MiR-29c regulates the expression of miR-34c and miR-449a by targeting DNA methyltransferase 3a and 3b in nasopharyngeal carcinoma

Abstract

Background: Nasopharyngeal carcinoma (NPC) is prevalent in South East Asia and Southern China particularly, despite the reported 5-year survival ratio is relative higher than other deadly cancers such as liver, renal, pancreas cancer, the lethality is characterized by high metastatic potential in the early stage and high recurrence rate after radiation treatment. MicroRNA-29c was found to be down-regulated in the serum as well as in the tissue of nasopharyngeal carcinoma tissue.

Methods: In this study, we found accidentally that the transfection of pre-miR-29c or miR-29c mimics significantly increases the expression level of miR-34c and miR-449a but doesn't affect that of miR-222 using real-time quantitative PCR in nasopharyngeal carcinoma cell lines. To explore the molecular mechanism of the regulatory role, the cells are treated with 5-Aza-2-deoxycytidine (5-Aza-CdR) treatment and the level of miR-34c and miR-449a but not miR-222 accumulated by the treatment. DNA methyltransferase 3a, 3b were down-regulated by the 5-Aza-CdR treatment with western blot and real-time quantitative PCR.

Results: We found that pre-miR-29c or miR-29c mimics significantly increases the expression level of miR-34c and miR-449a. We further found DNA methyltransferase 3a and 3b are the target gene of miR-29c. Restoration of miR-29c in NPC cells down-regulated DNA methyltransferase 3a, 3b, but not DNA methyltransferase T1.

Conclusions: The regulation of miR-29c/DNMTs/miR-34c\449a is an important molecular axis of NPC development and targeting DNMTs or restoring of miR-29c might be a promising therapy strategy for the prevention of NPC.

Keywords: DNA methyltransferase; Nasopharyngeal carcinoma; miR-29c; miR-34c; miR-449a.

Fig. 1

The expression of miR-29c is down-regulated in nasopharyngeal carcinoma cell lines and tissues. a Total RNA was extracted from normal nasopharyngeal epithelium cells (NP69) and NPC cell lines (HNE1, HNE2, CNE2, C666-1, HK-1) and reversely transcribed into cDNA. Q-PCR was performed and analyzed for the expression level of miR-29c normalized by RNAU6. b The same method and protocol was performed from NPC tissue and reversely transcribed, Q-PCR was performed and analyzed. N: normal nasopharyngeal epithelium tissue, 2, 3, 4 were presented for Clinical Stage 2, 3, 4, M was presented for the NPC tissue with metastasis. 30 samples were used, each group contains six samples

Fig. 2

MiR-29c increases the level of miR-34c and miR-449a but not miR-222 in nasopharyngeal carcinoma cell lines. The pSuper-pre-miR-29c was transformed into nasopharyngeal carcinoma cells HNE1 (a) and CNE2 (b) according to the protocol and the cells were cultured for 24 h. Total RNA was extracted and inversely transcribed into cDNA. Q-PCR was performed and analyzed for the miR-29c, miR-34c, miR-449a and miR-222 normalized by RNAU6

Fig. 3

miR-34b/c and miR-449a levels were regulated by the epigenetic factors but miR-222 wasn’t. Nasopharyngeal carcinoma cells were treated with 5-Aza-CdR for 72 h, and then with or without Trichostatin A (TSA) for another 24 h. a, b Expression of miR-34b/c and miR-449a were analyzed

Fig. 4

DNMT3a and 3b but not DNMT1 are down-regulated by pSuper-pre-miR-29c transfection. DNMT3a, 3b, 1 were predicted as tentative targeted genes of miR-29c. Cells were transformed with pre-miR-29c and cultured for 24 h for Q-PCR and western blots for 48 h. a, b Q-PCR analysis of DNMT3a, 3b, 1 regulated by miR-29c in HNE1 and CNE2. c Western blot analysis of DNMT3a, 3b and DNMT1 in different cell lines transfected with pre-miR-29c

Fig. 5

MiR-29c inhibits the growth of nasopharyngeal carcinoma cells HNE1 and CNE2 by MTT assay. Cells were transformed with pre-miR-29c and cultured for 24 h. 5 × 10⁴ cells were seeded into 96-well dish triplicately. 20ul MTT solution was added to each well and then 200ul DMSO was added to the well with cells. Read optical density at 490 nm and subtract background at 570 nm. The readout was recorded at 4 time points (24, 48, 72, 96,120 h)

Fig. 6

Representative image of IHC staining of DNMT3a in nasopharyngeal carcinoma tissue. a Negative (−) (a-c), weak (+) (d-f), positive (++) (g-i) staining of DNMT3a in NPC tissue. b The correlation of DNMT3a staining with 5-year survival (a) and total survival time (b). * p < 0.05 is considered to be significant statistically

Fig. 7

Representative image of IHC staining of DNMT3b in nasopharyngeal carcinoma tissue. a Negative (−) (a–c), weak (+) (d–f), positive (++) (g–i) staining of DNMT3b in NPC tissue. b The correlation of DNMT3b staining with 5-year survival (a) and total survival time (b). * p < 0.05 is considered to be significant statistically

Fig. 8

Representative image of IHC staining of DNMT1 in nasopharyngeal carcinoma tissue. a Negative (−) (a–c), weak (+) (d–f), positive (++) (g–i) staining of DNMT1 in NPC tissue. b The correlation of DNMT1 staining with 5-year survival (a) and total survival time (b). * p < 0.05 is considered to be significant statistically