcitabine prescription adhering to the principles of precision treatment are lacking. It is noteworthy that the present study reveals Gemcitabine as a potential therapeutic strategy for COAD in the presence of low GDF15 expression levels. While more studies are warranted, this finding may support the development thesis of a precision treatment for COAD based on GDF15 expression levels.

Conclusions
This study indicated the miR-216a/GDF15 axis as a diagnostic/prognostic panel for COAD. We further identified Th1, Treg, eosinophils, and NKTs as counteracting factors and indicated potential relationships underlying immunomodulation, mitochondrial organization, apoptotic signaling, and ER stress. Our study data also revealed that Gemcitabine may be a potential drug for the development of a treatment strategy when combined with GDF15 targeting.