Association between long noncoding RNA rs944289 and rs7990916 polymorphisms and the risk of colorectal cancer in a Chinese population

Long non-coding RNAs (LncRNAs) play vital roles in the tumorigenesis of many cancers. Single nucleotide polymorphisms (SNPs) of the lncRNA also play vital roles in tumorigenesis. We explored lncRNA rs944289 and rs7990916 polymorphisms and analyzed the relationship between these lncRNA polymorphisms with the colorectal cancer (CRC) risk in a Chinese population. We recruited 1003 CRC patients from the Affiliated People’s Hospital of Jiangsu University and the Fujian Medical University Union Hospital from October 2014 to August 2017. Genomic DNA was extracted using a DNA Kit from lymphocytes of peripheral blood and the genotyping was performed with a SNPscan method. We found that the rs944289 TT homozygote was associated with the decreased CRC risk in the overall population. LncRNA rs944289 TT decreased the CRC risk in the subgroup of female, male, age ≥ 61, without alcohol intake, smoking and BMI ≥ 24 by logistic regression. The subgroup analysis revealed that lncRNA rs7990916 was not associated with CRC risk except for age < 61. Logistic regression analysis revealed that lncRNA rs944289 TT homozygote was associated with the increased risk of rectum cancer (TT vs. CC + CT: adjusted OR = 1.29, 95% CI 1.10–1.66, P = 0.041) or colon cancer. In summary, we proved that lncRNA rs944289 might be significantly related to the decreased CRC risk in the Chinese Han populations and lncRNA rs7990916 was not associated with the CRC risk except for patients of age < 61. In the future, studies with larger samples should be conducted to validate our results.

www.nature.com/scientificreports/ Alzheimer's disease (AD) risk 10 . Papillary thyroid carcinoma susceptibility candidate 3 (PTCSC3) rs944289 polymorphism increased the risk of papillary thyroid cancer (PTC) 11,12 . Cao et al. also reported that lncRNA rs944289 may increase the risk of esophagogastric junction adenocarcinoma (EGJA) 13 . However, Xu et al. found that lncRNA rs944289 have no effect on the risk of breast cancer (BC) 14 . These data indicate SNPs in lncRNAs play important roles in tumorigenesis. Although lncRNA rs944289 and rs7990916 polymorphisms have a risk effect on some diseases, there are no studies concerning the relationship of these two variants with CRC risk. Based on previous studies, we explored lncRNA rs944289 and rs7990916 polymorphisms in CRC patients and analyzed the relationship between these lncRNA polymorphisms and CRC risk.

Materials and methods
Patients and samples. We recruited 1003 CRC patients from the Affiliated People's Hospital of Jiangsu University and the Fujian Medical University Union Hospital from October 2014 to August 2017. Patients with histologically confirmed CRC were enrolled and the major exclusion criteria were as follows: (1) presence of immunological diseases, (2) with other primary cancers, and (3) with other colonic diseases (e.g. Crohn's disease), (4) exposure to antitumor treatments before surgery. One thousand three hundred and three healthy subjects were selected as healthy controls from the departments of physical examination in these two hospitals.
The healthy controls were Chinese Han populations with the following exclusion criteria: (1) with history of any cancer, (2) with metabolic or autoimmune diseases, and (3) with liver/kidney dysfunction, (4) with any other systemic diseases. The demographic characteristics were recorded, including body mass index (BMI), age, gender, alcohol intake and smoking. The study was approved by the Ethics Committee of the Affiliated People's Hospital of Jiangsu University and conducted according to the Declaration of Helsinki. All participants signed an informed consent before enrolled in this research and all methods were performed in accordance with the relevant guidelines and regulations. SNP genotyping. We collected blood samples from each participant after their admission to the hospital.
The blood samples collected in a test tube containing EDTA were used for genotyping assay. Genomic DNA was extracted using a DNA Kit (Promega, Madison, USA) from lymphocytes of peripheral blood. The DNA was quantified by a measurement of OD260 and then was stored at -80℃. SNPs of lncRNA rs944289 and rs7990916 were analyzed by a SNPscan Kit (Genesky Biotechnologies Inc., Shanghai, China) 15 . 4% DNA samples were selected randomly and analyzed by PCR/Sanger sequencing. The rs944289 locus primers were: 5-TGG TTG AAA GAT AGT CAT TG-3 (forward) and 5-AGA TTT GTA ATA GCT GGG AA-3(reverse). The rs7990916 locus primers were: 5-CTT TGT ATC CTT CAT TCT TA-3 (forward) and 5-CAA GTT GAC ACT CAG AAT TA-3(reverse). For quality control, replicate blinded samples were included to check for the reproducibility of the results by another technician and the results were unchanged. Statistical analysis. All data were analyzed by using SPSS 23.0 (IBM Corporation, Armonk, NY, USA). The chi-square or Fisher's exact test was applied to compare the categorical variables, whereas the continuous variables were evaluated by the student's t-test or Wilcoxon signed-rank test. The relationship between the lncRNA rs944289/7990916 polymorphism and CRC risk was assessed by odds ratios (OR) and 95% confidence intervals (CIs). Logistic regression model was conducted to analyze the associations among lncRNA rs944289 and rs7990916 polymorphisms, the clinical characteristics and CRC risk. Hardy-Weinberg equilibrium (HWE) was used to analyze the genotype distributions of lncRNA rs944289/7990916. A P < 0.05 (two-tailed) was adopted as the statistically significant level.
Ethics approval and consent to participate. The Ethics Committee of the Affiliated People's Hospital of Jiangsu University approved the protocol of the study (K-20210105-W), and all participants signed an informed consent before enrolled in this research.

Consent for publication.
The authors appreciate all the patients in this work for their cooperation and permission for the publication of the article.

Results
Characteristics of CRC patients. In our study, 1003 CRC cases (431 colon cancer and 572 rectum cancer patients) and 1303 healthy controls were enrolled to investigate the correlation of the two SNPs (rs944289 and rs7990916) with CRC risk. Table 1 listed detailed demographics data. The mean age of CRC patients and controls were 61.10 ± 12.17 years and 61.40 ± 9.61 years, respectively. There were no statistically significant differences in age and gender between CRC patients and controls (both P > 0.05). However, smoking, alcohol intake and BMI increased the risk of CRC (both P < 0.05), so we adjusted these factors by multiple logistic regression analyses.
Primary information for lncRNA rs944289 and rs7990916. The genotypic frequencies of lncRNA rs944289 and rs7990916 met the HWE (P = 0.105 and P = 0.359, respectively). Minor allele frequency (MAF) of lncRNA rs944289 polymorphism was 0.28, which was similar to SNP database for Chinese populations (MAF = 0.24). MAF of lncRNA rs7990916 polymorphism was 0.23, which was similar to SNP database for Chinese populations. Table 2 summarized the corresponding information of lncRNA rs944289 and rs7990916.
Stratified analyses between lncRNA rs7990916 polymorphism and CRC risk. We further analyzed stratified effects of lncRNA rs7990916 on CRC risk by logistic regression model. Genotype distributions of rs7990916 were evaluated with age, sex, BMI, drinking and smoking. Results show that there was no significant association between CRC patients and controls except for age < 61 (CT vs. CC: adjusted OR = 0.67, 95% CI 0.48-0.93, P = 0.018) ( Table 5).

Discussion
LncRNAs play an essential role in various biological processes, including cell proliferation, apoptosis, genomic imprinting, transcriptional interference and other critical processes 16,17 . Furthermore, lncRNAs have been reported to participate in the process of tumorigenesis in CRC. For instance, lncRNA CCAL could activate Wnt/β-catenin signaling pathway and induce multidrug resistance in CRC 18 . LncRNA SPRY4-IT1 could promote invasion and proliferation as a ceRNA of miRNA-101-3p in CRC 19 22,23 . Considering this background, we performed a case-control study to determine the relationship between lncRNA rs944289/ rs7990916 polymorphism and CRC risk.
In this study, we found that lncRNA rs944289 TT homozygote could decrease CRC risk. After adjustment by multiple logistic regression, the TT genotype of lncRNA rs944289 was associated with the decreased CRC risk in the subgroups of female, male, age ≥ 61, BMI ≥ 24, smoking and never alcohol intake populations. LncRNA rs944289 have been confirmed to increase the risk of PTC 11,24 , and rs944289 predisposes to PTC by inhibiting the expression of PTCSC3 11,12 . PTCSC3 is a large intergenic noncoding RNA gene that is involved in the regulation of tumorigenesis. The SNP rs944289 is located 3.2 kb upstream of PTCSC3 and suppresses PTCSC3 by destroying a transcription factor-binding site in the promoter of PTCSC3 25,26 . Cao et al. also found that lncRNA rs944289 may increase the risk of EGJA in the smoking and age < 60 years populations 13 . However, in a Chinese population, Xu et al. discovered that lncRNA rs944289 have no significant effect on the risk of BC, which might be due to the specific tumorigenesis of BC 14 . However, up to now, no study has reported the association between lncRNA rs944289 polymorphism and CRC risk, and in this study, we observed significant relationships between the lncRNA rs944289 polymorphism and CRC risk.
Our results also revealed that there was no significant difference between lncRNA rs7990916 polymorphism and the CRC risk in an overall comparison. But when stratified by age < 61 years, lncRNA rs7990916 polymorphism decreased the risk of CRC. Similar to our results, Cao et al. observed no close relationship between lncRNA rs7990916 polymorphism and EGJA patients. In contrast, Jendrzejewski et al. found significant relationship between lncRNA rs7990916 and the risk of AD in the Europe populations. But the etiology of CRC is primarily different from that of AD 21 . Thus, these distinct conclusions on the lncRNA rs7990916 polymorphism may be mainly attributed to the heterogeneity of ethnics and disease types.
Nevertheless, the mechanism of lncRNA polymorphisms on the tumorigenesis of CRC is unclear. Previous studies reported that lncRNA GAS5 rs55829688 polymorphism increased CRC risk by modulating the binding affinity of the transcription factors YY1 to GAS5 promotor 27 . Moreover, MALAT1 rs664589 polymorphism increased CRC risk by binding to miRNA-194-5p, which resulted in an overexpression of MALAT1 28 . Taken together, lncRNA polymorphisms could induce the occurrence of tumor by binding to transcription factors, binding to miRNA and so on.
To the best of our knowledge, our research focused on the possible association of lncRNA rs944289 and rs7990916 polymorphisms with CRC risk for the first time. There still existed several limitations in the study. Firstly, this case-control study only focused on Chinese Han populations. Secondly, the two lncRNAs selected in our study may not be comprehensive because there may be other genetic polymorphisms that affected the risk of CRC. Thirdly, we did not perform the association of lncRNA rs944289 and rs7990916 polymorphisms with tumor stages, patients' prognosis, or other associated risk factors, which might affect the precision of estimating the effect of these two polymorphisms on the CRC risk. Finally, we did not investigate the mechanism of the two polymorphisms during the tumorigenesis of CRC. Nevertheless, our study provides clews for a further mechanism study and provides possible biomarkers for CRC diagnosis.

Conclusions
In summary, we proved that the lncRNA rs944289 might be significantly related to the decreased risk of CRC in the Chinese Han populations. However, there was no significant difference between lncRNA rs7990916 polymorphism and CRC risk except for the subgroup of age < 61. In the future studies, larger samples including detailed clinical information and different ethnic populations should be conducted to validate our findings.