Antisense oligonucleotide mediated knockdown of HOXC13 affects cell growth and induces apoptosis in tumor cells and over expression of HOXC13 induces 3D-colony formation

Abstract

HOXC13 is a homeobox containing gene that plays crucial roles in hair development and origin of replication. Herein, we investigated the biochemical functions of HOXC13 and explored its potential roles in tumor cell viability. We have designed a phosphorothioate based antisense-oligonucleotide that specifically knockdown HOXC13 in cultured cells. Cell viability and cytotoxicity assays demonstrated that HOXC13 is essential for cell growth and viability. Antisense-mediated knockdown of HOXC13 affected the cell viability and induced apoptosis in cultured tumor cells. HOXC13 regulates the expression of cyclins and antisense-mediated knockdown of HOXC13 resulted in cell cycle arrest and apoptosis in colon cancer cells. Finally over expression of HOXC13 resulted in 3D-colony formation in soft-agar assay indicating its potential roles in cell proliferation and tumorigenesis.

Figure 1

HOXC13 antisense oligonucleotide design and characterization. (A) Oligonucleotide sequences of HOXC13 antisense. Nucleotides are linked by phosphorothioate linkages instead of phosphodiester bonds. (B–D) Antisense–mediated of knockdown of HOXC13. SW480 cells were transfected with different concentration of HOXC13 (0.75 – 1.75 µg/mL) and scramble antisense (1.25 µg/mL) for 48 h. RNA was extracted, reverse-transcribed and subjected to PCR with primers specific to HOXC13 and actin (control) (in panel B). Real-time PCR for quantification of HOXC13 knockdown in control and antisense treated cells are shown in panel C (GAPDH was used as control). Bars are indicating standard error (n=6, p<0.05). (D) Protein extracts from the control and antisense-treated cells were analyzed by Western blotting using actin (control) and HOXC13 antibodies.

Figure 2

Effect of HOXC13 knockdown on cell viability. (A) Miscroscopic analysis: Different cell lines were transfected with HOXC13 (1.75 µg/mL) and scramble (1.75 µg/mL) antisenses separately for 48 h and visualized under a microscope. (B) Cytotoxicity analysis: Different cell lines were transfected with 1.75 µg/mL of HOXC13 and scramble antisenses (1.75 µg/mL) for 48 h and then subjected to MTT assay. The percent viability of HOXC13 antisense versus scramble antisense samples were plotted for different cell types. Bars indicate standard errors (n=8, p<0.05). (C) Effect of HOXC13 knockdown on cell growth: SW480 cells were transfected with 1.75 µg/mL HOXC13 and scramble antisenses (1.75 µg/mL) and harvested at different times (24, 48 and 72 h), stained with trypan blue and counted under microscope and plotted. Three replicates of each experiment were repeated for at least two times. Bars indicate standard errors.

Figure 3

HOXC13 knockdown affects cell cycle progression. SW480 cells were transfected with varying concentrations (0.75 – 1.75 µg/mL) of HOXC13 and scramble antisenses (1.25 µg/mL) for 48 h. Cells were harvested, fixed with 70% ethanol/PBS, stained with PI and subjected to flow cytometry analysis. Cell population percentage at each stages of the cell cycle are shown in the bottom panel.

Figure 4

HOXC13 knockdown affects cell viability, nuclear integrity and cell cycle regulatory gene expressions. (A)TUNEL assay: SW480 cells were transfected with 1.75 µg/mL of HOXC13 and scramble antisense (1.75 µg/mL) separatally for 48 h, fixed with 4% formaldehyde and then subjected to terminal dUTP nicked end labeling and stained with DAPI. The green speckles are showing apoptotic cells containing fragmented DNA. (B) Cytochrome-c release assay: HOXC13 knocked down and control cells were immuno-stained with cytochrome-c antibody and then FITC-labeled secondary antibody and visualized under fluorescence microscope. Nuclear staining was performed with DAPI. (C) Effect of HOXC13 knockdown on cyclin expression. SW480 cells were treated with 1.25 and 1.75 µg/mL of HOXC13 and scramble antisense (1.25 µg/mL) for 48 h. RNA from control and antisense treated cells were harvested, reverse-transcribed and subjected to PCR using specific primers.

Figure 5

HOXC13 over expression induces tumorigenesis. (A) Western blotting for HOXC13 overexpression analysis. Proteins were isloated from HEK293 cells and stable cells expressing Flag-HOXC13 and subjected to Westen blotting using actin(control) and flag antibodies. (B) 3D-colony formation ability of HOXC13 over expressed cells. HEK293 and Flag-HOXC13 stable cells were incubated in soft agar for 4–5 weeks stained with 0.005% crystal violet and analyzed under microscope. Magnified view of one HOXC13 over expressed colony is shown on the left side. (C) Number of colonies grown in soft agar cultures were counted under light microscope and plotted. Bars indicate standard errors.