NACC1, as a Target of MicroRNA-331-3p, Regulates Cell Proliferation in Urothelial Carcinoma Cells

Abstract

The nucleus accumbens-associated protein 1 (NACC1) is a transcription factor constitutively expressed in the urothelium, where it regulates cell growth, senescence, autophagy, and epithelial-mesenchymal transition. microRNA (miRNA) constitutes a class of small non-coding RNAs which are involved in cell proliferation, differentiation, and progression of tumors. miRNAs and their target molecules are utilized for molecular diagnosis of urothelial carcinoma. NACC1 is one of several putative target molecules of miR-331-3p, and is associated with cell proliferation in cancers such as prostate and cervical cancer. Functional experiments involving miR-331-3p and its target molecule NACC1 were conducted using the urothelial carcinoma (UC) cell lines, T24, UMUC6, and KU7. Furthermore, quantitative reverse transcription polymerase chain reaction and immunostaining were performed to evaluate the expression of NACC1 in UC derived from transurethral resection of bladder tumor (TUR-Bt) specimens. The methane thiosulfonate (MTS) assay revealed that cell proliferation was significantly reduced after transient transfection of miR-331-3p precursor and/or NACC1 siRNA in UC cells. Cell senescence via cell cycle arrest at the G1 phase was induced by NACC1 inhibition. On the other hand, suppression of NACC1 induced cell migration and invasion abilities. Immunohistochemical analysis of TUR-Bt specimens revealed that over 70% of UC cells presented strongly positive results for NACC1. In contrast, normal urothelial cells were weakly positive for NACC1. It was also found that NACC1 expression was lower in invasive UC cells than in non-invasive UC cells. Loss of NACC1 induced vessel invasion in invasive UC tissues. The present results indicate that NACC1 regulated by miR-331-3p contributes to cell proliferation, and is involved in cell migration and invasion. This suggests that NACC1 can serve as a potential target molecule for the prediction and prognosis of UC, and can contribute to effective treatment strategies.

Keywords: NACC1; cell cycle arrest; miR-331-3p; migration and invasion; urothelial carcinoma.

Figure 1

Expression of Nucleus accumbens-associated protein 1(NACC1) in Urothelial carcinoma (UC) of bladder tissues. UC were histologically classified in pTa, pTis, pT1 and >pT2. Hematoxylin and eosin (HE) stain and Immunohistochemistory,scale bar: ×200.

Figure 2

(A) The effect of transient NACC1 siRNA transfection in three UC cells. The left graph is qRT-PCR for NACC1 mRNA. The right image is western blot for NACC1 protein; (B) Methane thiosulfonate (MTS) assay in T24, UMUC6, and KU7 cells. Cell proliferation was suppressed by transient transfection of NACC1 siRNA (* p < 0.05).

Figure 3

(A) Senescence-Associated β-Galactosidase (SA-β-gal) assay. Senescent cells with SA-β-galactosidase activity were significantly induced, scale bar: ×100; (B) The cell cycle assay for UC cells. Cell cycle arrest at G0/G1 phase was induced in T24 cells. (* p < 0.05); (C) mRNA expression of cyclin A and B under suppression of NACC1 in T24 cells. (* p < 0.05).

Figure 4

(A) mRNA expression of NACC1, which are the putative target molecules of miR-331-3p in T24, UMUC6, and KU7 cells. The left graph shows ct value of three UC cell lines. The right graph shows relative fold value (NACC1/actin); (B) The effect of miR-331-3p pre transfection by qPCR; (C) NACC1 expression under overexpression of miR-331-3p in three UC cell lines. (* p < 0.05, pre: precursor) miR-331-3p down-regulates NACC1 mRNA expression in T24, UMUC6, and KU7 cells.

Figure 5

The effect of miR-331-3p on cell proliferation in three UC cell lines. (A) MTS assay. Cell proliferation was suppressed by overexpression of miR-331-3p. (* p < 0.05); (B) SA-β-gal assay for T24 cells. The Y-axis shows the number of positive cell counts per one high-power field (hpf). Positive cells were induced by transfection of miR-331-3p precursor (pre: precursor). Scale bar: ×100.

Figure 6

Cell invasion and migration assay in T24 cells. (A) Matrigel invasion assay showing promotion of invasion capability of T24 cells transfected with NACC1 siRNA; (B) Representative images and graphical display of rate of open space in wound closure (%) in T24 cells; (C) Immunohistochemistry for D2-40. In total, 22 cases of invasive UC were assessed. Scale bar: ×200.

Figure 7

Suppression of NACC1 induces cell cycle arrest at G0/G1 phase and cell senescence in UC. On the other hand, NACC1 promotes cell migration and invasion capability.