Over-expression of telomere binding factors (TRF1 & TRF2) in renal cell carcinoma and their inhibition by using SiRNA induce apoptosis, reduce cell proliferation and migration invitro

Abstract

Telomere binding factors viz. TRF1 and TRF2 are a part of sheltrin complex that are present exclusively at the ends of chromosomes. These factors play an important role in maintaining chromosomal integrity at the ends. However, their status and role are not clear in renal cell carcinoma (RCC). Therefore, the present study was conducted to evaluate TRF1 and TRF2 expressions in RCC tissues. Further, the role of these factors involved in tumorigenesis was elucidated by gene silencing using siRNA in RCC cell line (A498). The present study documented a significant over-expression of TRF1 (P = 0.005) and TRF2 (P = 0.0048) mRNAs by real time PCR in RCC tissues as compared with adjacent normal kidney tissues. Immunohistochemistry studies also revealed higher expression of TRF1 and TRF2 proteins in RCC. Moreover, TRF1 or TRF2 gene silencing using siRNA showed marked reduction in proliferation of RCC cells (P = 0.000). Further, significantly induced cell cycle arrest (P = 0.000) and apoptosis of RCC cells (P = 0.000) was documented upon TRF1 or TRF2 gene silencing. Henceforth, the results deduce that TRF1 or TRF2 inhibitions play an important role in the induction of apoptosis in A498 cells, which may serve as a potential therapeutic target in RCC.

Fig 1. mRNA and protein expression of TRF1 and TRF2 (a).

The result are presented as box plot showing TRF1 and TRF2 mRNA expression level (A) TRF1 (B) TRF2 in normal and RCC tissues were determined by quantitative real time PCR. The bottom and top edges of the box located at the 25^th and 75^th percentiles respectively of the sample. The centre horizontal lines are drawn at the median of the sample. β-actin mRNA levels were used to normalize TRF1 and TRF2 gene expression. Statistical analysis was done by one sample t- test. P<0.05 was considered significant. (b) & (c). Immunohistochemical staining of TRF1 and TRF2 protein respectively. IHC was performed for TRF1 in all the cases (92) of RCC. The representative figures of (A) Normal renal parenchyma; (B) grade I clear cell RCC; (C) grade II clear cell RCC; (D) grade III clear cell RCC; (E) grade IV clear cell RCC; (F) papillary RCC; (G) oncocytoma; (H) sarcomatoid were shown (x40).

Fig 2. Antiproliferative effect of TRF1 or TRF2 silencing on RCC cells.

Results were expressed as percentage of viable cells relative to untreated cells. Values are expressed as the percentage of control, which was defined as 100%. Each column represents the mean ± S.D. Results were analyzed by ANOVA. * P<0.05, ** P<0.01, *** P<0.001 was considered as significant.

Fig 3. Inhibitory effect of TRF1, TRF2 silencing on the migration of renal cell carcinoma cells.

Representative microscopic images are (A). Non-transfected cells (B). Cells transfected with scrambled siRNA (C). Cells transfected with TRF1 siRNA (D). Cells transfected with TRF2 siRNA. The solid lines define the area lacking cells.

Fig 4. The effect of TRF1, TRF2 silencing on cell cycle distribution measured by flow cytometry (B) Graphical representation of flow cytrometry results.

Values are expressed as the percentage of cells in each phase of cell cycle. Each column represents mean ± S.D. Results were analyzed by ANOVA. * P<0.05, ** P<0.01, *** P<0.001 was considered as significant.

Fig 5. (a). Cytofluorimetric analysis of apoptosis by Annexin V-FITC/PI after TRF1, TRF2 silencing in renal cell carcinoma cells.

(A) Viable cells are in the lower left quadrant, early apoptotic cells are in the lower right quadrant, late apoptotic or necrotic cells are in the upper right quadrant, and nonviable necrotic cells are in the upper left quadrant. (B) Graphical representation of flow cytometry results. Values are expressed as the percentage of cells in quadrant. Each column represents mean ± S.D. Results were analyzed by ANOVA. * P<0.05, ** P<0.01, *** P<0.001 was considered as significant. (b). H& E staining of cells transfected with TRF1 or TRF2 siRNA. (A) non-transfected cells (B) scrambled siRNA transfected cells (C) TRF1 siRNA transfected cells (D) TRF2 siRNA transfected cells.