N-methylnicotinamide and nicotinamide N-methyltransferase are associated with microRNA-1291-altered pancreatic carcinoma cell metabolome and suppressed tumorigenesis

Abstract

The cell metabolome comprises abundant information that may be predictive of cell functions in response to epigenetic or genetic changes at different stages of cell proliferation and metastasis. An unbiased ultra-performance liquid chromatography-mass spectrometry-based metabolomics study revealed a significantly altered metabolome for human pancreatic carcinoma PANC-1 cells with gain-of-function non-coding microRNA-1291 (miR-1291), which led to a lower migration and invasion capacity as well as suppressed tumorigenesis in a xenograft tumor mouse model. A number of metabolites, including N-methylnicotinamide, involved in nicotinamide metabolism, and l-carnitine, isobutyryl-carnitine and isovaleryl-carnitine, involved in fatty acid metabolism, were elevated in miR-1291-expressing PANC-1. Notably, N-methylnicotinamide was elevated to the greatest extent, and this was associated with a sharp increase in nicotinamide N-methyltransferase (NNMT) mRNA level in miR-1291-expressing PANC-1 cells. In addition, expression of NNMT mRNA was inversely correlated with pancreatic tumor size in the xenograft mouse model. These results indicate that miR-1291-altered PANC-1 cell function is associated with the increase in N-methylnicotinamide level and NNMT expression, and in turn NNMT may be indicative of the extent of pancreatic carcinogenesis.

Fig. 1.

The morphology of control (A) and miR-1291-expressing PANC-1 (B) cells showed no difference, whereas an increased miR-1291 expression (C; *P < 0.05, Student’s t-test) led to a significant suppression (*P < 0.05, two-way analysis of variance) of cell proliferation (D). Values are mean ± SD (N = 6 in each group).

Fig. 2.

Transwell migration (A) and Matrigel invasion (B) capacity of the PANC-1-expressing cells with gained miR-1291 function were sharply reduced. Values are mean ± SD (N = 6 in each group). *P < 0.05, as compared to the control.

Fig. 3.

Scores scatter plot and loading S-plot from the supervised OPLS analyses distinguished miR-1291-expressing PANC-1 cells from the control cells. The top six ions (P1–P6) showing significant differences in two groups are marked in the S-plot. (A) Score plot under ESI⁺-HILIC mode; (B) score plot under ESI⁻-RPLC mode; (C) S-plot under ESI⁺-HILIC mode; (D) S-plot under ESI⁻-RPLC mode.

Fig. 4.

Cellular concentrations of NMN (A), taurine (B), l-carnitine (C), isobutyryl-carnitine (D) and isovaleryl-carnitine (E) as well as the relative levels of hydroxybutyryl-carnitine (F) in the control and miR-1291-expressing PANC-1 cells. Data were normalized to protein concentration of individual samples. Values are mean ± SD (N = 6 in each group). *P < 0.05, compared to the control.

Fig. 5.

Cellular mRNA levels of genes associated with the metabolomics markers including (A) NNMT, (B) CPT1A, (C) CPT1B, (D) CPT1C, (E) CPT2 and (F) CRAT. β-Actin was used as an internal control. Values are mean ± SD (N = 6 per group). *P < 0.05, compared to the control.

Fig. 6.

NNMT mRNA level was inversely related to the size of xenograft tumor. (A) Representative mice bearing xenograft tumors derived from miR-1291-expressing or control PANC-1 cells. (B) Growth of xenograft tumors was significantly different (*P < 0.05, two-way analysis of variance) between miR-1291-expressing and control PANC-1 cells. (C) Comparison of tumors excised from xenograft mice at week 7 after inoculation. (D) Weight of xenograft tumors harvested from mice. (E) Inverse relationship between the xenograft tumor size and cancerous NNMT mRNA level. Data mean ± SD (N = 6). *P < 0.05, compared to the control.