Demographic and clinical characteristics of T2DM patients and healthy controls
In brief, the present study included 200 cases of type 2 diabetes mellitus and 200 healthy control subjects [Table 1]. Among the T2DM cases, 68% of patients were males, and 32% were females while in healthy controls, 70% were males, and 30% were females, more details mentioned in Table 1.
Table 1
Demographic and clinical characteristics of enrolled patients with Type 2 diabetes mellitus and healthy controls.
Variables
|
T2DM patients (%)
|
Controls subjects (%)
|
Total no.
|
200 (100)
|
200 (100)
|
Gender
|
Males
|
136 (68)
|
140 (70)
|
Females
|
64 (32
|
60 (30)
|
Age at diagnosis (Years)
|
≤ 40
|
94 (47)
|
100 (50)
|
> 40
|
106 (53)
|
100 (50)
|
Mean age (in years)
|
46.32 ± 7.18
|
47.63 ± 6.76
|
Hypertension
|
Yes
|
122 (61)
|
No
|
78 (39)
|
Increased urination
|
Yes
|
97 (48.5)
|
No
|
103 (51.5)
|
Weight loss
|
|
Yes
|
94 (47)
|
No
|
106 (53)
|
Fatigue
|
|
Yes
|
95 (47.5)
|
No
|
105 (52.5)
|
Wound healing
|
|
Yes
|
84 (42)
|
No
|
116 (58)
|
Blur vision
|
|
Yes
|
67 (33.5)
|
No
|
133 (76.5)
|
Loss of appetite
|
|
Yes
|
91 (45.5)
|
No
|
109 (54.5)
|
Smoking status
|
Yes
|
92 (46)
|
No
|
108 (54)
|
Alcoholism
|
Yes
|
102 (51)
|
No
|
98 (49)
|
Association of anthropometric data and biochemical parameters among T2DM and healthy subjects
Study recorded anthropometric data and biochemical data and depicted in Table 2. In T2DM, Hip, waist, and body mass index (BMI) were 35.97, 32.61, and 29.0 while in healthy subjects, 34.87, 31.65, and 24.82 and differences among them were observed to be statistically significant (p = 0.004, p = 0.02, p < 0.0001) respectively. Blood glucose parameters such as fasting, postprandial, and Hba1c were tested and differences among T2DM and healthy subjects were observed to be significant (p < 0.0001, p < 0.0001, p < 0.0001).
Systolic and diastolic blood presser differences among T2DM and healthy subjects were statistically significant (p < 0.0001, p = 0.0007). Lipid profile analysis such as High-density lipoprotein (HDL), Low-density lipoprotein (LDL), Triglycerides (TG), Very low-density lipoprotein (VLDL), cholesterol showed significant differences among T2DM and healthy control subjects (p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001) respectively.
Table 2
Comparison of anthropometric data and biochemical parameters among T2DM and healthy subjects.
Parameters
|
T2DM cases
(Mean ± SD)
|
Healthy control
(Mean ± SD)
|
p value
|
Age (years)
|
46.32 ± 7.18
|
47.63 ± 6.76
|
0.13
|
Hip (cm)
|
35.97 ± 3.59
|
34.87 ± 1.87
|
0.004
|
Waist (cm)
|
32.61 ± 3.84
|
31.65 ± 2.50
|
0.02
|
Weight (kg)
|
71.64 ± 13.63
|
70.74 ± 7.79
|
0.53
|
Body mass index
|
29.0 ± 5.51
|
24.82 ± 2.33
|
< 0.0001
|
Fasting blood glucose
|
240.0 ± 18.8
|
90.22 ± 7.10
|
< 0.0001
|
Postprandial blood glucose
|
349.0 ± 75
|
136.0 ± 5.0
|
< 0.0001
|
Hba1c
|
7.39 ± 0.77
|
5.22 ± 0.58
|
< 0.0001
|
Blood pressure (systolic)
|
129.4 ± 13.99
|
118.32 ± 10.78
|
< 0.0001
|
Blood pressure (diastolic)
|
86.76 ± 13.31
|
81.90 ± 6.18
|
0.0007
|
High density lipoprotein (HDL)
|
42.42 ± 12.9
|
49.78 ± 6.92
|
< 0.0001
|
Low density lipoprotein (LDL)
|
192.2 ± 27.0
|
124.54 ± 14.20
|
< 0.0001
|
Triglycerides (TG)
|
375.4 ± 117.7
|
140.98 ± 5.52
|
< 0.0001
|
Very low density lipoprotein (VLDL)
|
35.92 ± 5.18
|
26.52 ± 2.53
|
< 0.0001
|
Cholesterol
|
248.5 ± 13.45
|
152.63 ± 18.82
|
< 0.0001
|
LncRNA H19 expression in T2DM patients
It was found that T2DM patients had an overall 0.59-fold expression of lncRNA H19 compared to healthy controls [Table 3]. Among the T2DM patients, a significant difference in lncRNA H19 expression was observed between males and females [P = 0.004]. Males showed 0.70-fold lncRNA H19 expression while females had 0.56-fold lncRNA H19 expression. Hypertensive T2DM patients, had 0.41 fold changes in lncRNA H19 expression, while non-hypertensive patients showed 0.88 fold lncRNA H19 expression, and statistical differences between them were observed to be significant [P < 0.0001]. T2DM patients with weight-loss showed 0.41 fold lncRNA H19 expression, while patients without weight loss showed 0.79 fold lncRNA H19 expression [P = 0.02]. Besides, T2DM patients who had fatigue showed 0.47 fold lncRNA H19 expression, while patients with no fatigue showed 0.73 fold lncRNA H19 expression [P = 0.02].
Table 3
Long non-coding RNA H19 expression in type 2 Diabetes mellitus subjects.
Variables
|
H19 expression [Mean ± SD]
|
P-value
|
Overall expression
|
0.59 ± 0.89
|
-
|
Age [in years]
|
≤ 40 years
|
0.70 ± 1.07
|
0.82
|
> 40 years
|
0.56 ± 0.82
|
Gender
|
Male
|
0.41 ± 0.68
|
0.004
|
Female
|
0.81 ± 1.04
|
Hypertension
|
Yes
|
0.41 ± 0.85
|
< 0.0001
|
No
|
0.88 ± 0.87
|
Urination
|
Yes
|
0.62 ± 0.92
|
0.46
|
No
|
0.56 ± 0.82
|
Weight loss
|
Yes
|
0.41 ± 0.74
|
0.02
|
No
|
0.79 ± 0.99
|
Fatigue
|
Yes
|
0.47 ± 0.78
|
0.02
|
No
|
0.73 ± 0.98
|
Wound healing
|
Yes
|
0.59 ± 0.86
|
0.68
|
No
|
0.59 ± 0.91
|
Blur vision
|
Yes
|
0.50 ± 0.74
|
0.87
|
No
|
0.64 ± 0.95
|
Loss of appetite
|
Yes
|
0.55 ± 0.83
|
0.41
|
No
|
0.64 ± 0.93
|
Smoking
|
Yes
|
0.49 ± 0.78
|
0.13
|
No
|
0.68 ± 0.96
|
Alcoholism
|
Yes
|
0.48 ± 0.75
|
0.52
|
No
|
0.70 ± 1.0
|
Association of microRNA-29a among T2DM patients
T2DM patients showed an overall 5.62 mean fold expression of miRNA-29a compared to healthy controls [Table 4]. T2DM patients who were hypertensive showed 5.96-fold miRNA-29a expression, whereas non-hypertensive reported having 5.09-fold miRNA-29a expression. Besides, the differences between diabetic smoker and non-smoker patients in H19 expression were observed to be statistically significant [P < 0.0001]. We also studied whether cigarette smoking has an impact on miRNA-29a expression. We observed that the T2DM patients with smoking habit showed 5.98-fold miRNA-29a expression, and non-smokers showed 5.24-fold miRNA-29a expression [P = 0.04]. T2DM subjects with the habit of alcohol consumption showed 6.17-fold miRNA-29a expression, while non-alcoholic T2DM patients showed 5.04-fold miRNA-29a expression. Furthermore, a statistically significant correlation was observed in H19 expression between alcoholic and non-alcoholic diabetic patients [P < 0.0001]. Our observations illustrated that miRNA-29a expression in T2DM patients is directly associated with hypertension and alcohol consumptions.
Table 4
miR-29a expression in subjects with Type 2 Diabetes Mellitus.
Variables
|
miRNA-29a expression [Mean ± SD]
|
P-value
|
Overall expression
|
5.62 ± 1.92
|
-
|
Age [in years]
|
≤ 40 years
|
5.57 ± 1.55
|
0.64
|
> 40 years
|
5.63 ± 2.03
|
Gender
|
Male
|
5.69 ± 1.84
|
0.14
|
Female
|
5.53 ± 2.03
|
Hypertension
|
Yes
|
5.96 ± 1.84
|
< 0.0001
|
No
|
5.09 ± 1.95
|
Urination
|
Yes
|
5.58 ± 1.97
|
0.64
|
No
|
5.65 ± 1.89
|
Weight loss
|
Yes
|
5.81 ± 2.14
|
0.21
|
No
|
5.40 ± 1.63
|
Fatigue
|
Yes
|
5.62 ± 1.92
|
0.88
|
No
|
5.62 ± 1.93
|
Wound healing
|
Yes
|
5.67 ± 1.95
|
0.70
|
No
|
5.57 ± 1.92
|
Blur vision
|
Yes
|
5.68 ± 2.0
|
0.65
|
No
|
5.49 ± 1.78
|
Loss of appetite
|
Yes
|
5.74 ± 2.15
|
0.75
|
No
|
5.52 ± 1.72
|
Smoking
|
Yes
|
5.98 ± 1.90
|
0.04
|
No
|
5.24 ± 1.58
|
Alcoholism
|
Yes
|
6.17 ± 2.09
|
< 0.0001
|
No
|
5.04 ± 1.55
|
Association of microRNA-29b expression among T2DM patients
T2DM patients showed an overall 5.58 mean fold expression of miRNA-29b compared to healthy controls [Table 5]. T2DM patients who were hypertensive showed 5.85-fold miRNA-29b expression while non-hypertensive showed 5.17-fold miRNA-29b expression. Statistical analyses revealed differences between analyzed groups [P = 0.0001]. Furthermore, T2DM patients who had weight-loss showed 5.91-fold miRNA-29b expression, while those who did not show weight loss had 5.21-fold miRNA-29b expression [P = 0.002]. Smokers T2DM patients showed 6.16-fold miRNA-29b expression, while non-smokers showed 5.15-fold miRNA-29b expression [P = 0.0002]. Alcoholic T2DM patients showed 6.11-fold miRNA-29b expression, while non-alcoholic T2DM patients showed 5.05-fold miRNA-29b expression [P < 0.0001].
Table 5
miR-29b expression in Type 2 Diabetes Mellitus patients.
Variables
|
miR-29b expression [Mean ± SD]
|
P-value
|
Overall expression
|
5.58 ± 1.83
|
-
|
Age [in years]
|
≤ 40 years
|
5.39 ± 1.73
|
0.41
|
> 40 years
|
5.65 ± 1.86
|
Gender
|
Male
|
5.41 ± 1.79
|
0.25
|
Female
|
5.69 ± 1.97
|
Hypertension
|
Yes
|
5.85 ± 1.68
|
0.0001
|
No
|
5.17 ± 1.97
|
Urination
|
Yes
|
5.71 ± 2.03
|
0.80
|
No
|
5.47 ± 1.62
|
Weight loss
|
Yes
|
5.91 ± 1.86
|
0.002
|
No
|
5.21 ± 1.72
|
Fatigue
|
Yes
|
5.47 ± 1.87
|
0.20
|
No
|
5.69 ± 1.79
|
Wound healing
|
Yes
|
5.69 ± 1.84
|
0.41
|
No
|
5.51 ± 1.82
|
Blur vision
|
Yes
|
6.01 ± 2.13
|
0.09
|
No
|
5.37 ± 1.62
|
Loss of appetite
|
Yes
|
5.58 ± 1.79
|
0.97
|
No
|
5.59 ± 1.87
|
Smoking
|
Yes
|
6.16 ± 2.07
|
0.0002
|
No
|
5.10 ± 1.43
|
Alcoholism
|
Yes
|
6.11 ± 2.04
|
< 0.0001
|
No
|
5.04 ± 1.39
|
Relationship of biochemical parameters with lncRNA H19, miRNA-29a, and miRNA-29b expression among T2DM patients
Regression analysis was done to check the effect of all the parameters such as H19, VLDL, PPG, FPG, BP (Diastolic), Cholesterol, BP (systolic), Hba1c, HDL, TG, LDL with miRNA29a, miRNA29b expression, and the observed R2 value was 0.13 (p = 0.003) and 0.14 (p = 0.001) (Table 6). The beta coefficient was also calculated and the observed beta value for lncH19 to miRNA-29a was − 0.298 (p < 0.0001) and concerning miRNA-29a was-0.028 (p < 0.0001) (Table 7).
Table 6
Association of lncRNA H19 with miRNA29a and miRNA29b.
Parameters
|
R2
|
P-value
|
Dependent Variable: miRNA29a
|
0.13
|
.003
|
Dependent Variable: miRNA29b
|
0.14
|
.001
|
Predictors: (Constant), H19, VLDL, PPG, FPG, BP (Diastolic), Cholesterol, BP (systolic), Hba1c, HDL, TG, LDL.
|
Table 7
Association of lncRNA H19 and biochemical parameters with miRNA29a and miRNA29b.
Variables
|
Standardized Coefficients
|
P-value
|
Standardized Coefficients
|
P-value
|
Beta
|
Beta
|
H19
|
-0.298
|
0.0001
|
-0.285
|
0.0001
|
FPG
|
0.075
|
0.284
|
0.042
|
0.549
|
PPG
|
-0.015
|
0.848
|
-0.124
|
0.124
|
Hba1c
|
0.110
|
0.130
|
0.134
|
0.062
|
BP (systolic)
|
0.115
|
0.111
|
0.122
|
0.090
|
BP(Diastolic)
|
-0.109
|
0.120
|
-0.073
|
0.293
|
HDL
|
-0.106
|
0.189
|
0.102
|
0.202
|
LDL
|
-0.129
|
0.190
|
0.099
|
0.312
|
TG
|
0.089
|
0.354
|
0.013
|
0.893
|
Cholesterol
|
-0.015
|
0.856
|
-0.070
|
0.404
|
VLDL
|
-0.019
|
0.785
|
-0.026
|
0.702
|
Dependent Variable: miRNA29a
|
Dependent Variable: miRNA29b
|
Association of IncRNA H19 with miRNA-29a and miRNA-29b
Significant association of low [≤ 1 fold] and high [> 1 fold] expression of lncRNA H19 with miRNA29a and miRNA29b was observed [Figure 1]. It was analyzed that the T2DM patients who had low expression of lncRNA H19 showed 5.91-fold miRNA-29a expression and high expression of lncRNA H19 showed 4.60 fold changes in miRNA-29a expression and difference among them was found to be statistically significant [P = 0.005]. T2DM patients who had low expression of lncRNA H19 showed 7.21-fold miRNA-29b expression and high expression of lncRNA H19 showed 4.69-fold miRNA-29b expression was observed [P < 0.0001].
Correlation of lncRNA H19 with miRNA-29a and miRNA-29b
A negative correlation of lncRNA H19 with miRNA-29a and miRNA-29b was observed [Figure 2]. It was found that lncRNA H19 expression showed negative correlation with miRNA-29a [r= -0.27, P < 0.0001] as well as negative correlation with miRNA-29b [r= -0.47, P < 0.0001] was also observed.