This study found that a mutation (T) of RIPK1 rs17548629 was associated with a significantly increased risk of lung cancer in an adult population aged younger than 60 years, under the codominant and recessive genetic model, after adjustments for general demographic characteristics and environmental factors. Moreover, the bioinformatics analysis revealed that hsa-miR-1197 may be combined with the polymorphic site of rs17548629, with a role in post-transcriptional regulation. Plasmids were constructed of wildtype and mutant target genes, and transfected into A549 and 293T cells. Changes in RIPK1 protein levels in cells were observed with a dual luciferase reporter system. It was determined that rs17548629 is the target of hsa-miR-1197 when carrying the T allele. When a rs17548629 C > T mutation was present, a new binding site of hsa-miR-1197 was found in the 3′-UTR of RIPK1. The combination of the two was associated with low of RIPK1 protein levels, and thereby may promote the progression of lung cancer.
Currently, the number of lung cancer patients under the age of 60 is gradually increasing, and the mortality rate shows a clear upward trend. According to data in the National Central Cancer Registry of China (NCCR), in the year 2015 the incidence and mortality rate of men ages 45 to 59 years and with lung cancer were 122.0/100,000 and 88.5/100,000 respectively; for women of the same age range, the rates were 53.9/100,000 and 32.5/100,000 [2]. Because young and middle-aged people are the working force of the nation, lung cancer in this age group will bring great trauma and economic pressures to society, as well as the patients and their families. Therefore the exploration of risk factors, mechanisms of lung cancer, and public health screening for biomarkers in this age group is very important.
The most common histological pathological type of lung cancer in young and middle-aged patients appears to be adenocarcinoma [17]. The present study found that, among all pathological types of lung cancer, only adenocarcinoma was associated with the polymorphism of RIPK1 rs17548629 under the recessive genetic model. This possibility is real, but the number of cases is not sufficient to establish a strong statistical association in other pathological types.
RIPK1 was the first described member of the receptor-interacting protein (RIP) family, and has been a research hotspot in the field of cell signal transduction. RIPK1 participates in the conversion of apoptosis to necrotic apoptosis [18]. It is capable of interacting with the death receptor family member Fas and tumor necrosis factor receptor 1 (TNFR1). That is, it binds to the Fas-associated death domain (FADD) and the TNF receptor-associated death domain (TRADD) [19] to regulate the inflammatory response, apoptosis, and necrotic apoptosis signaling pathways. Tumor necrosis factor alpha-like (TNFα) is a potent pro-inflammatory cytokine that stimulates cells to cause the formation of a TNF-R1 signaling complex, to mediate downstream cell signaling. Activation of the TNF-R1 signaling complex is regulated by different types of ubiquitination, and may result in RIPK1-dependent and -independent cell death, including apoptosis and necrotic apoptosis [20].
Recently, RIPK1 has been proved to have anti-cancer effects, especially preventing proteasome degradation of TRAF2 to control the development of human liver cancer [21]. Yao et al. [22] studied the association between RIPK1 gene polymorphism and hepatocellular carcinoma. They found that the GG genotype of the SNP of RIPK1 rs2272990 positively correlated with tumor-node-metastasis stage and lymph node metastasis of hepatocellular carcinoma, and negatively correlated with hepatic ischemia-reperfusion injury and prognosis of liver resection.
There have been few reports regarding associations between RIPK1 or its polymorphisms and lung cancer. Because RIPK1 can affect tumorigenesis by regulating apoptosis, we speculate that the SNP of RIPK1 is likely related to lung cancer. In the present study, rs17548629 and rs77736895 of RIPK1 were selected, but the specific molecular mechanism remains unclear. After adjustments for general demographic characteristics and stable environmental factors, under the codominant genetic model the risk of lung cancer in carriers of the RIPK1 rs17548629 TT genotype was 1.671-fold that of CC genotype carriers (95% CI 1.01 to 2.766). Under the recessive genetic model, the risks of lung cancer and lung adenocarcinoma of TT genotype carriers were, respectively, 1.803- and 1.769-fold that of CC + CT genotype carriers (95% CI 1.104 to 2.944 and 1.007 to 3.109). There was no correlation between rs77736895, another site of RIPK1, and lung cancer in this population.
According to the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/snp), the global minor allele frequency of rs17548629 is 0.0931. However, the minor allele frequency of the Han Chinese is 0.268, which is higher than that of other ethnic groups. RIPK1 is located in the 6p25.2 region of the chromosome, and the positive site rs17548629 screened in this study is located in the 3′-UTR region of RIPK1. The bioinformatics analysis predicted that rs17548629 C > T will generate a new hsa-miR-1197 binding site on the 3′-UTR of RIPK1, which inhibits the translation of RIPK1 mRNA.
Hsa-miR-1197 was isolated from neuroblastoma by Afanasyeva et al. [23] in 2008 and belongs to the miR-379 gene family. Its biological function is not clear. The present study found that the RIPK1 rs17548629 C > T mutation in young and middle-aged Han can significantly increase the risk of lung cancer and lung adenocarcinoma. Combined with the predictions of the present bioinformatics analysis, we speculate that this may be due to the interaction between has-miR-1197 and 3′-UTR. This interaction inhibits transcription of the RIPK1 gene, affects its inhibition of inflammation or induction of programmed apoptosis and necrotic apoptosis, and then leads to malignant progression of lung cancer. Yu et al. [24] used a combined priority method to predict new microRNA-disease associations by maximizing network information flow, with considerable effectiveness and stability. Then this method was applied to lung and breast tumors, predicting the top 30 microRNA candidates, which were consistent with the published literature and databases.
To investigate whether the rs17548629 mutation will generate a new hsa-miR-1197 binding site on the 3′-UTR of RIPK1, a dual luciferase reporter gene assay was conducted in the present study. The results obtained by the 293T and A549 cell groups were similar. Although the absolute value of the fluorescence value changed little, the RIPK1-Mut group statistically did bind to miR-1197 to a certain extent; the RIPK1-Wt group did not bind to miR-1197. These results were consistent with the bioinformatics prediction. The rs17548629 C > T variant did produce a new binding site for hsa-miR-1197 on the 3′-UTR of RIPK1, but the binding may be less tight. Thus, we hypothesize that when the concentration of miR-1197 and RIPK1 mRNA is moderate, the rs17548629 C > T mutation increases the binding strength between them, and the protein levels of RIPK1 may decrease. Moreover, the inhibition of inflammation or induction of programmed apoptosis and necrotic apoptosis is weakened, leading to the development of lung cancer. To clarify the mechanism, it would be necessary to determine the concentration of miR-1197 in lung cancer cells. Western blot and real-time fluorescence quantitative PCR should be used to detect changes in protein and mRNA levels expressed by alleles of the target gene in lung cancer cells, after overexpression or inhibition of microRNA. Moreover, the ratio of miR-1197 and mRNA in lung cancer cells under natural conditions needs to be determined.
The present general population and cytology study preliminarily verified an association between SNP variants on RIPK1 rs17548629 and lung cancer. It provides new targets and ideas for the treatment of lung cancer and drug research, which has certain clinical significance. However, due to the multiplicity of RIPK1 protein functions, determining the specific molecular mechanism was beyond the scope of the present study. Moreover, the research was limited to the general population and the cytology, without in vivo validation. Therefore, generalized conclusions are not yet possible. However, the present results warrant experiments with animal models to confirm that SNP variants on RIPK1 rs17548629 are associated with the progression of lung cancer.