LncRNA H19 gene rs2839698 polymorphism is associated with a decreased risk of colorectal cancer in a Chinese Han population: A case‐control study

Abstract Background Long non‐coding RNA (lncRNA) H19 is involved in the carcinogenesis, progression, and metastasis of colorectal cancer (CRC). Recently, a few studies explored the relationship between lncRNA H19 gene rs2839698 polymorphism and CRC risk, but with conflicting findings. Materials and methods A case‐control study with 315 CRC cases and 441 controls was designed in a Chinese population. Genotyping was performed using PCR‐RFLP. Results It was found rs2839698 polymorphism was associated with a decreased risk of CRC (AA vs GG: OR, 0.73; 95% CI, 0.54‐0.98; P = .037; A vs G: OR, 0.78; 95% CI, 0.63‐0.96; P = .021). Stratified analyses indicated this positive association was also significant in the non‐smokers (AA vs GG: OR, 0.49; 95% CI, 0.25‐0.93; P = .029), non‐drinkers, those aged ≥ 60 years, and overweight individuals (BMI ≥ 24). In addition, rs2839698 polymorphism was also related to the lymph node metastasis (AA vs GG: OR, 0.43; 95% CI, 0.21‐0.88; P = .019) and tumor size (AA vs GG: OR, 0.42; 95% CI, 0.20‐0.88; P = .020) for patients with CRC. Conclusion To sum up, the lncRNA H19 gene rs2839698 polymorphism decreases the risk of CRC in Chinese individuals, especially among the non‐smokers, non‐drinkers, individuals aged ≥ 60 years, and overweight individuals (BMI ≥ 24). Thus, the lncRNA H19 gene rs2839698 polymorphism might be an important biomarker and diagnostic marker for predicting the susceptibility to CRC in Chinese Han population.

common cancer in women and men, 5 respectively. Treatments for CRC included endoscopic and surgical local excision, chemotherapy and radiotherapy, targeted therapy, and immunotherapy and so on. 3 The pathogenesis of CRC was complex and multifactorial, thus poorly understood. Although risk factors including smoking, excessive alcohol, excessive obesity, lack of physical exercise, and high consumption of red meat contributed to the risk of CRC, 3,6,7 there were still CRC patients without exposure to these risk factors, indicating genetic susceptibility might play an important role in the pathogenesis of CRC. 2 As reported, environmental factors, genetic factors, and their interactions contributed to the risk of CRC. 8 Many studies have identified novel risk variants in patients with CRC. [9][10][11][12][13][14] Long non-coding RNAs (lncRNAs), deemed as non-protein-coding transcripts longer than 200 nucleotides, were associated with crucial functions of various biological processes that affected cancer susceptibility. 15,16 LncRNAs played different roles in multiple physiological and pathological processes, including transcriptional regulation, posttranscriptional process, genome rearrangement, epigenetic control, chromatin modification, metabolic processes, and apoptosis. 17,18 Increasing evidence identified the key roles of lncRNAs in human diseases, especially in cancer. 19,20 The aberrant lncRNA expression may result in tumorigenesis. 21 H19 was the first discovered lncRNA and was recognized as an important imprinted gene. LcnRNAH19 was related to cancer, and it was regarded as an important cancer biomarker for human cancer diagnosis and detection. 22 Aberrant change in lncRNA H19 expression was shown in various cancers, suggesting a pivotal role of H19 in cancer progression. 23 Cui et al 24 indicated that H19 was upregulated in CRC.
Yokoyama et al showed that changes in H19 were considered for predicting the susceptibility to 5-FU-based neoadjuvant chemotherapy in rectal cancer. 54 H19 was also reported as a regulator of drug resistance in CRC. 25,26 The lncRNA H19 gene is located on human chromosome 11p15.5, which contains four small introns and five exons. 27 The single nucleotide polymorphisms (SNPs) in H19 gene may affect its gene expression and function, thereby conferring the susceptibility to CRC. Several studies explored the association between the H19 gene rs2839698 polymorphism and the risk of cancers, 18,28-33 but the findings concerning different types of cancers were conflicting. Among these studies, Li et al 31 found that H19 gene rs2839698 polymorphism increased the risk of CRC. However, no other studies investigated the relationship between this SNP and CRC risk.
Thus, we designed this case-control study to assess the effects of H19 gene rs2839698 polymorphism on the risk of CRC.

| Blood sampling and genotyping
Peripheral blood (2 mL) was taken from each of the cases and controls. Genomic DNA was extracted from peripheral blood leukocytes by a TIANamp Blood DNA kit (Tiangen Biotech). The quality and concentration of the extracted DNA were meas- carried out with an initial denaturation at 96°C for 5 minutes, followed by 35 cycles of 96°C for 30 seconds, annealing at 57°C for 40 seconds, and ending with a final elongation step at 72°C for 5 minutes. About 10% of the selected samples were randomly chosen for genotyping twice to ensure the genotyping accuracy, 34,35 and the results were 100% concordant.

| Statistical analysis
The epidemiological variables of cases and controls were calculated using either Student's t test (continuous variables) or chi-square (χ 2 ) test (categorical variables). The deviation between the observed and the expected frequencies among controls was evaluated by Hardy-Weinberg equilibrium (HWE) test through a goodness-offit chi-square test. 36,37 The relationship between the lncRNA H19 gene rs2839698 polymorphism and the CRC risk was evaluated by computing the crude and adjusted odds ratios (ORs) and their 95% confidence intervals (CIs) using logistic regression analysis. 38,39 Subgroup analyses were conducted according to gender, age, alcohol, BMI, and smoking. In addition, the association between the lncRNA H19 gene rs2839698 polymorphism and clinicopathologic features of patients with CRC was also investigated. All statistical analyses were analyzed on SPSS 22.0 (SPSS Inc) with the significance level at P < .05.

| Characteristics of the study population
Demographic information and clinical characteristics of the subjects are summarized in Table 1

| Relationship between lncRNA H19 gene rs2839698 polymorphism and CRC risk
The genotype and allele distributions for the lncRNA H19 gene rs2839698 polymorphism were significantly different between the patients with CRC and the controls (

| Correlation between lncRNA H19 gene rs2839698 polymorphism and clinicopathological characteristics of CRC patients
At last, the association between lncRNA H19 gene rs2839698 polymorphism and the clinicopathologic features of patients with CRC was investigated (

| D ISCUSS I ON
The H19 gene rs2839698 polymorphism was shown to decrease the risk of CRC in a Chinese population. Stratified analyses showed H19 gene rs2839698 polymorphism decreased the risk of CRC among non-smokers, non-drinkers, aged ≥ 60 years, and overweight individuals (BMI ≥ 24). In addition, the rs2839698 polymorphism was significantly correlated with lymph node metastasis and tumor size in patients with CRC.
LncRNAs are transcripts longer than 200 nucleotides, which could not be translated into proteins. 40 LncRNAs were localized in the nucleus 41 or cytoplasm. 42 There were approximately more than 60 000 lncRNAs found in humans. LncRNAs could be further classified into four types: long intergenic ncRNAs (lincRNAs), pseudogenes, antisense RNAs (asRNAs), and circular RNAs (cir-cRNAs). LncRNAs were the main components of the human transcriptome. 43 LncRNAs were involved in biological processes by interfering with gene expression in some cancer types. 44,45 Recently, increasing studies revealed the critical roles of lncRNAs in the development of cancers. 19,20 The dysregulated lncRNAs regulated cell proliferation and apoptosis, invasion, epithelial-to-mesenchymal transition, migration, and drug resistance. 46 In addition, some lncRNAs were biomarkers for the prognosis and diagnosis of some cancers. 40 32 Gong et al 30 did not obtain any significant association between H19 gene rs2839698 polymorphism and lung cancer susceptibility, but suggested this SNP was associated with a platinum-based chemotherapy response in lung cancer. In addition, two studies showed H19 gene rs2839698 polymorphism did not confer susceptibility to neuroblastoma in Chinese children. 18,33 Moreover, H19 gene rs2839698 polymorphism increased the risk of gastric cancer in a Chinese Han population and the rs2839698 CT and TT genotypes were also associated with higher serum H19 mRNA levels. 29  The stratified analyses uncovered a decreased CRC risk in non-smokers, non-drinkers, aged ≥ 60 years, and overweight individuals (BMI ≥ 24). The above data suggested that the individuals exposed to these risk factors were not prone to CRC. Next, we explored the relationship between this SNP and clinicopathologic features of patients with CRC and uncovered that the CRC patients with rs2839698 polymorphism genotypes were not prone to lymph node metastasis and showed a decreased risk in the subgroup with tumor size < 4 cm. However, we did not obtain positive findings in the anal- tumor site, the well-differentiated grade, and Duke's stage of C/D were significantly related to CRC risk. To sum up, our study indicated that lncRNA H19 gene rs2839698 polymorphism was correlated with lymph node metastasis and the tumor size of CRC.

TA B L E 2 Association between H19 gene rs2839698 polymorphism and colorectal cancer risk determined by logistic regression analyses
The advantages of this study included the following aspects: One, this is the first study to uncover the protective role of H19 gene rs2839698 polymorphism in CRC development; two, we observed that H19 gene rs2839698 polymorphism was related to a decreased risk for CRC in non-smokers, non-drinkers, and those aged ≥ 60 years; and three, the rs2839698 polymorphism was significantly correlated with lymph node metastasis and tumor size in patients with CRC. We think these abovementioned points were the innovations of this study, which were not found in other studies.
However, this present study had several limitations. First, the sample size was not large, which may yield false-positive results. Second, this case-control study may have some weakness in identifying the cause-effect relationship. Third, the lack of follow-up data on patients with CRC limited further analysis. Fourth, the controls from the two hospitals may not fully represent the general population.
Last, we only investigated one SNP of H19 gene.

| CON CLUS ION
The lncRNA H19 gene rs2839698 polymorphism is associated with deceased risk for CRC. Further studies with larger sample sizes are warranted to further verify this finding.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests. Abbreviations: MD, Moderately differentiation; PD, Poorly differentiation; WD, Well differentiation.