Wei Liu†
Jun Xiong- Department of Orthopedic Trauma, Hainan General Hospital, Hainan Affiliated Hospital of Hainan Medical University, Haikou, China
Background: Alcoholic osteonecrosis of the femoral head (ONFH) is a multifaceted illness that seriously disturbs the patients’ quality of life. The role of lncRNAs in alcoholic ONFH has attracted widespread attention in recent years. This study mainly explored whether MIR31HG polymorphism affects the risk of ONFH.
Methods: There were 733 males (308 alcohol-induced ONFH patients and 425 healthy controls). Seven single nucleotide polymorphisms from MIR31HG were genotyped using the Agena MassARRAY platform. Odds ratio (OR) and 95% confidence intervals (CI) via logistic regression was applied to assess the contribution of MIR31HG variants to alcoholic ONFH susceptibility.
Results: We found that rs10965059 was related to a lower risk of alcoholic ONFH in the overall, age, and necrotic sites analysis. Rs10965064 also showed a risk-reducing effect in the occurrence of alcoholic ONFH patients older than 40 years old.
Conclusions: We confirmed that MIR31HG variants have a significant correlation with the occurrence of alcoholic ONFH among the Chinese Han male population. our findings may provide new ideas for understanding the effect of MIR31HG on the prevention and diagnosis of alcoholic ONFH.
Introduction
Osteonecrosis of the femoral head (ONFH) is a common hip illness characterized by impaired microvascular circulation leading to the death of bone cells. It eventually causes structural changes, collapse of the femoral head, and joint dysfunction. It is reported that the age of patients with ONFH is mostly 30~50 years old, and the incidence rate is gradually increasing with the increase of age (1). There are 8.12 million ONFH patients in China, with approximately 150,000 to 200,000 newly diagnosed ONFH patients yearly (2). Some research showed that one of the common causes of ONFH was excessive drinking (3, 4). Besides, Cui et al. reported that alcoholic ONFH accounted for 30.7% of all femoral head necrosis in China (5). Nevertheless, it has been found in clinical practice that only some people who drink excessively suffer from femoral head necrosis, which suggested that genetic susceptibility may contribute to the occurrence of alcoholic ONFH. Meanwhile, a large number of reports confirmed that genetic variants were related to alcoholic ONFH predisposition (6–8).
Long non-coding RNAs (lncRNAs) refers to non-coding RNA with a length of over 200 nucleotides, which can regulate the physiological and pathological activities of organisms by participating in gene transcription and post-transcriptional regulation, epigenetic modification, and translation (9, 10). Recently, lncRNAs have aroused researcher’s concern in bone development, differentiation, and osteonecrosis. For example, Tang et al. found that lncRNA-OG could promote the differentiation of osteoblasts (11). Liu et al. indicated that lncRNA AK077216 facilitated bone resorption and osteoclastogenesis (12). Besides, a study performed by Xiang et al, which reported that lncRNA RP11-154D6 was reduced in ONFH patients and involved in the progression of ONFH (13).
MIR31HG (also named as lncHIFCAR, LOC554202) is described to participant in proliferation, differentiation, invasion of cancer cells (14, 15). In addition, researchers have discovered that MIR31HG silence promoted osteogenic differentiation and relieved the inflammation-induced inhibition of osteogenesis (16). However, the role of MIR31HG in alcoholic ONFH development remains elusive. However, the role of MIR31HG in alcoholic ONFH development remains elusive.
Here, we explored the association of MIR31HG genetic variations with alcoholic ONFH susceptibility among the Chinese Han male population. This will help to provide new understandings for MIR31HG into the pathogenesis of alcoholic ONFH.
Methods
Study subjects
A total of 733 Chinese Han males (308 alcoholic ONFH patients and 425 healthy controls) were enrolled. All patients were randomly selected from Hainan Affiliated Hospital of Hainan Medical University. The patients met the criteria as following: 1) exceeded 400 mL/week (17) alcohol intake for more than 6 months; 2) diagnosed ONFH within one year after drinking alcohol; 3) no rheumatoid arthritis, hyperlipidemia, spinal cord cavitation, decompression sickness, osteoporosis, cardiovascular disease, and virus infection; 4) no history of steroid use; 5) The diagnosis of alcoholic ONFH by X-ray, nuclear magnetic resonance imaging (MRI), and computed tomography (CT). The stage of alcoholic ONFH patients were identified by the Ficat Classification system (18). Healthy people were selected based on 1) drinking habits or greater than 400 mL alcohol intake per week for more than 6 months; 2) no history of the traumatic disease (ONFH, rheumatoid arthritis, hyperlipidemia, spinal cord cavitation, decompression sickness, osteoporosis, cardiovascular disease, etc…), and no steroid use. The study protocol was approved by the Ethics Committee of the Hainan Affiliated Hospital of Hainan Medical University, in compliance with the declaration of Helsinki. And the signature consent of participants was received.
SNP genotyping
Seven SNPs in MIR31HG were chosen with a minor allele frequency (MAF) >5% in 1000 Genomes Chinese Han Beijing population. Total DNA was isolated from peripheral blood cells by GoldMag blood DNA Kit (GoldMag Co. Ltd, Xi’an, China), and the concentration were evaluated via NanoDrop 2000 (Thermo Scientific, USA). SNP genotyping was completed through the Agena MassARRAY platform and the data were analyzed using the Agena Typer 4.0 software.
Data analysis
Student t-test was applied to evaluate age difference between the two groups. Hardy-Weinberg equilibrium (HWE) in controls was calculated using χ2 test. The linkage between MIR31HG polymorphisms and alcoholic ONFH susceptibility was examined by odds ratio (OR) and 95% confidence interval (CI) through logistic regression. Linkage disequilibrium (LD) was analyzed by Haploview software. Multifactor dimensionality reduction (MDR) was conducted to evaluate the SNP-SNP interactions in the risk of ONFH. False positive report probability (FPRP) values and statistical power were calculated (19). P < 0.05 was identified a significant difference.
Results
Participants and selected SNPs in MIR31HG
As listed in Table 1, this study included 308 alcoholic ONFH patients and 425 healthy controls, with an average age of 43.37 ± 11.34 years and 42.73 ± 12.88 years, respectively. The age distribution between the two groups was well-matched (p=0.478).
Table 1 Characteristics of ONFH patients and controls in this study.
We chose and genotyped seven SNPs (rs1332184, rs72703442, rs2025327, rs55683539, rs2181559, rs10965059, rs10965064) in the intron region of MIR31HG, and all SNPs in line with HWE (p>0.05). And we found that rs10965059 could reduce the susceptibility of alcoholic ONFH in the allele model (p < 0.001, OR = 0.48, 95%CI=0.35-0.66, Table 2).
Table 2 Basic information of the selected SNPs in MIR31HG.
Alcoholic ONFH risk assessment
The association of seven SNPs in MIR31HG and alcoholic ONFH risk was evaluated (Table 3). The results revealed that MIR31HG- rs10965059 was related to a lower risk of alcoholic ONFH under codominant (p<0.001, OR=0.43, 95%CI=0.30-0.62), dominant (p<0.001, OR=0.42, 95%CI=0.30-0.61) and additive models (p<0.001, OR=0.45, 95%CI=0.32-0.62).
Table 3 Association of MIR31HG polymorphisms with alcohol-induced ONFH.
Age stratification showed that rs10965059 decreased the susceptibility to alcoholic ONFH individuals older than 40 years in the allele (p<0.001, OR=0.36), codominant (p<0.001, OR=0.45), dominant (p<0.001, OR=0.31), and additive models (p<0.001, OR=0.33, 9 Table 4). Rs10965064 only reduced the alcoholic ONFH susceptibility under the dominant model (p=0.049, OR=0.67).
Table 4 Relationships between MIR31HG SNPs and alcohol-induced ONFH susceptibility based on stratification by age.
Besides, the necrotic sites stratification results (Table 5) indicated that rs10965059 played a protective role in alcoholic bilateral ONFH in the allele (OR=0.45, p<0.001), codominant (OR=0.40, p<0.001), dominant (OR=0.39, p<0.001), and recessive model (OR=0.42, p<0.001).
Table 5 Association between MIR31HG polymorphisms and ONFH risk stratified by necrotic sites.
Haplotype analysis and MDR analysis
We analyzed the haplotype of MIR31HG gene. Table 6 represented that there was no linkage between haplotypes and alcoholic ONFH susceptibility (p>0.05). Besides, we found a LD block formed by rs72703442, rs2025327, and rs55683539 (Figure 1).
Table 6 Haplotype analysis of MIR31HG SNPs with alcohol-induced ONFH.
Figure 1 Linkage disequilibrium (LD) plots containing three polymorphisms from MIR31HG. Block 1 includes rs72703442, rs2025327, and rs55683539. The numbers inside the diamonds indicate the D’ for pairwise analyses.
The Fruchterman-Rheingold of SNP-SNP interaction was presented in Figure 2. The results of Supplemental Table 1 revealed that rs10965059 was the best single locus model to prediction the ONFH susceptibility (testing accuracy=0.581, CVC=10/10, p < 0.0001).
Figure 2 The Fruchterman-Reingold of SNP-SNP interactions. Each SNP is reported in per cent the value of Information Gain (IG), while numbers in the connections indicate the entropy-based IG for the SNP pairs. Orange bar indicate the high-level synergies on the phenotype, while the brown indicate a medium-level interaction, green and blue connections with negative IG values indicate redundancy or lack of synergistic interactions between the markers.
FPRP analysis
As shown in Supplemental Table 2, all significant results of rs10965059 remained noteworthy, at the prior probability of 0.001 and FPRP threshold of 0.2.
Discussion
We illustrated the relationship of MIR31HG polymorphisms with alcoholic ONFH susceptibility in this study. Results of our research indicated that MIR31HG-rs10965059 decreased the susceptibility of alcoholic ONFH overall. In the age stratification, we also observed that rs10965059 and rs10965064 had protective effect on alcoholic ONFH occurrence older than 40 years old. Besides, rs10965059 could reduce the alcoholic bilateral ONFH risk. These data underline the importance of MIR31HG in alcoholic ONFH occurrence and may serve as a new biomarker for the early prevention and treatment of alcoholic ONFH.
MIR31HG is located at the chromosomal locus 9q21.3 in humans. It has been described to participant in cell proliferation, differentiation in many diseases (20–22). In recent years, the functional role of MIR31HG in bone-related diseases has been studied. For example, Sun et al. found that MIR31HG was higher in tissues of osteosarcoma patients compared with healthy controls and it could regulate osteosarcoma cell growth and migration via miR-361 (23). Ma et al. have shown that MIR31HG was elevated in chordoma patients and MIR31HG silence repressed the migration, growth, and invasion of chordoma cells (24). Besides, suppression of MIR31HG could facilitate the differentiation of osteoblast in human adipose-derived stem cell (16). Moreover, a genome-wide association studies uncovered that MIR31HG polymorphism was related to radius bone density and content in children (25). These evidences led us to hypothesize that MIR31HG may have pathogenic significance in alcoholic ONFH. Here, we firstly observed the contribution of MIR31HG polymorphisms to alcoholic ONFH susceptibility. Rs10965059 exerted a protective role in alcoholic ONFH occurrence in both overall and stratified analysis.
Rs10965059 polymorphism is in the intron region of MIR31HG. Zhao et al. confirmed that the common intronic WDFY4 rs877819 affects the expression of WDFY4 gene by affecting YY1 binding (26). Choi et al. also found that intronic SNP (rs2280964) significantly correlated with reduced the expression of CXCR3 gene, which resulted in changes of immune cell responses to chemokine-cytokine signaling in ex vivo and vitro (27). Based on the above studies, we speculatedthat rs10965059 may affect the susceptibility of alcoholic ONFH by altering the expression of MIR31HG gene. In follow-up studies, we will explore the functional consequence of intronic SNP rs10965059 in vitro and ex vivo to support our hypothesis.
Inevitably, there are some limitations to this study. First, we did not conduct a functional analysis, which is essential to understand the role of MIR31HG in alcoholic ONFH. Second, the subjects were all Chinese Han population, and there may be a certain selection bias. Therefore, we needed animal or cell experiments and more ethnic groups to verify our findings.
Conclusions
To sum up, we confirmed that MIR31HG variants have a significant correlation with the occurrence of alcoholic ONFH in a Chinese Han male population. This may provide new ideas for the prevention and diagnosis of alcoholic ONFH.
Data availability statement
The data presented in the study are deposited in the Zenodo repository, accession number DOI: 10.5281/zenodo.7349781.
Ethics statement
The studies involving human participants were reviewed and approved by Hainan Affiliated Hospital of Hainan Medical University. The patients/participants provided their written informed consent to participate in this study.
Author contributions
JX designed this study protocol; WL drafted the manuscript; XW performed the DNA extraction and genotyping; JC performed the data analysis; FZ performed the sample collection and information recording. JX conceived and supervised the study. All authors read and approved the final manuscript.
Funding
This study was supported by Hainan Provincial Natural Science Foundation of China (No. 822RC808) and Project supported by the Education Department of Hainan Province (No. Hnky2022ZD-13).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo.2022.976165/full#supplementary-material
Abbreviations
ONFH, Osteonecrosis of the femoral head; OR, odds ratio; CI, confidence intervals; MAF, minor allele frequency; HWE, Hardy-Weinberg Equilibrium; CT, computed tomography; MRI, nuclear magnetic resonance imaging; LD, linkage disequilibrium.
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Keywords: osteonecrosis of the femoral head, MIR31HG, polymorphism, case-control study, gene
Citation: Liu W, Wang X, Chen J, Zeng F and Xiong J (2022) The polymorphisms of MIR31HG gene is correlated with alcohol-induced osteonecrosis of the femoral head in Chinese Han male population. Front. Endocrinol. 13:976165. doi: 10.3389/fendo.2022.976165
Received: 05 July 2022; Accepted: 26 October 2022;
Published: 24 November 2022.
Edited by:
Chong Yin, Affiliated Hospital of North Sichuan Medical College, ChinaReviewed by:
Jacek Gagala, Medical University of Lublin, PolandVarun Sharma, NMC Healthcare (NMC Genetics), India
Copyright © 2022 Liu, Wang, Chen, Zeng and Xiong. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Jun Xiong, amllanVuYmluZ0AxMjYuY29t
†These authors share first authorship