LYAR Promotes Colorectal Cancer Progression by Upregulating FSCN1 Expression and Fatty Acid Metabolism

Colorectal cancer (CRC) is a highly malignant tumor associated with poor prognosis, yet the molecular mechanisms are not fully understood. In this study, we showed that LYAR, a nucleolar protein, is expressed at a higher level in CRC tissue than in adjacent normal tissue and that LYAR expression is closely associated with distant CRC metastasis. LYAR not only significantly promotes the migration and invasion of CRC cells in vitro, but knockdown (KD) of LYAR in CRC cells also inhibits xenograft tumor metastasis in vivo. Microarray analysis of LYAR KD cells combined with a chromatin immunoprecipitation (ChIP) assay, gene reporter assay, and rescue experiment indicated that FSCN1 (encoding fascin actin-bundling protein 1 (Fascin-1)) serves as a novel key regulator of LYAR-promoted migration and invasion of CRC cells. Knockdown of FSCN1 significantly inhibits subcutaneous tumorigenesis of CRC cells and leads to the downregulation of FASN and SCD, genes encoding key enzymes in fatty acid synthesis. In summary, this study reveals a novel mechanism by which LYAR promotes tumor cell migration and invasion by upregulating FSCN1 expression and affecting fatty acid metabolism in CRC.


Introduction
Colorectal cancer (CRC) is the third most frequently diagnosed cancer, with 1.85 million new cases in 2018, and is the second most common cause of cancer-related mortality worldwide [IARC World Cancer Report 2020] [1]. With advances such as prognostic techniques, neoadjuvant chemoradiotherapy, cytotoxic therapy, and surgical therapy, great progress in treat-ment has been made. The five-year survival rate for CRC, however, remains poor (~64%), and in metastatic cases, it is even worse (14%) [2,3]. Twenty-five percent of patients with CRC already have metastases at the time of diagnosis, and 50-60% of the remainder will develop metastases later in the course of the disease [4,5]. The formation of metastasis is a multistep process, in which tumor cells leave the primary tumor site, invade and penetrate the surrounding extracellular matrix and endothelium, enter the blood and lymph vessels, survive migration, and finally attach at a distant site, where tumor cells begin to proliferate, induce angiogenesis, evade apoptotic death, and form a new tumor [6,7]. These distant settlements of tumor cells, metastases, are the cause of 90% of human cancer deaths [6,[8][9][10]. Tumor recurrence and metastasis are the main causes of death in CRC. Approximately 30-50% of patients with CRC after curative surgery will relapse [11]. However, the molecular mechanism involved in the cascade of events during invasion and metastasis of CRC is still not fully understood.
LYAR was first identified in 1993 as a novel nucleolar oncoprotein, which consists of a zinc finger motif and three nuclear localization signals [12]. However, research was stagnant until a second LYAR article was published in 2009 [13]. Subsequently, there have been very few articles related to the function of LYAR [14][15][16][17][18], especially in the field of tumor research. In 2014, Ju et al. demonstrated that LYAR is a transcription factor with a DNA-binding motif (GGTTAT/G) that inhibits human fetal globin gene expression [19]. We further reported that LYAR promotes tumor cell migration and invasion by directly binding to the LYAR-binding motif (CTAACC; reverse complement GGTTAG) in the LGALS1 promoter to activate its expression in CRC cells [20].
LGALS1 encodes galectin-1, which is a member of the lectin family. Galectin-1 is a homodimer of 14 kD subunits and is characterized by its affinity for glycans containing β-galactosides. Galectin-1 has been detected in various malignancies such as colorectal, hepatocellular, prostate, ovarian, and breast cancers. Galectin-1 activation occurs via autocrine or paracrine sugar-dependent interactions with β-galactoside-containing glycoconjugates and participates in various key processes of carcinogenesis such as metastasis, angiogenesis, and immunosuppression [21].
The FSCN1 gene is located on chromosome 7p22 and encodes Fascin-1, a cytoskeleton protein with a relative molecular weight of 55kD [22]. Fascin-1 can promote the formation of filamentous pseudopodia, lamellar pseudopodia, and microspines of cell membrane after cross-linking with F-actin. This enhances the movement, metastasis, and invasion of tumor cells [23]. A mass spectrometry-based proteomic analysis predicted that FSCN1 may play an important role in metabolism, suggesting a new role of FSCN1 in tumors [24].
In the present study, we showed that LYAR, a key regulator of CRC, regulates a novel LYAR target, FSCN1, to promote the migration and invasion of CRC cells, which in turn positively regulates fatty acid metabolism. Our data demonstrated the presence of a LYAR/FSCN1/fatty acid metabolism axis which promoted the growth of CRC tumors in vivo. These findings suggest that LYAR could be used as a prognostic and therapeutic candidate target for the prevention and treatment of CRC.

Materials and Methods
2.1. Tissue Microarrays and Immunohistochemistry. CRC tissue arrays were purchased from Shanghai Outdo Biotech Co., Ltd., Shanghai, China. The immunohistochemistry exper-iment and analysis were carried out according to the procedure described in our previous literature [20]. The LYAR primary antibody (orb215217) and the anti-rabbit secondary antibody were purchased, respectively, from Biorbyt and Sigma-Aldrich. All researches involving human CRC tissues were approved by the Ethics Committee of Bengbu Medical College and were in accordance with the principles of the Declaration of Helsinki.

2.3.
Silencing of LYAR and FSCN1 by Transient Transfection siRNA. The siRNA sequences against human LYAR, FSCN1 and nonsilencing were designed and chemically synthesized by Genepharma (Shanghai, China). HCT15 cells were transfected with 100-200 pmol siRNA using 5-10 μL INTER-FERin® transfection reagent (Polyplus) in a 6-well plate. After 24-48 hours, cells were collected to perform cell proliferation, cell cycle, apoptosis, colony formation, adhesion, migration, and invasion assays. The siRNAs sequences were as follows: (1) Human LYAR-siRNA1: 5′-GGGAGGUGAAGAAG AAUAA-3′ (2) Human LYAR-siRNA2: 5 ′ -GCACUCGGAAGUUG AAACA-3 ′ FSCN1 siRNA and NC (negative control) were transfected into cells and cultured for 24-48 hours. Then, total RNA was isolated by the RNA pure Tissue and Cell Kit and the RNA was sequencing by the 150 bp paired method. Hisat2, samtools, and htseq-count packages were used to align 150 bp paired-end reads with hg19 (UCSC). The expression of genes and transcripts was quantified by using cufflinks tools. 2 Oxidative Medicine and Cellular Longevity We downloaded expression profiles of human colorectal cancer of TCGA and normal colon data of GTEx from UCSC Xena browser (https://xenabrowser.net/datapages/).

Plasmid Construction and Viral Infection.
To build stable knockdown cells, the small hairpin RNAs (LYAR-shRNAs, FSCN1-shRNAs, and Control-shRNA) were designed correspondingly according to the sense and antisense sequences of the two LYAR-specific siRNAs and FSCN1 siRNAs (see above) and a nonspecific siRNA and synthesized by Invitrogen (Shanghai, China). These chemically synthesized oligonucleotides were annealed to generate double-stranded oligonucleotides using the touchdown program in the PCR instrument and inserted into the Xho I/ Hpa I sites in the pLentiLox 3.7 vectors, which was subsequently confirmed by sequencing.
To overexpress FSCN1, the human FSCN1 coding sequence (CDS) was prepared by reverse transcriptionpolymerase chain reaction (RT-PCR) from normal colorectal tissues and cloned into the pLVX-IRES-mCherry Vector at Xba I and BamH I sites for overexpressing FSCN1 in LYAR-KD cells. Lentiviruses were packaged and produced in 293T cells. The supernatant of virus production was collected and filter-sterilized to infect the corresponding cells. The stably transfected cells were sorted and collected by flow cytometry using mCherry fluorescence.

Cell Migration and Invasion
Assays. Equal amounts of cells (1 × 10 5 in 200 μL medium without FBS) were seeded on a fibronectin-coated polycarbonate membrane insert in a Transwell apparatus (Corning Costar), and medium supplemented with 20% FBS was added to the lower chamber. After incubation for 48 h at 37°C, the insert was washed with phosphate-buffered saline, and cells on the upper surface of the insert were removed by wiping with a wet cotton swab. Cells migrating to the lower membrane surface were fixed by an equal volume mixture of methanol and acetone and stained with 0.4% crystal violet and counted under a microscope (Nikon). The invasion assay was executed as described in the migration assay using upper chamber precoated with 50 μL matrigel solution that original matrigel (BD Biosciences) was diluted with FBS-free medium according to the ratio of 1 : 10.

2.7.
Quantitative RT-PCR (RT-qPCR). Total RNA was isolated from cells with TRIzol® Reagent (Life Technologies). cDNA was synthesized using HiScript® II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme). The RT-qPCR primers were designed by Primer Premier 6.0 software. RT-qPCR was carried out in a Rotorgene 6000 (Corbett Research) using the Fas-tStart Universal SYBR Green Master Mix (Roche) in a final volume of 20 μL. The relative quantification was executed for the following genes using GAPDH as an internal reference in CRC cells. Each reaction was performed in triplicate and repeated at least two times.

Chromatin Immunoprecipitation (ChIP).
ChIP assays were performed following standard ChIP procedures [26]. Chromatin fractions from HCT15 cells were immunoprecipitated with LYAR primary antibody (a kind gift from the State Key Laboratory of Pharmaceutical Biotechnology,

Oxidative Medicine and Cellular Longevity
School of Life Sciences, Nanjing University, Nanjing, China). Normal rabbit immunoglobulin G (IgG, Beyotime) served as the controls. The ChIP samples were analyzed by quantitative real-time PCR using the FastStart Universal SYBR Green Master Mix (Roche) and specific primers (Table S2) spanning the FSCN1 promoter. A standard curve was prepared for each set of primers using serial titrations of the input DNA. The percentage of ChIP DNA was calculated relative to the input DNA from primer-specific standard curves using the Rotor-Gene 6000 Series Software 1.7. Each experiment was performed at least two independent times.
2.11. Rescue Experiments. The supernatant of virus production of pLVX-IRES-mCherry (empty vector) and pLVX-IRES-mCherry-FSCN1-OE (exogenous expression of FSCN1) were used, respectively, to infect the stable LYAR-KD and LYAR-Control (only infected by empty vector) cells. Those stably infected cells were sorted and collected using mCherry fluorescence by flow cytometry, followed by cell migration and invasion assays (see above).    For the tumor xenograft assay, 8 six-week-old female nude mice were processed by subcutaneous implantation of 1 × 10 6 HCT15 cells expressing either control shRNA or FSCN1-shRNA. Mice were maintained for 30 days, and tumor volumes were measured at indicated time points. At the end of experiments, mice were euthanized and xenograft tumors were dissected for further analyses.
2.13. Statistical Analysis. All statistical analyses were performed using SPSS 22.0 software (SPSS Inc. Chicago, IL, USA). The experimental results were statistically evaluated using Student's t -test for comparisons between two groups or ANOVA for comparisons between more than two groups. Kaplan-Meier survival curves were generated to determine the relationship between LYAR levels and the overall survival of CRC patients, and the differences between the curves were calculated using the log-rank test. Multiple Cox proportional hazards regression was carried out to identify the independent factors with a significant impact on patient survival. p values < 0.05 were considered statistically significant.

LYAR Is Highly Expressed in Human CRC Tissue and
Promotes CRC Metastasis. Based on data from the Oncomine database (https://www.oncomine.org/) and a previous study [20], we found that LYAR was highly expressed and promoted cell mobility in CRC cells. However, the relationship between the high expression level of LYAR and the survival rate of CRC patients is unclear. To answer this question, we performed immunohistochemistry (IHC) analysis of LYAR on tissue arrays of 166 paraffin-embedded adjacent sections of normal colorectal tissues and CRC tissues. Tissues that displayed moderate or strong immunostaining were classified as having high LYAR expression and those that displayed negative or weak immunostaining as having low LYAR expression. There was strong LYAR staining in only 4.8% (8/166) of the adjacent normal epithelial tissues, whereas~47.0% (78/166) of total CRC tissues had high expression levels of LYAR (Figure 1(a), Table 1,  and Table S1). Furthermore, high LYAR expression was significantly correlated with poor prognosis (Figure 1(b)), which was consistent with the survival curve analysis of CRC data from The Cancer Genome Atlas (TCGA) database ( Figure S1). Notably, LYAR expression was positively correlated with a higher metastasis status (Figure 1(c) and Table S1). Taken together, these results indicate that LYAR is highly expressed in CRC tissues, particularly metastatic tissues. This suggests that LYAR may be involved in metastasis of CRC cells, with the potential to be a novel prognostic biomarker for CRC.
LYAR has zinc finger DNA-binding motifs and binds to DNA at the GGTTAT/G consensus sequence [12,19]. To perform a systematic analysis and to take our previous research a step forward, we verified that LYAR promoted migration and invasion of HCT15 cells in vitro (Figures 2(a)-2(c) and Figure S2-4). More importantly, we found that LYAR enhanced the lung and liver metastasis of CRC cells through a mouse tail vein assay in vivo. (Figures 2(d)-2(f)).

FSCN1 Is a Novel Target of LYAR.
Heterogeneity is a distinctive feature of solid tumors. Therefore, we speculated that there is more than one molecular mechanism by which LYAR promotes CRC migration and invasion, in addition to upregulating LGALS1. In order to comprehensively identify regulatory roles of LYAR in metastatic CRC, a whole-genome microarray analysis of gene expression was performed in LYAR knockdown (KD) and control HCT15 cells (Figure 3(a)). Gene Set Enrichment Analysis (GSEA) showed that differentially expressed genes (DEGs) in the LYAR-KD line were significantly enriched in cholesterol homeostasis, a lipid metabolism-related pathway (Figure 3(b)). We then screened for genes associated with metastasis and metabolism among the DEGs. The upstream promoter region (2000 bp upstream of the transcription start site) of each candidate gene was searched for the GGTTAT/G motif, which is the consensus DNA-binding site of LYAR. Candidate genes with this motif were selected for further analysis in HCT15 cells. Quantitative RT-PCR (RT-qPCR) revealed that the 7 Oxidative Medicine and Cellular Longevity expression of FSCN1, one of the candidate genes, was consistently lower in LYAR-KD cells compared to the control (Figure 3(c)). Downstream involvement of FSCN1 in LYAR-regulated CRC would be a novel finding; we therefore conducted further experiments to verify this relationship between LYAR and FSCN1. We detected FSCN1 expression after LYAR knockdown by siRNA in all three cell lines and found that FSCN1 was only downregulated in HCT15 (Figure 3(d) and Figure S5). We therefore performed the subsequent experiments only in HCT15 cells. Together, these results indicated that FSCN1 is a potential target of LYAR in CRC cells.   Table S2). Chromatin immunoprecipitation (ChIP) analysis spanning the promoter of the FSCN1 gene was performed to determine if LYAR binds to the FSCN1 promoter and regulates its expression. LYAR indeed bound to the FSCN1 promoter region between -337 and -183 bp in HCT15 cells (Figure 4(b)). When LYAR was knocked down in HCT15 cells with shRNAs, LYAR enrichment on the FSCN1 promoter was significantly reduced (Figure 4(c)). Furthermore, a luciferase reporter assay showed that the extent of LYAR binding to the FSCN1promoter corresponded to changes in LYAR expression in HCT15 cells transfected with a wild-type FSCN1 promoter-driven luciferase construct (Figures 4(d) and 4(e)). Mutating the LYAR binding motif from GGTT AG to GACTAG abolished corresponding FSCN1 promoter activity changes (Figures 4(d) and 4(e)). In summary, these results demonstrated that LYAR directly regulates FSCN1 gene expression and that the LYAR-binding motif (GGTT AG) in the promoter of FSCN1 plays a critical role in LYAR-mediated transcriptional activation of FSCN1.

Ectopic Expression of FSCN1 Restores Migration and
Invasion Potential of LYAR-KD Cells. FSCN1 has been reported to increase CRC migration and invasion in cell cultures and cause cell dissemination and metastasis in vivo [27]. In this study, we found that knockdown of LYAR by shRNAs in HCT15 cells led to downregulation of FSCN1 (Figures 3(c) and 3(d)) and that the cell migration and invasion potential were correspondingly decreased (Figures 2(c) and S4). To investigate the role of FSCN1 in LYAR-promoted CRC cell migration and invasion, rescue experiments were performed in LYAR-KD cells in which FSCN1 was overexpressed by a stably transfected FSCN1coding sequence. Overexpression of FSCN1 and knockdown of LYAR were confirmed by Western blot in HCT15 cells ( Figure 5(a)). Compared with the control LYAR-KD cells (LYAR-KD-Vector), the migration and invasion potential of the HCT15 cells with FSCN1 overexpression was significantly increased (Figures 5(b) and 5(c)), indicating that exogenous expression of FSCN1 partially restored cell migration and invasion potential of LYAR-KD cells. To test this effect in vivo, stable FSCN-KD or control HCT15 cells were subcutaneously adoptively transferred to nude mice. Xenografts in the FSCN1-KD group were significantly inhibited compared to those in the control group ( Figures 5(d)-5(f)). In summary, these results indicated that FSCN1 plays a key role in mediating LYAR-promoted CRC cell migration and invasion.

Reduction of FSCN1 Expression in CRC Cells Inhibits the Expression of Some Key Enzymes in Fatty Acid Metabolism.
To clarify the mechanism of FSCN1 in promoting tumor invasion and metastasis, RNA-Seq was used to search for potential FSCN1 targets and signaling pathways. In the FSCN1-KD line, metabolic pathways, including lipid metabolism, were enriched ( Figure 6(a)). Because our previous analysis showed that LYAR knockdown caused a reduction in cholesterol homeostasis, which is also a lipid metabolism pathway (Figure 3(b)), we further investigated the influence of FSCN1 inhibition on key enzymes in lipid metabolism. Among the lipid metabolism-related genes, the expression of fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) were consistently decreased in FSCN1-KD HCT15 cells compared to the control at both the mRNA (Figure 6(b)) and protein (Figure 6(c)) levels. It was previously reported that de novo synthesis of palmitic acid from acetyl-CoA is mainly catalyzed by FASN; moreover, saturated fatty acids (SFAs) are converted into monounsaturated fatty acids (MUFAs) by SCD, and their chains are elongated by elongases [28]. These data suggest that FSCN1 positively regulates fatty acid metabolism.
In xenograft samples, FASN and SCD were also downregulated in the FSCN1-KD line (Figure 6(d)). Intriguingly, the analysis of TCGA CRC data revealed that the expression levels of LYAR, FSCN1, FASN, and SCD in CRC tissues were all higher than those in normal tissues ( Figure S6). This suggests that LYAR may promote CRC progression by upregulating FSCN1 expression and subsequent fatty acid metabolism, which is logically consistent with the results of our in vitro experiments (Figures 6(b)-6(d)). In conclusion, our data demonstrated the presence of a LYAR/FSCN1/fatty acid metabolism axis which promoted the growth of CRC tumors in vivo.

Discussion
The formation of metastasis is a complex, multistep process, and the molecular mechanisms of CRC metastasis are still not fully understood. In recent years, some studies have found that LYAR plays an important role in different biological processes, such as RNA synthesis and the development of erythroid cells [14-18, 29, 30]. LYAR may be involved in the MYCN signaling pathway in the development of medulloblastoma [31] and induces neuroblastoma cell proliferation and survival [32], and LYAR together with other five genes (PDIA3, NOP14, NCALD, MTSS1, and CYP1B1) can be used as potential prognostic biomarkers for curative and postoperative supportive therapies for ovarian cancer [33]. However, aside from these, there have been no other reports on the function of LYAR in cancer. We previously found that LYAR was expressed at a higher level in metastatic CRC tissues and that it promotes tumor cell migration and invasion by upregulating LGALS1 expression in CRC cell lines [20]. However, that study lacked in vivo experiments; the potential of LYAR to promote metastasis could not be verified, and retrospective analysis could not be conducted on the survival of clinical CRC patients to hypothesize whether LYAR was an independent prognostic factor. In addition, the heterogeneity of solid tumors makes it unlikely that upregulating the expression of LGALS1 is the only molecular mechanism by which LYAR promotes CRC migration and invasion among all colorectal cancers with high LYAR expression.
In this study, we used large-scale immunohistochemical tissue microarrays and found that LYAR is highly expressed in CRC tissues. This high expression is positively correlated with the metastatic rate of CRC and is significantly correlated with poor CRC prognosis. Moreover, we performed a genome-wide expression profile analysis to further study heterogeneity in molecular mechanisms through which LYAR participates in the progression of colorectal cancer, demonstrating that LYAR plays a role in the promotion of CRC invasion and metastasis by upregulating FSCN1 expression. However, we detected the expression of FSCN1 after LYAR siRNA knockdown in three different cell lines and found that FSCN1 was only downregulated in HCT15. This suggested that LYAR regulates FSCN1 expression in only a subset of colorectal cancers. This phenomenon may be due to differences in race, gender, and age of the patients from whose tissue different colorectal cancer cell lines are derived; these can lead to differences in molecular features between cell lines. This result again illustrates that solid tumors exhibit significant heterogeneity.
FSCN1 has recently been shown to promote cancer cell migration and invasion through its role in formation of cellular protrusions such as filopodia and invadopodia [34]. Forced expression of FSCN1 in CRC cells increased their migration and invasion in vitro and caused cell dissemination and metastasis in vivo [27]. Another recent study showed that imipramine, a novel FSCN1 inhibitor, has been confirmed to have significant antitumor effects both in vivo and in vitro, which lays the foundation for molecular targeted therapy of serrated adenocarcinoma (SAC) and other FSCN1-overexpressing tumors [35]. In this study, we revealed that LYAR positively regulates FSCN1 expression. We also found that LYAR knockdown in CRC cells led to the reduction of FSCN1 levels and inhibited metastasis to the lungs and liver in NOD/SCID mice. This is consistent with the report that forced expression of FSCN1 in CRC cells caused cell dissemination and metastasis in vivo.
There is previous evidence in the literature of FSCN1 involvement in tumor metabolism. In lung cancer, FSCN1 promotes cancer growth and metastasis by enhancing gly-colysis and PFKFB3 expression through YAP1 activation [36]. In the context of PIK3CA mutation or amplification, high expression of FSCN1 is associated with poor prognosis and radiotherapy response in cancer patients; mutant PIK3CA (E542K and E545K) can enhance glucose metabolism and cell proliferation in cancer cells [37,38]. In the present study, separate from glucose metabolism, we demonstrated that FSCN1 was related to lipid metabolism in CRC. FSCN1 knockdown reduced the expression of FASN and SCD, which are key genes in de novo fatty acid synthesis, suggesting that FSCN1 promoted CRC by affecting lipid metabolism.
This regulatory action of FSCN1 may be because CRC is closely related to obesity and lipid metabolism [39,40]. Fatty acids are indispensable for the biosynthesis of most lipids, such as membrane lipids and lipid signaling molecules, in addition to acting as substrates for energy production [41]. De novo synthesis of palmitic acid from acetyl-CoA (acetyl-coenzyme A) is mainly catalyzed by fatty acid synthase (FASN). SFAs are converted into MUFAs by stearoyl-CoA desaturase 1 (SCD), and their chains are elongated by elongases [28]. A high-fat diet increases palmitic acid levels, which in turn increase β2AR expressions in a Sp-1 dependent manner. Subsequently, the cAMP/PKA axis is activated and hormone sensitive lipase (HSL) is phosphorylated at S552 to increase energy production, which promotes CRC growth [42]. In addition, there may be another FSCN1-lipid metabolism axis independent of LYAR, which would require future studies to confirm.
In summary, we demonstrated that FSCN1 is a direct target of LYAR. A novel pathway for function of the transcription factor LYAR in CRC has also been revealed: LYAR promotes tumor migration and invasion by upregulating FSCN1 expression, which in turn positively regulates fatty acid metabolism. These findings suggest that LYAR could be used as a prognostic and therapeutic candidate target for the prevention and treatment of CRC.

Data Availability
The data generated or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval
All research involving human CRC tissues was approved by the Ethics Committee of Bengbu Medical College and was in accordance with the principles of the Declaration of Helsinki. All animal studies were approved by the ethical regulations of Institutional Animal Care and Use Committee (IACUC) of Suzhou University. Figure S1: Overall survival curve of those with colorectal cancer. Data were obtained from TCGA database and analyzed to generate Kaplan-Meier curves, which show a correlation between patients with tumors expressing low levels of LYAR and higher survival rates. Figure S2: LYAR expression in stable LYAR knockdown HCT15 cells. (a) RT-qPCR analysis of LYAR gene expression normalized to GAPDH gene expression in stable LYAR knockdown HCT15 cells. * * p < 0:01 compared with the scrambled control. (b) Western blot assay showing LYAR protein expression in stable LYAR knockdown HCT15 cells. GAPDH served as the loading control. Figure S3: Detection of tumor cell phenotype in transient LYAR knockdown HCT15 cells. LYAR knockdown had no effect on cell proliferation (a), cell adhesion (b), cell cycle (c), apoptosis (d), or colony formation (e) of HCT15 cells. Parental refers to the control, nonsilencing refers to cells transfected with a nonspecific siRNA, and LYAR-siRNA1 and LYAR-siRNA2 refer to cells transfected with LYAR-specific siRNA1 and LYAR-specific siRNA2, respectively. Figure S4: Detection of migration and invasion in stable LYAR knockdown HCT15 cells. (a) Representative photos of haptotactic migration assay and matrigel chemoinvasion assay using stable LYAR knockdown HCT15 cells. Original magnification, 200x. (b) Results of migration and invasion assays. Data shown are the mean ± standard deviation (n = 3). * * p < 0:01 compared to the scrambled control. Figure S5: Western blot assay showing FSCN1 expression after LYAR knockdown by siRNA. (a) LYAR and FSCN1 expression after LYAR knockdown in HCT8 cells. (b) LYAR and FSCN1 expression after LYAR knockdown in HCT116 cells. KD1 and KD2 refer to the stable LYAR knockdown lines LYAR-siRNA1 and LYAR-siRNA2, respectively. Figure S6: Expression of LYAR and downstream genes in colorectal cancer. The expression profiles of human colorectal cancer and normal colon tissue were obtained from TCGA and GTEx databases, respectively. The data show differential expression of LYAR (a), FSCN1 (b), FASN (c), and SCD (d) in colorectal cancer tissues compared to adjacent normal tissues. * * * p < 0:001 and * * * * p < 0:0001.