Polymorphisms in miR-17-92 cluster promoter region is associated with risk and prognosis of endometrial cancer.

Endometrial carcinoma (EC) is the sixth most frequent female tumor and the third most common cause of gynecological cancer-related death. [ 1 ] At present, the incidence and mortality of EC is still rising, especially in developing countries, and the incidence of EC is getting younger. [ 2 , 3 ] Despite advances in diagnostic techniques, surgery, radiotherapy, and chemotherapy, there are still a large number of EC patients with poor prognosis. [ 4 ] Abnormal activation of various gene mutations and related signaling pathways are closely related to the development of EC, [ 5 , 6 ] but there are no commonly used molecular biomarkers to identify EC patients. Therefore, it is necessary to identify potential molecular markers that could help to predict the risk and prognosis of EC. MicroRNAs (miRNAs) are highly conserved small noncoding RNAs, that degrading the target mRNAs or suppressing translation by binding to the 3′-untranslated region (3′-UTRs), thereby regulating gene expression at the post-transcriptional level and performing biological functions. [ 7 ] MiRNAs can be used as a new oncogene or tumor suppressor genes to participate in the occurrence and development of tumors and then affect metastasis and recurrence of cancers, and the abnormal expression of miRNAs is one of the markers of cancers. [ 8 ] More and more studies have shown that miRNAs are involved in the occurrence and progression of EC. [ 9 – 11 ] MiR-17-92 cluster is located on the third intron 13q31.3 and C13orf25 gene of the human chromosome, which consists of 6 miRNAs (miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a). [ 12 ] Previously, it was reported that the miR-17-92 cluster was differentially expressed in EC, and regulated cell proliferation, apoptosis, migration, and invasion via target genes. [ 13 – 16 ] These results suggest that miR-17-92 cluster may be the key molecule regulating the occurrence and development of EC. However, the molecular mechanism of the effect of miR-17-92 cluster abnormalities on EC remains unclear. Single nucleotide polymorphisms (SNPs) may influence gene expression and finally affect the susceptibility to human diseases, [ 17 , 18 ] including EC. [ 19 , 20 ] Previous studies have demonstrated the association between polymorphisms in the promoter of miR-17-92 cluster and susceptibility to human diseases, such as systemic lupus erythematosus, [ 21 ] ischemic stroke. [ 22 ] Recently, rs9588884G and rs982873C in the miR-17-92 cluster promoter have been reported to have protective effects against colorectal cancer, [ 23 ] cervical cancer [ 24 ] and breast cancer, [ 25 ] respectively. In this study, we evaluated the role of these 2 polymorphisms (rs9588884 and rs982873) in EC risk and prognosis in patients with EC in a northern Chinese population. We also detected miR-17-92 mRNA levels in EC tissues of patients carrying different genotypes of these 2 polymorphisms.

Data curation: Ping Li, Huili Wu, Jianlei Wu. Funding acquisition: Zhihui Jie, Jianlei Wu. Investigation: Yan Zhou. Methodology: Zhihui Jie, Ping Li, Huili Wu, Yan Zhou, Jianlei Wu. Project administration: Huili Wu, Yan Zhou, Jianlei Wu. Supervision: Yan Zhou, Jianlei Wu. Writing – original draft: Zhihui Jie, Ping Li, Jianlei Wu. Writing – review & editing: Zhihui Jie, Jianlei Wu.

This case-control study included 398 cases of EC from The Second Affiliated Hospital of Shandong First Medical University between 2008 and 2018. All patients were diagnosed as EC by pathology. Inclusion criteria included EC of women of any age confirmed histologically. Exclusion criteria include patients with any other previous cancers, adjuvant chemotherapy, preoperative radiotherapy or hormone therapy, and metastatic cancers from other sources. A total of 420 female patients as control subjects were confirmed by surgical exploration without any malignant disease, due to benign diseases such as adenomyosis, uterine leiomyoma, cervical intraepithelial neoplasia III (CINIII) or uterine prolapse, and pathologically confirmed endometrial tissue after hysterectomy. Women with any family or personal history of cancer were excluded from our study. All control subjects were chosen from the same hospital in the same period. All subjects selected were Han Chinese in northern China and unrelated to each other. A total of 398 EC patients were followed up by telephone, clinic, and access to medical records. The median follow-up time was 56 months. A total of 189 cases were followed-up for over 5 years. Overall survival (OS) was defined as the time from study entry until death from any cause. This study was approved by the Ethics Committee of the Second Affiliated Hospital of Shandong First Medical University (No. 1019102), followed the Declaration of Helsinki, and obtained informed consent from the participants. Five milliliters of ethylenediaminetetraacetic acid (EDTA) anticoagulated venous blood samples were collected from all subjects and stored at 4°C. Genomic DNA is extracted by the classic Miller salting out method within 1 week after sampling. [ 21 ] The extracted genomic DNA samples were preserved at − 20°C. Genotypes of the 2 SNPs were determined by Shanghai Generay Biotech Co., Ltd. ( http://www.generay.com.cn ) using the polymerase chain reaction/ligase detection reaction (PCR-LDR) method. PCR was carried out in a 15-μL final volume containing 50 ng of DNA template, 1.5 μL of 10 × PCR buffer, 1.5 μL of 25 mM MgCl 2 , 2.5 pmol of each primer, 0.3 μL of 10 mM dNTPs, and 1.25 U of Taq DNA polymerase (TaKaRa, Biotechnology Co. Ltd, Dalian, China). Reaction conditions were 94°C for 2 minutes, 35 cycles of 94°C for 15 seconds, 55°C for 15 seconds, and 72°C for 25 seconds, followed by 72°C for 3 minutes. The primers used for PCR amplification of rs9588884 were F: 5′-TCACAAGCATTCTTATACACC-3′ and R: 5′-TCCACTATCAGAGGCATTTTC-3′. The primer sequences for rs982873 were F: 5′-TGTCCGGCAATCATGAAGTA-3′ and R: 5′-GCTGTATTACGTCTGGAAAGTGCC-3′. [ 23 ] After each PCR product was subjected to a ligase detection reaction (LDR), LDR products were analyzed on an ABI 3730XL DNA sequencer (Applied Biosystems). To evaluate the impact of the 2 polymorphisms on miR-17-92 expression, we examined the mRNA levels of miR-17-92 in EC tissues from patients with different genotypes of rs9588884 and rs982873. Total RNA was isolated from EC tissue samples using TRIzol reagent (Generay Biotech (Shanghai), Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. 1 μg of RNA was converted to cDNA using a commercial kit according to the manufacturer’s manual (Ribobio, Guangzhou, China). RT-qPCR was carried out using sequence-specific primers from the TaqMan MicroRNA Assays. The reaction conditions were as follows: 95°C for 30 seconds, and 40 cycles of 95°C for 5 seconds, 60°C for 30 seconds, and 72°C for 5 seconds. The primer sequences, designed and synthesized by Ribobio Co., Ltd. (Guangzhou, China), were as follows: miR-17-92, forward 5′-CAGTAAAGGTAAGGAGAG CTCAATCTG-3′ and reverse 5′-CAGTAAAGGTAAGGAGAGCTCAATCTG-3′; and U6 (used as an internal reference gene), forward 5′-GCTTCGGCAOCACATATACTAAAAT-3′ and reverse 5′-CGCTCACGAATTTG CGTCAT-3′. The relative expression of miR-17-92 was calculated using the 2 −ΔΔCT method and each reaction was performed in triplicate. As previously reported in the literature, rs9588884 and rs982873 polymorphisms affect the expression of hsa-miR-17 and hsa-miR-20a, respectively. We evaluated prognostic significance of hsa-miR-17 and hsa-miR-20a expression for EC patients using the Kaplan–Meier plotter ( http://www.kmplot.com ), an online database which contained gene expression data and clinical data of 537 clinical EC. In this study, we estimated the OS, risk ratio (HR) and 95% confidence interval (CI) and log-rank P value of EC patients by Kaplan–Meier survival plot. The threshold of each gene was set as follows: P value < .05. SPSS 24.0 software package (SPSS Inc., Chicago) and Graph Prism 8.0 was used for statistical analysis. Chi-square test was used to evaluate the Hardy–Weinberg equilibrium of genotype frequency. Differences in genotype and allele distribution between case and control groups were compared using the Chi-square test. The odds ratio (OR) and 95% CI were calculated by unconditional logistic regression models. Kaplan–Meier analysis and log-rank test were used to draw a survival curve. Cox proportional hazard regression model was used for univariate and multivariate survival analysis. P  < .05 means the difference is statistically significant.

The age of case group is 54.1 ± 10.6 years, while the age of control group is 53.8 ± 9.4 years. There was no significant difference in age distribution between the 2 groups ( P  = .668). The demographic characteristics and clinical data of the study population are listed in Table 1 . The frequency of rs9588884 and rs982873 genotypes in the control group was not significantly different from the expected frequency of Hardy–Weinberg equilibrium ( P  = .347, .348). The clinical characteristics of patients stratified by the 2 polymorphisms are shown in Table 2 . Characteristics of the studied population. BMI = body mass index. Association between the 2 polymorphisms and clinical characteristics of EC patients. EC = endometrial cancer, FIGO = International Federation of Gynecology and Obstetrics. The genotype distribution and EC risk of rs9588884 and rs982873 polymorphisms are shown in Table 3 . Compared with the CC genotype, rs9588884 GG genotype was associated with a decreased risk of EC (GG vs CC: OR = 0.56, 95% CI = 0.36–0.87, P  = .009). Carriers of the G allele have a significantly lower risk of developing EC than those of the C allele (G vs C: OR = 0.89, 95% CI = 0.81–0.97, P  = .017). Compared with TT genotype, rs982873 CC genotype had a reduced EC risk (CC vs TT: OR = 0.53, 95% CI = 0.34–0.82, P  = .004). Compared with T allele carriers, C allele carriers had a significantly decreased risk of developing EC (C vs T: OR = 0.87, 95% CI = 0.79–0.95, P  = .003). Association between the polymorphisms in the promoter of miR-17-92 and risk of EC. CI = confidence interval, EC = endometrial cancer, OR = odds ratio. Adjusted for BMI, menopausal status. One hundred and eighty nine patients were followed up for >5 years. In the 5-year follow-up study, Kaplan–Meier survival analysis and log-rank test were performed to further estimate the association between rs9588884 and rs982873 polymorphisms and EC patient prognosis. Figure 1 A,B showed overall survival curve. The mean OS of EC patients with CC, CG and GG genotypes of rs9588884 was 36, 48, and 54 months, respectively. Compared with those patients carrying the CC genotype, patients with the CG or GG genotypes had longer OS durations ( P  < .001). The mean OS of EC patients with rs982873 TT, TC, and CC genotypes was 43, 44, and 53 months, respectively. Survival analysis showed that the rs982873 polymorphism was not correlated with prognosis of EC patients ( P  = .080). Furthermore, after adjusting for prognostic factors (age, histology, FIGO grade, stage, myometrial invasion, lymph node metastasis), GG genotype of rs9588884 was significantly higher than that of CC genotype in the 5-year follow-up period (OS [HR = 0.43, 95% CI = 0.27–0.70]). However, the rs982873 polymorphism was not found to affect the 5-year overall survival rate. These results indicate that the rs9588884 polymorphism may play an important role in prognosis in EC (Table 4 ). Multivariate analysis of prognostic factors for 5-year overall survival of EC patients. CI = confidence interval, EC = endometrial cancer, FIGO = International Federation of Gynecology and Obstetrics, OR = odds ratio. Overall survival curves of EC patients according to genotypes of rs9588884 C/G (A) and rs982873 T/C (B) polymorphisms. EC = endometrial cancer, OS = overall survival. RT-qPCR results showed that the expression level of miR-17-92 mRNA in EC tissues with the rs9588884 GG genotype was significantly lower than those with the GC + CC genotype (Fig. 2 A, P  = .030). However, there were no significant differences in miR-17-92 mRNA levels among different rs982873 genotypes (Fig. 2 B, P  = .343). Effects of miR-17-92 cluster polymorphisms on the expression of miR-17-92 cluster mRNA in tumor tissue of EOC patients. (A) rs9588884, (B) rs982873. We used the Kaplan–Meier plotter to evaluate whether hsa-miR-20a and hsa-miR-17 are related to the 5-year OS of EC patients, as shown in Figure 3 A,B. High hsa-miR-20a expression was significantly associated with poor OS (HR = 1.63, 95% CI = 1.02–2.61, P  = .039). In EC patients, the high expression of hsa-miR-17 was not correlated with OS (HR = 1.49, 95% CI = 0.93–2.41, P  = .098). The prognostic value of mRNA level of miR-17-92 cluster members in EC patients (OS in Kaplan–Meier plotter database), (A) has-miR-20a (B) has-miR-17. EC = endometrial cancer, HR = hazard ratio, OS = overall survival.

In the present study, we investigated the influence of rs9588884 and rs982873 in the promoter region of the miR-17-92 cluster on the risk and clinical outcome in EC patients. The results showed that the rs9588884 and rs982873 were associated with the risk of EC. The rs9588884 G allele and rs982873 C allele are significantly related to a lower risk of the risk of EC in northern Chinese women. Moreover, survival analysis revealed that rs9588884 CC genotype may significantly decrease the OS of EC patients. To the best of our knowledge, this is the first molecular epidemiologic study to evaluate the potential relationship between the genetic variants of miR-17-92 cluster and EC in Northern Chinese women. To date, numerous studies have shown that there are differences in the expression levels of miRNA between EC and normal endometrial tissue. Detailed studies have shown that miRNAs affect the occurrence and development of EC through the regulation of target mRNA expression and can be used as a biomarker for diagnosis and prognosis. [ 22 – 24 ] miR-17-92 cluster, also known as oncomiR-1, is overexpressed in several malignant tumors, including hematological tumors and solid tumors, such as diffuse large B-cell lymphoma, [ 25 ] lung cancer, [ 26 ] gastric cancer, [ 27 ] colorectal cancer, [ 28 ] ovarian cancer [ 29 ] and cervical cancer, [ 30 ] which is suspected to be associated with the development and progression of tumors. Studies have shown that members of the miR-17-92 cluster are abnormally expressed in endometrial cancer. Chung et al [ 13 ] used qRT-PCR to detect differentially expressed miRNAs in EC tissues and normal endometrium and found that miR-19b expression was increased in endometrial cancer. Snowdon et al [ 14 ] used a microarray to screen for differentially expressed miRNAs in EC tissues and normal endometrial tissues and found that 11 miRNAs, including miR-18a, exhibited increased expression in EC, and used qRT-PCR to detect miR-18a expression. LU et al [ 15 ] utilized high-throughput sequencing technology to obtain comprehensive miRNA maps of type I (Ishikawa) and type II (HEC-1B) endometrial adenocarcinoma cell lines. In Ishikawa cell line, miR-20a and miR-18a were upregulated, while miR-19a and miR-92a were downregulated compared to their expression in the HEC-1B cell line. [ 15 ] These studies indicate that miR-17-92 is closely related to the occurrence and development of EC, but the mechanism by which abnormal miR-17-92 expression affects EC is not clear. Previous studies have demonstrated an association between polymorphisms in the promoter of miR-17-92 cluster and susceptibility to several diseases. In one of the studies, Huang et al found that the rs9301654 G allele in the promoter of miR-17-92 cluster may be associated with a decreased risk of ischemic stroke in the Chinese population. [ 17 ] In another study, Wang et al reported that the rs9515692 CT + TT genotype was a protective factor for the susceptibility of systemic lupus erythematosus by increasing the expression of miR-17. [ 18 ] More importantly, Sun et al found that the rs9588884 and rs982873 polymorphisms in the promoter of miR-17-92 cluster are associated with a reduced risk of colorectal cancer. [ 19 ] Huang et al observed a reduced risk of the rs9588884 GG genotype in the promoter of miR-17-92 with cervical squamous cell carcinoma occurrence. [ 20 ] However, to our knowledge, no studies have examined the association between polymorphisms of the promoter miR-17-92 cluster and EC risk. In this study, we investigated the distributions of 2 Polymorphisms (rs9588884 and rs982873) among 398 EC patients and 410 age-matched controls. We found that the rs9588884 G and rs982873 C were associated with a reduced risk of EC. Notably, Sun et al found that rs9588884 G and rs982873 C in the promoter of miR-17-92 decreased the transcriptional activity and expression levels of miR-20a and miR-17. [ 19 ] Similarly, Huang et al reported that rs9588884 GG genotype was associated with lower levels of miR-20a. [ 20 ] In our study, the results also demonstrated that EC patients with the rs9588884 GG genotype had significantly lower expression of miR-17-92 mRNA in cancer tissues than those with the GC + CC genotype by RT-qPCR. These results suggest that rs9588884 may affect the expression of miR-17-92 and is a functional polymorphism. Therefore, we hypothesized that rs9588884 G in the promoter of miR-17-92 cluster may decrease the expression of miR-17-92 mRNA to protect against EC in northern Chinese women. Therefore, we hypothesized that rs9588884 G and rs982873 C in the promoter of miR-17-92 cluster may decrease the expression of miR-20a and miR-17 to protect against EC, respectively, in northern Chinese women. In addition, we found that rs9588884 and rs982873 polymorphisms were associated with EC patient outcomes. The OS in the EC patients with the rs9588884 CC was significantly shorter than that in EC patients with the GG or CG genotype during the 5-year follow-up. At the same time, we assessed the prognostic value of hsa-miR-17 and hsa-miR-20a in EC patients using the Kaplan–Meier plotter database. The results indicated that high expression of hsa-miR-20a was significantly associated with poor OS in EC patients but that the hsa-miR-17 expression level was not significantly related to OS in EC patients. Therefore, we speculated that rs9588884 may influence the clinical prognosis of EC patients by influencing the expression of miR-17-92 cluster members. There are several limitations to this study. First, although we identified that rs9588884 and rs982873 SNPs were associated with decreased EC risk, we could not reveal the underlying molecular mechanisms. Further studies need to be focused on these points. Second, our study was conducted in the Han Chinese population, and the sample size is small. A larger sample size including other ethic populations would increase the statistical power and the credibility of the conclusions. In summary, this study was the first to determine that the polymorphisms in the promoter of miR-17-92 cluster act as risk factors for EC development in the Chinese population. Analyses suggested that rs9588884 and rs982873 decreased the risk of developing EC and rs9588884 is associated with EC prognosis. This study will help elucidate the molecular epidemiology of EC. In the future, the mechanism of action of these genetic variants should be verified in vitro. Thus, the underlying molecular mechanisms by which rs9588884 and rs982873 regulate miR-17-92 cluster expression and their involvement in EC need to be further studied.