Association of MIR17HG&nbsp;and MIR155HG&nbsp;gene variants with steroid-induced osteonecrosis of the femoral head in the population of northern&nbsp;China

doi:10.21203/rs.3.rs-374738/v1

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Research article

Association of MIR17HG and MIR155HG gene variants with steroid-induced osteonecrosis of the femoral head in the population of northern China

https://doi.org/10.21203/rs.3.rs-374738/v1

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Introduction

Steroid-induced osteonecrosis of the femoral head (ONFH ) is a disease of bone metabolism, and genetic factors are generally considered to play an important role. The purpose of this study was to investigate the relationship between single nucleotide polymorphisms (SNPs) in MIR17HG and MIR155HG and the risk of steroid-induced ONFH in the population of northern China.

Methods

A total of 199 steroid-induced ONFH patients and 506 healthy controls were recruited for the study. Four SNPs of MIR17HG and seven SNPs of MIR155HG were genotyped by Sequenom MassARRAY. ORs and 95% CIs were used to evaluate the relationship between these SNPs and steroid-induced ONFH.

Results

In the codominant model, patients with the MIR17HG SNPs(rs7318578) AA genotype had an increased risk of steroid-induced ONFH (OR = 1.79, p = 0.039), in the recessive model, patients with the MIR17HG SNP(rs7318578) AA genotype had an increased risk of steroid-induced ONFH (OR = 1.78, p = 0.032). Stratified analysis showed that a MIR17HG SNP (rs7318578) and MIR155HG SNPs(rs77218221, rs11911469, rs34904192 and rs4143370) were closely related to different unornamented phenotypes of steroid-induced ONFH. Analysis of the clinical indicators revealed significant differences in high-density lipoprotein (HDL-C) levels between the ONFH group and the control group (p = 0.005). MIR17HG SNP(rs75267932) and MIR155HG SNPs(rs77699734, rs1893650 and rs34904192) were correlated with different lipid indexes.

Conclusion

Our results confirm that MIR17HG and MIR155HG gene mutations are associated with steroid-induced ONFH susceptibility in the population of northern China, providing new evidence for the early detection and prevention of ONFH.

Orthopedic Surgery

Steroid-induced osteonecrosis of the femoral head

MIR17HG

MIR155HG

Single nucleotide polymorphism

Case-control study

Osteonecrosis of the femoral head (ONFH), also known as avascularity necrosis of the femoral head, occurs mostly in young adults aged 30–50 and is a refractory disease in the Department of Orthopedics(Wang et al., 2017). Due to hidden early symptoms, most patients with osteonecrosis develop to the middle and late stages when they are diagnosed with osteonecrosis, and there are varying degrees of femoral head collapse or loss of hip joint function, which seriously affect the quality of life of patients(Xu et al., 2017; Zhang et al., 2017; Yuan et al., 2018). ONFH has different causes including steroids, alcohol, trauma, and idiopathic origins. Among them, steroid-induced ONFH accounts for the highest proportion and has become the main cause of femoral head necrosis(Cui et al., 2016). A national epidemiological study on ONFH in Japan showed that steroid-induced ONFH accounted for 51% of the total cases(Fukushima et al., 2010). Steroid-induced ONFH is a complex and multifactorial disease. A variety of internal and external factors jointly promote intramural microvascular lesions;thrombosis leads to insufficient blood supply and oxygen supply to the femoral head, and finally leads to bone cell death. However, the exact pathogenesis is still unclear. Clinical observations have found that not all patients treated with glucocorticoids have experienced femoral head necrosis, suggesting that there may be other risk factors or individual differences in the occurrence of glucocorticoid induced ONFH. Some studies have shown that the difference between individuals may be related to gene polymorphism(Yuan et al., 2018; Tian et al., 2019).

Osteoblasts (OBs) and osteoclasts (OCs) are the main cells regulating bone homeostasis. Both cell types play a key role in bone tissue reconstruction, bone system integrity, mineral homeostasis in bone tissue and bone metabolism balance(Chacon-Cortes et al., 2015; Moussa et al., 2020). In the ONFH study, one of the pathogeneses was due to the imbalance of the OB/OC ratio and activity in bone tissue, which disrupted the dynamic balance between bone resorption and bone formation(Zhang et al., 2013; Wang et al., 2019).

MicroRNAs (miRNAs) are small, single stranded, endogenous noncoding RNAs with a length of 21–25 nucleotides. They play biological roles by inhibiting the expression of target proteins at the posttranscriptional level(Chacon-Cortes et al., 2015; Wu and Sun, 2019; Zou et al., 2020). According to conformation analysis, more than one-third of human genes are regulated by miRNAs, which indicates that miRNAs play an important role in regulating gene expression(Liu et al., 2014). MiR-17 and miR-155 are transcripts of MIR17HG and MIR155HG, respectively. MiR-17 and miR-155 are both multifunctional miRNAs, that play a consequential role in the occurrence, development and prognosis of tumors (Wu et al., 2019b; Zou et al., 2020). They also have important regulatory effects on bone metabolism. MiR-17-92 is located on chromosome 13q31.3 of the host gene MIR17HG (also known as c13orf2 or mirage). Its expression is downregulated with the differentiation of OBs, and its expression is the lowest in mature OBs. These results indicate that miR-17-92 regulates the proliferation, differentiation and apoptosis of normal OBs(Zhou et al., 2014). In the study of OCs, it was found that miR-155 inhibits osteoclast differentiation by mediating IFN-b(Zhang et al., 2012).

Single nucleotide polymorphisms(SNPs) are the most common type of genetic variation. At present, there is no study on the relationship between MIR17HG and MIR155HG SNPs and steroid-induced ONFH. In this study, we conducted a case-control study in a Chinese Han population to analyze the relationship between 11 SNPs and the risk of steroid-induced ONFH. Our results can be used as molecular markers for the diagnosis of osteonecrosis and provide a new theoretical basis for the early detection and prevention of osteonecrosis.

2.1 Study participants

From 2018 to 2020, a total of 199 blood samples from steroid-induced ONFH patients were collected at Zhengzhou's Traditional Chinese Medicine Hospital trauma center. The diagnosis of steroid-induced ONFH was defined as prednisolone with an average routine daily dose of 16.6 mg or a maximum daily common dose of 80 mg for at least one year(Zhang et al., 2014). All patients underwent anterior, posterior and frog X-ray examinations. The diagnosis of ONFH was confirmed by MRI. All patients with steroid-induced ONFH had no history of chemotherapy or radiotherapy.

At the same time, 506 unrelated healthy people were recruited from the Zhengzhou Traditional Chinese Medicine Hospital as a control. All the subjects were of Han nationality, and lived in Zhengzhou and its surrounding areas. Steroid-induced ONFH was excluded as follows: traumatic dislocation of the hip or other hip diseases, more than 400 milliliters of alcohol per week or having a clearly familial hereditary disease. In general, subjects with chronic diseases and diseases involving the brain, heart, liver, lungs and other vital organs were excluded from the study.

The Ethical Committee of Zhengzhou Traditional Chinese Medicine Traumatology Hospital agreed to the study, and all participants signed written informed consent forms.

2.2 Genotyping

Five milliliter blood samples were collected in EDTA test tubes, centrifuged at 2000 rpm for 10 minutes, and stored at minus 80 degrees Celsius for future experiments. Genomic DNA was extracted by GoldMag extraction (GoldMag, China) and stored at 20 degrees. The multichannel SNP quality extension detection method was developed using Agena MassARRAY Assay Design 4.0 software. The Agena MassARRAY system was used for SNP genotyping. Agena Bioscience Typer 4.0 software was used for data management and analysis. In addition, approximately 10% of the total samples were randomly selected for repeat genotyping, and the reproducibility was 100%.

2.3 Statistical analysis

SPSS20.0 software (SPSS, Chicago, IL) was used for statistical analysis of the data. A chi-squared test was used to analyze whether the genotype distribution of the control group accorded with Hardy Weinberg's equilibrium (HWE). In this study, P values less than 0.05 were statistically significant. We used a chi-squared test to calculate the distribution differences of alleles, genotypes and haplotypes. We analyzed the relationship between genotypes of MIR17HG and MIR155HG polymorphisms, and the risk of steroid-induced ONFH using different genetic models including codominant, dominant, recessive and log-additive models; and calculated the odds ratios (ORs) and 95% confidence intervals (CIs) of logistic regression adjusted for age and sex. Then, stratified analysis was conducted according to age, gender, hip joint disease and course of disease. Finally, linkage disequilibrium (LD) and haplotype structure were estimated by half software (version 4.2).

2.4 KEGG analysis of downstream genes regulated by MIR155HG

We searched out the downstream genes regulated by MIR155HG in (http://www.bio-bigdata.net/LncACTdb/index.html) and (http://mirtarbase.cuhk.edu.cn/) databases, using R software KEGG analysis was performed on these genes.

Information on steroid-induced ONFH in patients and healthy individuals is shown in Table 1. A total of 199 patients (116 males and 83 females) were enrolled, with an average age of 41.21 ± 12.90 years; 506 healthy persons (423 males and 83 females) with an average age of 42.58 ± 13.15 years were included. Basic features, included gender, age, hip lesions (unilateral or bilateral) and course (> 25 months or ≤ 25 months). Steroid-induced ONFH patients were matched with the age of the control group (p = 0.212).

The basic information of 11 SNPs in MIR17HG and MIR155HG, including gene, chromosome, location, allele, MAF, etc., is shown in Table 2. All these SNPs were in Hardy Weinbergequilibrium in the control group. The Χ² test was used to calculate the difference in allele frequency distribution between the case and the control group. We did not find any loci that affect the genetic susceptibility of steroid-induced ONFH (Table 2).

We further evaluated the association between SNPs and the risk of steroid-induced ONFH in four genetic models (codominance, dominant, recessionary, and additive) by logistic regression analysis adjusted for gender and age. Table 3 shows that SNP rs7318578 was associated with an increased risk of steroid-induced ONFH in the age, gender adjusted codominance model (OR = 1.01, 95% CI: 0.70–1.46, p = 0.039) and recessionary model (OR = 1.78, 95% CI: 1.05-3.00, p = 0.032) (Table 3).

The data were stratified according to age, gender, unilateral or bilateral hip joint diseaseand course of disease(> 25 months or ≤ 25 months). We further used the Χ² test to calculate the difference in allele frequency distribution between the case and the control group in different subgroups,and the results are shown in Table 4. Above age45, rs77218221 in the MIR155HG polymorphism was associated with an increased risk of steroid-induced ONFH (OR = 2.03, 95% CI: 1.02–4.04, p = 0.041). In males, rs11911469 in the MIR155HG polymorphism was associated with an increased risk of steroid-induced ONFH(OR = 1.53, 95% CI: 1.01–2.30, p = 0.0396), whereas rs34904192 reduced the risk of steroid-induced ONFH(OR = 0.67, 95% CI: 0.47–0.96, p = 0.0297). In women, rs11911469 in the MIR155HG polymorphism reduced the risk of steroid-induced ONFH(OR = 0.50, 95% CI: 0.26–0.94, p = 0.0289), and rs4143370 was associated with an increased risk of steroid-induced ONFH(OR = 1.86, 95% CI: 1.04–3.32, p = 0.035). In the course of disease, rs77218221 (OR = 2.73,95% CI: 1.16–6.40, p = 0.017) and rs34904192 (OR = 1.72, 95% CI: 1.06–2.78, p = 0.027) in the MIR155HG polymorphism were associated with an increased risk of steroid-induced ONFH(Table 4). The effects of allele and SNP genotypes in different subgroups on steroid-induced ONFH risk were further evaluated, as shown in Table 5. Stratified analysis by age showed that rs77218221 was associated with an increased risk of steroid-induced ONFH in the group older than 45 years of age (OR = 2.75, 95% CI: 1.24–6.08, p = 0.013), in the dominant model (OR = 2.65, 95% CI: 1.20–5.82, p = 0.016), and in the additive model (OR = 2.39, 95% CI: 1.12–5.08, p = 0.023). Rs7318578 was associated with risk reduction of steroid-induced ONFH in patients senior than 45 years of age in the codominate model (OR = 0.82, 95% CI: 0.43–1.55, p = 0.042) and the recessive model (OR = 0.83, 95% CI: 1.61–6.92, p = 0.022). In the male group, rs34904192 reduced the risk of steroid-induced ONFH in the codominant model (OR = 0.61, 95% CI: 0.39–0.95, p = 0.030), the dominant model (OR = 0.61, 95% CI: 0.40–0.94, p = 0.024) and the additive model (OR = 0.67, 95% CI: 0.47–0.98, p = 0.037). The codominate model of rs11911469 (OR = 0.47, 95% CI: 0.23–0.94, p = 0.034), the dominant model (OR = 0.45, 95% CI: 0.22–0.91, p = 0.026) and the additive model (OR = 0.45, 95% CI: 0.23–0.88, p = 0.021) were associated with reduced risk of steroid-induced ONFH in the female group.In the course group, rs77218221 was associated with an increased risk of steroid-induced ONFH in the dominant model (OR = 2.64, 95% CI: 1.06–6.55, p = 0.037), additive model (OR = 2.64, 95% CI: 1.06–6.55, p = 0.038) and rs34904192 under the co-dominant model (OR = 1.22, 95% CI: 0.63–2.35, p = 0.043).(Table 5).

Linkage analysis showed that MIR17HG SNPs (rs75267932, rs7336610, rs7318578) (Fig. 1)and MIR155HG SNPs (rs4143370, rs77218221, rs12482371, rs77699734, rs11911469, rs189365) (Fig. 2)exhibited significant linkage disequilibrium.

The relationship between serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), apolipoprotein A1 (ApoA1), apolipoprotein B (ApoB) and MIR17HG and MIR155HG gene polymorphisms. Analysis of clinical indicators revealed significant differences in HDL-C levels between the steroid-induced ONFH group and the control group (p = 0.005). As shown in Fig. 3(A). The correlation analysis between rs77699734 and rs34904192 genotypes and serum lipid levels showed that there were differences in serum LDL-C levels among the three genotypes of rs77699734 GG, GC and CC (p = 0.018), and the serum LDL-C level of ONFH patients with the GG genotype was significantly higher than that of patients with the GC or CC genotype. There were relevant differences in serum HDL-C levels among ONFH patients with rs34904192 AA, AG and GG genotypes (p = 0.022), as shown in Fig. 3 (B).

There were significant differences in serum TG between GG + GA and AA (p = 0.027) in the rs75267932 governing model, in the LDL-C level between CC + CT and TT in the rs1893650 governing model (p = 0.048), and in the HDL-C level in three genotypes of ONFH patients with AA + AG and GG in the rs34904192 dominant model (p = 0.035),as shown in Fig. 3(C). In the recessionary model carrying rs77699734, the serum LDL-C levels of patients with ONFH between GG and GC + CC were unusual (p = 0.013), and the serum TG levels of patients with ONFH between CC and CT + TT in the rs1893650 recessive models were distinct (p = 0.047), as shown in Fig. 3(D).

In the database (http://www.bio-bigdata.net/LncACTdb/index.html), we found that MIR155HG has a regulatory effect on miR-155-5p. In the (http://mirtarbase.cuhk.edu.cn/) database, we found 919 target genes regulated by miR-155-5p. Through KEGG analysis, found that the signal pathway that regulates the pluripotency of stem cells and osteoclast differentiation pathway is involved in the occurrence of ONFH has an important role in it. Among them, six miR-155-5p regulated genes (IFNGR1, AKT1, TAB2, MITF, SOCS1, SOCS3) were found in the osteoclast differentiation pathway.as shown in Fig. 4–5.

This study is the first to investigate the selective SNPs in MIR17HG and MIR155HG and their relationship with steroid-induced ONFH. In the population of northern China, we found that MIR17HG SNPs(rs7318578) were associated with an increased risk of steroid-induced ONFH. MIR17HG SNPs(rs7318578) and MIR155HG SNPs(rs77218221, rs11911469, rs34904192 and rs4143370) were closely related to distinctive clinical phenotypes of steroid-induced ONFH. MIR17HG SNP(rs75267932) and MIR155HG SNPs(rs77699734, rs1893650 and rs34904192) were correlated with different lipid indexes.

MIR17HG is located on chromosome 13and has been found to play a prominent role in a variety of human diseases. Studies have confirmed that abnormal expression of miR-17 has been detected in the local tissues or serum of patients with ONFH(Jia et al., 2014; Wu and Sun, 2019). MiR-17-5p (located in the miR-17-92 cluster) delayed the differentiation and proliferation of OBs by downregulating the partial expression of SMAD7, leading to the occurrence of nontraumatic ONFH(Jia et al., 2014; Wei et al., 2017). Studies also found that the miR-17-92 cluster was downregulated in the OB differentiation of ES cells and the bone progenitor cell line Mc3t3-e1(Song et al., 2019). In the analysis of miRNA expression in OBs exposed to apoptotic inducers, it was found that miR-17-92a played a consequential role in protecting estrogen anti-bone loss by regulating Bim expression(Guo et al., 2013). These findings suggest that the miR-17-92 cluster plays a key role in the occurrence and development of steroid-induced ONFH. Previous studies have shown an increased risk of multiple myelomas in patients with the rs7336610 allele of MIR17HG, and the three SNPs of MIR17HG (rs7336610, rs7318578, and rs17735387) were associated with a risk of colorectal cancer(Chen et al., 2019; Wu et al., 2019b). In this study, rs7318578 was identified as a genetic susceptibility factor for steroid-induced ONFH. To date, only the SNPs (rs7336610, rs7318578, rs17735387, and rs75267932) of MIR17HG and the risk of steroid-induced ONFH have been investigated. Therefore, more samples are needed for correlation studies to confirm the results.

OCs are one of the most important cells in maintaining homeostasis, and their excessive bone resorption often leads to bone loss. According to the literature, miR-155 is involved in the regulation of OCs. In osteoporotic mice, miR-155 inhibited OC activation by targeting the leptin receptor (LEPR) via the AMPK pathway(Mao et al., 2019). MiR-155 can target proto-oncogene SPI1, microsomia-related transcription factor and cytokine signal transduction inhibitor protein 1 to inhibit macrophage activation and OC differentiation, thus inhibiting the process of bone resorption(Song et al., 2019). As an intron gene with higher transcription levels, miRNA is involved in physiological processes such as development, cell proliferation, differentiation, and metabolism(Li et al., 2018). Research on mesenchymal stem cells of patients with steroid-induced femoral necrosis showed that the overexpression of miR-155-5p in their cells can directly inhibit GSK3B to promote cell proliferation and bone differentiation(Wu et al., 2020). MiR-155 is the transcription product of the host gene MIR155HG, and its expression may be affected by genetic variation in the MIR155HG gene. In a study of SNPs in MIR155HG and colorectal cancer, rs12482371, rs1893650, rs92888 rs11911469 and rs34904192 increased the risk of colorectal cancer(Wu et al., 2019a). In our study, the four SNPs of MIR155HG (rs77218221, RS11911469, rs34904192 and rs4143370) were associated with a risk of steroid-induced ONFH. Our study suggests that MIR155HG may be a new susceptibility gene for steroid-induced ONFH, providing new evidence for the early detection of ONFH.

In a study of the pathogenesis of femoral head necrosis, the lipid metabolism theory is currently the most concerning. Hyperlipidemia can affect the microcirculation of the femoral head from multiple sources (coagulation system, bone fat embolism and bone microthrombosis), and then lead to femoral head necrosis.(Phemister, 2005; Guo et al., 2013).With further research, increasing number of genes have been found to participate in the regulation of lipid metabolism. SREBP2 can activate target gene transcription and gene expression of cholesterol biosynthesis pathways, which play an important role in lipid homeostasis. Through the study of the relationship between SREBP2 gene polymorphism and ONFH, it was concluded that SREBP2 gene polymorphism and function could cause lipid metabolism disorder in ONFH patients(Song et al., 2017). Apolipoprotein is considered to be a sensitive indicator to estimate the disorder of lipid metabolism in the ONFH population(Hao et al., 2018). ApoAI is the main component of HDL. ApoB is a component of lipoprotein, chylomicrons, very low-density lipoprotein and medium-density lipoprotein, and is the only structural protein of low-density lipoprotein. Both are essential for lipid transport and metabolism. The study found that the − 75G/A polymorphism of the ApoAI gene may be associated with susceptibility to osteonecrosis in the Chinese population(Yin et al., 2014). We studied the genotypes and lipid levels of ONFH patients, and found their relationship with clinical phenotype and the development of ONFH. MIR17HG and MIR155HG may have some effects on lipid metabolism.

In summary, our study demonstrates that MIR17HG SNP(rs7318578) are significantly associated with an increased risk of steroid-induced ONFH in the population of northern China. A MIR17HG SNP(rs75267932) and MIR155HG SNPs (rs77699734, rs1893650 and rs34904192)were associated with abnormal lipid metabolism.These findings provide evidence for the early screening and prevention of femoral head necrosis.However, more samples from different regions are needed to confirm the results.

Ethical Approval

Ethics committee approval was received for this study from Zhengzhou Traditional Chinese Medicine Traumatology Hospital.All participants provided written informed consent.

Conflicts of Interest

The authors declare there are no conflicts of interest.

Acknowledgments

We gratefully acknowledge all of the participants, the clinicians, and all the hospital staff for their contributions to this study. We are also thankful to Zhengzhou TCMT raumatology Hospital, Zhengzhou, Henan Province, China, for their support.

Funding

This work received the support of the National Natural Science Foundation of China (nos. 81160228, 81260284,81660378 and 81860401) and Natural Science Foundation of Inner Mongolia Autonomous Region（2017MS08119）

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Authors' Contributions

WJZ and DQM conceived and designed the project.

WTT ,HCX and SMH recruited and collected study samples.

CYJ and AFM performed the experiments.

MC and LTT selected the SNPs and designed primers.

WJZ and HCX performed and analyzed the data.

LTT and WJZ wrote and revised the manuscript.

Chacon-Cortes, D., Smith, R.A., Lea, R.A., Youl, P.H. and Griffiths, L.R., 2015. Association of microRNA 17-92 cluster host gene (MIR17HG) polymorphisms with breast cancer. Tumour Biol 36, 5369-76.

Chen, P., Bai, Y., Li, Y., Yuan, Y., Cheng, Y., Pang, J., Zhu, H. and Chen, C., 2019. Association between polymorphisms of MIR17HG and risk of colorectal cancer in the Chinese Han population. Mol Genet Genomic Med 7, e667.

Cui, L., Zhuang, Q., Lin, J., Jin, J., Zhang, K., Cao, L., Lin, J., Yan, S., Guo, W., He, W., Pei, F., Zhou, Y. and Weng, X., 2016. Multicentric epidemiologic study on six thousand three hundred and ninety five cases of femoral head osteonecrosis in China. Int Orthop 40, 267-76.

Fukushima, W., Fujioka, M., Kubo, T., Tamakoshi, A., Nagai, M. and Hirota, Y., 2010. Nationwide epidemiologic survey of idiopathic osteonecrosis of the femoral head. Clin Orthop Relat Res 468, 2715-24.

Guo, L., Xu, J., Qi, J., Zhang, L., Wang, J., Liang, J., Qian, N., Zhou, H., Wei, L. and Deng, L., 2013. MicroRNA-17-92a upregulation by estrogen leads to Bim targeting and inhibition of osteoblast apoptosis. J Cell Sci 126, 978-88.

Hao, Y., Guo, H., Xu, Z., Qi, H., Wang, Y., Lu, C., Liu, J. and Yuan, P., 2018. The relationship between apolipoprotein genes polymorphisms and susceptibility to osteonecrosis of the femoral head: a meta-analysis. Lipids Health Dis 17, 192.

Jia, J., Feng, X., Xu, W., Yang, S., Zhang, Q., Liu, X., Feng, Y. and Dai, Z., 2014. MiR-17-5p modulates osteoblastic differentiation and cell proliferation by targeting SMAD7 in non-traumatic osteonecrosis. Exp Mol Med 46, e107.

Li, Z., Yang, B., Weng, X., Tse, G., Chan, M.T.V. and Wu, W.K.K., 2018. Emerging roles of MicroRNAs in osteonecrosis of the femoral head. Cell Prolif 51.

Liu, B., Li, J. and Cairns, M.J., 2014. Identifying miRNAs, targets and functions. Brief Bioinform 15, 1-19.

Mao, Z., Zhu, Y., Hao, W., Chu, C. and Su, H., 2019. MicroRNA-155 inhibition up-regulates LEPR to inhibit osteoclast activation and bone resorption via activation of AMPK in alendronate-treated osteoporotic mice. IUBMB Life 71, 1916-1928.

Moussa, F.M., Cook, B.P., Sondag, G.R., DeSanto, M., Obri, M.S., McDermott, S.E. and Safadi, F.F., 2020. The role of miR-150 regulates bone cell differentiation and function. Bone, 115470.

Phemister, D.B., 2005. Repair of bone in the presence of aseptic necrosis resulting from fractures, transplantations, and vascular obstruction. 1930. J Bone Joint Surg Am 87, 672.

Song, H., Li, X., Zhao, Z., Qian, J., Wang, Y., Cui, J., Weng, W., Cao, L., Chen, X., Hu, Y. and Su, J., 2019. Reversal of Osteoporotic Activity by Endothelial Cell-Secreted Bone Targeting and Biocompatible Exosomes. Nano Lett 19, 3040-3048.

Song, Y., Du, Z., Chen, B., Ren, M., Yang, Q., Sui, Y., Wang, Q., Wang, A., Zhao, H., Qin, Y. and Zhang, G., 2017. Association of SREBP2 gene polymorphisms with the risk of osteonecrosis of the femoral head relates to gene expression and lipid metabolism disorders. Mol Med Rep 16, 7145-7153.

Tian, Y., An, F., Wang, J., Liu, C., Wu, H., Cao, Y. and Wang, J., 2019. MMP2 and MMP10 Polymorphisms Are Related to Steroid-Induced Osteonecrosis of the Femoral Head among Chinese Han Population. Biomed Res Int 2019, 8298193.

Wang, C., Sun, W., Ling, S., Wang, Y., Wang, X., Meng, H., Li, Y., Yuan, X., Li, J., Liu, R., Zhao, D., Lu, Q., Wang, A., Guo, Q., Lu, S., Tian, H., Li, Y. and Peng, J., 2019. AAV-Anti-miR-214 Prevents Collapse of the Femoral Head in Osteonecrosis by Regulating Osteoblast and Osteoclast Activities. Mol Ther Nucleic Acids 18, 841-850.

Wang, Y., Yang, X., Shi, J., Zhao, Y., Pan, L., Zhou, J., Wang, G. and Wang, J., 2017. Combination analysis of NOS3, ABCB1 and IL23R polymorphisms with alcohol-induced osteonecrosis of the femoral head risk in Chinese males. Oncotarget 8, 33770-33778.

Wei, B., Wei, W., Zhao, B., Guo, X. and Liu, S., 2017. Long non-coding RNA HOTAIR inhibits miR-17-5p to regulate osteogenic differentiation and proliferation in non-traumatic osteonecrosis of femoral head. PLoS One 12, e0169097.

Wu, F., Huang, W., Yang, Y., Liu, F., Chen, J., Wang, G. and Sun, Z., 2020. miR-155-5p regulates mesenchymal stem cell osteogenesis and proliferation by targeting GSK3B in steroid-associated osteonecrosis. Cell Biol Int. 1065-6995

Wu, H., He, G., Han, H., Xiong, W., Song, T., Chen, H., Chen, X., Wu, X., Huang, G., Zhang, Y., Sun, C., Zhao, C. and Chen, Y., 2019a. Analysis of MIR155HG variants and colorectal cancer susceptibility in Han Chinese population. Mol Genet Genomic Med 7, e778.

Wu, H., Huang, T., Ye, Z., Fu, X., Hu, K. and Yang, X., 2019b. Correlation of MicroRNA 17-92 Cluster Host Gene (MIR17HG) Polymorphisms With Susceptibility and Prognosis for Multiple Myeloma. Clin Lymphoma Myeloma Leuk 19, e359-e366.

Wu, X. and Sun, W., 2019. Noncoding RNAs in Steroid-Induced Osteonecrosis of the Femoral Head. Biomed Res Int 2019, 8140595.

Xu, B., Hu, Q.H., Zhao, B. and Zhou, Z.K., 2017. Variation and significance of serum leptin, blood lipid level, adiponectin, NO and TNF-α for patients with non-traumatic ischemic necrosis of the femoral head. Saudi J Biol Sci 24, 1763-1766.

Yin, J.M., Liu, Z., Zhao, S.C., Guo, Y.J. and Liu, Z.T., 2014. Relationship between the Apolipoprotein AI, B gene polymorphism and the risk of non-traumatic osteonecrosis. Lipids Health Dis 13, 149.

Yuan, L., Li, W., Wang, X., Yang, G., Yu, H. and Sun, S., 2018. The relationship between genetic polymorphisms in apolipoprotein E (ApoE) gene and osteonecrosis of the femoral head induced by steroid in Chinese Han population. Genes Genomics 40, 225-231.

Zhang, J., Zhao, H., Chen, J., Xia, B., Jin, Y., Wei, W., Shen, J. and Huang, Y., 2012. Interferon-β-induced miR-155 inhibits osteoclast differentiation by targeting SOCS1 and MITF. FEBS Lett 586, 3255-62.

Zhang, L., Zheng, W., Wang, Y., Wang, Y. and Huang, H., 2013. Human bone marrow mesenchymal stem cells support the derivation and propagation of human induced pluripotent stem cells in culture. Cell Reprogram 15, 216-23.

Zhang, Y., Kong, X., Wang, R., Li, S., Niu, Y., Zhu, L., Chen, W. and Lin, N., 2014. Genetic association of the P-glycoprotein gene ABCB1 polymorphisms with the risk for steroid-induced osteonecrosis of the femoral head in Chinese population. Mol Biol Rep 41, 3135-46.

Zhang, Y., Sun, R., Zhang, L., Feng, L. and Liu, Y., 2017. Effect of blood biochemical factors on nontraumatic necrosis of the femoral head : Logistic regression analysis. Orthopade 46, 737-743.

Zhou, M., Ma, J., Chen, S., Chen, X. and Yu, X., 2014. MicroRNA-17-92 cluster regulates osteoblast proliferation and differentiation. Endocrine 45, 302-10.

Zou, W., Li, X., Li, C., Liu, D., Lv, Y., Yang, Y., Ye, N., Guo, D. and He, S., 2020. Analysis of the relationship between MIR155HG variants and gastric Cancer susceptibility. BMC Gastroenterol 20, 17.

Table 1: Characteristics of the individuals in controls and steroid-induced ONFH patients.

Variables	Cases (n=199)	Controls (n=506)	p
Variables	N ( % )	N ( % )	p
Age, years
Mean ± SD	41.21±12.90	42.58±13.15	0.212^a
≤45	129(65%)	275(54%)
>45	70(35%)	231(46%)
Gender			< 0.001^b
male	116(58%)	423(84%)
female	83(42%)	83(16%)
Hip lesions
Unilateral	142(71%)
Bilateral	55(28%)
Course, months
>25	67(34%)
≤25	132(66%)

P < 0.05 indicates statistical significance.

^a Independent samples t test.

^bTwo-sided Chi-squared test.

Table 2: Basic information of candidate SNPs in this study.
SNP	Gene	Chromosome	Position	Alleles	MAF		HWE	ORs	95% CI		p^b
SNP	Gene	Chromosome	Position	A/B	case	control	p^a	ORs	95% CI		p^b
rs75267932	MIR17HG	13	91351812	A/G	0.114	0.124	0.411	0.90	0.63	1.30	0.575
rs7336610	MIR17HG	13	91352883	C/T	0.528	0.481	0.129	1.21	0.95	1.52	0.116
rs7318578	MIR17HG	13	91353215	A/C	0.327	0.292	0.389	1.18	0.92	1.51	0.206
rs17735387	MIR17HG	13	91353800	A/G	0.191	0.176	0.358	1.1	0.81	1.48	0.537
rs4143370	MIR155HG	21	25564661	C/G	0.136	0.146	0.597	0.92	0.66	1.28	0.610
rs77218221	MIR155HG	21	25565063	C/T	0.058	0.046	1.000	1.26	0.75	2.10	0.377
rs12482371	MIR155HG	21	25566041	C/T	0.332	0.323	0.104	1.04	0.81	1.33	0.758
rs77699734	MIR155HG	21	25566995	C/G	0.090	0.103	0.636	0.87	0.58	1.29	0.486
rs11911469	MIR155HG	21	25567971	A/C	0.138	0.130	0.545	1.12	0.80	1.57	0.522
rs1893650	MIR155HG	21	25568503	C/T	0.196	0.202	0.491	0.97	0.72	1.29	0.813
rs34904192	MIR155HG	21	25569623	A/G	0.226	0.251	0.288	0.87	0.66	1.15	0.328

SNP: single nucleotide polymorphism, HWE: Hardy-Weinberg equilibrium, OR: odds ratio, 95%CI: 95%confidence interval, and MAF: minor allele frequency.

^ap values were calculated by exact test.

^bp values were calculated by Pearson Chi-squared test.

Table 3: Genotypic model analysis of relationship between SNPs and steroid-induced ONFH.
SNP	Model	Genotype	Group = control	Group = case	OR (95% CI)	p
rs7318578	Codominant	C/C	47(9.3%)	27(13.6%)	1	0.039*
		C/A	200(39.5%)	76(38.2%)	1.01(0.70-1.46)
		A/A	256(50.6%)	96(48.2%)	1.79(1.03-3.09)
	Dominant	C/C	47(9.3%)	27(13.6%)	1	0.427
		C/A-A/A	456(90.1%)	172(86.4%)	1.15(0.82-1.62)
	Recessive	C/C-C/A	247(48.8%)	103(51.8%)	1	0.032*
		A/A	256(50.6%)	96(48.2%)	1.78(1.05-3.00)
	Log‐additive	-	-	-	1.23(0.95-1.58)	0.115

^*P < 0.05 indicates statistical significance.

Table 4:The subgroup information of the MIR17HG gene and MIR155HG gene
Subgroup	SNP	Alleles	MAF		HWE-p^a	ORs	95% CI		p^b
Subgroup	SNP	A/B	case	control	HWE-p^a	ORs	95% CI		p^b
Age,＞45	rs77218221	C/T	0.1	0.052	0.467	2.03	1.02	4.04	0.041
Gender，male	rs11911469	A/C	0.164	0.113	1.000	1.53	1.02	2.3	0.040
	rs34904192	A/G	0.19	0.259	0.312	0.67	0.47	0.96	0.030
Gender，female	rs11911469	A/C	0.1	0.19	0.282	0.50	0.26	0.94	0.029
Course, months	rs4143370	C/G	0.187	0.11	0.186	1.86	1.04	3.32	0.035
case，>25	rs77218221	C/T	0.097	0.038	1.000	2.73	1.16	6.40	0.017
control，≤25	rs34904192	A/G	0.291	0.193	0.783	1.72	1.06	2.78	0.027

SNP: single nucleotide polymorphism, HWE: Hardy-Weinberg equilibrium, OR: odds ratio, 95%CI: 95%confidence interval, and MAF: minor allele frequency.

^aP values were calculated by exact test.

^bP values were calculated by Pearson Chi-squared test.

Table 5:The relationship between MIR17HG and MIR155HG gene polymorphism and steroid-induced ONFH subgroup analysis
Subgroup analysis	SNP	Model	Genotype	Control	Case	OR (95% CI)	p
Subgroup analysis	SNP	Model	Genotype	Control	Case	OR (95% CI)	p
Age,＞45	rs77218221	Codominant	C/C	1(0.4%)	0	1	0.013*
			C/T	22(9.5%)	14(20.0%)	2.75(1.24-6.08)
			T/T	208(90.0%)	56(80.0%)	-
		Dominant	C/C	1(0.4%)	0	1	0.016*
			C/T-T/T	230(99.6%)	70(100.0%)	2.65(1.20-5.82)
		Recessive	C/C-C/T	23(10.0%)	14(20.0%)	1	0.999
			T/T	208(90.0%)	56(80.0%)	-
		Log-additive	-	-	-	2.39(1.12-5.08)	0.023*
Age, ＞45	rs7318578	Codominant	C/C	17(7.4%)	10(14.3%)	1	0.042*
			C/A	90(39.0%)	23(32.9%)	0.82(0.43-1.55)
			A/A	121(52.4%)	37(52.9%)	2.62(1.04-6.60)
		Dominant	C/C	17(7.4%)	10(14.3%)	1	0.826
			C/A-A/A	211(91.3%)	60(85.7%)	1.07(0.60-1.91)
		Recessive	C/C-C/A	107(46.3%)	33(47.1%)	1	0.022*
			A/A	121(52.4%)	37(52.9%)	2.83(1.61-6.92)
		Log-additive	-	-	-	1.30(0.84-2.01)	0.238
Male	rs34904192	Codominant	A/A	24(5.7%)	5(4.3%)	1	0.030*
			A/G	171(40.4%)	34(29.3%)	0.61（0.39-0.95）
			G/G	228(53.9%)	77(66.4%)	0.62（0.23-1.69）
		Dominant	A/A	24(5.7%)	5(4.3%)	1	0.024*
			A/G-G/G	339(80.1%)	111(95.7%)	0.61(0.40-0.94)
		Recessive	A/A-A/G	195(46.1%)	39(33.6%)	1	0.557
			G/G	228(53.9%)	77(66.4%)	0.74(0.27-2.00)
		Log-additive	-	-	-	0.67(0.47-0.98)	0.037*
Female	rs11911469	Codominant	C/C	1(1.2%)	0	1	0.034*
			C/A	29(34.9%)	17(20.5%)	0.47(0.23-0.94)
			A/A	53(63.9%)	66(79.5%)	-
		Dominant	C/C	1(1.2%)	0	1	0.026*
			C/A-A/A	82(98.8%)	83(100.0%)	0.45(0.22-0.91)
		Recessive	C/C-C/A	30(36.1%)	17(20.5%)	1	0.999
			A/A	53(63.9%)	66(79.5%)	-
		Log-additive	-	-	-	0.45(0.23-0.88)	0.021*
Course, months	rs77218221	Codominant	C/C	0	0	1
case，>25			C/T	10(7.6%)	13(19.4%)	-
control，≤25			T/T	122(92.4%)	54(80.6%)	-
		Dominant	C/C	0	0	1	0.037*
			C/T-T/T	132(100.0%)	67(100.0%)	2.64(1.06-6.55)
		Recessive	C/C-C/T	10(7.6%)	13(19.4%)	1	-
			T/T	122(92.4%)	54(80.6%)	-
		Log‐additive	-	-	-	2.64(1.06-6.55)	0.038*
Course, months	rs34904192	Codominant	A/A	4(3.0%)	7(10.4%)	1	0.043*
case，>25			A/G	43(32.6%)	25(37.3%)	1.22(0.63-2.35)
control，≤25			G/G	85(64.4%)	35(52.2%)	3.84(1.04-14.15)
		Dominant	A/A	4(3.0%)	7(10.4%)	1	0.240
			A/G-G/G	128(97.0%)	60(89.6%)	1.45(0.78-2.69)
		Recessive	A/A-A/G	47(35.6%)	32(47.8%)	1	0.052
			G/G	85(64.4%)	35(52.2%)	3.56(0.99-12.82)
		Log‐additive	-	-	-	1.55(0.95-2.55)	0.082

^*P < 0.05 indicates statistical significance.

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Editorial decision: Major revision
09 May, 2021
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Reviewer #2 agreed at journal
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You are reading this latest preprint version

Association of MIR17HG and MIR155HG gene variants with steroid-induced osteonecrosis of the femoral head in the population of northern China

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Study participants

2.2 Genotyping

2.3 Statistical analysis

2.4 KEGG analysis of downstream genes regulated by MIR155HG

3. Results

4. Discussion

5. Conclusion

Declarations

References

tables

Status:

Version 1