hepatocellular carcinoma ( hcc ) is the third most common cause of cancer - related mortality and the fifth most common cancer worldwide 1 , 2 . in taiwan chitinase 3-like 1 ( chi3l1 ) , also known as ykl-40 and human cartilage glycoprotein 39 , is a secreted 40-kd mammalian glycoprotein encoded by the chitinase 3-like 1 gene located on human chromosome 1q32.1 4 . chi3l1 is a nonspecific inflammatory biomarker of disease and is secreted by activated macrophages and neutrophils in various tissues exhibiting inflammation , arthritic chondrocytes , cancer cells and vascular smooth muscle cells 5 - 11 . because chi3l1 is expressed and secreted by activated neutrophils and macrophages , previous studies have indicated that chi3l1 may play an essential role in the inflammatory processes of tumors , such as stimulating angiogenesis and the remodeling of the extracellular matrix 7 , 12 - 14 . high expression of chi3l1 has been shown to be closely connected with the recurrence and metastasis of various human tumors such as breast cancer , nonsmall cell lung cancer , glioblastoma , and gastric cancer 15 - 20 . in addition , pretreatment levels of chi3l1 were reported to be elevated in cervical cancer 21 . recently , chi3l1 was shown to correlate positively with p - akt cell signaling but negatively with e - cadherin expression in hcc 22 , and serum chi3l1 was shown to be an independent prognostic factor for overall and recurrence - free survival in hcc patients receiving curative resection , indicating that elevated serum chi3l1 levels predict poor prognosis in hcc after surgery 23 . single - nucleotide polymorphisms ( snps ) are the most common type of dna sequence variation , and may affect the expression of specific genes 24 , 25 . snps in the chi3l1 promoter region have been associated with elevated serum chi3l1 levels and a higher risk of schizophrenia 26 , 27 , differential gene expression 27 , and elevated transcript levels 28 . elevated circulating chi3l1 levels might be a biomarker for asthma and declining lung function 29 . moreover , elevated serum chi3l1 levels and chi3l1 overexpression are found in and linked to liver injury , advanced liver fibrosis , and poor prognosis in hcc 22 , 23 , 30 . zhu et al . also found that serum chi3l1 level was an independent prognostic biomarker in hcc patients after transcatheter arterial chemoembolization 31 . however , the connection between chi3l1 snp expression and hcc regulation is not well established . determining the mechanism of chi3l1 regulation and expression in hcc requires information on the genetic variant of chi3l1 snps involved in hepatocarcinogenesis . we performed a case - control study involving four chi3l1 snps located in the promoter region and exon 5 to analyze the contribution of these polymorphisms of chi3l1 to susceptibility to hcc and its pathological development . in this study , we recruited 343 patients with hcc between 2012 and 2016 at the chung shan medical university hospital , taiwan . demographic characteristics and medical information of the patients , including tnm staging , tumor size , lymph - node metastasis , vascular invasion , distant metastasis , presence of hbv surface antigen ( hbsag ) and liver cirrhosis , were obtained from their medical records . the blood samples which obtained from the controls and hcc patients were stored in edta tubes , centrifuged immediately and stored at -80c . the institutional review board of chung shan medical university hospital approved this study ( csmuh no : cs15099 ) , and informed written consent was obtained from each participant . three snps rs6691378 ( -1371 , c / t ) , rs10399805 ( -247 , c / t ) , and rs4950928 ( -131 , g / c ) in the promoter region and snp rs880633 ( + 2950 , t / c ) in exon 5 were selected based on the chinese hapmap ( han chinese in beijing , china ) data . the snps rs6691378 , rs10399805 and rs4950928 in the promoter region of the chi3l1 gene exhibit strong association of schizophrenia and the snp rs4950928 gc transversion impairs the myc / max - regulated transcriptional activity 27 . snp rs10399805 has been reported to disrupt the c / ebp - aml-1 binding site in the gene promoter and is predicted to increase chi3l1 expression 32 . the chi3l1 snps rs6691378 and rs10399805 and chi3l1 haplotypes all correlated with the development of cervical pre - cancerous lesions and invasive cancer 33 . genomic dna was extracted using qiaamp dna blood mini kits ( qiagen , valencia , usa ) according to the manufacturer 's instructions as described previously 34 . the final dna prepared was stored at -20c and used as templates for the following experiments . allelic discrimination of the rs880633 ( + 2950 , t / c ) , rs6691378 ( -1371 , g / a ) , rs4950928 ( -131 , c / g ) and rs10399805 ( -247 , g / a ) polymorphisms was analyzed and asscessed by using abi stepone real - time pcr system ( applied biosystems , foster city , ca , usa ) . the final volume for each reaction mixture was 5 l , containing 2.5 l taqman genotyping master mix , 0.125 l taqman probe mix , and 10 ng genomic dna . the reaction conditions included an initial denaturation step at 95 c for 10 min followed by 40 cycles at 95c for 15 s and 60c for 1 min as described previously33 , 35 . the distributions of demographic characteristics and genotype frequencies for different genotypes between the hcc group and control group were analyzed using the chi - square test for categories of variables . the odds ratios ( ors ) and their 95% confidence intervals ( cis ) of the association between the genotype frequencies and hcc were estimated using multiple logistic regression models by controlling for covariates . in this study , we recruited 343 patients with hcc between 2012 and 2016 at the chung shan medical university hospital , taiwan . demographic characteristics and medical information of the patients , including tnm staging , tumor size , lymph - node metastasis , vascular invasion , distant metastasis , presence of hbv surface antigen ( hbsag ) and liver cirrhosis , were obtained from their medical records . the blood samples which obtained from the controls and hcc patients were stored in edta tubes , centrifuged immediately and stored at -80c . the institutional review board of chung shan medical university hospital approved this study ( csmuh no : cs15099 ) , and informed written consent was obtained from each participant . three snps rs6691378 ( -1371 , c / t ) , rs10399805 ( -247 , c / t ) , and rs4950928 ( -131 , g / c ) in the promoter region and snp rs880633 ( + 2950 , t / c ) in exon 5 were selected based on the chinese hapmap ( han chinese in beijing , china ) data . the snps rs6691378 , rs10399805 and rs4950928 in the promoter region of the chi3l1 gene exhibit strong association of schizophrenia and the snp rs4950928 gc transversion impairs the myc / max - regulated transcriptional activity 27 . snp rs10399805 has been reported to disrupt the c / ebp - aml-1 binding site in the gene promoter and is predicted to increase chi3l1 expression 32 . the chi3l1 snps rs6691378 and rs10399805 and chi3l1 haplotypes all correlated with the development of cervical pre - cancerous lesions and invasive cancer 33 . genomic dna was extracted using qiaamp dna blood mini kits ( qiagen , valencia , usa ) according to the manufacturer 's instructions as described previously 34 . the final dna prepared was stored at -20c and used as templates for the following experiments . allelic discrimination of the rs880633 ( + 2950 , t / c ) , rs6691378 ( -1371 , g / a ) , rs4950928 ( -131 , c / g ) and rs10399805 ( -247 , g / a ) polymorphisms was analyzed and asscessed by using abi stepone real - time pcr system ( applied biosystems , foster city , ca , usa ) . the final volume for each reaction mixture was 5 l , containing 2.5 l taqman genotyping master mix , 0.125 l taqman probe mix , and 10 ng genomic dna . the reaction conditions included an initial denaturation step at 95 c for 10 min followed by 40 cycles at 95c for 15 s and 60c for 1 min as described previously33 , 35 . the distributions of demographic characteristics and genotype frequencies for different genotypes between the hcc group and control group were analyzed using the chi - square test for categories of variables . the odds ratios ( ors ) and their 95% confidence intervals ( cis ) of the association between the genotype frequencies and hcc were estimated using multiple logistic regression models by controlling for covariates . table 1 shows the statistical analysis of the demographic characteristics and clinical parameters . the 686 controls and 343 patients with hcc showed significant differences in their level of alcohol consumption ( p < 0.001 ) ( table 1 ) . genotype distributions and associations between hcc and chi3l1 gene polymorphisms are shown in table 2 . the alleles with the highest distribution frequency for rs6691378 , rs10399805 , rs4950928 , and rs880633 genes of chi3l1 in both the controls and hcc patients were homozygous for g / g , g / g and c / c and heterozygous for t / c . after the adjustment of several variables , we found that people with the rs6691378 , rs10399805 , and rs4950928 polymorphisms of the chi3l1 gene did not have a significantly increased risk of hcc relative to wild - type ( wt ) carriers ( table 2 ) . however , subjects with the chi3l1 polymorphic rs880633 tc+cc genotypes exhibited significantly ( p < 0.05 ) higher risks ( odds ratio [ or ] = 1.329 95% ci = 1.009 - 1.750 ) of hcc than did their corresponding wt homozygous subjects ( table 2 ) . by estimating the distribution frequency of clinical statuses and chi3l1 genotype frequencies in the hcc patients , we clarified the role of chi3l1 gene polymorphisms in hcc clinicopathologic statuses such as clinical stage ( tumor , node , metastasis ) , tumor size , lymph node metastasis , distant metastasis , vascular invasion , child - pugh score , hbsag , antihepatitis c virus ( anti - hcv ) , and liver cirrhosis . no significant associations of rs10399805 , rs4950928 , and rs880633 gene polymorphisms with clinicopathologic statuses were observed ( data not shown ) . however , among the 343 hcc patients , those with the ga+aa polymorphic rs6691378 gene had a lower risk of distant metastasis ( or = 0.314 ; 95% ci = 0.109 - 0.900 , p = 0.024 ) and a lower level of positive hbsag ( or = 0.567 ; 95% ci = 0.368 - 0.875 , p = 0.010 ) than the patients with rs6691378 wt did ; however , no difference was found regarding the clinical stage , tumor size , lymph node metastasis , vascular invasion , child - pugh score , anti - hcv , and liver cirrhosis ( table 3 ) . moreover , in 200 hbsag - negative hcc patients , we observed that those showing the chi3l1 rs4950928 genetic variant cg+gg had a higher risk of vascular invasion ( or = 2.710 ; 95% ci = 1.267 - 5.796 , p = 0.009 ) relative to those showing the rs4950928 wt ; no difference was observed regarding the clinical stage , tumor size , lymph node metastasis , child - pugh score , anti - hcv , and liver cirrhosis ( table 4 ) . alpha - fetoprotein ( afp ) , aspartate aminotransferase , and alanine aminotransferase are common clinical pathological markers of hcc . we analyzed the levels of these pathological markers , which are associated with chi3l1 genotypic frequencies , to clarify the relationship between the progression of clinical status and level of clinical pathological markers in hcc patients . table 5 shows the association between the chi3l1 genotypic frequencies and hcc laboratory status ; no significant association with the rs4950928 and rs880633 gene polymorphisms was observed . however , patients with the rs6691378 ga+aa ( p = 0.026 ) or rs10399805 ga+aa ( p = 0.047 ) genetic variant exhibited higher levels of afp ( table 5 ) compared with those with the wild - type gene polymorphisms . this study provides novel information regarding the effects of chi3l1 snps on hcc susceptibility and clinicopathology . alcohol can cause progressive perivenous injury , impaired hepatocyte function , and endothelial cell pore loss 36 , 37 . chi3l1 serum levels have been shown to potentially provide prognostic information because they are elevated in alcoholic patients and are related to the presence of liver fibrosis 38 . we observed that alcohol consumption was more common in the hcc patients than in the controls ( table 1 ) . chi3l1 might secrete by hepatic stellate cells 39 , which are believed to be the main effector cell in liver fibrogenesis 36 , 37 . elevated chi3l1 levels have been found to be associated with an increased risk of liver fibrosis and to be involved in the activation of the innate immune system 40 . thus , alcohol - consuming hcc patients may sustain high levels of baseline serum chi3l1 , thereby triggering liver fibrosis and cirrhosis . the chi3l1 snp rs880633 is located in the promoter region exon 5 of the chi3l1 gene . we found that chi3l1 snp rs880633 tc+cc genetic variants occur more frequently in hcc patients than in controls ( table 2 ) . the rs880633 polymorphism has been shown to modulate age - adjusted lung function in cystic fibrosis lung disease , and chi3l1 might be a potential biomarker for this condition 41 . however , the detailed mechanism and influence of chi3l1 rs880633 genetic variants have not been thoroughly investigated . the promoter snp -131cg ( rs4950928 ) in the chi3l1 gene was found to be involved in chi3l1 serum level modulation and asthmatic lung disease 29 ; it is located in the core promoter of chi3l1 within a binding site for myc and max transcription factors . the minor allele ( -131 g on the forward strand ) was found to disrupt binding and to be associated with lower messenger rna levels in peripheral blood cells , reduced levels of circulating chi3l1 protein , and , on the basis of a luciferase assay , reduced transcription 27 . . found that the rs4950928 cc genotype is associated with greater levels of circulating chi3l1 
 42 . moreover , in a previous report , abd el - fattah et al . shown that chi3l1 rs4950928 snps have no significant association with colorectal cancer 43 . in the present study , we found that the chi3l1 rs4950928 cg+gg genotype carriers among the 200 hbsag - negative hcc patients indicate a greater risk of vascular invasion ( p = 0.009 ) ( table 4 ) . chi3l1 has been identified as a promoter of angiogenesis in neoplasms and is involved in activation of the mitogen - activated protein kinase / extracellular signal - regulated kinase pathway in endothelial cells 44 - 46 . chi3l1 has been shown to modulate vascular endothelial cell morphology and stimulate migration of endothelial cells by promoting the formation of branching tubules 47 , 48 . an in vivo study by shao et al . found that chi3l1 contributes to the growth of primary and metastatic tumors 49 . our results are consistent with these findings in correlating chi3l1 rs4950928 snps with malignancy and poor prognosis in hcc . afp , desgamma - carboxy prothrombin time , and afp - leptin 3 , have been identified as potential candidates for evaluating the prognosis of hcc patients 50 . however , these candidates are not always sufficient for the prediction of prognosis and recurrence in hcc 51 . in the present study , we found that hcc patients with rs6691378 ga+aa ( p = 0.026 ) or rs10399805 ga+aa ( p = 0.047 ) genetic variant have lower levels of afp ( table 5 ) . chi3l1 levels in both healthy adults and in diseased patients have been found to be affected by chi3l1 genetic variations 26 , 29 , 52 , 53 . the rs10399805 snp is located at position -247 and disrupts the c / ebp - aml-1 binding site in the gene promoter ; it has been speculated that it increases chi3l1 expression 32 . however , the rs10399805 polymorphism has been found to be associated with diseases such as atopy and schizophrenia 32 , 54 . regarding rs6691378 and rs10399805 , the genetic contribution of chi3l1 to schizophrenia has been shown to vary , despite it being mechanistically involved in the disease process 55 . in addition , our previous study demonstrated that the chi3l1 snps rs6691378 and rs10399805 and the chi3l1 haplotype correlate with the development of cervical precancerous lesions and invasive cancer 33 . the precise roles of rs6691378 and rs10399805 in chi3l1 expression and regulation in hcc remain unclear . this may be a result of the variation and coordination of the regulation of different chi3l1 polymorphisms in chi3l1 serum levels . in conclusion , our study shows that the snps of the chi3l1 gene are potential tumor markers for hcc . carriers of chi3l1 genetic variants of rs880633 polymorphisms show various correlations with chi3l1 modulation , hcc severity , and hcc prognosis .
hepatocellular carcinoma ( hcc ) is the second leading cause of cancer death in taiwan . 
 multiple risk factors , such as chronic hepatitis b or c virus infection , carcinogen exposure , cirrhosis , and various single - nucleotide polymorphisms ( snps ) , are considered to contribute to hepatocarcinogenesis . 
 chitinase-3-like protein 1 ( chi3l1 ) , a biomarker implicated in inflammation and tissue remodeling , plays a promoting role in angiogenesis , antiapoptosis , and cell proliferation . 
 this study investigated the role of chi3l1 snps in hcc susceptibility and clinicopathology . 
 real - time polymerase chain reaction was used to analyze four snps of chi3l1 in 343 patients with hcc and 686 cancer - free controls . 
 we found associations with hcc susceptibility in chi3l1 rs880633 polymorphism carriers with genotypes ( tc+cc ) . 
 we observed that hcc patients had lower frequencies of chi3l1 rs6691378 polymorphisms with the variant genotype ga+aa than the wild - type carriers with distant metastasis and positive hbsag did . in 200 
 hbsag negative hcc patients , we observed that the chi3l1 rs4950928 polymorphisms carriers with the variant genotype cg+gg had higher frequencies of vascular invasion . 
 finally , carriers of chi3l1 rs6691378 and 10399805 polymorphisms with the variant genotypes ga+aa showed lower levels of alpha - fetoprotein in hcc laboratory status . in conclusion , 
 our results indicate that patients with chi3l1 rs880633 variant genotypes tc+cc are at a higher risk of hcc . 
 chi3l1 polymorphisms rs880633 or rs4950928 may be potential candidates for predicting poor hcc prognosis and clinical status .