Evaluation of microRNAs as potential biomarkers in circulating HPV-DNA-positive non-small cell lung cancer patients

Figures & data

Figure 1. Detection of HPV-DNA in the peripheral blood of patients with non-small cell lung cancer. (a)HPV universal primer amplification electrophoresis map; (b)HPV16-specific primer amplification electropherogram; (c)HPV18-specific primer amplification electropherogram. Note: M: marker; PC: positive control

Table 1. Comparison of clinical-pathological parameters betweenHPV16-DNA-positive and HPV16-DNA-negative non-small cell lung cancer patients

parameters	HPV16-DNA(+)	HPV16-DNA(-)	P value
Age,y
≥60	9	50	0.81
<60	7	34
Gender
Men	4	39	0.17
Women	12	45
Pathological type
Squamous cell carcinoma	2	39	<0.05
Adenocarcinoma	14	45
Tumor grade
poor	9	24	<0.05
Moderate/high	7	60
Tumor diameter
≥3 cm	12	45	0.17
<3 cm	4	39
Tumor stage
I–II	3	41	<0.05
III–IV	13	43
Lymph node metastasis
Yes	15	47	<0.01
No	1	37

Table 2. Differentially expressed miRNAs in lung adenocarcinoma

miRNA	P value	FDR value	Change in expression level
hsa-miR-183	6.21E-39	1.09E-36	up
hsa-miR-9-3	1.67E-32	2.82E-30	up
hsa-miR-200a	2.83E-25	4.70E-23	up
hsa-miR-210	7.15E-25	1.17E-22	up
hsa-miR-182	3.43E-19	5.35E-17	up
hsa-miR-141	1.06E-15	1.55E-13	up
hsa-miR-142	2.29E-15	3.29E-13	up
hsa-miR-3607	2.25E-14	3.19E-12	up
hsa-miR-29b-1	7.00E-14	9.80E-12	up
hsa-miR-21	2.41E-13	3.35E-11	up
hsa-miR-486-2	1.26E-18	1.93E-16	down
hsa-miR-486-1	1.48E-18	2.27E-16	down
hsa-miR-451a	2.28E-10	2.81E-08	down
hsa-miR-144	5.22E-09	6.05E-07	down

Table 3. Differentially expressed miRNAs in lung squamous cell carcinoma

miRNA	P value	FDR value	Change in expression level
hsa-miR-183	3.73E-48	6.53E-46	up
hsa-miR-224	1.04E-42	1.80E-40	up
hsa-miR-130b	9.11E-42	1.57E-39	up
hsa-miR-9-3	1.15E-38	1.94E-36	up
hsa-miR-205	4.20E-35	6.97E-33	up
hsa-miR-149	3.52E-34	5.74E-32	up
hsa-miR-210	8.69E-33	1.39E-30	up
hsa-miR-182	4.63E-31	7.32E-29	up
hsa-miR-345	4.40E-30	6.74E-28	up
hsa-miR-33a	4.95E-20	5.74E-18	up
hsa-miR-375	7.82E-38	1.31E-35	down
hsa-miR-30a	2.56E-30	4.00E-28	down
hsa-miR-139	2.91E-30	4.52E-28	down
hsa-miR-338	3.52E-30	5.43E-28	down
hsa-miR-3065	3.15E-29	4.73E-27	down
hsa-miR-486-1	1.78E-25	2.44E-23	down
hsa-miR-486-2	3.81E-25	5.18E-23	down
hsa-miR-30d	7.19E-25	9.71E-23	down
hsa-miR-144	8.33E-22	1.03E-19	down
hsa-miR-451a	2.65E-20	3.16E-18	down

Figure 2. Heat map clustering of differentially expressed miRNA microarray data. (a) Heat map of differentially expressed miRNAs in lung adenocarcinoma; (b) heat map of differentially expressed miRNAs in lung squamous cell carcinoma. Red represents upregulated miRNAs; the darker the color, the more pronounced the upregulation. Green represents downregulated miRNAs; the darker the color, the more pronounced the downregulation

Figure 3. The expression levels of eight miRNAs in the plasma of normal human subjects, HPV16-DNA (-) NSCLC patients, and HPV16-DNA (+) NSCLC patients were measured. RT-qPCR was used to analyze the plasma levels of miR-182 (a), miR-183 (b), miR-9-3 (c), miR-210 (d), miR-144 (e), miR-451-a (f), miR-486-1 (g), and miR-486-2 (h) in 52 healthy subjects, 84 HPV16-DNA (-) NSCLC patients, and 16 HPV16-DNA (+) NSCLC patients. Note: *: P< .05, **: P < .01, ns: P > .05; the smaller the ΔCt value, the higher the expression

Table 4. The efficacy of plasma miR-182, miR-183, miR-210 and miR-144 to distinguish HPV16-DNA-positive NSCLC cases from healthy controls

	AUC	Cutoff	Sensitivity(%)	Specificity(%)	Yoden index
miR-182	0.858	2.70	93.75	71.15	0.6490
miR-183	0.843	2.44	75.00	82.69	0.5769
miR-210	0.950	2.61	93.75	86.54	0.8029
miR-144	0.881	2.93	81.25	80.77	0.6202
miR-182+ miR-183	0.881	0.2825	81.25	82.69	0.6394
miR-182+ miR-210	0.953	0.1763	93.75	86.54	0.8029
miR-182+ miR-144	0.936	0.22945	87.50	90.38	0.7788
miR-183+ miR-210	0.952	0.24153	93.75	92.31	0.8606
miR-183+ miR-144	0.945	0.4238	81.25	100.00	0.8125
miR-210+ miR-144	0.987	0.17859	93.75	98.08	0.9183

Figure 4. Analysis of the value of four miRNAs for the diagnosis of HPV16-DNA-positive NSCLC and healthy individuals. (a-d) The ROC curve was used to assess the value of miR-182, miR-183, miR-210 and miR-144 in the diagnosis of HPV16-DNA-positive NSCLC. (miR-182, 0.858, P < .0001; miR-183, 0.843, P < .0001; miR-210, 0.950, P < .0001; and miR-144, 0.881, P < .0001.)

Figure 5. Analysis of the value of combined miRNAs for the diagnosis of HPV16-DNA-positive NSCLC and healthy individuals. (a-f) The ROC curve shows that for the distinction between HPV16-DNA-positive NSCLC and healthy individuals, the combined detection of the two miRNAs is more accurate than the single miRNA

Table 5. Efficacy of plasma miR-182, miR-183, miR-210 and miR-144 to distinguish HPV16-DNA-positive NSCLC patients from HPV16-DNA-negative NSCLC patients

	AUC	Cutoff	Sensitivity(%)	Specificity(%)	Yoden index
miR-182	0.746	2.10	87.50	60.71	0.4821
miR-183	0.753	2.44	75.00	70.24	0.4524
miR-210	0.865	1.05	68.75	92.86	0.6161
miR-144	0.729	5.33	50.00	91.67	0.4167
miR-182+ miR-183	0.781	0.13392	81.25	64.29	0.4554
miR-182+ miR-210	0.864	0.32247	68.75	91.67	0.32247
miR-182+ miR-144	0.875	0.08631	87.50	80.95	0.6845
miR-183+ miR-210	0.878	0.10537	93.75	67.86	0.6161
miR-183+ miR-144	0.818	0.10322	81.25	90.48	0.7173
miR-210+ miR-144	0.938	0.04541	93.75	94.05	0.8780

Figure 6. Value analysis of four miRNAs for the diagnosis of HPV16-DNA-positive NSCLC patients and HPV16-DNA-negative NSCLC patients. (a-d) The ROC curve was used to assess the value of miR-182, miR-183, miR-210 and miR-144 in the diagnosis of HPV16-DNA-positive NSCLC. (miR-182, 0.746, P< .0001; miR-183, 0.753, P < .0001; miR-210, 0.865, P < .0001; miR-144, 0.729, P < .0001.)

Figure 7. Analysis of the value of combined miRNAs for the diagnosis of HPV16-DNA-positive NSCLC patients and HPV16-DNA-negative NSCLC patients. (a-f) The ROC curve shows that for the distinction between HPV16-DNA-positive NSCLC and HPV16-DNA-negative NSCLC patients, the combined detection of the two miRNAs is more accurate than the single miRNA

Table 6. HPV universal primers, HPV16 and HPV18 nested-PCR primers

Primers	Sequence of primers(5ʹ-3ʹ)	Product(bp)
HPV consensus primer (SPF1/GP6++)	SPF1A:GCiCAGGGiCACAATAATGG SPF1B:GCiCAGGGiCATAACAATGG SPF1C:GCiCAGGGiCATAATAATGG SPF1D:GCiCAAGGiCATAATAATGG GAAAAATAAACTGTAAATCATATTCTTC GAAAAATAAACTGTAATTCATATTCCTC GAAAAATAAACTGTAAATCATATTCCTC GAAAAATAAATTGTAAATCAAATTCCTC	180
HPV-16 first round	GAACTGCAATGTTTCAGGACC CGTGTTCTTGATGATCTGC	422
HPV-16 nested	CAATGTTTCAGGACCCACAGG GCAACAAGACATACATCGACCG
HPV-18 first round	CAATGTTTCAGGACCCACAGG GCAACAAGACATACATCGACCG	477
HPV-18 nested	CTACAAGCTACCTGATCTGTGC CAATGTTGCCTTAGGTCCATGC

i: secondary jaundice.

Table 7. The sequence of miRNA-specific forward primer

miRNA	forward primer (5ʹ-3ʹ)
miR-183	TATGGCACTGGTAGAATTCACT
miR-9-3	TCTTTGGTTATCTAGCTGTATGA
miR-210	CTGTGCGTGTGACAGCGGCTGA
miR-182	TTTGGCAATGGTAGAACTCACACT
miR-144	TACAGTATAGATGATGTACT
miR-451a	AAACCGTTACCATTACTGAGTT
miR-486-1	GCGGCGGGCATCCTGTACTGAG
miR-486-2	GCGGCGGTCCTGTACTGAGCTG