Modulation of Colorectal Tumor Behavior via lncRNA TP53TG1-Lipidic Nanosystem

Abstract

Long non-coding RNAs (lncRNAs) are an emerging group of RNAs with a crucial role in cancer pathogenesis. In gastrointestinal cancers, TP53 target 1 (TP53TG1) is an epigenetically regulated lncRNA that represents a promising therapeutic target due to its tumor suppressor properties regulating the p53-mediated DNA damage and the intracellular localization of the oncogenic YBX1 protein. However, to translate this finding into the clinic as a gene therapy, it is important to develop effective carriers able to deliver exogenous lncRNAs to the targeted cancer cells. Here, we propose the use of biocompatible sphingomyelin nanosystems comprising DOTAP (DSNs) to carry and deliver a plasmid vector encoding for TP53TG1 (pc(TP53TG1)-DSNs) to a colorectal cancer cell line (HCT-116). DSNs presented a high association capacity and convenient physicochemical properties. In addition, pc(TP53TG1)-DSNs showed anti-tumor activities in vitro, specifically a decrease in the proliferation rate, a diminished colony-forming capacity, and hampered migration and invasiveness of the treated cancer cells. Consequently, the proposed strategy displays a high potential as a therapeutic approach for colorectal cancer.

Keywords: colorectal cancer; emulsions; epigenomics; long non-coding RNAs; nanocarriers.

Figure 1

Association of pc(mCherry) to DSNs. (A) Association of pc(mCherry) (1 to 10 µg) to DSNs, prepared with 1% or 10% of DOTAP (DSNs1% and DSNs10%), determined by agarose gel electrophoresis. (B) Physicochemical properties of DSNs1% associating 1 µg of pc(mCherry) and DSNs10% associating 1, 5 and 10 µg of pc(mCherry). (C) Colloidal stability of DSNs10% associating 10 µg of pc(mCherry) in 1% FBS-supplemented DMEM, up to 4 h. (nm, nanometers; PdI, polydispersity index; zeta pot., zeta potential; mV millivolts).

Figure 2

Nanosystems cytotoxic profile and internalization capacity on HCT-116 cells. (A) Blank nanosystems (SNs, DSNs1%, DSNs10%) and DSNs10% associating 10 µg of the model plasmid encoding mCherry (pc(mCherry)-DSNs10%) were incubated for 24 h with HCT-116 cells up to 5 mg/mL and cell cytotoxicity was determined by MTT. (B) Confocal microscopy images of internalized blank nanosystems labeled with DiR (red channel) and pc(TP53TG1)-DSNs10% (associating 10 µg plasmid) labeled with NBD-SM (green channel). Cell nuclei were counterstained with DAPI (blue channel). Scale bars correspond to 25 µm. (C) FACS analysis of HCT-116 cells positive to pc(TP53TG1)-DSNs labeled with NBD-SM (blue line) upon 4 h of incubation at 37 °C. Ultra-pure water was used as control (red line).

Figure 3

Transfection efficiency of DSNs in HCT116 cells. (A) Fluorescence microscopy images of mCherry (red signal) expression in cells transfected with a pc(mCherry)-DSNs1% and -DSNs10% for 24 h at 37 °C; (B) Expression of TP53TG1 and its mutated form (MutTP53TG1), in cells transfected with DSNs10% without plasmid (blank) or associating 10 µg of pc(empty), pc(TP53TG1), pc(MutTP53TG1), determined by qRT-PCR using specific primers. The relative expression of each gene was calculated as mean ± SEM of 2^-ddCT normalized to GAPDH. (*** p < 0.001; **** p < 0.0001).

Figure 4

Tumor suppressor features of pc(empty)-DSNs10%, pc(TP53TG1)-DSNs10% and pc(MutTP53TG1)-DSNs10% in vitro. (A) HCT-116 cells’ viability determined each 24 h up to 72 h, after treatment with DSNs10%, associating different pcDNAs, for 4 h. Data is normalized to time point 0 h. (B) Effect of DSNs10%, associating different pcDNAs on the ability of HCT-116 cells to form colonies. HCT-116 cells were incubated with the nanosystems for 4 h and after 11 days the number of formed colonies was determined. (C) Wound healing assay was performed to evaluate the effect of the nanosystems on HCT-116 cell ability to migrate and close the wound. After creating a wound, the cells were treated with the nanosystems for 4 h (n = 3). The evolution of the wound was monitored up to 96 h through direct observation under light microscope and photos were taken. (D) The migration rate (wound healing assay) was calculated using the acquired photos and data was normalized against time point 0 h. The effects of pc(TP53TG1)-DSNs10% and pc(MutTP53TG1)-DSNs10% were compared to the ones produced by pc(empty)-DSNs10% at each time point. (ns, not significant; * p < 0.05, ** p < 0.01).