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Indian Journal of Clinical Biochemistry logoLink to Indian Journal of Clinical Biochemistry
. 2023 Mar 15;39(3):421–428. doi: 10.1007/s12291-023-01131-6

Osteocalcin, miR-143, and miR-145 Expression in Long-Standing Type 1 Diabetes Mellitus and Their Correlation with HbA1c

Nabil A Hasona 1,5,✉, Adel Abdel Moneim 2, Esraa A Mohammed 3, Nabila A Abdul Twab 3, Aya A Abdel Azeem 3, Gehad M Teryak 3, Sheryhan Sh Ewiss 3, Rehab G Khalil 4
PMCID: PMC11239619  PMID: 39005871

Abstract

Inadequate management and control of hyperglycemia predisposes diabetic patients to a wide range of complications. Thus, this opens new windows for exploring and scrutinizing novel candidate biomarkers. This study was designed to scrutinize the relationship between HbA1c, osteocalcin, calcium, phosphorus, and expression levels of miR-143 and miR-145 in individuals with T1DM and explore their correlations and diagnostic potential for T1DM. 120 unrelated participants were included (i.e., 90 participants with type 1 diabetes mellitus and 30 healthy controls) and were allocated into two groups. Participants with T1DM were allocated into three subgroups (i.e., below 1 year, 1–8 years, and over 8 years) based on diabetic duration. Participants with T1DM experienced noticeable HbA1c elevation. However, osteocalcin, phosphorus, and calcium profiles notably declined in participants with diabetes compared with those in healthy controls. Moreover, the expression levels of miR-143 and miR-145 decreased in participants with diabetes with a significant difference between participants with diabetes and healthy controls. Additionally, significant alterations in HbA1c, osteocalcin, phosphorus, and calcium profiles and expression levels of miR-143 and miR-145 were observed with increasing diabetic duration (T1DM > 8 years compared with those with a diabetes duration of less than 1 year). This study suggests that miR-143 and miR-145 are prospective biomarkers of diabetes mellitus, which may help predict the progression of complications.

Keywords: Osteocalcin, miR-143, miR-145, HbA1c, T1DM

Introduction

Diabetes mellitus is a frequent endocrine disease correlated with high mortality and morbidity, and has become a health issue worldwide and expected to affect 642 million individuals by 2040 [1, 2]. Due to the elevation of the biosynthesis of proteins and lipids in their glycated forms and the direct effects of hyperglycemia and insulin deficiency, hyperglycemia is linked to dysfunction and alterations in the functions of numerous systems, including the cardiovascular, skeletal, renal, and nervous systems [3, 4]. Continuous exposure to high levels of sugar, fats, insulin, and inflammation causes cellular dysfunction in various organs, including the pancreas, liver, kidney, brain, retina, and heart [5]. As a result, to monitor the progression of diabetes and organ failure, efficacious bioassays for disease prognosis, diagnosis, and surveillance are needed.

Osteocalcin (carboxyglutamic acid bone protein) is a crucial non-collagenous protein produced and secreted by osteoblasts [6]. Osteocalcin consists of uncarboxylated and carboxylated form, and is necessary for normal bone mineralization, inhibiting abnormal hydroxyapatite formation, and reducing growth cartilage mineralization [7, 8]. Calcium and phosphorus are two electrolytes found in the serum, which are essential for cell and organism functions. These electrolytes play a vital role in intermediate metabolism and cell function, such as enzyme activities, electric gradients, and the maintenance of acid-base homeostasis, and their levels are influenced by blood glucose levels [9, 10].

In numerous studies, microRNAs (miRNAs) were new potential biomarkers for diseases owing to their specificity of expression and constancy in body fluids [11]. miRNAs are non-coding intrinsic RNAs with 19–24 nucleotides, which could modulate several gene expressions by controlling the translation process of target mRNAs. Consequently, the expression of abnormal actions of miRNA may imply pathological disorders, such as metabolic syndromes, diabetes, and cancer [12, 13].

Both miR-143 and miR-145, found on chromosome 5, are two major controllers of multiple genes in the insulin signaling route and pancreatic function in the biosynthesis and secretion of insulin [14]. MiR-143/-145 dysregulation was implicated in the pathogenesis of various disorders, such as cardiac and hypertension disorders [15]. MiR-143/-145 has been demonstrated to inhibit the growth of some cancers, including colorectal and breast cancer [16]. According to a recent study [17], The insulin resistance pathway has been linked to miR-145 by Akt/protein kinase B. Because few clinical studies have focused on miR-143 and miR-145 in individuals with T1DM, more research is needed to assess their relative expression patterns and approve their potential use as screening biomarkers.

To our knowledge, miR-143 and miR-145 relative expression levels in individuals with T1DM with varying diabetic durations and their correlation with OC and electrolytes (i.e., calcium and phosphorus) have not been explored. Thus, this study was designed to scrutinize the relationship between HbA1c, osteocalcin, calcium, phosphorus, and expression levels of miR-143 and miR-145 in individuals with T1DM and explore their correlations and diagnostic potential for T1DM.

Subjects and Methods

Patients and Study Design

In this study, 120 unrelated participants were analyzed; the participants were divided into two main groups: Group I contained healthy controls; who have no significant health-related issues and glycated hemoglobin (HbA1c) < 6% and Group II consisted of individuals diagnosed with T1DM, according to the criteria of the World Health Organization and glycated hemoglobin (HbA1c) > 6%. Informed written consent was received from the parents of all children, or from adult participants. The study protocol was performed as per the declaration of Helsinki and good clinical practice guidelines, and the General Institutions of Healthy Insurance Committee gave its approval (GIHIC/2020/11).

Based on the diabetic duration, T1DM enrolled participants were divided into three subgroups. Group IIa consisted of T1DM participants whose diabetic duration below 1 year. Group IIb included T1DM participants whose diabetic duration 1–8 years. Group IIc comprised T1DM participants whose diabetic duration more than 8 years. Meanwhile, individuals with diabetes who were pregnant, those who had anemia, and those with liver and renal dysfunction were excluded from the study.

Laboratory Assays

Venous blood was collected in the morning after overnight fasting before the insulin injections were administered. In order to determine the glycated haemoglobin (HbA1c%) level, blood samples were collected on ethylenediaminetetraacetic acid (EDTA). The reagent kit obtained from Cobas Integra 800(Roche, Basel, Switzerland) was used to estimate HbA1c%. Moreover, serum ionized calcium, total calcium, and phosphorus levels were assessed using the auto-analyzer technique (Cobas Integra 6000 from Roche Diagnostics, Germany) and measured using standard methods. Epitope Diagnostics's equipment (San Diego, CA92121, USA) was used to measure the levels of serum osteocalcin utilising the enzyme-linked immunosorbent assay method, according to Nagasue et al. [18].

miR-143 and miR-145 Expression Analysis

The total RNA was isolated from the serum samples using the Direct-zol RNA Miniprep Plus kit (Cat # R2072, Zymo Research, Irvine, CA, USA), and RNA quality was assessed using a Beckman dual spectrophotometer (Brea, CA, USA). qRT-PCR was used to assess the relative expression of miR-143 and miR-145. RNA was isolated using an RNA purification kit (Qiagen, Valencia, CA). The isolated RNA was measured, and cDNA was synthesised using a high-capacity cDNA reverse transcription kit (Qiagen, Valencia, CA), and the acquired cDNA was amplified using the Maxima SYBR Green master mix (Qiagen) and the primers specific for miR-143 F: 5′-AAGCTTAAGGGTTTCCGGTACTT-3′; and R 5′ -GCAAATTCGTGAGCGTTCCATA-3′ for U6, and 5′ -CGCTTCACGAATTTGCGTGTCAT-3′ for R. The acquired data were analysed using the 2−ΔΔCt method [19].

Statistical Analysis

The results data were presented as mean ± standard deviation (SD). SPSS 22(SPSS, Chicago,IL) Software was used for statistical analysis and multiple comparisons. One-way ANOVA and Duncan’s post-hoc test were used. Shapiro-test using the R-programming packages to check the normality of the results data. The diagnostic power of miR-143 and miR-145 was assessed using the area under the ROC curve (AUC), sensitivity, and specificity. P values less than 0.05 were considered significant.

Results

In this study, we included 30 participants with T1DM whose diabetes duration was more than 8 years and 30 healthy individuals. Table 1 summarizes the characteristics of the study participants. Regarding glycemic status, all participants with diabetes had significantly higher rates of HbA1c than healthy individuals (P < 0.05). However, osteocalcin, phosphorus, ionized calcium, and total calcium levels in participants with T1DM were considerably different from those in healthy subjects (Table 1). Likewise, HbA1c exhibited non-significant elevation in Groups IIb and IIc HbA1c as compared to Group IIa. However, significant differences in osteocalcin and total calcium levels were observed between Groups IIb and IIc and Group IIa. Moreover, phosphorus, ionized calcium, and total calcium levels differed significantly between Groups IIb and IIc (Table 1).

Table 1.

Demographics and characteristics of the participants in the study

Variables Healthy controls All diabetic subjects Group 1 Group 2 Group 3
Age 18.00 ± 2.69 16.11 ± 6.46 11.47 ± 4.80 15.37 ± 4.48 21.50 ± 5.65
Gender (M/F) 18/12 43/47 17/13 14/16 12/18
Duration – 5.95 ± 5.47 0.42 ± 0.24 4.93 ± 2.03 12.50 ± 3.27
HbA1C (%) 4.14 ± 0.15a 9.38 ± 2.33b 9.14 ± 1.91b 9.16 ± 2.25b 9.84 ± 2.75b
Phosphorus (mg/dl) 5.61 ± 0.73d 3.71 ± 0.55b 4.11 ± 0.42c 3.91 ± 0.31bc 3.10 ± 0.24a
Ionized Calcium (mg/dl) 1.15 ± 0.13c 0.90 ± 0.10b 0.93 ± 0.06b 0.93 ± 0.08b 0.84 ± 0.11a
Total calcium (mg/dl) 10.00 ± 1.07c 8.02 ± 0.82b 8.51 ± 0.77c 8.05 ± 0.42b 7.50 ± 0.87a
Osteocalcin (ng/ml) 38.20 ± 5.64d 23.30 ± 4.70b 28.49 ± 2.84c 21.52 ± 3.33ab 19.88 ± 2.31a
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Data were expressed as mean ± SD; According to the Duncan multiple range test, the different letters indicate statistical significance different means. G1: Type-1 diabetic patients with a diabetes duration of less than one year; G2: Type-1 diabetic patients with diabetes duration of one to eight years; G3: Type-1 diabetic patients with a diabetes duration of more than eight years; HbA1c: glycated hemoglobin

To determine whether miR-143 and miR-145 were differentially expressed in the serum of individuals with diabetes and control individuals, their expression was measured using quantitative real-time PCR. T1DM participants had lower miR-143 and miR-145 expression levels. Furthermore, the expression levels of miR-143 and miR-145 in Groups IIb and IIc showed a substantial decline as compared to Group IIa (Table 2).

Table 2.

Expression fold of miR-143 and miR-145 regarding diabetic participants

Variables Healthy control All diabetic patients Group 1 Group 2 Group 3
miR-143 0.98 ± 0.07d 0.38 ± 0.24b 0.68 ± 0.10c 0.33 ± 0.05b 0.14 ± 0.07a
miR-145 1.07 ± 0.05d 0.46 ± 0.23b 0.75 ± 0.06c 0.43 ± 0.07b 0.22 ± 0.05a
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Data were expressed as mean ± SD; According to the Duncan multiple range test, the different letters indicate statistical significance different means

Pearson correlation analysis was used to evaluate the association between the expression of miR-143 and miR-145, glycemic status (HbA1c), electrolyte profile (calcium and phosphorus), and osteocalcin. Negative significant correlation between electrolyte profile, osteocalcin, miR-143, and miR-145 were found with HbA1c among study participants (Fig. 1). Additionally, Negative significant correlation between electrolyte profile, osteocalcin, miR-143, and miR-145 were found with diabetic duration among enrolled participants (Fig. 2).

Fig. 1.

Fig. 1

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Correlations between osteocalcin, phosphorus, total calcium, miR-143, and miR-145 with HbA1c level among enrolled participants

Fig. 2.

Fig. 2

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Correlation between electrolyte profile, osteocalcin, miR-143, and miR-145 with diabetic duration among enrolled participants

Serum miR-143 discriminate T1DM enrolled participants from healthy controls with a cut-off value of 0.83, sensitivity was 96.67% sensitivity, 100% specificity, area under the ROC curve [AUC] = 0.998; and 95% confidence interval [CI] = 0.994–1.002 (Fig. 3a). Likewise, serum miR-145 with a cut-off value of 0.930, had 100% sensitivity and 100% specificity in discrimination T1DM enrolled participants from healthy controls, with AUC = 1.00, P < 0.001 (Fig. 3a). Additionally, serum miR-143 distinguished Group IIc from T1DM enrolled participants, with AUC = 0.982, cut-off value of 0.193, sensitivity of 90%, and specificity of 100%. Likewise, serum miR-145 had AUC = 0.994, cut-off value 0.28, 93.33% sensitivity, and 100% specificity for discrimination Group IIc from T1DM enrolled participants (Fig. 3c, d).

Fig. 3.

Fig. 3

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a, b ROC analysis regarding miR143 and miR145 between diabetic and healthy participants. c, d ROC analysis regarding serum miR-143 and miR145 diabetic group with diabetes duration more than 8 years

Discussion

Inadequate management and control of hyperglycemia predisposes diabetic patients to a wide range of complications [20]. This opens new windows for exploring and scrutinizing novel candidate biomarkers. Thus, this study was designed to scrutinize the relationship between HbA1c, osteocalcin, calcium, phosphorus, and expression levels of miR-143 and miR-145 in individuals with T1DM and explore their correlations and diagnostic potential for T1DM.

Glycosylated hemoglobin (HbA1c) is considered a credible biomarker for evaluating glycemic control efficacy over the three previous months, referring to the lifetime of erythrocytes [21, 22]. HbA1c levels showed a remarkable increase in T1DM participants with healthy controls. Our results are compatible with Maggio et al. [23] and Pater et al. [24], which reported that T1DM patients exhibited a noticeable elevation in HbA1c compared with healthy controls.

Our study revealed considerable alterations in OC and the electrolyte bone matrix profile (calcium and phosphorus) in T1DM patients when compared to healthy controls in terms of both. Moreover, OC was negatively correlated with HbA1c, indicating that the decline in OC is linked to bone and electrolyte (calcium and phosphorus) alterations. Meanwhile, levels of serum phosphorus and calcium were lower in participants with diabetes than in healthy subjects. Similar findings have been reported in patients with diabetes where osteocalcin levels declined as compared to healthy participants [25]. Moreover, our findings agreed with the results obtained by Starup-Linde [26], Maddaloni et al. [27], and Napoli et al. [28], who demonstrated a correlation between biomarkers of bone turnover, particularly OC, and glycemic status, such as HbA1c. Additionally, Wang et al. [7] proposed that low serum osteocalcin levels were linked to hyperglycemia and had an effect on insulin resistance. Moreover, our findings corroborate the findings of Leão et al. [29], who reported that lower levels of osteocalcin in patients with T1DM were associated with lower bone mass.

In T1DM patients, changes in bone mass are brought on by two types of pathophysiological factors, those that delay the formation of bone and those that elevate the risk of falls and other physical trauma. Persistent high glucose, as well as low insulin levels, and the biosynthesis of advanced glycosylation end products are some factors that may stimulate cells responsible for the synthesis of organic components of bone matrix, collagen, and glycoproteins and cells responsible for the resorption of the bone matrix and remodeling of bone tissues [30]. Thus, the inabilities of bone cells in hyperglycemia are linked to suspicious behavior of endothelial progenitors, adipocytes, and mesenchymal cells and may have a greater impact on adipogenesis and deposition of fat, resulting in bone quality deterioration and an increased osteoporosis risk [31]. Calcium is a divalent metal that helps muscle contraction, nerve excitability, and blood clots and affects the secondary messenger system. Furthermore, blood levels of calcium should maintained within a tight range; otherwise, unfavorable physiological changes will occur quickly. Low calcium levels in peripheral cells have linked to decrease insulin secretion and action [32].

The miR-143/miR-145 expression levels was assessed in patients with T1DM and compared with that in healthy participants. The miR-143 and miR-145 expression levels showed a significant decrease in T1DM participants. Likewise, the expression levels of miR-143/miR-145 was significantly lower in T1DM participants who had diabetes for more than eight years than in those who had diabetes for less than a year. Pearson’s correlations revealed a negative relationship between the expression levels of miR-143/miR-145 and HbA1c.

To assess the potential role of miR-143/miR-145 in diagnosing T1DM, an ROC curve was developed. According to our findings, to distinguish participants with T1DM from healthy participants, the AUC was 0.861 and 1.00, respectively (P < 0.001). This implies the impact of the differential expression of miR-145 as a suitable diagnostic biomarker for predicting the progress of diabetes status. Our findings support the study by He et al. [33] and previous studies of Shahrokhi et al. [34] and Shyu et al. [35], which reported that the relative expression of miR-145 in cultured pancreatic cells differs from that in controls under hyperglycemic circumstances. Furthermore, our findings corroborate those of Ge et al. [36]; Lan, Albinsson [37], who found that the relative expression of miR-145 reduced diabetic vascular smooth muscle cells.

Circulating non-coding RNAs have been linked to the pathogenesis of hypertension, atherosclerosis, cancer, and coronary artery disease owing to their high stability in various bodily fluids, so non-coding RNAs have been pointed out as diagnostic biomarkers [38]. Therapeutic strategies using miRNA mechanisms have been viewed as a promising route for clinical trials in treating many disorders [11, 39]. miR-143 and miR-145 have previously been identified as regulators of insulin signaling and glucose absorption in vascular smooth muscle cells [36, 37]. Therefore, miR-143/miR-145 targeting is a promising therapeutic option for managing diabetes mellitus and its complications. Downregulation of miR-143 and miR-145, which are linked to hyperglycemia, causes widespread damage to the macro-and microvasculature in various organs and tissues and disrupts the endogenous vascular repair mechanism, influencing OC decline and altering levels of calcium and phosphorus. Thus, miR-143 and miR-145 could be considered potency factors for diabetes mellitus in predicting the progression of diabetic complications, which could help determine how diabetic complications evolve.

As far as we know, this is the first study that has assessed the relationship between HbA1c and OC, calcium profile, phosphorus, and expression levels of microRNAs (i.e., miR-143 and miR-145) in T1DM patients with different diabetic durations. However, this study has numerous limitations, including the sample size and the assessment of bone density score and inflammatory biomarkers. Therefore, further large-scale studies and clinical trials are needed; moreover, the functional impacts of these markers on putative target genes and pathways should be evaluated.

Conclusion

The study revealed that patients with T1DM have a trend toward significant alterations in HbA1c, osteocalcin, calcium, phosphorus, and molecular expression levels of miR-143 and miR-145 with increasing diabetic duration (T1DM > 8 years compared with those with a diabetes duration of less than 1 year). Thus, the increasing duration of diabetes without respectable management has been associated with a higher incidence of diabetic complications. This study also revealed that miR-143 and miR-145 are promising candidates for the potential diagnosis of T1DM, which may alleviate the progression of diabetic complications, and pending further studies to explore their therapeutic target in diabetic complications.

Data availability

All data generated or analysed during this study are included in this published article.

Declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This study was conducted in compliance with the Declaration of Helsinki, and the General Institutions of Healthy Insurance Committee provided its approval (GIHIC/2020/11).

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Data Availability Statement

All data generated or analysed during this study are included in this published article.

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