{'fraction_non_alphanumeric': 0.03492134831460674, 'fraction_numerical': 0.018202247191011236, 'mean_word_length': 5.151783245783799, 'pattern_counts': {'":': 0, '<': 2, '<?xml version=': 0, '>': 0, 'https://': 4, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 7, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Development of a novel necroptosis-associated miRNA risk signature to evaluate the prognosis of colon cancer patients

Background Necroptosis is a recently discovered caspase-independent form of cell death which plays an important role in the occurrence and development of cancer. As an important regulatory factor in necroptosis, microRNAs (miRNAs) are important for the development of colon cancer. This study established a novel necroptosis-related miRNA risk signature to evaluate the prognosis of patients with colon adenocarcinoma (COAD). Methods The necroptosis-related miRNAs were selected by assessing the differential expression of miRNAs in 459 COAD patient samples and 8 control samples from The Cancer Genome Atlas (TCGA). Selection operator Cox analyses and survival analyses were used to establish the risk signature of 7 miRNAs related to necroptosis. Functional enrichment analysis and nomograms were used to explore the potential effects of necroptosis-related miRNAs on prognosis and metastasis. The target genes of the necroptosis-related miRNAs were predicted using online databases and the genes related to overall survival (OS) were screened. Results The risk signature was based on 7 necroptosis-related miRNAs. Nomograms showed that the risk signature was effective at predicting the prognosis and TNM stage of COAD patients. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses demonstrated that these miRNAs play an important role in cancer development, metastasis, and prognosis. A total of 38 target genes for these miRNAs were found to be associated with the OS in COAD patients. Conclusions This study provided novel evidence that necroptosis-related miRNAs are associated with the prognosis of COAD patients. A risk signature established based on these miRNAs could effectively predict the prognosis and metastasis of COAD in patients.


Introduction
Colon adenocarcinoma (COAD) is among the most common tumor of the digestive system. At present, COAD is the fourth most common cancer type in the United States and is the second leading cause of cancer-related mortality (1). In China, COAD is also one of the top five leading causes of cancer and cancer-associated deaths. Furthermore, the incidence of COAD has risen substantially over the past two decades, surpassing the rate of increase for rectal cancer (2). While the specific mechanisms of COAD remain to be fully elucidated, its incidence has been associated with dietary and genetic factors, inflammatory bowel disease, and diverticulosis (3,4). Currently, the main forms of treatments include surgery, chemotherapy, and immunotherapy. However, within 5 years of treatment, an estimated 30-50% of COAD patients ultimately develop recurrent or metastatic disease (5). Therefore, it is vital to develop novel approaches for diagnosing and evaluating the prognosis of COAD patients, thereby facilitating improved individualized treatment regimens.
Necroptosis is a recently identified form of caspaseindependent regulated cell death (6). Necroptosis has been linked to a range of inflammatory and malignant diseases including ischemic, cardiovascular, and cerebrovascular diseases, as well as cancers (7,8). Studies have shown that necroptosis can inhibit tumor proliferation, metastasis, and progression (9), while also promoting reactive oxygen species (ROS) production and the cancer microenvironment (10)(11)(12). However, the mechanisms by which necroptosis promote tumorigenesis and progression in COAD remain to be fully elucidated.
MicroRNAs (miRNAs) are small transcripts of about 19-24 nucleotides long (13). Recent studies have found that although miRNAs lack coding ability, they can bind to tumor-related genomic regions or vulnerable sites in the genome, thereby regulating the expression of tumor suppressor genes and oncogenes (14). Moreover, particular miRNAs can influence oncogenesis by regulating necroptotic cell death. Visalli et al. found that miR-371-5p, miR-373, and miR-543 were upregulated in hepatocellular carcinoma (HCC) tissues compared to adjacent noncancerous tissues. These miRNAs bound to their target casp-8 mRNA transcripts, thereby promoting necroptosis of HCC cells (15). Another study suggested that miR-381-3p may be a biomarker for predicting the sensitivity of cells to apoptosis and necroptosis and may be a potential therapeutic target for renal cell carcinoma (RCC) (16). Cai et al. found that miR-148a-3p was up-regulated in acute pancreatitis and its deficiency inhibited necrosis, AP progression and inflammatory infiltration while inducing apoptosis in vivo (17).
Given the existing findings, we know that necroptosis plays an important role in the development of tumours and antitumour processes; however, its specific functions in COAD have been less studied. This present study utilized miRNA sequencing data from The Cancer Genome Atlas (TCGA) to define necroptosis-related miRNA expression profiles associated with COAD to define novel biomarkers with potential clinical and/or therapeutic relevance.
We present the following article in accordance with the REMARK reporting checklist (available at https://dx.doi. org/10.21037/atm-21-6576).

Data sources
The TCGA database was used to obtain the miRNA-seq data of patients with COAD. Mature miRNA sequences in the Fasta format were obtained from miRbase and utilized for miRNA annotations. The miRNA-seq data pertaining to 467 patient samples (459 tumors and 8 control samples) were analyzed. In addition, patient data including age, gender, stage, TNM classification, survival outcome, and survival duration were collated for analysis ( Table 1). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

Identification of differentially expressed necroptosisassociated miRNAs
Based on prior review studies, 16 miRNAs known to be associated with necroptosis (18) were examined (Table S1). The expression of these miRNAs in COAD tumors and normal tissue samples was compared using the R 'reshape2' and 'limma' packages. Associations among necroptosisassociated miRNAs were assessed using Pearson correlation coefficients and the R 'ggplot2' package.

Generation of the prognostic necroptosis-associated miRNA model
The association between necroptosis-related miRNA expression and overall survival (OS) in patients was examined using the TCGA-COAD cohort. Univariate Stage: a combination of T, N and M stages is used to determine colon cancer Stage; I: the cancer cells grow into the second or third layer of the colon wall, and there are no cancer cells in the nearby lymph nodes or the extracolonic area; II: the cancer cells have grown to or beyond the fourth layer of the colon wall, and there are no cancer cells in the nearby lymph nodes or outside the colon; III: the cancer has spread from the colon to nearby lymph nodes or there is tumor deposition. Tumor deposits are small tumors in the fat around the colon. IV: the cancer has spread beyond the colon and to nearby lymph nodes; Tis: carcinoma in situ; T1: tumor invasion into the submucosa; T2: the tumor invaded the muscularis propria of the intestinal wall; T3: the tumor penetrated into the muscularis propria and invaded subserosal layer, or when the primary lesion was located in the colon and rectum without serosal layer, the tumor had invaded paracolonic or rectal tissues; T4: the tumor has penetrated the peritoneum or directly invaded other organs; N0: no metastasis to regional lymph nodes; N1: 1-3 regional lymph node metastases; N2: ≥4 regional lymph node metastases; M0: no distant metastasis; M1: there are distant metastases.
and multivariate Cox regression analyses were performed with a P value <0.01 as the cutoff criteria. Analyses were performed with the R 'survival' and 'forestplot' packages. Kaplan-Meier curves were utilized for survival analyses, with log-rank tests and univariate Cox proportional hazard regression models used to generate p-values as well as hazard ratios (HRs) with 95% confidence intervals (CIs). Those necroptosis-associated miRNAs that exhibited prognostic relevance in the initial analyses were retained for further assessment. A risk score was defined based on these prognostic miRNAs using the TCGA dataset following centralization and standardization with the R 'scale' function. Risk scores were calculated using the following formula: risk score = ∑7iXi×Yi, where X and Y correspond to coefficients and miRNA expression levels, respectively. The median risk scores were used to stratify patients from the TCGA-COAD cohort into low-and high-risk groups, after which OS was compared between these groups using the Kaplan-Meier approach. The 1and 5-year OS of patients were analyzed using receiver operating characteristic (ROC) curves generated with the R 'survival, 'ROCR', and 'timeROC' packages. The prognostic relevance of necroptosis-associated miRNAs was further assessed through Cox regression analyses. Kaplan-Meier analyses were conducted as above and prognostic miRNAs were chosen for subsequent evaluation.

Prognostic analyses
Demographic and clinical data, including patient age, gender, and TNM staging, were extracted from the TCGA dataset. Univariate and multivariate Cox regression models were used to analyze the prognostic value of these variables and the risk scores generated above. Cox regression analysis was subsequently used to create prognostic and metastatic nomograms, which were validated using the consistency index (C-index) and calibration plots in the "Rms" R package v5.1.

RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)
Total cellular RNA was isolated from colon cancer cell lines (SW480, SW620, HCT116, and LOVO) and a human colon tissue cell line (CCD18-Co) using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA using a cDNA synthesis kit (Takara, Dalian, China) according to the manufacturers' instructions. qPCR amplification was performed using the SYBRGreen PCR Kit (Takara) in the ABI 7900 Sequence Detection System (Applied Biosystems, USA). The primers are shown in Table S2. U6 was used as the internal control. The PCR parameters were set for an initial cycle of 95 ℃ for 2 minutes, followed by 40 cycles at 95 ℃ for 30 seconds, 57 ℃ for 45 seconds, and 72 ℃ for 45 seconds. The relative expression of each gene was analyzed using the 2 ΔΔCt method. Experiments were performed in duplicates and repeated 3 times.

miRNA target gene enrichment analysis
Putative target genes associated with prognostic miRNAs were identified using the TargetScan, miRDB, and miRTarBase databases. Genes that were predicted by at least two of these three databases were chosen as miRNA target genes. A miRNA and target gene interaction network were generated using Cytoscape (19), while gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using the R software.

Statistical analysis
Single-factor analysis of variance was applied to compare the gene expression levels between the normal ovarian and OC tissues. To assess the independent prognostic value of the risk model, we used univariate and multivariate Cox regression models. All statistical analyses were performed on SPSS v22.0. All of the experiments in our study were independently performed in triplicate, P<0.05 was considered statistically significant.

Risk-related miRNA identification
Considering the potential prognostic value of some necroptosis-associated miRNAs, experiments were conducted to identify the risk-related miRNAs that may be able to predict outcomes in COAD patients. Kaplan-Meier analyses revealed that elevated expression of hsa-mir-16-5p, hsa-mir-141-3p, and hsa-mir-148a-3p were significantly correlated with decreased survival of patients (Figure 2A-2C). This suggested that these miRNAs may play a deleterious role in the context of COAD-related necroptosis such that patients who express high levels of these miRNAs are likely to experience inferior oncologic outcomes.

Univariate and multivariate analyses of the risk model
Univariate and multivariate Cox regression analyses were conducted to clarify whether the risk scores were independent predictors of patient prognosis. In univariate analyses, risk scores, pT stage, and pN stage were associated with COAD patient prognosis. Multivariate analysis showed that after adjusting for potential confounding variables, age, stage, and risk scores were independent predictors   Tables 4,5). Moreover, the expression of these 7 miRNAs was significantly elevated in colon cancer cell lines compared to normal colon cells ( Figure 4A-4G).

Development of the necroptosis-associated miRNA nomogram models
Nomograms for OS at 1-, 3-, and 5-year were created based on age, stage, pathologic T stage, and risk scores ( Figure 5A). The variable score is based on the intersection of the vertical line and point axis associated with each variable, and the total risk score is the sum of the scores of each variable. These total scores identify predictors of OS for each patient. The C-index of the nomogram was 0.772, indicating that the predicted results were generally consistent with the actual OS of COAD patients. Finally, the calibration plots visually demonstrated that the predicted outcomes of the nomograms were in strong agreement with the actual prognostic outcomes at 1-, 3-, and 5-year ( Figures 5B-5D).

Evaluation of miRNA target genes
The TargetScan, miRDB, and miRTarBase databases were used to predict the putative target genes of the hsa-miR-16-5p, hsa-miR-141-3p and hsa-miR-148a-3p These three miRNAs were statistically significant in the prognosis of COAD. Target genes that overlapped in different databases were further analyzed ( Figure 6A and available online: https://cdn.amegroups.cn/static/public/atm-21-6576-01.pdf). The association between these miRNAs and their target genes were assessed with Cytoscape ( Figure 6B). GO enrichment analyses of these target genes revealed that they were enriched in biological processes (BPs) including mitotic cell cycle, dephosphorylation, deubiquitination, and epithelial cell proliferation. They were also enriched for molecular function terms (MFs) including protein serine/threonine kinase activity and transforming growth factor-beta receptor binding, as well as cellular component terms (CCs) including recycling endosome, protein kinase complex, transferase complex, transferring phosphorus-containing groups, focal adhesion, and cell-substrate junction ( Figure 6C and available online: https://cdn.amegroups.cn/static/ public/atm-21-6576-02.pdf). KEGG pathway enrichment analyses indicated that these target genes were enriched in the MAPK, mTOR, FOXO, p53, and PI3K-Akt signaling pathways ( Figure 6D and available online: https://cdn. amegroups.cn/static/public/atm-21-6576-03.pdf).

Analysis of the prognostic relevance of miRNA target genes
Analysis of the prognostic relevance of the miRNA target genes revealed significant associations between the expression of 38 genes and the OS of COAD patients (Figure 7 and Figure S1).

Discussion
Necroptosis is a form of regulated cell death that is often considered to be secondary to apoptosis in a range of pathological conditions (20). During cell necroptosis, the volume of the cell increases, the organelles expand, ultimately leading to the rupture of the plasma membrane and cell death (21,22). Interestingly, necroptosis can promote the occurrence and progression of tumors b y t r i g g e r i n g a p r o -o n c o g e n i c t u m o r i m m u n e microenvironment (12,23,24) and modulating stromal cell responses (25,26). However, in certain cases, necroptosis can inhibit tumor progression. Indeed, experimental studies on tumor immunology have found that necroptosis may play an important role in triggering immunogenicity and promoting natural or treatment driven anti-cancer immune monitoring (27,28). Several necroptosis-associated miRNAs have been suggested to play important roles in various cancers (29). In glioblastoma, mesenchymal glioblastoma subtypes exhibit extensive necrosis, and low miR-218 expression is a characteristic feature of the highly necrotic and hypoxic mesenchymal glioblastoma subset (30). Furthermore, the expression of miR-193a-3p is significantly reduced in malignant pleural mesothelioma, and it has been shown to regulate necrotic cell death by targeting the apoptosisrelated gene MCL-1 (31).
This current study evaluated the expression of 16 miRNAs associated with necroptosis and demonstrated that 7 miRNAs were upregulated in COAD tumor tissues compared with normal colon tissues. In addition, there was Relative expression of hsa-miR-148a-3p Relative expression of hsa-miR-200a-5p Relative expression of hsa-miR-223-3p Relative expression of hsa-miR-425-5p Relative expression of hsa-miR-7-5p Relative expression of hsa-miR-16-5p a significant positive correlation among the 7 miRNAs, suggesting that these may work together to regulate necroptosis in COAD. A risk score and prognostic model based on the 7 miRNAs was constructed and shown to accurately predict the 1-and 5-year OS in COAD patient. These risk scores, together with patient age and tumor stage, were found to be independently associated with COAD patient prognosis. Other studies have also explored factors that are predictive of COAD prognosis. Zhou et al.
confirmed the role of immune-related prognostic models in COAD (32). Autophagy-associated prognostic features have also been identified as reliable predictors of COAD patient prognosis (33). Our novel necroptosis-associated miRNA-based model may represent a valuable tool for predicting prognosis and survival in COAD patients.
To further evaluate the prognostic value of these necrosis-related miRNAs, univariate regression analyses were performed. The miRNAs hsa-miR-141-3p, hsa-miR-148a-3p, and hsa-miR-425-5p were correlated with the prognosis of COAD patients. However, other researchers have reported that upregulation of hsa-miR-141-3p inhibited the growth of colon tumors (34). Li et al. showed that in necrotizing enterocolitis, the expression of has-miR-141-3p can inhibit ripk1-mediated necrosis, thereby protecting small intestinal epithelial cells from injury (35). Upregulation of miR-148a-3p in gliomas has been  (36). miR-148a-3p has also been found to be upregulated in distant metastatic tissue from osteosarcoma patients, and its upregulation is inversely correlated with distant metastasis, tumor size, and OS (37). In line with our findings, Kjersem et al. reported that plasma miR-148a expression was associated with decreased progression free survival (38). Previous studies have suggested that miR-425-5p may promote the development and metastasis of colorectal cancer by activating the CTNND1-mediated β-catenin pathway (39). In sepsis, the miR-425-5p/RIP1 axis can repair related liver injury through necroptosis  and inflammation (40). We found that hsa-miR-16-5p was highly expressed in colon cancer and was associated with poor prognosis of colon cancer. Interestingly, previous studies have found that the expression and role of mir-16 in cancer are uncertain. Hasáková et al. found that the expression level of mir-16 was up-regulated in colon cancer tissues compared with normal tissues (41). It has also been found that miR-16 was down regulated in colon cancer and was associated with late TNM stage, poor histological grade, and better OS (42,43).
Our study confirmed that risk signatures can effectively classify COAD patients into subgroups with different risk levels. The risk signatures were combined with clinical factors to construct a new nomogram to predict  The potential target genes of the differentially expressed necroptosis-related miRNAs were identified and functional analyses were conducted. In KEGG pathway analyses, these target genes were found to be enriched in the FOXO, p53, Hippo, PI3K/Akt/mTOR, MAPK, ErbB, AMPK, apelin, RAS, PD-L1, TGF-β, focal adhesion, and stem cell pluripotency regulatory signaling pathways. The PI3K/Akt/ mTOR pathway is a key regulator of metastatic progression and chemoresistance in colorectal cancer (44). Hu and colleagues reported that PI3K can promote necroptotic cell death by interacting with RIP3 (45). Similarly, p53 can promote necroptotic cell death by upregulation of apoptosisinducing factors, interacting with Bak, and directly binding to Bcl-XL and Bcl-2 (46). The Hippo signaling pathway is an important regulator of colon cancer but its role in the context of necrotic cell death remains unclear (47). Kim et al. reported the ability of bee venom to disrupt focal adhesion and the integrity of the cell membrane, inducing necroptotic pluripotent stem cells death (48). The apelin signaling pathway is related to the invasion and migration of colon cancer cells. While its association with necroptosis in COAD has not been fully eludicated, it may be a promising anticancer therapeutic target (49). Furthermore, a link between necroptotic cell death and immune checkpoint activity has been identified. Lomphithak et al. reported a positive correlation between necroptosis and the expression of PD-L1. The researchers found that high expression of PD-L1 was correlated with longer patient survival (50). Additional investigations are needed to verify this in COAD. Target gene-based survival analyses revealed that 38 of these genes were associated with COAD patient OS. High expression of 20 of these genes was associated with poor survival outcomes, while elevated expression of 16 of these genes was associated with improved patient prognosis. Therefore, these target genes may be involved in cancer development and may be potential biomarkers for the prognosis and treatment of patients with COAD.
In conclusion, the results of this analysis suggested that the occurrence and progression of COAD are closely related to necroptosis. There are some limitations to this research. This study was conducted using the TCGA-COAD data set, and other data sets should be similarly analyzed to validate our findings. Moreover, future in vitro and in vivo experiments are warranted to support the clinical findings.