Aberrant miR-362-3p is Associated with EBV-Infection and Prognosis in Nasopharyngeal Carcinoma and Involved in Tumor Progression by Targeting JMJD2A

Figures & data

Table 1 Association of miR-362-3p with the Clinicopathological Characteristics of NPC Patients

Characteristics	Total Cases (n = 112)	miR-362-3p Expression		P value
		Low (n = 60)	High (n = 52)
Age (years)
<50	40	21	19	0.865
≥50	72	39	33
Gender
Female	43	23	20	0.989
Male	69	37	32
EBV infection
Negative	60	20	40	< 0.001
Positive	52	40	12
Smoking
No	46	24	22	0.804
Yes	66	36	30
Differentiation
Well/moderate	57	24	33	0.013
Poor	55	36	19
TNM stage
I–II	62	25	37	0.002
III–IV	50	35	15

Abbreviations: miR, microRNA; NPC, nasopharyngeal carcinoma; EBV, Epstein–Barr virus.

Figure 1 Expression of miR-362-3p in NPC patients. (A) The levels of miR-362-3p in 497 tumor tissue samples and 44 normal tissue samples from TCGA HNSC cohort. (B) Expression of miR-362-3p in the serum of NPC patients and healthy controls from our study cohort. (C) Expression of miR-362-3p in NPC tissues and normal tissues of NPC patients. (D) There was a significant positive correlation between serum miR-362-3p levels and miR-362-3p levels in NPC tissues (r = 0.823, P < 0.001). (E) ROC curve indicated the high diagnostic value of miR-362-3p for screening NPC patients from healthy controls. ***P < 0.001 vs HNSC tissue samples from TCGA HNSC cohort or Healthy controls from our study cohort or Normal tissues.

Abbreviations: miR, microRNA; HNSC, head and neck squamous cell carcinoma; NPC, nasopharyngeal carcinoma; AUC, area under the ROC curve; ROC, receiver operating characteristic.

Table 2 Cox Regression Analysis Results for Patients with NPC

Variables	HR	95% CI	P value
Age (≥50 vs <50)	1.256	0.825–1.779	0.374
Gender (male vs female)	1.113	0.756–1.502	0.652
EBV infection (Positive vs negative)	1.401	0.881–1.968	0.112
Smoking (Yes vs no)	1.108	0.840–1.443	0.285
Differentiation (Poor vs well/moderate)	1.667	1.103–2.217	0.039
TNM stage (III–IV vs I–II)	2.152	1.374–3.015	0.006
miR-362-3p (low vs high)	2.374	1.417–3.338	0.003

Abbreviations: NPC, nasopharyngeal carcinoma; EBV, Epstein–Barr virus; miR, microRNA; HR, hazard ratio; CI, confidence interval.

Figure 2 Relationship of miR-362-3p expression with NPC patient survival. (A) Expression levels of miR-362-3p in NPC tissues from non-survivors and survivors in patients. (B) Low miR-362-3p expression was associated with short survival of NPC patients (log-rank P = 0.026). ***P < 0.001 vs Survivors.

Abbreviations: miR, microRNA; NPC, nasopharyngeal carcinoma.

Figure 3 Different miR-362-3p expression in NPC between EBV negative and positive cases. (A) Expression of miR-362-3p in EBV+ patients and EBV- patients. (B) Expression of miR-362-3p in normal cell line (NP69), EBV+ (C666-1) and EBV- (SUNE1) NPC cell lines. (C) ROC analysis indicated the ability of miR-362-3p to discriminate between EBV infection positive and negative patients. ***P < 0.001 vs EBV- patients or NP69 cells. ^##P < 0.01 vs SUNE1 cells.

Abbreviations: miR, microRNA; EBV, Epstein–Barr virus; NPC, nasopharyngeal carcinoma; AUC, area under the ROC curve; ROC, receiver operating characteristic.

Figure 4 Effects of miR-362-3p on NPC cell proliferation, migration and invasion. (A and B) In SUNE1 and C666-1 cells, miR-362-3p mimic promoted, and miR-362-3p inhibitor suppressed the levels of miR-362-3p. (C and D) In SUNE1 and C666-1 cells, miR-362-3p overexpression inhibited, and miR-362-3p knockdown promoted cell proliferation. (E and F) miR-362-3p overexpression inhibited, and miR-362-3p knockdown promoted the migration and invasion of SUNE1 and C666-1 cells. *P < 0.05, **P < 0.01 ***P < 0.001 vs Control.

Abbreviations: miR, microRNA; NPC, nasopharyngeal carcinoma; NC, negative control; OD, optical density.

Figure 5 miR-362-3p directly inhibited JMJD2A in NPC. (A) Complementary binding sequences between miR-362-3p and JMJD2A. (B and C) Relative luciferase activity in the JMJD2A-WT group was inhibited by miR-362-3p overexpression and facilitated by miR-362-3p knockdown. However, there was no change in luciferase activity in the JMJD2A-MUT group. (D and E) JMJD2A mRNA levels were inhibited by miR-362-3p overexpression and promoted by miR-362-3p knockdown in both SUNE1 and C666-1 cell lines. (F) The mRNA levels of JMJD2A in NPC tissues and normal tissues of patients. (G) There was a significant negative correlation between JMJD2A mRNA levels in tissues and miR-362-3p levels in tissues (r = −0.564, P < 0.001). *P < 0.05 vs Control; ***P < 0.001 vs Control or Normal tissues.

Abbreviations: JMJD2A, Jumonji C domain 2A; miR, microRNA; NPC, nasopharyngeal carcinoma; NC, negative control; WT, wild type; MUT, mutant type.