Detecting the FLJ22447 lncRNA in Ovarian Cancer with Cyclopentane-Modified FIT-PNAs (cpFIT-PNAs)

Abstract

Ovarian cancer (OC) is one of the most lethal gynecologic cancers that is typically diagnosed at the very late stage of disease progression. Thus, there is an unmet need to develop diagnostic probes for early detection of OC. One approach may rely on RNA as a molecular biomarker. In this regard, FLJ22447 lncRNA is an RNA biomarker that is over-expressed in ovarian cancer (OC) and in cancer-associated fibroblasts (CAFs). CAFs appear early on in OC as they provide a metastatic niche for OC progression. FIT-PNAs (forced intercalation-peptide nucleic acids) are DNA analogs that are designed to fluoresce upon hybridization to their complementary RNA target sequence. In recent studies, we have shown that the introduction of cyclopentane PNAs into FIT-PNAs (cpFIT-PNA) results in superior RNA sensors. Herein, we report the design and synthesis of cpFIT-PNAs for the detection of this RNA biomarker in living OC cells (OVCAR8) and in CAFs. cpFIT-PNA was compared to FIT-PNA and the cell-penetrating peptide (CPP) of choice was either a simple one (four L-lysines) or a CPP with enhanced cellular uptake (CLIP6). The combination of CLIP6 with cpFIT-PNA resulted in a superior sensing of FLJ22447 lncRNA in OVCAR8 cells as well as in CAFs. Moreover, incubation of CLIP6-cpFIT-PNA in OVCAR8 cells leads to a significant decrease (ca. 60%) in FLJ22447 lncRNA levels and in cell viability, highlighting the potential theranostic use of such molecules.

Keywords: FLJ22447 lncRNA; RNA biomarker; cancer-associated fibroblasts; cell-penetrating peptide; cyclopentane modified FIT-PNA; ovarian cancer.

Scheme 1

The general design of FIT-PNAs and cpFIT-PNAs.

Figure 1

Fluorescence emission spectrum of FIT-PNAs and cpFIT-PNAs towards synthetic RNA. (λex = 580 nm; λem = 590 nm). [FIT-PNA] = 0.5 μM and [RNA] = 1 μM.

Figure 2

cpPNA and CLIP6 enhance fluorescence of FIT-PNAs in OVCAR8 living cells. (A) OVCAR8 cells treated with 2 μM FIT-PNA/cpFIT-PNA for 3 h at 37 °C were washed twice with 1× PBS, followed by nuclei staining with Hoechst stain (1 μg/mL) and washing twice with 1× PBS. Confocal imaging at 20 × magnification. (a) OVCAR8 cells as control; OVCAR8 cells treated with (b) K4 scr-cpFIT-PNA; (c) K4 FIT-PNA; (d) K4 cpFIT-PNA; (e) CLIP6 FIT-PNA; (f) CLIP6 cpFIT-PNA, respectively. (B) Quantification of the mean fluorescence intensity signal of OVCAR8 cells treated with 2 μM FIT-PNA/cpFIT-PNA using ImageJ 1.53t software. Statistical analysis performed using Student’s t-test; error bars represent mean ± SEM; ** p < 0.01, *** p < 0.001. (C) Magnified images (40×) for cellular uptake for K4 cpFIT-PNA and CLIP6 cpFIT-PNA in OVCAR8 cells.

Figure 2

cpPNA and CLIP6 enhance fluorescence of FIT-PNAs in OVCAR8 living cells. (A) OVCAR8 cells treated with 2 μM FIT-PNA/cpFIT-PNA for 3 h at 37 °C were washed twice with 1× PBS, followed by nuclei staining with Hoechst stain (1 μg/mL) and washing twice with 1× PBS. Confocal imaging at 20 × magnification. (a) OVCAR8 cells as control; OVCAR8 cells treated with (b) K4 scr-cpFIT-PNA; (c) K4 FIT-PNA; (d) K4 cpFIT-PNA; (e) CLIP6 FIT-PNA; (f) CLIP6 cpFIT-PNA, respectively. (B) Quantification of the mean fluorescence intensity signal of OVCAR8 cells treated with 2 μM FIT-PNA/cpFIT-PNA using ImageJ 1.53t software. Statistical analysis performed using Student’s t-test; error bars represent mean ± SEM; ** p < 0.01, *** p < 0.001. (C) Magnified images (40×) for cellular uptake for K4 cpFIT-PNA and CLIP6 cpFIT-PNA in OVCAR8 cells.

Figure 3

FIT-PNA/cpFIT-PNA uptake into OC cells (OVCAR8) as analyzed by flow cytometry. (A) Histogram of flow cytometry analysis in OVCAR8 cells treated with 2 μM FIT-PNA/cpFIT-PNA for 3 h at 37 °C. Histogram illustrates the mean fluorescence intensity plotted in horizontal axis against the number of cell events detected in the vertical axis. Both cp and CLIP6 enhance fluorescence of FIT-PNAs in OVCAR8 cells. (B) Mean fluorescence intensity of FIT-PNA/cpFIT-PNA in OVCAR8 cells. Statistical analysis performed using FlowJo 10.10 software and Student’s t-test; error bars represent mean ± SEM; *** p < 0.001.

Figure 4

FIT-PNA/cpFIT-PNA uptake into CAF primary cells as analyzed by flow cytometry. (A) Histogram of flow cytometry analysis in CAF primary cells treated with 2 μM FIT-PNA/cpFIT-PNA for 3 h at 37 °C. Histogram illustrates the mean fluorescence intensity plotted in horizontal axis against the number of cell events detected in the vertical axis. Both cp and CLIP6 enhance fluorescence of FIT-PNAs in CAF primary cells. (B) Mean fluorescence intensity of FIT-PNA/cpFIT-PNA in CAF primary cells. Statistical analysis performed using FlowJo 10.10 software and Student’s t-test; error bars represent mean ± SEM; *** p < 0.001.

Figure 5

(A) RT-qPCR analysis of FLJ22447 lncRNA expression in normal fibroblast (MRC5) and ovarian cancer cell OVCAR8. (B) RT-qPCR analysis of FLJ22447 lncRNA expression in normal fibroblast (NF) and CAF primary cells. Statistical analysis performed using Student’s t-test; error bars represent mean ± SEM; ** p < 0.01, *** p < 0.001.

Figure 6

RT-qPCR analysis of FLJ22447 lncRNA and HIF-1α expression in OVCAR8 cells treated with 2 μM K4 cpFIT-PNA and CLIP6 cpFIT-PNA for 48 h at 37 °C. cpFIT-PNA treated cells show down-regulation of FLJ22447 lncRNA as well as reduction in HIF-1α expression. Statistical analysis performed using Student’s t-test; error bars represent mean ± SEM; ** p < 0.01, *** p < 0.001.

Figure 7

The effect of cpFIT-PNAs on the cell viability of OVCAR8 cells analyzed by MTT assay. Cells were incubated with cpFIT-PNAs (2 μM) for 3 h and 48 h. The viability of untreated OVCAR8 cells were considered as 100%. Statistical analysis performed using Student’s t-test; error bars represent mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001.