LncRNA GAS5 contributes to lymphatic metastasis in colorectal cancer

Colorectal cancer (CRC) ranks the third most common type of cancer worldwide. However, the detailed molecular mechanisms underlying these processes are poorly understood. Recent studies have shown that lncRNAs play important roles in carcinogenesis and progression of CRC. The lncRNA growth arrest special 5 (GAS5), was previously identified to be down-regulated and functions as a tumor suppressor gene in many kinds of cancers. In current two-stage, case-control study, we systematically evaluated the potential role of lncRNA GAS5 and its genetic variation rs145204276 in the development and metastasis process of CRC in a Chinese population. We found the allele del of rs145204276 was significantly associated with 21% decreased risk of CRC (OR=0.79; 95% CI=0.70-0.89; P value = 5.21×10−5). Compared with the genotype ins/ins, both the genotype ins/del (OR=0.78; 95% CI=0.68-0.91) and del/del (OR=0.64; 95% CI=0.49-0.84) showed decreased susceptibility. For both in colon and rectum cancers, the associations kept statistically significant (OR=O.78 and 0.80, while P value = 4.56×10−4, and 3.80×10−3, respectively). The results also showed that the carriers of allele del are less likely to get lymph node metastasis (OR=0.80; 95% CI=0.68-0.95; P value = 0.010). Taken together, our findings provided strong evidence for the hypothesis that GAS5 rs145204276 were significantly associated with the susceptibility and progression of CRC.


INTRODUCTION
Colorectal cancer (CRC) remains one of the leading cause of cancer-related death and the third most commonly diagnosed cancer in males and the second in females, with an estimated 1.4 million cases and 693,900 deaths occurring in 2012 worldwide [1,2]. In united States, the estimated new CRC cases and deaths were 134,490 and 49,190 respectively in 2016 [3]. While in China, the estimated new cancer cases and deaths were 376. 3 and 191.0 thousands respectively, according to the Chinese National Office for Cancer Prevention and Control [4]. About 11.0% of CRC patients had synchronous lung metastases, which caused that the 3-year relative survival rate was 11.3% [5]. All evidence above suggest that CRC is still a big threat to human health and public health problem. Although some genetic factors, body weight, physically activity, consumption of red and processed meat and alcohol, and smoking has been identified to be associated with the outcomes of CRC patients, relapse and metastasis happened in many CRC patients, however, the detailed molecular mechanisms underlying these processes are poorly understood [6][7][8].

Research Paper
in hepatocellular cell lines, which indicated the differential roles of GAS5 in carcinogenesis of different cancer types. In current study, we aim to systematically evaluate the potential role of lncRNA GAS5 and its genetic variation rs145204276 in the development and metastasis process of CRC in a Chinese population.

Population characteristics
As shown in Table 1, characteristics of the subjects included in the two stage of current study were generally comparable. No significant difference were detected for age group, gender, alcohol status and smoking status between CRC cases and healthy controls (all the P value > 0.05). Table 1 also present the percentages of different tumor sites (Colon and Rectum), lymph node metastasis and distant metastasis, which are similar between the two stages in current study. The distribution of genotypes of rs145204276 in healthy controls in the two stage was in accordance with Hardy-Weinberg equilibrium (HWE, P > 0.05). Table 2 presents the association between GAS5 rs145204276 and CRC susceptibility in two independent stages. In stage I, the allele del was significantly associated with decreased risk of CRC (OR=0.81; 95% CI=0.68-0.96; P value = 0.016). Thus, we replicated the association in an independent stage (stage II), which also presented a significant association (OR=0.78; 95% CI=0.67-0.91; P value = 1.24×10 -3 ). When pooled together, the allele del was significantly associated with 21% decreased risk of CRC (OR=0.79; 95% CI=0.70-0.89; P value = 5.21×10 -5 ). Compared with the genotype ins/ins, both the genotype ins/ del (OR=0.78; 95% CI=0.68-0.91) and del/del (OR=0.64; 95% CI=0.49-0.84) showed decreased susceptibility.

Associations between GAS5 rs145204276 and CRC susceptibility stratified by Tumor site
Then the associations between GAS5 rs145204276 and CRC susceptibility were analyzed stratified by Tumor site (Table 3). For both in colon and rectum cancers, the associations kept statistically significant (OR=O.78 and 0.80, while P value = 4.56×10 -4 , and 3.80×10 -3 , respectively).

Associations between GAS5 rs145204276 and Lymph node metastasis and Distant metastasis of CRC
To determine whether GAS5 rs145204276 can affect the progression of CRC, we also explored the associations between GAS5 rs145204276 and Lymph node metastasis and Distant metastasis of CRC. As shown in Table  4, the carriers of allele del are less likely to get lymph node metastasis (OR=0.80; 95% CI=0.68-0.95; P value = 0.010). Compared with those owing the genotype ins/ ins, both subjects with the genotype ins/del (OR=0.86; 95% CI=0.68-1.08) and genotype del/del (OR=0.58; 95% CI=0.37-0.90) showed decreased possibility of lymph node metastasis. Due to limited the sample size and statistical power, the association between GAS5 rs145204276 and distant metastasis of CRC was marginal significant (OR=0.80; 95% CI=0.63-1.02; P value = 0.07).

Relative expression of GAS5
we also examined expression level of GAS5 in CRC tumor tissues and adjacent normal tissues of 50 CRC cases ( Figure 1). Among all the pairs of CRC patients, the expression levels of lncRNA GAS5 in CRC tissues were significantly lower than those in the corresponding normal tissues (P<0.001).

DISCUSSION
In this two-stage, case-control study with large sample size, we systematically evaluated the association of GAS5 rs145204276 with the susceptibility and progression of CRC among Chinese population. We found the allele del of rs145204276 was significantly associated with 21% decreased risk of CRC (OR=0.79; 95% CI=0.70-0.89; P value = 5.21×10 -5 ). The associations kept statistically significant in colon and rectum cancers, and the results also showed that the carriers of allele del are less likely to get lymph node metastasis. To the best of our knowledge, this should be first study which systematically evaluated the genetic association of GAS5 with the susceptibility and progression of CRC.
The GAS5 gene, which was first reported by Coccia et al in 1992, was isolated from mouse genomic DNA and structurally characterized [49]. The transcriptional unit is www.impactjournals.com/oncotarget     divided into 12 exons that span around 7 kb [49]. Then Nakamura et al [50] found that The GAS5 gene fuses to BCL6 as a result of t(1;3)(q25;q27) in a patient with B-cell lymphoma. Also GAS5, whose transcript levels were significantly reduced in breast cancer samples relative to adjacent unaffected normal breast epithelial tissues, could controls apoptosis and down-regulated in breast cancer [31]. Further literatures reported the potential role of GAS5 in the human T-lymphocytes, renal cell carcinoma, prostate cancer, pancreatic cancer, bladder cancer, nonsmall-cell lung cancer, gastric cancer, hepatocellular carcinoma, cervical cancer, and so on [27][28][29][30][51][52][53][54][55]. Yin et al [56] found that GAS5 could also affects cell proliferation and predicts a poor prognosis in patients with CRC, although the sample size of recruited patients were limited (only 66 CRC patients).
Conclusively, in this two-stage, case-control study integrating bioinformatics analysis and large-samplesize, we highlighted a potential functional locus, GAS5 rs145204276, for susceptibility and progression of CRC. Systematic researches on different population and more susceptibility loci are warranted to identify causal variants and elaborate the genetic etiology for susceptibility and progression of CRC.

Subjects
This study was a two-stage, case-control sets including 600 newly diagnosed incident CRC cases and 600 healthy controls in stage I, and 800 newly diagnosed incident CRC cases and 800 healthy controls in stage II, which were recruited between 2010 and 2015. All patients had never received any medical treatments, and the diagnosis of patients was validated through pathologic examination by two different senior pathologists. Healthy controls free of any type cancers were selected from health check-up programs at the same hospital during the same period. Then they were matched to the CRC cases by gender and age group, gender, alcohol and smoking status. Then, 5 ml peripheral blood was collected from each subject, and demographic and pathological information were face to face collected by interviewers. The study was approved by appropriate Research Ethics Committee (REC), and written informed consent was obtained from all patients.

Genotyping
Extraction of the genomic DNA from blood samples and HCC tumor tissues was conducted using Qiagen genomic DNA purification kit. DNA fragments containing the indel polymorphism were amplified using the following genotyping primers: F-TCCCGACTGAGGAGGAAGAGCA; R-AACACC GTCCCGGAAGTGAAA. The PCR products were analyzed by 7% non-denaturing polyacrylamide gel electrophoresis and visualized by silver staining. Quality control was performed by direct sequencing 5% duplicate samples in blind, with a concordance rate of 100%. Furthermore, a 5% random selected sample was tested in duplicate by different persons, and the concordance rate was 100%.

Statistical analyses
Two-sided Student's t-test was selected to compare the differences in the quantitative data, while χ2-test was used to analyze the differences of categorical data between the two groups. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were selected to estimate the strength of association between rs145204276 and risk of CRC and its Lymph node metastasis and Distant metastasis by unconditional multivariable logistic regression, adjusted by age group, gender, alcohol and smoking status. HWE of genotypes was evaluated in controls by a goodness-of-fit χ 2 test. All statistics were performed using SPSS software 19.0 (SPSS Inc., Chicago, IL, USA), and P values were two sided with the statistical significance criteria of P < 0.05 all through the study.