A study of microRNA Prescreening that targeted and regulated Vitamin D rceptor in tuberculosis patients

doi:10.3877/cma.j.issn.1674-6902.2020.06.003

Abstract

Abstract:

Objective

MicroRNA with significantly differential expression and targeted regulation of Vitamin D receptor(VDR) in Tuberculosis were screened by High-throughput sequencing and bioinformatics methods, whichprovided the further study of Vitamin D anti-tuberculosis mechanism and clinical application.

Method

According to the inclusion and exclusion criteria, 5 patients with definite diagnosis of tuberculosis and 5 non-tuberculosis volunteers were selected as the study group and control group. Peripheral blood mononuclear cells(PBMC) of the two groups were collected as clinical samples. After total RNA was extracted, high-throughput sequencing technology was used to detect the differential expression of miRNA between the two groups.

Result

The clinical baseline data of 5 tuberculosis patients and 5 non-tuberculosis volunteers were collected, and the homogeneity and heterogeneity of 23 items between the two groups were obtained by statistical analysis. It was found that there were no significatly statistical differences in gender, age, smoking and drinking habits between the two groups. Body mass index (BMI), total cholesterol (TC), hemoglobin (Hb) albumin (ALB), globulin (GLB) and total protein (TP) in the study group were lower than those in the control group with significatly statistical differences(P<0.05). A total of 1560 miRNAs were screened out by miRNA sequencing. Through stratified cluster and comprehensive volcanic map analysis, 23 miRNAs with the most significant (P<0.05) differences were found in the case group, among which 15 were down-regulated(∣LOG2FC∣>1) and 8 were up-regulated(FC>1). In short, Combined with bioinformatics predictions, two miRNAs targeted VDR that differentially expressed in tuberculosis patients were screened out: hsa-miR-326(P<0.05、FC=1.37)、hsa-miR-654-5p(P<0.05、∣LOG2FC∣=1.06).

Conclusion

These two differentially expressed miRNAs mentioned abovemay be potential diagnostic markers and new therapeutic targets for tuberculosis.This found provides a new strategy and feasible direction for the prevention and treatment of Tuberculosis.

Key words: Tuberculosis, microRNA, Vitamin D, High-throughput sequencing, Bioinformatics

Min Xiao, Song Yang, Yang Chen, Tongxin Li, Shiming Yang, Hui Lin. A study of microRNA Prescreening that targeted and regulated Vitamin D rceptor in tuberculosis patients[J]. Chinese Journal of Lung Diseases(Electronic Edition), 2020, 13(06): 731-736.

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Figures/Tables 5

References 21

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临床资料	研究组(n＝5)	对照组(n＝5)	P值
年龄(岁)	44.2±9.24	51.60±20.29	0.491
性别[男(%)]	100.00	100.00	1.000
身高(cm)	165.6±5.86	162.8±11.54	0.642
体重(kg)	55.5±5.77	63.1±14.09	0.242
体重指数(BMI)	20.19±0.79	23.53±3.2	0.036
吸烟史	0.8±0.45	0.2±0.45	0.067
饮酒史	0.2±0.45	0.4±0.55	0.545
WBC(10⁹/L)	6.442±1.52	6.93±1.71	0.641
Hb(g/L)	114±15.24	148.60±27.64	0.042
PLT(10⁹)	297.6±86.11	246.40±74.84	0.345
TC(mmol/L)	2.72±0.63	5.12±0.78	0.001
TG(mmol/L)	0.99±0.24	2.10±1.74	0.197
HDL(mmol/L)	1.13±0.20	1.32±0.18	0.147
LDL(mmol/L)	1.91±0.25	2.69±0.82	0.077
ALT(IU/L)	17.4±14.10	27.90±9.99	0.211
AST(IU/L)	26.40±10.78	23.24±3.86	0.554
ALB(g/L)	35.9±5.78	51.98±3.38	0.001
GLB(g/L)	24.26±1.91	27.88±1.51	0.010
TP(g/L)	60.16±4.78	79.86±4.10	0.000
Cr(μmol/L)	63.54±12.96	77.42±18.29	0.204
BUN(mmol/L)	4.92±0.69	5.18±2.00	0.790
TBILI(μmol/L)	13.62±4.23	15.74±4.94	0.487
GLU(mmol/L)	7.14±4.16	4.96±0.82	0.283

临床资料	研究组(n＝5)	对照组(n＝5)	P值
年龄(岁)	44.2±9.24	51.60±20.29	0.491
性别[男(%)]	100.00	100.00	1.000
身高(cm)	165.6±5.86	162.8±11.54	0.642
体重(kg)	55.5±5.77	63.1±14.09	0.242
体重指数(BMI)	20.19±0.79	23.53±3.2	0.036
吸烟史	0.8±0.45	0.2±0.45	0.067
饮酒史	0.2±0.45	0.4±0.55	0.545
WBC(10⁹/L)	6.442±1.52	6.93±1.71	0.641
Hb(g/L)	114±15.24	148.60±27.64	0.042
PLT(10⁹)	297.6±86.11	246.40±74.84	0.345
TC(mmol/L)	2.72±0.63	5.12±0.78	0.001
TG(mmol/L)	0.99±0.24	2.10±1.74	0.197
HDL(mmol/L)	1.13±0.20	1.32±0.18	0.147
LDL(mmol/L)	1.91±0.25	2.69±0.82	0.077
ALT(IU/L)	17.4±14.10	27.90±9.99	0.211
AST(IU/L)	26.40±10.78	23.24±3.86	0.554
ALB(g/L)	35.9±5.78	51.98±3.38	0.001
GLB(g/L)	24.26±1.91	27.88±1.51	0.010
TP(g/L)	60.16±4.78	79.86±4.10	0.000
Cr(μmol/L)	63.54±12.96	77.42±18.29	0.204
BUN(mmol/L)	4.92±0.69	5.18±2.00	0.790
TBILI(μmol/L)	13.62±4.23	15.74±4.94	0.487
GLU(mmol/L)	7.14±4.16	4.96±0.82	0.283

编　号	miRNA名称	miRNA序列	上调/下调	倍数(FC)	倍数(log2FC)	P值(T检验)
1	hsa-miR-1291_L+1R+1	GTGGCCCTGACTGAAGACCAGCAGTT	up	3.91	1.97	0.004
2	hsa-miR-27a-3p_R-1	TTCACAGTGGCTAAGTTCCG	up	1.37	0.45	0.006
3	hsa-miR-339-3p_R-2	TGAGCGCCTCGACGACAGAGC	up	1.52	0.61	0.016
4	hsa-miR-326_R+1	CCTCTGGGCCCTTCCTCCAGT	up	1.37	0.45	0.021
5	hsa-mir-6516-p3	TGTATGATACTGCAAACAGGA	up	3.13	1.65	0.034
6	hsa-miR-301a-3p	CAGTGCAATAGTATTGTCAAAGC	up	6.26	2.65	0.037
7	hsa-miR-30b-5p	TGTAAACATCCTACACTCAGCT	up	1.46	0.55	0.046
8	hsa-miR-30d-3p	CTTTCAGTCAGATGTTTGCTGC	up	2.9	1.54	0.047
9	hsa-miR-432-5p	TCTTGGAGTAGGTCATTGGGTGG	down	0.4	-1.34	0.000
10	hsa-miR-134-5p	TGTGACTGGTTGACCAGAGGGG	down	0.46	-1.11	0.001
11	hsa-miR-485-5p_R+1	AGAGGCTGGCCGTGATGAATTCG	down	0.5	-1.01	0.008
12	hsa-miR-4667-3p_R+1	TCCCTCCTTCTGTCCCCACAGT	down	0.27	-1.91	0.011
13	hsa-miR-127-3p	TCGGATCCGTCTGAGCTTGGCT	down	0.43	-1.21	0.012
14	hsa-miR-433-3p	ATCATGATGGGCTCCTCGGTGT	down	0.44	-1.18	0.012
15	hsa-miR-493-5p	TTGTACATGGTAGGCTTTCATT	down	0.47	-1.08	0.012
16	hsa-miR-382-5p	GAAGTTGTTCGTGGTGGATTCG	down	0.47	-1.1	0.014
17	hsa-miR-323a-3p_L+1	GCACATTACACGGTCGACCTCT	down	0.48	-1.07	0.018
18	hsa-miR-431-5p_R-1	TGTCTTGCAGGCCGTCATGC	down	0.4	-1.33	0.018
19	hsa-miR-654-5p	TATGTCTGCTGACCATCACC	down	0.48	-1.06	0.020
20	hsa-miR-941	CACCCGGCTGTGTGCACATGTGC	down	0.29	-1.81	0.021
21	hsa-miR-409-3p	GAATGTTGCTCGGTGAACCCCT	down	0.49	-1.03	0.022
22	hsa-miR-758-3p_R-1	TTTGTGACCTGGTCCACTAAC	down	0.5	-1.01	0.032
23	hsa-miR-550a-3-5p_R+1	AGTGCCTGAGGGAGTAAGAGA	down	0.23	-2.11	0.048

编　号	miRNA名称	miRNA序列	上调/下调	倍数(FC)	倍数(log2FC)	P值(T检验)
1	hsa-miR-1291_L+1R+1	GTGGCCCTGACTGAAGACCAGCAGTT	up	3.91	1.97	0.004
2	hsa-miR-27a-3p_R-1	TTCACAGTGGCTAAGTTCCG	up	1.37	0.45	0.006
3	hsa-miR-339-3p_R-2	TGAGCGCCTCGACGACAGAGC	up	1.52	0.61	0.016
4	hsa-miR-326_R+1	CCTCTGGGCCCTTCCTCCAGT	up	1.37	0.45	0.021
5	hsa-mir-6516-p3	TGTATGATACTGCAAACAGGA	up	3.13	1.65	0.034
6	hsa-miR-301a-3p	CAGTGCAATAGTATTGTCAAAGC	up	6.26	2.65	0.037
7	hsa-miR-30b-5p	TGTAAACATCCTACACTCAGCT	up	1.46	0.55	0.046
8	hsa-miR-30d-3p	CTTTCAGTCAGATGTTTGCTGC	up	2.9	1.54	0.047
9	hsa-miR-432-5p	TCTTGGAGTAGGTCATTGGGTGG	down	0.4	-1.34	0.000
10	hsa-miR-134-5p	TGTGACTGGTTGACCAGAGGGG	down	0.46	-1.11	0.001
11	hsa-miR-485-5p_R+1	AGAGGCTGGCCGTGATGAATTCG	down	0.5	-1.01	0.008
12	hsa-miR-4667-3p_R+1	TCCCTCCTTCTGTCCCCACAGT	down	0.27	-1.91	0.011
13	hsa-miR-127-3p	TCGGATCCGTCTGAGCTTGGCT	down	0.43	-1.21	0.012
14	hsa-miR-433-3p	ATCATGATGGGCTCCTCGGTGT	down	0.44	-1.18	0.012
15	hsa-miR-493-5p	TTGTACATGGTAGGCTTTCATT	down	0.47	-1.08	0.012
16	hsa-miR-382-5p	GAAGTTGTTCGTGGTGGATTCG	down	0.47	-1.1	0.014
17	hsa-miR-323a-3p_L+1	GCACATTACACGGTCGACCTCT	down	0.48	-1.07	0.018
18	hsa-miR-431-5p_R-1	TGTCTTGCAGGCCGTCATGC	down	0.4	-1.33	0.018
19	hsa-miR-654-5p	TATGTCTGCTGACCATCACC	down	0.48	-1.06	0.020
20	hsa-miR-941	CACCCGGCTGTGTGCACATGTGC	down	0.29	-1.81	0.021
21	hsa-miR-409-3p	GAATGTTGCTCGGTGAACCCCT	down	0.49	-1.03	0.022
22	hsa-miR-758-3p_R-1	TTTGTGACCTGGTCCACTAAC	down	0.5	-1.01	0.032
23	hsa-miR-550a-3-5p_R+1	AGTGCCTGAGGGAGTAAGAGA	down	0.23	-2.11	0.048

编　号	miRNA名称	3′非翻译区位置	种子匹配数	上下文得分
1	hsa-miR-654-5p	491-498	8mer	99
2	hsa-miR-541-3p	491-498	8mer	99
3	hsa-miR-302a-3p	3157-3164	8mer	98
4	hsa-miR-302b-3p	3157-3164	8mer	98
5	hsa-miR-302d-3p	3157-3164	8mer	98
6	hsa-miR-372-3p	3157-3164	8mer	98
7	hsa-miR-373-3p	3157-3164	8mer	98
8	hsa-miR-520e	3157-3164	8mer	98
9	hsa-miR-520a-3p	3157-3164	8mer	98
10	hsa-miR-520b	3157-3164	8mer	98
11	hsa-miR-520c-3p	3157-3164	8mer	98
12	hsa-miR-520d-3p	3157-3164	8mer	98
13	hsa-miR-532-3p	1859-1866	8mer	98
14	hsa-miR-302e	3157-3164	8mer	98
15	hsa-miR-556-3p	3130-3137	8mer	97
16	hsa-miR-10a-5p	1694-1701	8mer	95
17	hsa-miR-10b-5p	1694-1701	8mer	95
18	hsa-miR-3619-5p	3052-3059	8mer	95
19	hsa-miR-1913	251-258	8mer	87
20	hsa-miR-3194-5p	2937-2943	7mer-m8	84
21	hsa-miR-520d-5p	2605-2612	8mer	80
22	hsa-miR-4640-5p	2638-2644	7mer-m8	80
23	hsa-miR-2467-3p	115-121	7mer-m8	79
24	hsa-miR-524-5p	2605-2612	8mer	78
25	hsa-miR-326	285-292	8mer	76
26	hsa-miR-4726-5p	2638-2644	7mer-m8	70
27	hsa-miR-3918	2534-2540	7mer-m8	68
28	hsa-miR-324-3p	1081-1087	7mer-m8	62
29	hsa-miR-761	316-322	7mer-1A	45
30	hsa-miR-214-3p	2542-2548	7mer-m8	43
31	hsa-miR-330-5p	1058-1064	7mer-m8	39
32	hsa-miR-515-5p	902-908	7mer-1A	21
33	hsa-miR-519e-5p	902-908	7mer-1A	21

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