Cancer Management and Research Dovepress open access to scientific and medical research

Open Access Full Text Article ORIGINAL RESEARCH miRNA Expression Profile in the N2 Phenotype Neutrophils of Colorectal Cancer and Screen of Putative Key miRNAs

This article was published in the following Dove Press journal: Cancer Management and Research

Liang Wang1 Objective: Colorectal cancer (CRC) is one of the most common malignant tumors in Jun Yang1 the digestive tract, which accounts for 10% of all the malignant tumors in the world. Jian Huang1 The aim of this study was to identify key genes and miRNAs in CRC diagnosis, Zheng-Qi Wen1 prognosis, and therapy and to further explore the potential molecular mechanisms of Ning Xu1 CRC. Methods: The infiltration and metastasis of neutrophils in primary colorectal cancer tissue Xuan Liu1 2 and paracancerous tissue were observed by immunohistochemical staining. After inducing Jian-Hua Zhang β 1 N2 neutrophils with TGF- 1 in vitro, exosomes were extracted and sequenced, and then the Wen-Liang Li expression differences of miRNAs were screened by using Agilent miRNA microarrays. The 1Department of Oncology, The First data were imported to the Web CARMA for differential expression analysis. The GO and fi Af liated Hospital of Kunming Medical KEGG enrichment analysis were performed using DIANA-MirPath v3.0 using TargetScan University, Kunming, Yunnan Province 650032, People’s Republic of China; database. And the corresponding targets were imported into Gephi for network analysis. The 2Department of General Surgery, Hong expression level of differentially expressed miRNA using quantitative real-time polymerase He Prefecture Third People’s Hospital, chain reaction (RT-PCR) was validated. Gejiu City 661100, People’s Republic of China Results: A total of 2 miRNAs were found to be associated with N2 neutrophils, in which the expression of hsa-miR-4780 was upregulated and the expression of hsa-miR-3938 was downregulated in N2 neutrophils, compared with the neutrophils. In addition, the results of miRNA-targets networks showed that the hsa-mir-3938 and hsa-mir-4780 could regulate TUSC1 and ZNF197. The expression level of hsa-miR-4780 and hsa-miR-3938 wase validated in accordance with the results of RT-PCR. Conclusion: The hsa-mir-3938 and hsa-mir-4780 were differentially expressed between N2 neutrophils and neutrophils. Moreover, the regulation of TUSC1 and ZNF197 by these DEmiRNA established the theoretical basis for the mechanism of N2 type neutrophils regulating the invasion and metastasis of CRC cells and provided the potential biomarker for prognosis for clinical treatment of CRC. Keywords: miRNA, N2 phenotype neutrophils, colorectal cancer

Introduction Colorectal cancer (CRC) is one of the most common malignant tumors in the 1 Correspondence: Wen-Liang Li digestive tract, which accounts for 10% of all the malignant tumors in the world. Department of Oncology, The First With the rapid development of social and economic life and the change of diet Affiliated Hospital of Kunming Medical University, No. 295 Xichang Road, lifestyle and diet structure, the incidence and prevalence of colorectal cancer are Kunming 650051, People’s Republic of increasing gradually.2 Although the treatment of CRC has improved significantly in China 3 Email [email protected] recent decades, the five year survival rate is only 8–10%. Therefore, it is more submit your manuscript | www.dovepress.com Cancer Management and Research 2020:12 5491–5503 5491 DovePress © 2020 Wang et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. – http://doi.org/10.2147/CMAR.S251427 php and incorporate the Creative Commons Attribution Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Wang et al Dovepress important to study the pathogenesis mechanism of CRC, therapeutic target for CRC, and provided the theoretical and to find the right drug target to promote the effect of basis for individualized treatment of CRC. treatment and prolong the survival time. Tumor microenvironment includes tumor cells them- Materials and Methods selves, various immune cells and interstitial cells. Immune Immunohistochemical Staining cells mainly include neutrophils, macrophages, NK cells, Tissue samples were obtained from 20 patients diagnosed lymphocytes and dendritic cells.4 Many recent researches with colorectal cancer between July 2019 and December have denoted that the neutrophils play important roles in 2019 at the First Affiliated Hospital of Kunming Medical the formation of the tumor microenvironment.5 Tumor- University (degree of differentiation: I: n=6; II: n=14). associated neutrophils (TANs) secrete various proteases, Twenty specimens of primary colorectal cancer tissues reactive oxygen species and cytokines, which play an and 20 specimens of paracancerous tissues were taken. important role in tumor growth, metastasis, angiogenesis Formalin-fixed, paraffin-embedded colon biopsy sections and immune regulation.6 TANs can be further divided into were deparaffinized in xylene for 20 min and rehydrated in two subtypes as N1 TANs and N2 TANs. N1 TANs have a graded ethanol series (100–70%) for 8 min. Endogenous antitumor effect, while N2 TANs can promote the occur- peroxidase were deactivated with 3% hydrogen peroxide rence, development and metastasis of tumors.7 In addition, for 5–10 min at room temperature after deparaffinization. a number of studies revealed that the increase of neutro- Antigen retrieval was performed with a sodium citrate phil count in peripheral blood was related to poor clinical buffer (10 mM, pH = 6) for 10 min. A mouse IgM antihu- outcomes in CRC, but the mechanism of this process was man monoclonal antibody specific for CD11b, CD66b unclear.8–11 (ab197678) and mmp9 (BD Biosciences, San Diego, miRNAs are small molecule RNA with a length of California) was used to identify neutrophils. Negative 20–25 nucleotides, which are involved in the regulation controls were stained with a rabbit IgG negative control of various physiological and biochemical processes in prediluted in phosphate-buffered saline (Life organisms.12 It has been found that miRNAs played an Technologies). Slides were counterstained with hematox- important regulatory role in the occurrence and develop- ylin. The staining protocol was the same for all samples ment of diseases, especially tumors. If the expression of with regard to processing, incubation times, and tempera- miRNAs which can regulate cell proliferation and differ- ture. In addition, this study was approved by the ethics entiation was abnormal, it might lead to the occurrence of committee of the first affiliated hospital of Kunming tumor.13 In addition, previous studies showed that Medical University, and this study was conducted in accor- miRNAs expression were different in various tissues of dance with the Declaration of Helsinki. CRC, and it were related to the proliferation and metasta- sis of CRC tumor cells. For example, studies showed that β long chain noncoding RNA MIR17HG promoted metasta- TGF- 1 Induced Neutrophils to N2 sis of CRC via miR-17,14 and the increased expression of Polarization in vitro miR-17 also promotes metastasis of CRC.15 Another The acute promyelocytic leukemia (APL) cell line (NB-4) research showed that the miR-200 families were key factor was cultured in RPMI-1640 culture medium containing in the occurrence of epithelial to mesenchymal transition 10% fetal bovine serum at 37°C in the presence of 5% (EMT) in tumor cells and abnormal expression of miR-200 CO2 (NB-4 cells were provided by Shanghai Bioleaf might promote EMT in tumor cells and lead to Biotech Co., Ltd). Then, using all-trans retinoic acid metastasis.16 However, the relationship between miRNA (ATRA) induced the differentiation of NB-4 cells into in type N2 neutrophils and CRC is still unclear and neutrophils. After NB-4 cells were differentiated by a considerable amount of research is needed in this area. 1μmol/L ATRA on the 4th or 5th day, we verified by Based on the above background, we used transforming flow cytometry. A mouse IgM antihuman monoclonal anti- growth factor-β1 (TGF-β1) to induce N2 phenotype neu- body specific for CD11b (BD Biosciences, San Diego, trophils in vitro, extracted exosomes for sequenced, and California) was used to identify neutrophils. The verifica- screened miRNAs with different expression. We analyzed tion results were shown in the supplementary materials the mechanism of N2 neutrophil miRNA regulating the (Supplementary Figure 1). According to different polarity invasion and metastasis of CRC cells, which provided new disposal conditions, neutrophils were divided into 2

5492 submit your manuscript | www.dovepress.com Cancer Management and Research 2020:12 DovePress Dovepress Wang et al groups, normal control group and N2 polarization group Hybridization buffer was added to dilute the labeled (neutrophil group and neutrophil+TGF-β1 group). cRNA. 100 μL of hybridization solution was dispensed Neutrophils in the normal control group were intact. into the gasket slide and assembled to the gene expression Neutrophils in the N2 polarization group were incubated microarray slide. The slides were incubated for 17 hours at with 10ng/mL TGF-β1 for 24h. Then, the miRNAs in 65°C in an Agilent Hybridization Oven. The hybridized neutrophil-derived exosomes were extracted, used sequen- arrays were washed, fixed and scanned by using the cing to screen out the differentially expressed miRNAs, Agilent Microarray Scanner (part number G2505C). and then bio-informatics analysis was performed. Data Analysis Exosome Purification and The normalized circRNA and miRNA expression data Characterization were imported to the WebCARMA for differential expres- Exosomes were prepared from neutrophil supernatants sion analysis. Moderated t-test (from limma package in R) using differential centrifugation. Supernatants had been was used to detect the difference between neutrophil β – – – centrifuged at 2000g for 30 min to remove any contam- +TGF- 1 group (2 1, 2 2, 2 3) and neutrophil group – – – “ inating exosomes. Add 1/3 (400ul) volume of exosome (1 1, 1 2, 1 3). The results were stored in miRNA_DE. ” “ ” extraction reagent (Ribo™ Exosome Isolation Reagent) csv in the DE_analysis fold, where the column rawp is “ ” to the supernatants after centrifugation, mix well and put the raw p value, the column BH is BH adjusted FDR and “ ” it at 4°C overnight. Finally, centrifuged at 1500g for meanM is the mead difference between TEST group and 30min, discarded the supernatant, and the off-white pre- CONTROL group. cipitate at the bottom of the tube was the exosome. Differentially expressed miRNAs with statistical sig- nificance between the two groups were identified through fi Total RNA Extraction Volcano Plot ltering. Differentially expressed miRNAs between the two samples were identified through Fold Total RNA was extracted from samples using a TRIzol® Change filtering. Hierarchical Clustering was performed Reagent (Invitrogen life technologies) according to the using the R scripts. manufacturer’s introductions. RNA quantity and quality were measured by NanoDrop ND-1000. The integrity of RNA was assessed by standard denaturing agarose gel GO and KEGG Pathway Analysis electrophoresis. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed MiRNA Microarrays using DIANA-miRPath v3.0 using TargetScan database. The target prediction was also conducted using DIANA-miRPath The Whole Human MiRNA Microarray was a broad view v3.0 (TargetScan database) and the top 10 miRNAs (based on that represents all known miRNAs in the human transcrip- raw p-value). GO results were mainly classified into three tome. Sequences were compiled from a broad source sur- subgroups, namely, biological process (BP), cellular compo- vey, and then verified and optimized by alignment to the nent (CC), and molecular function (MF). assembled human transcriptome. Construction of the miRNA-Targets RNA Labeling and Array Hybridization Sample labeling and array hybridization were performed Network Analysis The corresponding targets were imported into Gephi for according to the Agilent miRNA Microarray System with network analysis. The network is shown in Figure 1 (red miRNA Complete Labeling and Hyb Kit protocol (Agilent nodes represent the miRNA and green nodes represent Technology). Briefly, total miRNA from each sample was targets). labeled with Cyanine 3-pCp under the action of T4 RNA ligase. The labeled cRNA over the procession of inspissa- tion and desiccation and then redissolved with water. 1 μg Construction of hsa-miR-3938 and of each labeled cRNA was fragmented by adding 11 μL10 hsa-miR-4780 Lentiviral Vector × Blocking Agent and 2.2 μL of 25×Fragmentation Buffer, CRCs were transfected with miR-4780 mimic, miR-4780 then heated at 60°C for 30 min, and finally 55 μL2×GE mimic NC, miR-3938 inhibitor, miR-3938 inhibitor NC

Cancer Management and Research 2020:12 submit your manuscript | www.dovepress.com 5493 DovePress Wang et al Dovepress

Figure 1 Heat map generated by hierarchical clustering of differentially expressed miRNAs in neutrophil and N2 neutrophil samples. (A) Group 1: neutrophi samples, (B) Group 2: N2 neutrophil samples, the highly-expressed and lowly-expressed miRNAs are represented in red and green respectively.

using siRNA and siRNA-MateTM transfection reagent AGGAATG-3′, and reverse primer was 5′-ATAATTCTC (ribo FECT ™ CP Transfection Kit). Briefly, 50–100 nM CAGCATCAC-3′. siRNA and 5ug/mL polybrene were mixed together and then added to cells that were 50–60% confluent. At Statistical Analysis 24–36 h after transfection, cells were collected for subse- All data were presented as mean ± SD. The comparison quent experiments. between two groups was conducted using unpaired Student’s t-test. p-value less than 0.05 were considered fi qRT-PCR Assay statistically signi cant. The content of the differentially expressed miRNAs in β Results neutrophil group and neutrophil+TGF- 1 group were fi detected by utilizing real-time PCR. RT-PCR was per- Comparison of the Neutrophil In ltration formed using the Applied Biosystems 7300 Sequence Among the Different Groups Detection System (Ambion, Austin TX, USA). As shown in Figure 2, the expression of CD11b (CD11b is Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) the most important adhesion molecule on neutrophil mem- were quantified as an internal control for data normaliza- brane) and CD66b (CD66b is a surface-specific molecular tion. The sequences of targets primers were as follows: marker of tumor-infiltrating neutrophils (TINs)) were sig- TUSC1 forward primer was 5′-GACTTGAGAAGCTGG nificantly increased in primary colorectal cancer. The AA-3′, and reverse primer was 5′-TCGGGTTCCTGTAG matrix metalloproteinase-9 (mmp9) is a tumor angiogen- AG-3′. ZNF197 forward primer was 5′-CTTCACTTCAG esis protein produced by tumor-associated neutrophils,

5494 submit your manuscript | www.dovepress.com Cancer Management and Research 2020:12 DovePress Dovepress Wang et al

Figure 2 Representative immunohistochemical images (A) and IOD analysis (B) of neutrophil infiltration in primary colorectal cancer and paracancerous tissues. Both CD66b, CD11b and mmp9 showed positive staining (arrows).*P < 0.05. regarding the result of immunohistochemical staining, the Expression Profiles of miRNAs in N2 expression of mmp9 was significantly increased in primary Neutrophils colorectal cancer (Figure 2A). Furthermore, we performed All DEmiRNAs were displayed in the hierarchical clustering an IOD analysis on the number of CD11b, CD66b and (Figure 1), with red color representing high read counts and mmp9 positive cells in primary colorectal cancer tissues green color representing low read counts of miRNAs. We and paracancerous tissues. The results were consistent identified a total of 799 upregulated miRNAs and 902 down- with immunohistochemical staining (Figure 2B). These results suggested that neutrophils were associated with regulated miRNAs in all samples. There were 33 miRNAs tumor cell adhesion, invasion and metastasis in colorectal withrawp-valuelowerthan0.05(only2haveFDRlowerthan cancer. 0.05). These 33 miRNAs were used for downstream analysis.

Cancer Management and Research 2020:12 submit your manuscript | www.dovepress.com 5495 DovePress Wang et al Dovepress

Table 1 Deferential Expression miRNA (Based on Raw p-value) discovery rate (FDR), thereby reducing the proportion of false

Systematic_Name Mirbase Raw BH- MeanM positives in the results. Therefore, based on the value of BH- Accession p FDR FDR, we chose hsa-mir-3938 and hsa-mir-4780 for further experiments. hsa-miR-3938 MIMAT0018353 0 0 −5.312 hsa-miR-4780 MIMAT0019939 0 0.001 5.171 In addition, as shown in Figure 3, enrichment of total hsa-miR-513b-3p MIMAT0026749 0.002 0.965 0.672 miRNAs in the 3 neutrophils and 3 N2 neutrophils was hsa-miR-5703 MIMAT0022496 0.008 0.965 0.437 estimated in the Box Plot. In the volcano plot and Scatter hsa-miR-630 MIMAT0003299 0.012 0.965 0.413 Plot, the up-regulated miRNAs were represented as the red − hsa-miR-6089 MIMAT0023714 0.015 0.965 0.306 dots, while the down-regulated miRNAs were represented hsa-miR-4684-3p MIMAT0019770 0.018 0.965 0.552 as the green dots (Figure 4A and B). hsa-miR-2114-3p MIMAT0011157 0.021 0.965 −0.492 hsa-miR-1301-5p MIMAT0026639 0.022 0.965 −0.281 hsa-miR-6736-3p MIMAT0027374 0.022 0.965 0.379 GO and KEGG Analysis of miRNAs hsa-miR-1307-3p MIMAT0005951 0.024 0.965 −0.606 The top 10 enriched GO-BP terms, the top 9 enriched GO- hsa-miR-5010-5p MIMAT0021043 0.027 0.965 −0.392 MF and the top 5 enriched GO-BP terms that may be − hsa-miR-4439 MIMAT0018957 0.028 0.965 0.411 associated with the mechanism of N2 neutrophils in hsa-miR-3196 MIMAT0015080 0.029 0.965 −0.4 tumor tissue infiltration are shown in Figure 5. hsa-miR-5694 MIMAT0022487 0.035 0.965 −0.621 hsa-miR-1273g-5p MIMAT0020602 0.035 0.965 −3.713 According to the results, the most enriched and meaning- hsa-miR-5587-5p MIMAT0022289 0.035 0.965 −3.713 ful GO-BP terms were related to “cellular nitrogen com- hsa-miR-7159-5p MIMAT0028228 0.036 0.965 −0.38 pound metabolic process,”“biosynthetic process,”“small hsa-miR-4738-3p MIMAT0019867 0.036 0.965 0.266 molecule metabolic process,”“symbiosis encompassing hsa-miR-6754-5p MIMAT0027408 0.037 0.965 0.278 mutualism through parasitism,”“viral process,”“neuro- hsa-miR-3928-5p MIMAT0027037 0.037 0.965 −0.453 trophic TRK receptor signaling pathway,”“gene expres- hsa-miR-4754 MIMAT0019894 0.037 0.965 0.381 ”“ fi ”“ hsa-miR-3151-5p MIMAT0015024 0.038 0.965 −0.544 sion, cellular protein modi cation process, cellular hsa-miR-6125 MIMAT0024598 0.039 0.965 −0.265 protein metabolic process” and “catabolic process.” hsa-miR-6818-5p MIMAT0027536 0.04 0.965 −0.675 Within the GO-CC terms, the largest number of predicted hsa-miR-615-3p MIMAT0003283 0.041 0.965 0.564 target genes were involved in organelle, cellular compo- − hsa-miR-4730 MIMAT0019852 0.042 0.965 0.905 nent, protein complex, cytosol and nucleoplasm. hsa-miR-8060 MIMAT0030987 0.042 0.965 0.288 Regarding the GO-MF terms, the predicted target genes hsa-miR-5588-3p MIMAT0022296 0.045 0.965 0.348 hsa-miR-4421 MIMAT0018934 0.045 0.965 0.561 were mainly associated with ion binding, enzyme binding, hsa-miR-3136-5p MIMAT0015003 0.046 0.965 0.318 nucleic acid binding transcription factor activity, molecu- hsa-miR-627-5p MIMAT0003296 0.048 0.965 0.338 lar function, cytoskeletal protein binding, enzyme regula- hsa-miR-6836-3p MIMAT0027575 0.048 0.965 −0.225 tor activity, transmembrane transporter activity, protein Notes: Raw p, raw p value; BH-FDR, adjusted false discovery rate (FDR) using binding transcription factor activity and small conjugating Benjamin & Hochberg method; meanM, the different normalized value between Test group and Control group (neutrophil+TGF-β1 group minus neutrophil group). protein binding. Regarding the KEGG analyses, the most significantly enriched pathways of the miRNA host genes were metabo- All the significantly different miRNAs are listed in Table 1. lism of xenobiotics by cytochrome P450, Glycosphingolipid Based on raw p value (neutrophil+TGF-β1groupVS.neutro- biosynthesis-lacto and neolacto series, Chagas disease phil group), the top 5 up-regulated miRNA were hsa-miR (American trypanosomiasis), sphingolipid signaling pathway -4780, hsa-miR-513b-3p, hsa-miR-5703, hsa-miR-630, and cell adhesion molecules (CAMs) (Figure 5). hsa-miR-4684-3p, and the top 5 down-regulated miRNA were hsa-miR-3938, hsa-miR-6089, hsa-miR-2114-3p, Construction of miRNA-Targets Networks hsa-miR-1301-5p, hsa-miR-6736-3p. Moreover, there were These 10 miRNAs in total were used for prediction of their 6/33 miRNA containing chromosomal distribution informa- targets and a network containing these miRNAs and genes tion: hsa-miR-2114-3p (chX), hsa-miR-1307-3p (ch10), hsa- was constructed (Figure 6). In addition, the target genes miR-1273g-5p (ch1), hsa-miR-3151-5p (ch8), hsa-miR-3136- regulated by DEmiRNAs were listed, as shown in Table 2, 5p (ch3), hsa-miR-627-5p (ch15). As shown in Table 1,we which showed that 80 target genes were regulated by hsa- used the Benjamin-Hochberg (BH) method to adjusted false miR-3938, and 101 target genes were regulated by hsa-

5496 submit your manuscript | www.dovepress.com Cancer Management and Research 2020:12 DovePress Dovepress Wang et al

Figure 3 The box plot shows the enrichment of total miRNAs in each sample. Box plot was used for intuitive analysis and comparison of the average level and degree of variation of multiple sets of data, and was a convenient method for quickly observing the data distribution. The y-axis shows the total miRNA enrichment as the log2-ratio. In the resulting profile, all enrichment > 0 are shown.

Figure 4 The volcano plot and Scatter Plot. (A) The volcano plot represented the differentially expressed miRNAs with statistical significance; (B) Scatter plot illustrated the normalized circRNA expression in both groups. The x-axis represented the miRNA level in neutrophil group, the y-axis represented the miRNA level in N2 neutrophil group. In the volcano plot and Scatter Plot, the up-regulated miRNAs were represented as the red dots, while the down-regulated miRNAs were represented as the green dots.

Cancer Management and Research 2020:12 submit your manuscript | www.dovepress.com 5497 DovePress Wang et al Dovepress

AB

CD

Figure 5 Gene Ontology and KEGG Pathway enrichment for the host genes encoding DEmiRNAs. miR-4780. The results showed that 2 target genes, Discussion including TUSC1 and ZNF197, were regulated by hsa- CRC is the most common malignant tumor of digestive mir-3938 and hsa-mir-4780 simultaneously. tract in the world, which seriously endangers people’s health and quality of life. However, its pathogenesis has The Expression Levels of DEmiRNA, not been completely elucidated, and progression of CRC is TUSC1 and ZNF197 a process that involves multiple genetic changes, multi- Figure 7A and B showed the expression levels of DEmiRNA factor, multi-step process.17,18 Previous studies indicate in neutrophil group and neutrophil+TGF-β1 group. As that one-third of CRC patients may develop liver metas- shown in Figure 7, in the neutrophil+TGF-β1 group, the tases, and most of CRC-related death is usually attributed expression levels of hsa-miR-3938 were significantly to distant metastasis. More and more evidences showed decreased (P = 0.0130), while the hsa-miR-4780 expression that neutrophils played an important role in tumor tissue increased significantly (P = 0.0188). In addition, as shown in infiltration to promoting the growth, invasion, angiogen- Figure 7C, the expression level of DEmiRNA targets also esis and metastasis of various types of cancer.19,20 In showed significant difference in the two groups. Regarding addition, studies showed that exosomes as a novel the neutrophil+TGF-β1 group, the expression levels of mechanism of intercellular communication, could lead to TUSC1 were significantly decreased (P = 0.0024), while the exchange of genetic information and reprogramming fi the ZNF197 expression increased signi cantly (P = 0.0058). of recipient cells and transport various biomolecules, such as proteins, messenger RNAs (mRNAs), miRNAs, and The Correlation Between hsa-mir-3938 long non-coding RNAs (lncRNAs).21,22 Recent studies and hsa-mir-4780 and TUSC1 and showed that exosomes play multiple roles in promoting ZNF197 in CRC Cells tumor growth, metastasis and drug resistance. However, Figure 8 shows the effects of hsa-mir-3938 and hsa-mir the function of neutrophil-derived exosomes in develop- -4780 on expression of TUSC1 and ZNF197. As shown in ment, invasion and metastasis of CRC has not been well Figure 8, compared with the NC group, down-regulation characterized. In this study, we reported miRNA profiles in of hsa-mir-3938 expression and up-regulation of hsa-mir the neutrophils of CRC for the first time. Additionally, -4780 expression both resulted in decreased expression of a network between miRNAs in neutrophils and targets, TUSC1 gene (Figure 8A) and increased ZNF197 gene which may participate in tumor tissue infiltration in (Figure 8B) expression. In addition, CRC cells co- CRC, was constructed. transfected with low-expressing hsa-mir-3938 and high- In this study, a total of 33 differentially expressed expressing hsa-mir-4780 showed more decreased TUSC1 miRNAs were identified in TGF-β1-infected neutrophils. gene expression and more increased ZNF197 gene KEGG pathway and GO category analysis demonstrated expression. that the target miRNAs were associated with many

5498 submit your manuscript | www.dovepress.com Cancer Management and Research 2020:12 DovePress Dovepress Wang et al

TM4SF18 LAMC2 CCDC63 YAF2 SALL1 KCNIP4 ZMYM6 ZNF131 PPP1R15B HTR2C RNF150 C1ORF27 PIK3C2G FCGR2B BNIP2 CLEC3A AL353791.1 CNTNAP3B RBM4B WDR33 C17ORF74UBALD2 BAI1 GSTA1 SDR42E1 ELOVL4 FCER1A RASGRP3 CDC37 FNIP2 PDZD9 SP4 GRAP2 DENND1B SIGLEC10 BNIP3L AL953854.2 CDH23 DGCR6 HYAL4 GSTA3 SLC9C1 MID1 CHRNA6 ATP8B2 NPHP4 FADS2 C2ORF54TBC1D7 PLA2G4F POLR3H ZNF37A GSTA2 PTHLH SOX11 FFAR2 P4HB TARBP2 GAA KLHDC7A TSPYL4 AKIRIN1 NWD2 TBCA EVC2 C17ORF70 CD93 MRO FNBP4 SSTR1 UGT1A3 ZBTB2 GXYLT2 HSA-MIR-4684-3P RLN2 IMPG1 TPSG1 MTMR4 SULT1A1 MSTO1 CTB-186H2.3 SYTL2 USP22 NOL3 LRRC36 USP18 PLCB1 TMEM86A C17ORF96 IFFO1 SCAMP5 PPP1R11 PRR27 MSANTD1 FAM186B GINS3 PEX26 RBM39 EIF2A VCL HSPB8 C21ORF37 RRP36 ITGB3 HIBADH CABP2 PTAFR DDX49 SPANXN2 HNRNPDL RP11-347C12.3 EXTL2 ZNF133 DEF6 F9 TAS2R14 CEP72 NTN5 ATG10 GPR77 MTAP PCDH9 CYB561 NLRP1 NKX3-1 BCL2L12 TTC6 OLFML3 PLA2G4C VEZT CDK18 AC104942.1 LPXN C16ORF47 HTRA4 AHCYL2 FBXO28 CD22 S1PR5 RAD9A SBF2 MOXD1 CCDC23 BOK EGR2 NFYA PALD1 KLHDC3 SART3 CACNA1B ZNF250 AC099489.1 FOXP2 C1ORF106 WDR11 MYO18B DDR1 TRIOBP PACSIN3 SLC22A11RP11-345J4.3 C19ORF47 RHO STRC PPP1R16A NCKAP1L SYNPO2L GAS2 TMEM127 OTX1 CCDC183 SERPINA3 TTLL1 TBCD SLC22A3 ZNF462 RP11-12J10.3 FBXW7 RNF39 RP11-65D24.2 C17ORF62 GIT1 IL2RB SLC24A4 LAPTM5 S100A14 GIMAP1 PPP1R14C SKIV2L MARK4 SMIM11 HSA-MIR-5703 PIGZ DDIT4 AC107021.1 TSPEAR KNSTRN LRRC45 ZNF74 RANBP6 ERC1 SULT1A3 ABCD4 CDH7 TMED5 ADAMTS15 HIST2H2BE PRAMEF8 CPN2 MROH9 WNT8B GIMAP5 SLC25A12 ITFG3 USP6NL PIK3C2A PCYT1B DCDC1 GFPT2 PTCHD2 UGT1A9 FAM219A ST6GAL1 LALBA SLC4A5 RSPO4 STRA6 GBGT1 SRRD RASGRP2 PIEZO1 DHRS2 CASP1 DEGS1 ENGASE EGFLAM BCL2L2 APOC4-APOC2 GALNT13 TNNI1 CD300E AP1G1 TRIM25 PIGT NFATC1 EEPD1 TMCC2 RP11-51F16.8 GRHL3 EPHA1 LAMTOR1 TK2 APOPT1 NFE2 ADCK1 CCDC141 C7ORF55-LUC7L2 TMEM243 RNF157 TOP3B JPH1 ST6GALNAC5SPATA5L1 MRPS34 SH3BGR KCNAB1 NYNRIN BAIAP2 OTULIN C10ORF32TAF1B KCND1 DNAJC3 HMGCL TSPAN12 DDX56 CPEB2 CDC73 ZFYVE26 ITGA10 ARHGEF11 LGR6 TOMM22 NMRAL1 APOL3 PAPPA2 SPDYE2B COG4 FAT3 SLC6A4 LMBR1L HSA-MIR-6736-3P PDCD6 ALDH8A1 SULT1A4 CORO7-PAM16 DHDDS TCP11 TMEM237 GRAMD2 WDR6 ANGPTL5 DAZL SPCS3 IL17RD RHOBTB2 KANSL3 RASGRF1 PRKRA NRP1 CD6 KRT72 IL17RB SLC7A10 DMC1 C1ORF116 BLZF1 KAT2A DLD CACNA1G CHRNA1 CD46 QRICH2 PROKR2 FOXD2 C3ORF18 ZNF300 GCNT2 PLCB2 PTPN7 TLDC1 PTGER3 FLT3 MYH15 DUSP11 UGT1A10 AL033381.1 FGD2 CTB-50L17.14 UBAP2L PNISR SEPT11 PHF8 LRWD1 LONP1 IRX4 THUMPD3 MPP3 KLK3 AMOT RNF121 ACKR3 C15ORF32 MTX1 ABCA10 ZDHHC2 PWWP2A MSN CLEC4G SLC16A7 MTSS1L TBX4 PPP2R2C IFIT2 AC008394.1 EFNA1 MIB1 ZMYND12 FOXL2 GTF2F2 SLC27A3 LRRC46 SMPD1 CCSAP H2AFJ IL17A ZNF629 TMEM75 ANKRD54 TNNT1 CCDC120 SLC16A1 ZNF18 STRADA DCTN2 RPS19BP1RNF165 UBR5 SYT16 DGCR6L TMEM53 UGT1A5 ASAH2 MITD1 NPTX1 KDM4A PLCXD1 NPEPL1 CDC27 IRF4 SPDYE2 HMGXB3 ADCYAP1R1 WBP1L ISM1 LRP2 TUSC3 AIPL1 CLIP2 CCDC73 PHLDA3 IMP4 STAMBP KIAA1755 IPO7 MAP2K3 RELL1 FBXO48 NANP NOL6 HTRA2 RP5-966M1.6 ETV3 PDHX METTL1 AC006372.1 KIF18B GPD2 RNMT MRPL57 BCL10 SYTL5 BCL2L13 CYTH2 NUP85 C2ORF91 GPS1 CLTCL1 ZC2HC1A LCLAT1 SYMPK SPDYE1 UGT1A6 DECR1 HSA-MIR-3938 EIF2AK2 TOP2A AFMID FANCG BTBD19 STXBP2 TXNL1 MTMR7 SLC22A6 LPIN3 MFAP5 HBEGF GPATCH2L ARHGEF18 SS18 ST8SIA4 EHD1 MIEF2 RP11-181C3.1 SCG5 RP11-514P8.6 ARHGAP19 AOX1 THUMPD1 PRAMEF7 GPR155 GABRA4 B3GNT2 COL8A2 MTM1 MAB21L3 FAM216B MCMBP MEF2C IGFBP7 FAM78B FBXO38 RNF10 ACTL10 CUL9 ZNF793 ARCN1 NUDT15 RRP8 TSPAN31 EPSTI1 SH3BP1 ASRGL1 CTPS2 ACPP METTL16 C1D EVA1C ATP5A1 SETD6 SNX4 DYNC1I1 SPNS3 SOAT1 CTDSPL LAX1 FKBP5 TIFAB DERL3 AL137145.1 RHOT2 TROVE2 MRPS16 RBM4 SPANXN3 GADD45B TNF MTMR12 SLC35E4 FARP1 SLC17A4 MAPK11 AC010536.2 TMEM199CCDC24 CEP164 ICAM3 HTT CCDC169 IL36B AMDHD2GOLGB1 ZNF480 TXNDC12 OR13G1 C5ORF30HSA-MIR-1301-5P WBSCR17 TTC23L YBX1 TMEM206 ATP6V0A2 PYGO1 TTBK2 WDR72 GSDMB OSBPL7 GGA3 SNTN MNAT1 ZNF354C KDELC2 SLC16A14 BORA RASSF7 DDX19A LRRC74ZMYND10 DDX11 CAMSAP2 WIF1 WDR3 CPOX CDRT4 POU6F2 SYK HMGN3 STK32A OAS3 ERI1 TRIM8 UBE2Q1 AP4M1 FGF18 SUV39H2 NTRK2CDK5RAP2 IER5 RP11-362K2.2 DLG5 TXLNA UGT1A8 WDFY2 BRWD1 DDTL OR2L13 ITK HNF1A-AS1 GBP4 SLC38A4 ZNF514 TOX ABTB1 UBE3B HHEX CADPS BIRC3 ARIH2OS TRPC1 NUDT7 ARL13A ZNF319 NSMF TMEM135 DSG3 ITGAE ATE1 MTMR9 ZNF211 SRP68 RAD52 MLLT6 TMEM119RP11-324D17.1 CSGALNACT2C8ORF34C9ORF40 ZBTB34 C1ORF110 ABCA13 GCSAM POLE ZBTB49 VNN1 SUSD5 TAX1BP3MROH7-TTC4 HSA-MIR-513B-3P SERHL2 EHHADH BMP6 CNTN2 ZNF197 RABEP1 CDYL2 SENP2 COQ4 LBH PRMT9 CCDC112 ADAM9 SLC6A1 TMC3 NODAL P2RY8 LRPAP1 MTMR6 PIK3R1 BCL7A NCBP2 MAPK6 LRRC28 CD83 TUSC1 TRMT12 RNASEL ZMAT5 SHFM1 LMAN1 ANK3 NRG3 PTPRE PAPOLB GRAP TNFRSF11B EBAG9 ASB18 C6ORF120 TOMM5 ALG14 CBX3 PPIF MIER3 MTO1 CTXN2 CSMD1 CYP2D6 ZNF860 BTN2A2 TRPV1 ZCCHC4 PRR34 PIAS3 IQUB NR1I3 VGLL2 PCSK2 DHRSX TBC1D5 NDUFC2-KCTD14 SMIM3 SIGLEC15 RNF111 BBS7 UBBP4 SIGLEC1 MDM2 NCALD MED14 ALDH1L1 FAM86B1 RRAD PHKA1 TAF8 BIRC6 TRIM50 RBM46 CRHBP RSBN1 GRTP1 GPR156 SVIL GJB2 TMED4 HELZ TPST2 COPS6 GOLGA2 C11ORF80 FXR1 RECQL ARHGAP6 XRCC5 LRTM2 DDX3Y SLCO2A1 SIX5 AGTR2 STK4 KLF3 KCNJ14 METTL22 PPEF1 PTBP3 TOR1AIP1 VPS53 TDO2 FRMPD3 ENPP4 EPC1 SREK1 FAM102B KCNE2 CHRNA4 SIRPG MFSD11 MGLL CDHR5 KNDC1 LYPD6 DEPDC1B SH3PXD2B SF3B1 DCLK1 SLC29A1 STK36 OR5AU1 PSMD14 CTD-2583A14.9 LCE1C CTBS SPHKAP SRSF12 GEMIN2 ZNF770 SCML1 MMP13 SLC24A3 MMAB SFTPC BARD1 CCDC71L HSA-MIR-2114-3P EFTUD2 MMRN2 PTPRD S100A7A NDP PTGDR ENO3 RTN4RL1 AC140481.2C10ORF85 AMIGO1 CCDC163P CDC7 HSA-MIR-630 BAIAP2L1 SERGEF PLP1 PPP1R12B SPATC1 CD163 NNMT HSA-MIR-6089 TMTC3 GPR126 ZNF276 CA4 RPTOR ATG13 PGBD5 ELL2 ATG2B TUBB NLN KIAA1407 KHK INPP5B DLGAP5 TOM1L2 DHRS7B BAHCC1 IGSF23 FZR1 MRM1 ZNF655 HNMT PRIMA1 ANKRD33B COMMD10 CFHR3 MTG1 DPP10 POLD3 PARL KCNK10 ERCC1 SMPD3 PDZK1IP1 HSA-MIR-4780 KCNE1L CELF4 VRK2 FOXE1 ACRBP FAM104B ZNF678 LSM11 ATXN7 RP11-108K14.8 SPATS2L XPNPEP3 AC079341.1 CCHCR1 GABPB1 CIPC TMEM132C MRPS27 FANCL LRRCC1 N4BP3 RAB43 COL6A6 DLEU2L P2RX5-TAX1BP3 MPP1 CLNK PAQR4 ORAI2 SPRED3 RASGEF1A GAL3ST3 IL6ST TCP11X2 GJA5 APOBEC3C RELA COBLL1 STIM1 XKR3 BBIP1 ARHGAP31 TUBE1 MEGT1 CNNM2 ATXN2 DAO CCNB3 ZBTB8OS MVK SCYL3 DGKH PSMC3IP CTD-2583A14.10 PRDM7 NAPEPLD BTNL3 PKM ITGAV SLC25A29 CXCL12 TMEM167B CAMSAP1 LYSMD1 LDLRAD4 CHST15 FAM171B TOMM40L TMEM156 TRMT13 TSPAN3 NFATC3 GFOD1 COL21A1 C9ORF114 EPM2AIP1 C22ORF42 METTL8 ZSCAN29 CELF5 LCN10 KCNMB4 PID1 ZNF620 ERBB2IP GSDMA RP11-15E18.4 C14ORF180VPS26B FGF8 ASAP1 ZSCAN23 ZADH2 ROCK1 MAU2 SFRP1 LL0XNC01-19D8.1 SRPX2 GART RP11-296A16.1 OSBP CD28 COL24A1 SH2B1 EFCAB2 UBQLN1 MTHFD2L FAM135B ILDR2 NAGPA SHISA5 ANGEL2 HOXC8 SERINC4 ZNF75D C10ORF53 SNX22 CNNM1 ZNF552 FUT10 PDIA3 HEXIM1 LRRC75B COQ3 AP3M2 CHRNE ZNF646 TMEM246 KPNA6 ENOX2 RD3 PRSS16KIAA1429 MCEMP1 MTERF

Figure 6 The predicted downstream miRNA-targets network of the validated DEmiRNAs.

physiological processes. GO analysis implied that the These results suggested that the TUSC1 and ZNF197 DEmiRNAs were presumably involved in the biosyn- gene regulated by DEmiRNAs in N2 neutrophils might thetic and small molecules metabolic process of orga- be related to biosynthesis, metabolism and signal trans- nelles, nuclei and cytoplasm in cells. The KEGG duction in colorectal cancer cells. pathway analysis showed enrichment in the biological Tumor suppressor candidate 1 (TUSC1) gene is processes of cytochrome P450 metabolism xenobiotics, located within the region of chromosome 9p that harbors Glycosphingolipid biosynthesis-lacto and neolacto series, tumor suppressor genes critical in carcinogenesis. It is sphingolipid signaling pathway and CAMs. The results of an intronless gene which is downregulated in non-small- miRNA-targets networks showed that the hsa-mir-3938 cell lung cancer and small-cell lung cancer cell lines, and hsa-mir-4780 could regulate TUSC1 and ZNF197. suggesting that it may play a role in lung tumorigenesis

Cancer Management and Research 2020:12 submit your manuscript | www.dovepress.com 5499 DovePress Wang et al Dovepress

Table 2 DEmRNAs Target Genes

miRNA Target N

hsa-miR-3938 AMOT,ANGPTL5,ANKRD54,ARCN1,ASAH2,ATP6V0A2,BCL10,BORA,BRWD1,CCDC141,CCDC169,CCDC73, 80 CCSAP,CDC73,CDRT4,CPOX,CTPS2,DAZL,DDTL,DECR1,DLD,EIF2AK2,FAM216B,FARP1,FAT3,FBXO38,FGF18, GCNT2,GCSAM,GPR155,IL17RB,ISM1,KAT2A,KDELC2,KIAA1755,LAX1,LPIN3,LRP2,MIB1,MITD1,MRPS34,MTMR12, MTMR7,MYH15,NANP,NUDT15,PDHX,PNISR,PWWP2A,RELL1,RNF121,RP11-362K2.2,SLC16A1,SLC16A14,SLC16A7, SLC17A4,SLC35E4,SNX4,SS18,ST8SIA4,SYTL5,TLDC1,TMEM206,TMEM237,TMEM75,TOMM22,TROVE2,TSPAN31, TUSC1,TXNDC12,TXNL1,UBR5,WDFY2,WDR3,WIF1,ZMYND12,ZNF197,ZNF354C,ZNF629,ZNF793

hsa-miR-4780 AMIGO1,ANGEL2,AP3M2,ARHGAP31,ARHGAP6,ATG13,ATXN7,BAIAP2L1,BBIP1,BBS7,C6orf120,CD163,CFHR3, 101 CIPC,CLNK,COBLL1,COL21A1,COL24A1,COL6A6,COQ3,CRHBP,DCLK1,DDX3Y,DEPDC1B,DLEU2L,DLGAP5, ENOX2,EPM2AIP1,ERBB2IP,FUT10,GFOD1,GPR126,HELZ,HEXIM1,HNMT,IGSF23,IL6ST,ILDR2,ITGAV,KCNJ14,KLF3, LCN10,LL0XNC01-19D8.1,LSM11,MCEMP1,MED14,MEGT1,MFAP5,MFSD11,MMP13,MMRN2,MPP1,MTERF,MTHFD2L, MVK,NAPEPLD,NCALD,NR1I3,OR5AU1,PARL,PGBD5,PID1,PKM,POLD3,PRDM7,PRIMA1,PRR34,PSMD14,PTBP3, PTGDR,PTPRD,ROCK1,RP11-296A16.1,SCML1,SERINC4,SLC25A29,SMIM3,SREK1,SRPX2,SRSF12,SUV39H2,SVIL, TCP11X2,TMEM132C,TMEM156,TMEM246,TMTC3,TOMM40L,TRMT13,TUBB,TUSC1,UBQLN1,VRK2,XPNPEP3, XRCC5,ZADH2,ZBTB8OS,ZNF197,ZNF620,ZNF75D,ZSCAN23

Notes: The genes were regulated by hsa-mir-3938 and hsa-mir-4780 were listed in the table and were bold.

Table 3 List of Abbreviations gene in the CRC. In the current study, the results fi Abbreviations showed that TUSC1 protein decreased signi cantly in N2 type neutrophils compared with neutrophils. Another Acute promyelocytic leukemia APL All-trans retinoic acid ATRA result showed that in N2 type neutrophils, hsa-mir-3938 Biological process BP expression decreased significantly, while hsa-mir-4780 Cellular component CC expression increased significantly. In N2 neutrophils, Colorectal cancer CRC TUSC1 gene might inhibit the adhesion, invasion and Epithelial to mesenchymal transition EMT metastasis of colorectal cancer cells, which might be Gene Ontology GO Glyceraldehyde 3-phosphate dehydrogenase GAPDH related to the downregulation of hsa-mir-3938 expres- Kruppel-associated box KRAB sion and the upregulation of hsa-mir-4780 expression in Kyoto Encyclopedia of Genes and Genomes KEGG the tumor microenvironment. Long non-coding RNAs lncRNAs Zinc finger protein 197 (ZNF197) gene product Matrix metalloproteinase-9 MMP9 belongs to the zinc finger protein superfamily, members Messenger RNAs mRNAs MircRNAs miRNAs of which are regulatory proteins characterized by nucleic Molecular function MF acid-binding zinc finger domains. The encoded protein Real-time polymerase chain reaction RT-PCR contains 20 tandemly arrayed C2H2-type zinc fingers, Transforming growth factor-β1 TGF-β1 a Kruppel-associated box (KRAB) domain, and a SCAN Tumor-associated neutrophils TANs box. This gene is located in a cluster of zinc finger genes at Tumor infiltrating neutrophils TINs Tumor suppressor candidate 1 TUSC1 3p21. It is overexpressed in some thyroid papillary carci- 25 Zinc finger protein 197 ZNF197 nomas (provided by RefSeq, May 2017). However, there is no report about biological functions and regulatory mechanisms of the ZNF197 gene in the CRC. In our (provided by RefSeq, Jul 2008).23 Furthermore, study, the results showed that ZNF197 protein increased a previous study provided further evidence that TUSC1 significantly in N2 type neutrophils group. According to expression was downregulated in lung cancer cell lines the results of miRNA-targets networks, also suggesting and a trend towards higher expression of TUSC1 was that the ZNF197 gene regulated by hsa-mir-3938 and hsa- correlated with longer survival times for lung cancer mir-4780 in N2 neutrophils might be related to the tumor patients.24 However, there is no report about biological cell growth, invasion, metastasis and occurrence/develop- functions and regulatory mechanisms of the TUSC1 ment of CRC. The current research on this area is

5500 submit your manuscript | www.dovepress.com Cancer Management and Research 2020:12 DovePress Dovepress Wang et al

Figure 7 The expression levels of DEmiRNA, TUSC1 and ZNF197. (A) The expression levels of hsa-miR-3938 in neutrophil group and neutrophil+TGF-β1 group; (B) The expression levels of hsa-miR-4780 in neutrophil group and neutrophil+TGF-β1 group; (C) The expression levels of TUSC1 and ZNF197 in neutrophil group and neutrophil +TGF-β1 group.

Figure 8 (A) The effects of hsa-mir-3938 and hsa-mir-4780 on expression of TUSC1; (B) The effects of hsa-mir-3938 and hsa-mir-4780 on expression of ZNF197. ***P < 0.001. relatively less, there is much research work needs to be the theoretical basis for the mechanism of N2 type neu- done in future. trophils regulating the invasion and metastasis of CRC cells, and provided experimental evidence for future clin- Conclusion ical research and also provided the potential biomarker for Our study indicated that the regulation of TUSC1 and clinical treatment of CRC, but more studies should be ZNF197 by hsa-mir-3938 and hsa-mir-4780 established conducted to support this new hypothesis.

Cancer Management and Research 2020:12 submit your manuscript | www.dovepress.com 5501 DovePress Wang et al Dovepress

List of abbreviations 9. Pedrazzani C, Mantovani G, Fernandes E, et al. Assessment of neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio and pla- A list of abbreviations was provided in Table 3. telet count as predictors of long-term outcome after R0 resection for colorectal cancer. Sci Rep 2017;7:1494. 10. Li H, Zhao Y, Zheng F, et al. Prognostic significance of elevated Ethics Approval and Informed preoperative neutrophil-to-lymphocyte ratio for patients with color- Consent ectal cancer undergoing curative surgery: a meta-analysis. Medicine. 2019;98(3):e14126. doi:10.1097/MD.0000000000014126 This study received a documented review and approval 11. Tanio A, Saito H, Uejima C, et al. A prognostic index for colorectal from a formally constituted review board (The ethics cancer based on preoperative absolute lymphocyte, monocyte, and committee of the first affiliated hospital of Kunming neutrophil counts. Surg Today. 2019;49(3):245–253. doi:10.1007/ s00595-018-1728-6 Medical University). Written informed consent have been 12. Ma X, Becker Buscaglia LE, Barker JR, et al. miRNAs in provided by the patients. NF-kappaB signaling. J Mol Cell Biol. 2011;3(3):159–166. doi:10.1093/jmcb/mjr007 13. Lai H, Zhang J, Zuo H-Q, et al. Advances in research on the Acknowledgment correlation between miRNA and colorectal cancer metastasis. Chin – This work was supported by Yunnan Fundamental J New Clin Med. 2019;12:1168 1171. 14. Xu J, Meng Q, Li X, et al. Long noncoding RNA MIR17HG pro- Research Projects (NO. 2019FA039), National Natural motes colorectal cancer progression via miR-17-5p. Cancer Res. Science Foundation of China (No.31660312), and leading 2019;79:4882–4895. 15. Fu F, Jiang W, Zhou L, et al. Circulating exosomal miR-17-5p and medical talents in Yunnan (NO. L-2017001). miR-92a-3p predict pathologic stage and grade of colorectal cancer. Transl Oncol. 2018;11(2):221–232. doi:10.1016/j.tranon.2017.12.012 16. Diaz-Riascos ZV, Ginesta MM, Fabregat J, et al. Expression and role Author Contributions of miRNAs from the miR-200 family in the tumor formation and All authors contributed to data analysis, drafting or revising metastatic propensity of pancreatic cancer. Mol Ther Nucleic Acids. the article, gave final approval of the version to be published, 2019;17:491–503. doi:10.1016/j.omtn.2019.06.015 17. Caraglia M, Correale P, Giannicola R, et al. GOLFIG and agree to be accountable for all aspects of the work. chemo-immunotherapy in metastatic colorectal cancer patients. A critical review on a long-lasting follow-up. Front Oncol. 2019;9:1102. doi:10.3389/fonc.2019.01102 Disclosure 18. Arai H, Battaglin F, Wang J, et al. Molecular insight of regorafenib The authors report no conflicts of interest in this work. treatment for colorectal cancer. Cancer Treat Rev. 2019;81:101912. doi:10.1016/j.ctrv.2019.101912 19. Mizuno R, Kawada K. The role of tumor-associated neutrophils in References colorectal cancer. Int J Mol Sci. 2019;20:529. 20. Fridlender ZG, Albelda SM. Tumor-associated neutrophils: friend or 1. Swiderska M, Choromańska B, Dąbrowska E, et al. The diagnostics of foe? Carcinogenesis. 2012;33(5):949–955. doi:10.1093/carcin/bgs colorectal cancer. Contemp Oncol (Pozn). 2014;18(1):1–6. doi:10.51 123 14/wo.2013.39995 21. Zhang X, Shi H, Yuan X, et al. Tumor-derived exosomes induce N2 2. Hu J-M, Shen C-J, Chou Y-C, et al. Risk of colorectal cancer in polarization of neutrophils to promote gastric cancer cell migration. patients with periodontal disease severity: a nationwide, Mol Cancer. 2018;17(1):146. doi:10.1186/s12943-018-0898-6 population-based cohort study. Int J Colorectal Dis. 2018;33 22. Han Q, Zhao H, Jiang Y, et al. HCC-derived exosomes: critical player (3):349–352. doi:10.1007/s00384-018-2965-2 and target for cancer immune escape. Cells. 2019;8(6):558. doi:10.33 3. Haggar FA, Boushey RP. Colorectal cancer epidemiology: incidence, 90/cells8060558 mortality, survival, and risk factors. Clin Colon Rectal Surg. 2009;22 23. Shan Z, Parker T, Wiest JS, et al. Identifying novel homozygous (04):191–197. doi:10.1055/s-0029-1242458 deletions by microsatellite analysis and characterization of tumor 4. Shaul ME, Fridlender ZG. Neutrophils as active regulators of the suppressor candidate 1 gene, TUSC1, on chromosome 9p in human immune system in the tumor microenvironment. J Leukoc Biol. – 2017;102(2):343–349. doi:10.1189/jlb.5MR1216-508R lung cancer. Oncogene. 2004;23(39):6612 6620. doi:10.1038/sj. 5. Joyce JA, Pollard JW. Microenvironmental regulation of metastasis. onc.1207857 Nat Rev Cancer. 2009;9(4):239–252. doi:10.1038/nrc2618 24. Shan Z, Shakoori A, Bodaghi S, et al. TUSC1, a putative tumor 6. Masucci MT, Minopoli M, Carriero MV, et al. Tumor associated suppressor gene, reduces tumor cell growth in vitro and tumor growth neutrophils. Their role in tumorigenesis, metastasis, prognosis and in vivo. PLoS One. 2013;8(6):e66114. doi:10.1371/journal.pone.00 therapy. Front Oncol. 2019;9:1146. doi:10.3389/fonc.2019.01146 66114 7. Allavena P, Sica A, Garlanda C, et al. The Yin-Yang of 25. Li Z, Wang D, Na X, Schoen SR, Messing EM, Wu G. The VHL tumor-associated macrophages in neoplastic progression and immune protein recruits a novel KRAB-A domain protein to repress – surveillance. Immunol Rev. 2008;222(1):155–161. doi:10.1111/j.1600- HIF-1alpha transcriptional activity. EMBO J. 2003;22(8):1857 18 065X.2008.00607.x 67. doi:10.1093/emboj/cdg173 8. Stojkovic Lalosevic M, Pavlovic Markovic A, Stankovic S, et al. Combined diagnostic efficacy of Neutrophil-to-Lymphocyte Ratio (NLR), Platelet-to-Lymphocyte Ratio (PLR), and mean Platelet Volume (MPV) as biomarkers of systemic inflammation in the diag- nosis of colorectal cancer. Dis Markers. 2019;2019:6036979. doi:10.1155/2019/6036979

5502 submit your manuscript | www.dovepress.com Cancer Management and Research 2020:12 DovePress Dovepress Wang et al

Cancer Management and Research Dovepress Publish your work in this journal Cancer Management and Research is an international, peer-reviewed The manuscript management system is completely online and includes open access journal focusing on cancer research and the optimal use of a very quick and fair peer-review system, which is all easy to use. preventative and integrated treatment interventions to achieve improved Visit http://www.dovepress.com/testimonials.php to read real quotes outcomes, enhanced survival and quality of life for the cancer patient. from published authors.

Submit your manuscript here: https://www.dovepress.com/cancer-management-and-research-journal

Cancer Management and Research 2020:12 submit your manuscript | www.dovepress.com 5503 DovePress