脂肪酸结合蛋白4（FABP4）在结直肠癌中的高表达及其临床意义

High expression of FABP4 in colorectal cancer and its clinical significance 脂肪酸结合蛋白4（FABP4）在结直肠癌中的高表达及其临床意义 1 equal first-author 2 equal 2 corresp Min XIA, xmzb1013@163.com Min XIA, xmzb1013@163.com xmzb1013@163.com https://orcid.org/0000-0002-6394-9294 2 2 2 2 2 Department of Gastroenterology, the Second Affiliated Hospital of Nantong University, Nantong226001, China Department of Gastroenterology, the Second Affiliated Hospital of Nantong University, Nantong226001, China Department of Gastroenterology, Wuxi People’s Hospital Affiliated to Nanjing Medical University, Wuxi214023, China Department of Gastroenterology, Wuxi People’s Hospital Affiliated to Nanjing Medical University, Wuxi214023, China Objective 2 To investigate the relationship between the fatty acid-binding protein 4 (FABP4) and colorectal cancer (CRC). Methods 2 Using an enzyme-linked immunosorbent assay (ELISA), we measured the expression of FABP4 in plasma of 50 patients who underwent surgery for CRC from October 2017 to May 2018 and 50 healthy controls. The content of the visceral fat area (VFA) as seen with abdominal computed tomography (CT) scanning was measured by ImageJ software. The expression levels of FABP4, E-cadherin, and Snail proteins in CRC and adjacent tissues were determined by immunohistochemistry. Results 2 The mean concentration of plasma FABP4 of CRC patients was higher than that of the control group (22.46 vs. 9.82 ng/mL; P < 0.05). The concentration of plasma FABP4 was related to the tumor, node, metastatis (TNM) stage and lymph node metastasis and was independent of age, body mass index (BMI), tumor size and location, and the degree of differentiation of CRC. The concentration of plasma FABP4 was positively correlated with high VFA and lipoprotein-a (LPA) ( P < 0.05); but it was not correlated with total cholesterol (TG), total triglyceride (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or apolipoprotein AI (Apo-AI). The expression of FABP4 protein in CRC tissues was positively correlated with the degree of CRC differentiation, tumor stage, and lymph node metastasis. The level of FABP4 protein was negatively correlated with E-cadherin protein ( r =-0.3292, P =0.0196) and positively correlated with Snail protein ( r =0.5856, P < 0.0001). Conclusions 2 High LPA and VFA were risk factors for increased plasma FABP4 in CRC patients. FABP4 protein was highly expressed in CRC tissues and associated with TNM stage, differentiation, and lymph node metastasis of CRC. The level of FABP4 in CRC tissue was correlated with E-cadherin and Snail expression, suggesting that FABP4 may promote CRC progression related to epithelial-mesenchymal transition (EMT). 目的 2 探讨脂肪酸结合蛋白4（FABP4）与结直肠癌（CRC）的关系。 创新点 2 结直肠癌患者血浆FABP4浓度明显高于对照组（22.46ng/mL vs.9.82ng/mL， P < 0.05）。血浆FABP4浓度与肿瘤、淋巴结、转移灶（TNM）分期和淋巴结转移有关，与年龄、身体质量指数（BMI）、肿瘤大小、部位和结直肠癌分化程度无关。血浆FABP4浓度与高内脏脂肪区（VFA）和脂蛋白a（LPA）呈正相关（ P < 0.05），与总胆固醇（TG）、总甘油三酯（TC）、低密度脂蛋白（LDL）、高密度脂蛋白（HDL）以及载脂蛋白AI无相关性。FABP4蛋白在结直肠癌组织中的表达与大肠癌分化程度、肿瘤分期和淋巴结转移呈正相关。FABP4蛋白水平与E钙粘蛋白呈负相关（ r =?0.3292， P =0.0196），与SNAIL蛋白呈正相关（ r =0.5856， P < 0.0001）。 方法 2 采用酶联免疫吸附试验（ELISA）检测了2017年10月至2018年5月50例大肠癌手术患者和50例健康对照者血浆FABP4的表达。用ImageJ软件测量腹部CT扫描时VFA的含量。免疫组化法检测结直肠癌及癌旁组织中FABP4、E-钙粘蛋白（E-cadherin）和Snail蛋白的表达水平。 结论 2 高LPA和VFA是大肠癌患者血浆FABP4升高的危险因素。大肠癌组织中FABP4蛋白高表达与淋巴结转移密切相关。大肠癌组织中FABP4水平与E-cadherin和Snail表达相关，说明FABP4可能与上皮间充质转化（EMT）有关。 Fatty acid-binding protein 4 (FABP4) Colorectal cancer (CRC) Epithelial-mesenchymal transition 脂肪酸结合蛋白4（FABP4） 结直肠癌（CRC） 上皮间充质转化 1 Introduction 1 Colorectal cancer (CRC) is the second most common cancer in women and the third most common cancer in men. Every year, there are 746 000 new cases of CRC in males and 614 000 in females worldwide ( Furuhashi et al., 2014 Furuhashi et al., 2014 ), representing a serious health threat. Due to its concealed onset and unclear pathogenesis, in the early stage of CRC, there are often no obvious symptoms. Most symptoms occur in the middle and late stages, and thus the prognosis is poor. At present, serum carcinoembryonic antigen (CEA) is used for the screening and follow-up of CRC in clinic, but its sensitivity and specificity are low. Therefore, using CEA to diagnose CRC does not produce satisfactory outcomes ( Lee et al., 2012 Lee et al., 2012 ). Fatty acid-binding protein 4 (FABP4), a carrier protein for fatty acids, is widely expressed in adipocytes, macrophages, dendritic cells, and microvascular endothelial cells. It participates in lipid transport, metabolism, and intracellular signal transduction. It is believed that FABP4 levels may be an effective circulating biomarker for some metabolic diseases such as obesity, metabolic syndrome, and type 2 diabetes. Recently, more and more studies have shifted the focus to the connection between FABP4 and tumors. A study on non-small-cell lung cancer found that the expression of FABP4 messenger RNA (mRNA) and protein in lung cancer tissues was significantly increased, and FABP4 was also associated with tumor stage and 5-year survival rate ( Tang et al., 2016 Tang et al., 2016 ). However, FABP4 protein is lowly expressed in liver cancer and negatively correlated with tumor stage ( Zhong et al., 2018 Zhong et al., 2018 ). Recently, some researchers have studied FABP4 in CRC. Wang (2017) Wang (2017) found different plasma FABP4 levels between a colorectal adenoma group and normal control group, but no significant difference between a colorectal carcinoma group and normal control group. Nieman et al. (2011) Nieman et al. (2011) found that the overexpression of FABP4 in CRC tissues could promote tumor growth through activating the fatty acid oxidation pathway. Epithelial-mesenchymal transition (EMT) is an important mechanism for initiating the invasion and metastasis of malignant tumors ( Wan et al., 2020 Wan et al., 2020 ). Expressed in epithelial cells, E-cadherin can form tight junction barriers between cells and maintain epithelial cell polarity, stability, and integrity. When normal epithelial mucosal cells are damaged, the expression of E-cadherin decreases or even disappears. Snail protein is the earliest and most important transcription inhibitor found in the EMT pathway. Snail can down-regulate E-cadherin transcriptional activity, inhibit its expression, and promote distant metastasis of tumors by binding with E-box elements in the E-cadherin promoter region ( Zheng et al., 2015 Zheng et al., 2015 ). It can also weaken the tight junction of epithelial cells, making them lose polarity, enhancing their mobility, and then encouraging tumor cells to penetrate the mucosal barrier and to acquire distant invasive ability and metastasis. Jin et al. (2018) Jin et al. (2018) found that FABP4 could induce EMT through the protein kinase B (AKT)/glycogen synthase kinase-3β (GSK-3β)/Snail signaling pathway, and then promote metastasis. In this study, we investigated the expression of FABP4 in CRC patients and explored the possible effect of FABP4 on the occurrence of EMT in CRC. 2 Materials and methods 1 2.1 Collection and storage of plasma and tissue samples 2 The plasma and tissue specimens of 50 CRC patients who underwent surgery and 50 healthy volunteers were collected from Wuxi People's Hospital affiliated to Nanjing Medical University (Wuxi, China) between October 2017 and May 2018. All blood samples were collected in ethylenediaminetetraacetic acid (EDTA) anticoagulation vacuum blood collection tubes (Improve Medical, Guangzhou, CHA) and stored at 4 ℃. All samples were centrifuged within 2 h after collection (4 ℃, 1000 g , 15 min). Then the supernatant was placed in a dry Eppendorf (EP) tube and stored at -80 ℃. All specimens were fresh CRC and paracancerous tissues (normal mucosal tissues with a margin of more than 5 cm from the edge of the tumor). The specimens were placed in the prepared EP tubes treated with 0.1% diethyl pyrocarbonate (DEPC) water and stored in a liquid nitrogen tank in a low temperature refrigerator at -80 ℃. All patients’ surgical specimens were diagnosed by pathologists and no radiotherapy or chemotherapy had been conducted prior to the time of sampling. Exclusion criteria were: (1) inflammatory bowel disease or familial colon adenoma; (2) connective tissue diseases; (3) other malignant tumors; (4) acute phase of inflammatory diseases, especially sepsis. 2.2 Enzyme-linked immunosorbent assay (ELISA) 2 All reagents, working standards, and samples were placed at room temperature for at least 30 min. First, 100.0 μL of assay diluent was added to each well; then 50.0 μL of standard was added per standard well; next 2.5 μL of sample and 47.5 μL of calibrator diluent were added per sample well. The wells were covered with adhesive strips and incubated for 2 h at room temperature. Each well was filled with washing water buffer for washing, and the liquid was completely removed at each step. After the last wash, any remaining wash buffer was removed by aspirating or decanting. Human FABP4 (200 μL) was conjugated to each well and the wells were then covered with a new adhesive strip for 2 h at room temperature. Then, they were washed as before. Substrate solution (200 μL) was added to each well and the samples were incubated for 30 min at room temperature and kept from light. Stop solution was then added to each well. The optical density of each well was determined within 30 min using a microplate reader set to 450 nm. 2.3 Visceral fat measurement 2 ImageJ software (ImageJ64, SAAinc.lnk) was used to measure the subcutaneous fat area (SFA) and visceral fat area (VFA) in umbilical computed tomography (CT) images of patients. 2.4 Immunofluorescence staining 2 The staining step was performed according to the streptavidin perosidase (SP) kit instructions (CW2035, CoWin Biosciences (CWBIO)). The primary antibody was FABP4 (1:100), in addition to E-cadherin (1:40 000) and Snail (1:1000) antibodies (Cell Signaling Technology, USA). The slides were deparaffinized and rehydrated using routine methods, and then boiled in citrate buffer (pH=6.0) to retrieve antigenicity. The endogenous peroxidase blocking solution was incubated at room temperature for 10 min and then washed. After incubation with goat serum for 1 h at room temperature, the slides were incubated with primary antibody FGF9 (1:100) overnight at 4 ℃ and then with the secondary antibody at room temperature for 1 h. Then horseradish peroxidase (HRP)-labeled streptavidin was added and the slides were incubated for 10 min at room temperature. Diaminobenzidine (DAB) chromogenic reagent was used for staining. Two senior pathologists calculated the expression of FABP4, E-cadherin, and Snail using the proportion score and intensity score. The proportion score of positive cells ( B ) was set as follows: 0, < 5% positive cells; 1, 5%‒ < 25% positive cells; 2, 25%‒ < 50% positive cells; 3, 50% ‒ < 75% positive cells; and 4, ≥75% positive cells. The staining intensity score ( A ) was set as follows: 0, negative; 1, weak expression; 2, moderate expression; and 3, strong expression. The total score was calculated as A × B . A total score of 1 was regarded as no expression. A total score ranging between 1 and 4 was regarded as a low level of expression; a total score ranging between 5 and 8 was classified as a medium level of expression; a total score ranging between 9 and 12 was classified as a high level of expression. 2.5 Statistical analysis 2 All results were represented as the median±interquartile range for non-normally distributed data using SPSS software (IBM SPSS statistics, Version 21.0). The Mann-Whitney U test was used to determine the statistical significance of continuous data. Chi-square test, a contingency table, or the Fisher exact test was used to determine the statistical significance of categorical data. Multiple stepwise regression analysis was used to test the association between FABP4 and the other factors. Correlations were detected using the Spearman’s test. A P -value of < 0.05 was considered to be statistically significant in all analyses. 3 Results 1 3.1 Concentration of plasma FABP4 in CRC 2 We collected blood samples from 50 CRC patients and 50 healthy volunteers for the plasma FABP4 test. Plasma FABP4 concentration in the CRC group was significantly higher than that in the normal control group ((20.19±15.87) ng/mL vs. (8.69±5.26) ng/mL, P < 0.0001; Fig. 1 Fig. 1 ). 3.2 Correlation of plasma FABP4 levels and CRC 2 A total of 33 males and 17 females with CRC were recruited in our study for testing. The plasma FABP4 concentration in female CRC patients was higher than that in male CRC patients ((25.92±12.44) ng/mL vs. (14.11±21.34) ng/mL, P < 0.05). The plasma FABP4 concentration in the non-lymph node metastasis group was higher than that in the lymph node metastasis group ((25.53±16.27) ng/mL vs. (15.62±8.33) ng/mL, P < 0.05). The plasma FABP4 concentration in the tumor, node, metastatis (TNM) stage I+II group was higher than that in the III+IV group ((25.53±16.27) ng/mL vs. (15.62±8.33) ng/mL, P < 0.05). There was no significant correlation between plasma FABP4 concentration and age, tumor location or size, or CRC differentiation ( P > 0.05; Table 1 Table 1 ). P values for survival were determined with the Mann-Whitney U test. # Data were expressed as median±interquartile range. Variable n FABP4 (ng/mL) # P value Gender Male 33 14.11±21.34 0.0007 Female 17 25.92±12.44 Age ≤60 years 11 14.56±16.53 0.4697 >60 years 39 20.36±14.89 Differentiation Low 15 17.52±12.85 0.5629 Medium 28 30.93±20.58 High 7 17.89±15.26 Lymphatic metastasis Yes 25 15.62±8.33 0.0045 No 25 25.53±16.27 TNM stage I+II 25 25.53±16.27 0.0045 III+IV 25 15.62±8.33 3.3 Correllation of FABP4 plasma concentration with VFA and lipid metabolism in CRC patients 2 The concentration of plasma FABP4 in VFA of > 100 cm 2 was higher than that in VFA of ≤100 cm 2 ((26.71±15.91) ng/mL vs. (15.62±9.36) ng/mL, P < 0.05). The concentration of plasma FABP4 in lipoprotein-a (LPA) of > 300 mg/L was higher than that in LPA of ≤300 mg/L ((25.92±26.79) ng/mL vs. (20.39±15.50) ng/mL, P < 0.05). There was no significant difference in plasma FABP4 among different groups based on body mass index (BMI), total cholesterol (TG), total triglyceride (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or apolipoprotein AI (Apo-AI). However, monofactor analysis found that plasma FABP4 was associated with high LPA and high VFA ( Table 2 Table 2 ; Fig. 2 Fig. 2 ). We established a multivariate linear regression using FABP4 as a dependent variable, and TC, LPA, BMI, and VFA as independent variables. Stepwise analysis showed that the regression equation was y =0.138VFA+0.045LPA-7.297, suggesting that VFA and LPA were independent correlative factors of serum FABP4 ( Table 3 Table 3 ). P values for survival were determined with the Spearman’s test. Apo-AI: apolipoprotein AI; BMI: body mass index; CRC: colorectal cancer; HDL: high-density lipoprotein; LDL: low-density lipoprotein; LPA: lipoprotein-a; TG: triglyceride; TC: total cholesterol; VFA: visceral fat area. Variable r P value TG 0.157 0.277 TC 0.231 0.106 LDL 0.150 0.297 HDL 0.122 0.397 LPA 0.465 0.001 Apo-AI 0.130 0.368 BMI 0.270 0.058 VFA 0.544 <0.001 1,2; BMI: body mass index; CI: confidence interval; CRC: colorectal cancer; LPA: lipoprotein-a; VFA: visceral fat area; Sig: signficance. Parameter B 95% CI t Sig Constant -7.297 (-16.929, 2.334) -1.524 0.134 LPA 0.045 (0.024, 0.066) 4.369 <0.001 VFA 0.138 (0.084, 0.192) 5.161 <0.001 3.4 Positive correlation of FABP4 protein expression and CRC 2 FABP4 protein was mainly expressed in the cytoplasm and nucleus of CRC epithelial cells ( Fig. 3 Fig. 3 ). The expression rate of FABP4 protein was 82% (41/50) in CRC tissues and 18% (9/50) in normal adjacent tissues ( Table 4 Table 4 ). The expression of FABP4 protein in CRC was significantly higher than that in normal adjacent tissues ( <math id="M1"><msup><mrow><mi>χ</mi></mrow><mrow><mn mathvariant="normal">2</mn></mrow></msup></math> http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27608850&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27608848&type= 2.96333337 3.72533321 =40.960, P < 0.001). CRC: colorectal cancer. 2 Variable 2 n 2 FABP4 2 E-cadherin 2 Snail - + - + - + CRC 50 9 41 29 21 10 40 Normal 50 41 9 2 48 34 16 <math id="M7"><msup><mrow><mi>χ</mi></mrow><mrow><mn>2</mn></mrow></msup></math> http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27608847&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27608846&type= 2.62466669 3.13266659 2 40.960 2 34.081 2 23.377 P value 2 <0.001 2 <0.001 2 <0.001 The positive expression of FABP4 protein was higher in low-differentiated tissues than in high-differentiated tissues ( <math id="M2"><msup><mrow><mi>χ</mi></mrow><mrow><mn mathvariant="normal">2</mn></mrow></msup></math> http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27608880&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27608866&type= 2.96333337 3.72533321 =6.730, P =0.035). The positive expression of FABP4 protein in TNM stage III+IV was higher than that in stage I+II in CRC ( <math id="M3"><msup><mrow><mi>χ</mi></mrow><mrow><mn mathvariant="normal">2</mn></mrow></msup></math> http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27608883&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27608881&type= 2.96333337 3.72533321 =4.878, P =0.027). We also found that FABP4 expression with lymph node metastasis was higher than that without lymph node metastasis of CRC ( <math id="M4"><msup><mrow><mi>χ</mi></mrow><mrow><mn mathvariant="normal">2</mn></mrow></msup></math> http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27608874&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27608873&type= 2.96333337 3.72533321 =4.878, P =0.027). Thus, the expression of FABP4 protein was correlated with tumor differentiation, lymph node metastasis, and TNM stage, but not correlated with age, gender, tumor location, or tumor size ( P =0.644, 0.263, 0.591, and 0.287, respectively; Table 5 Table 5 ). CRC: colorectal cancer; TNM: tumor, node, metastatis. 2 Variable 2 n 5 FABP4 2 <math id="M8"><msup><mrow><mi>χ</mi></mrow><mrow><mn>2</mn></mrow></msup></math> http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27608876&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27608888&type= 2.87866688 3.72533321 2 P value - + ++ +++ Positive rate (%) Total 50 9 21 15 5 82.0 Age ≤60 years 11 3 2 4 2 72.7 0.214 0.644 >60 years 39 6 19 11 3 84.6 Gender Male 33 4 15 10 4 65.9 1.252 0.263 Female 17 5 6 5 1 70.6 Location Colon 29 4 12 9 4 86.2 0.288 0.591 Rectum 21 5 9 6 1 76.2 Size >3 cm 8 3 2 2 1 62.5 1.133 0.287 ≤3 cm 42 6 19 13 4 85.7 Differentiation Low 15 2 1 8 4 86.7 6.730 0.035 Medium 28 3 18 6 1 89.3 High 7 4 2 1 0 42.9 Lymph node metastasis Yes 25 1 11 11 2 96.0 4.878 0.027 No 25 8 10 4 3 68.0 TNM stage I+II 25 8 10 4 3 68.0 4.878 0.027 III+IV 25 1 11 11 2 96.0 3.5 Expression of E-cadherin and Snail protein in CRC 2 E-cadherin protein was mainly expressed on the cell membrane ( Fig. 4 Fig. 4 ). As shown in Table 4 Table 4 , the expression rate in the adjacent normal tissues was 96% (48/50), significantly higher than the rate in CRC tissues (42%, 21/50; <math id="M5"><msup><mrow><mi>χ</mi></mrow><mrow><mn mathvariant="normal">2</mn></mrow></msup></math> http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27608901&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27608897&type= 2.96333337 3.72533321 =34.081, P < 0.001). Snail protein was mainly expressed in the nucleus of cancer tissues ( Fig. 5 Fig. 5 ). As shown in Table 4 Table 4 , the expression rate of Snail in CRC was 80% (40/50), also significantly higher than that in the adjacent normal tissues (32%, 16/50; <math id="M6"><msup><mrow><mi>χ</mi></mrow><mrow><mn mathvariant="normal">2</mn></mrow></msup></math> http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27608916&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=27608904&type= 2.96333337 3.72533321 =23.377, P < 0.001). 3.6 Correlation of FABP4 with Snail and E-cadherin proteins in CRC 2 In the E-cadherin-positive staining group, seven samples showed negativity for FABP4. Two samples had both FABP4 and E-cadherin-negative staining. In the Snail-negative group, positivity for FABP4 was observed in four samples, and negativity for FABP4 was seen in six samples. Further Spearman's correlation analysis revealed that FABP4 had a positive correlation with Snail and a negative correlation with E-cadherin, with correlation coefficient ( r ) values of 0.5856 and -0.3292, respectively (Figs. 6 6 a and 6 6 b). E-cadherin had a negative correlation with Snail ( r =-0.3371, P =0.0167; Fig. 6 Fig. 6 c). 1,2,3; 4 Discussion 1 FABP4 is a lipid chaperone protein that is mainly expressed in adipocytes, macrophages, dendritic cells, and microvascular endothelial cells. It can specifically bind to free fatty acids and transport fatty acids, inhibit fat decomposition, and thereby affect lipid transport, cholesterol balance, and TC formation. Our study showed that the concentration of plasma FABP4 in CRC patients was significantly higher than that in healthy controls ( P < 0.05). This is consistent with the results of Zhang et al. (2019) Zhang et al. (2019) , who found that serum FABP4 concentrations in CRC patients were higher than those of the control group and decreased significantly two weeks after surgery. In our study, the expression of plasma FABP4 in TNM stage I+II was higher than that in stage III+IV; the concentration of plasma FABP4 in the non-lymph node metastasis group was higher than that in the lymph node metastasis group. We speculate that these results could be attributed to more participation of plasma FABP4 in lipid transport and metabolism in CRC stage III+IV, and more FABP4 being consumed and utilized by tumor tissues, resulting in low plasma FABP4 concentration in circulating blood. FABP4 is widely expressed throughout the body, but mainly in adipocytes ( Furuhashi et al., 2014 Furuhashi et al., 2014 ). Studies have confirmed that FABP4 positively regulates pre-adipocyte differentiation and stimulates its maturation ( Gan et al., 2015 Gan et al., 2015 ), which in turn affects lipid transport and the cholesterol level. Our study showed different plasma FABP4 concentrations between VFA and LPA groups. Spearman's correlation analysis revealed that plasma FABP4 in CRC patients was positively correlated with LPA and VFA ( P =0.001 and P < 0.001), but not correlated with TG, TC, LDL, HDL, or Apo-AI ( P > 0.05). A multivariate linear stepwise regression model showed that VFA and LPA are both significant influencing factors of plasma FABP4. The relationships of plasma FABP4 concentration with VFA and LPA in CRC are rarely reported and the mechanism remains unclear. The possible explanation could be as follows: since visceral fat mainly distributes in mesentery and omentum, it has more active metabolism. It secretes various proteins such as adipokines (adiponectin, endotrophin, leptin, and fatty acid-binding protein), inflammatory cytokines (interleukin-6 (IL-6), IL-8, tumor necrosis factor (TNF)), angiogenic factors, and extracellular matrix components, which promote the proliferation and spread of tumor cells through vascularization mediated by vascular endothelial growth factor ( Liesenfeld et al., 2015 Liesenfeld et al., 2015 ). A previous study showed that the accumulation of visceral fat is a strong determinant of insulin resistance and hyperinsulinemia ( Nam et al., 2010 Nam et al., 2010 ), which induces hyperglycemia, increases insulin-like growth factors, stimulates the proliferation of intestinal epithelial cells, and then promotes the occurrence and development of CRC. LPA is a special lipoprotein composed of a hydrophobic core rich in sterol lipids, TG, phospholipids, and cholesterol. LPA has been confirmed as an independent risk factor for coronary heart disease and proven to be associated with cardiovascular disease ( Kooijman et al., 2010 Kooijman et al., 2010 ). However, few studies have reported the association between LPA and CRC. LPA could reverse the pro-apoptotic behavior of 5-fluorouracil-induced apoptosis in colon cancer cell line HT29. This anti-apoptotic effect is mainly achieved by affecting the related proteins in the mitochondrial apoptosis pathway, inhibiting the expression of proapoptotic protein Bax and up-regulating the anti-apoptotic protein Bcl-2 ( Gao, 2013 Gao, 2013 ). It has been reported that FABP4 protein is highly expressed in cholangiocarcinoma ( Nie et al., 2017 Nie et al., 2017 ), breast cancer ( Guaita-Esteruelas et al., 2017 Guaita-Esteruelas et al., 2017 ), and ovarian cancer ( Nieman et al., 2011 Nieman et al., 2011 ), but lowly expressed in hepatocellular carcinoma ( Zhong et al., 2018 Zhong et al., 2018 ). In our study, the expression of FABP4 protein in CRC tissues was significantly higher than that in adjacent normal tissues. FABP4 protein overexpression in CRC tissues was significantly correlated with lymph node metastasis, tumor differentiation, and TNM stage ( P < 0.05), suggesting that FABP4 might promote CRC infiltration and metastasis. There were no significant differences in FABP4 protein expression based on participants’ age or gender, or on the size and location of tumors. However, Zhang et al. (2019) Zhang et al. (2019) found that tumors with high or low FABP4 expression have no significant correlation with distant organ and lymph node metastasis or TNM classification. FABP4 is a potential therapeutic target for metastatic prostate cancer ( Uehara et al., 2014 Uehara et al., 2014 ) and bladder cancer ( Mathis et al., 2018 Mathis et al., 2018 ). Tang et al. (2016) Tang et al. (2016) reported that high FABP3 or FABP4 protein expression as well as high concurrent FABP3 and FABP4 protein expression in non-small-cell lung cancer was all significantly associated with higher TMN stage. The five-year prognostic survival rate of early CRC is close to 90% ( Miller et al., 2019 Miller et al., 2019 ), while the five-year survival rate of late CRC is less than 10% ( Moriarity et al., 2016 Moriarity et al., 2016 ). Nearly 90% of CRC patients died of metastasis ( Li et al., 2017 Li et al., 2017 ). EMT is considered to be an important mechanism for initiating invasion and metastasis of malignant tumors ( Wan et al., 2020 Wan et al., 2020 ). Our study confirmed that the positive expression of E-cadherin protein in normal tissues was higher than that in CRC tissues. The positive expression of Snail protein in CRC tissues was higher than that in normal tissues adjacent to cancer. Our Spearman's correlation analysis showed that the expression of FABP4 protein was negatively correlated with E-cadherin protein ( r =-0.3292, P =0.0196) and positively correlated with Snail protein in CRC ( r =0.5856, P < 0.0001). E-cadherin protein was also negatively correlated with Snail protein level ( r =-0.3371, P =0.0167). The results suggest that FABP4 promotes malignant characteristics and metastasis of CRC highly likely to be related to EMT. Jin et al. (2018) Jin et al. (2018) found that the level of FABP4 protein was significantly increased in cervical carcinoma tissues and that FABP4 could activate Snail protein expression through the AKT/GSK-3β pathway in human cervical cancer cells. In summary, plasma FABP4 concentrations were higher in CRC patients than in the control group and positively correlated with VFA and lipid LPA. The expression of FABP4 protein in CRC tissues was increased and correlated with tumor differentiation, lymphatic metastasis, and TNM stage. 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Cancer Med , 7 ( 6 ): 2629 - 2640 . https://doi.org/10.1002/cam4.1511 https://doi.org/10.1002/cam4.1511 FABP4 suppresses proliferation and invasion of hepatocellular carcinoma cells and predicts a poor prognosis for hepatocellular carcinoma Min XIA, Yan ZHANG, and Wenjia ZHANG conceived and designed the experiments. Yan ZHANG wrote and edited the manuscript. Yan ZHANG, Zhujun XIE, Wenying TIAN, and Tianyue ZHU performed the experiments. Yan ZHANG, Wenjia ZHANG, and Zhujun XIE analyzed the data. Min XIA, Fangmei AN, and Qiang ZHAN revised the manuscript. All authors have read and approved the fnal manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the Science Foundation of Wuxi Health and Family Planning Commission (No. Z201713 to Min XIA), and the Special Project of Clinical Medicine of Nantong University (No. 2019JQ006 to Yan ZHANG), China. 10.1631/jzus.B2000366 1673-1581（2021）02-0136-10 ZHANG Yan,ZHANG Wenjia,XIA Min et al., 2021. High expression of FABP4 in colorectal cancer and its clinical significance. Journal of Zhejiang University-SCIENCE B(Biomedicine & Biotechnology), 22(2):136-145. ZHANG Yan,ZHANG Wenjia,XIA Min et al., 2021. High expression of FABP4 in colorectal cancer and its clinical significance. Journal of Zhejiang University-SCIENCE B(Biomedicine & Biotechnology), 22(2):136-145. 2020-07-14 2020-10-29 2021-01-18 2021-02-15 2022-05-09 © Zhejiang University Press 2021 Yan ZHANG, Wenjia ZHANG, Min XIA, Zhujun XIE, Fangmei AN, Qiang ZHAN, Wenying TIAN, and Tianyue ZHU declare that they have no conflict of interest. The present study was approved by the Ethics Committee of Wuxi People's Hospital Affiliated to Nanjing Medical University (Wuxi, China) and with the Helsinki Declaration of 1975, as revised in 2008 (5). All the patients and volunteers provided written informed consent for participation in this study. Additional informed consent was obtained from all patients for which identifying information is included in this article. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 2 22