Upregulated MicroRNA-483-3p is an Early Event in Pancreatic Ductal Adenocarcinoma (PDAC) and as a Powerful Liquid Biopsy Biomarker in PDAC

Figures & data

Table 1 Frequency of miR-483-3p Expression in PanIN Lesions and PDAC by LNA-ISH

Group	miR-483-3p Expression		Total
	Negative (−)	Positive (+)
PanIN-1	42 (35.9%)	75 (64.1%)	117
PanIN-2	15 (15.5%)	82 (84.5%)	97
PanIN-3	2 (3.4%)	56 (96.6%)	58
PDAC	0 (0.0%)	107 (100.0%)	107
Total	59	320	379

Notes: Chi-square test, p<0.0001.

Figure 1 miR-483-3p expression by LNA-ISH and SMAD4 protein expression by immunohistochemistry in PanIN lesions and PDAC tissues. (A) Expression of miR-483-3p was detected in the cytoplasm of normal pancreatic acini but not in normal pancreatic ducts. An increase in staining intensity for miR-483-3p was observed across the progression of normal pancreatic ducts to PanIN-1 (D), PanIN-2 (G), PanIN-3 (J) and PDAC (M) lesions. Protein expression of the putative miR-483-3p target gene SMAD4 was detected in normal pancreatic acini and ducts (B), as well as PanIN-1 (E) and PanIN-2 (H) lesions, but decreased in PanIN-3 (K) and PDAC (N) lesions. (C, F, I, L, O) Hematoxylin and eosin (HE) staining for normal pancreas (C), PanIN-1 (F), PanIN-2 (I), PanIN-3 (L) and PDAC (O).

Figure 2 Relative miR-483-3p expression levels by RT-PCR in serum and serum-derived exosomes from 63 PDAC patients and 22 healthy controls. (A) Serum and (B) serum-derived exosomal miR-483-3p levels in PDAC patients compared with those in healthy subjects. Mann–Whitney U-test, ** p<0.0001, * p<0.01. (C) Serum exosomal miR-483-3p levels in grade 1, 2, and 3 PDAC patients. Kruskal–Wallis test was used. (D) Relationship of serum and serum-derived exosomal miR-483-3p levels. Linear regression was used.

Figure 3 Diagnostic and predicted performance of circulating miR-483-3p levels. (A–E) ROC curve analysis for circulating miR-483-3p levels. (F–G) ROC curve analysis for CA 19–9, CA 19–9 combined with serum miR-483-3p level. PDAC, pancreatic ductal adenocarcinoma; N, normal controls. (H–I) Kaplan–Meier survival analysis in PDAC patients. Log rank test was used.

Figure 4 miR-483-3p promotes pancreatic cancer cell migration and invasion in vitro. (A) Transwell migration and invasion assays of PANC1 cells transiently transfected with a miR-483-3p inhibitor (Inhibitor) or negative control (NC), as indicated. (B) Transwell migration and invasion assays of AsPC1 cells transiently transfected with a miR-483-3p mimic (Mimic) or negative control (NC), as indicated. Histograms show the quantification of the cells that traversed the chamber in each assay.

Table 2 Correlation Between Circulating miR-483-3p Expression and Clinicopathologic Characteristics of PDAC Patients

Variables	Patients Number	Exosomal miR-483-3p			Serum miR-483-3p
		High Level	Low Level	p-value	High Level	Low Level	p-value
Age				1			1
≤65 years	46	23	23		23	23
>65 years	17	9	8		9	8
Gender				0.131			0.45
Male	35	21	14		16	19
Female	28	11	17		16	12
Differentiation				0.023*			0.248
Well	6	0	6		1	5
Moderate	38	20	18		21	17
Poor	19	12	7		10	9
Tumor Size				0.237			0.356
≤2 cm	14	5	9		6	8
>2cm	49	27	22		26	23
pT classification				0.085			0.578
pT1	13	4	9		5	8
pT2	29	19	10		15	14
pT3	21	9	12		12	9
pT4	0	0	0		0	0
pN classification				0.019*			0.718
pN0	27	11	16		12	15
pN1	29	14	15		16	13
pN2	7	7	0		4	3
Stage (AJCC)				0.022*			0.443
I	18	8	10		7	11
II	37	16	21		20	17
III	6	6	0		3	3
IV	2	2	0		2	0

Notes: Chi square test or Chi-square test, *p<0.05.

Table 3 Multivariate Analysis of Circulating miR-483-3p Levels and Other Clinicopathologic Factors in PDAC Patients

Variable	Multivariate Analysis
	Hazard Ratio	95% CI	p-value
Tumor Size (> 2.0cm)	5.067	0.650 to 39.522	0.122
Stage (> I)	3.350	0.756 to 14.837	0.111
Exo miR-483-3p level (High)	3.307	1.104 to 9.903	0.033