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, 307 (1), 194-203

Antisense Inhibition of Hyaluronan synthase-2 in Human Osteosarcoma Cells Inhibits Hyaluronan Retention and Tumorigenicity

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Antisense Inhibition of Hyaluronan synthase-2 in Human Osteosarcoma Cells Inhibits Hyaluronan Retention and Tumorigenicity

Yoshihiro Nishida et al. Exp Cell Res.

Abstract

Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion.

Figures

Figure 1
Figure 1. RT-PCR analysis of HAS mRNA expression in MG-63 cells
(A) Total RNA isolated from MG-63 cells was reverse-transcribed and PCR amplified for 35 cycles with HAS-1, HAS-2 or HAS-3 specific primers. The products were separated on 1.5% agarose gels and visualized by SYBR Green I staining. Lanes 1–3 represent conventional PCR amplification of HAS-1, HAS-2 or HAS-3, respectively. Lane Std represents 600-bp of 100 base pair DNA ladder marker band. (B) Aliquots (0.125 μg) of total RNA were reverse-transcribed and PCR co-amplified with HAS-2 or HAS-3 DNA mimics for 26 cycles with HAS-2 or HAS-3 specific primers, respectively. The slower migrating bands in all lanes are the mimics. Lanes 1–4 represent co-amplification of sample cDNA with 50, 25, 12.5, and 6.25 attomoles of HAS-2 DNA mimic and primers. Lanes 5–8 represent aliquots of the same RNA PCR co-amplified with 2, 1, 0.5, and 0.25 attomol of HAS-3 DNA mimic and HAS-3 primers. The stained products were scanned and quantified using a fluoroimaging system. Std represents 100 base pair DNA ladder markers. HAS-2 target, 409 bp; mimic, 523 bp. HAS-3 target, 331 bp; mimic, 421 bp.
Figure 2
Figure 2. The effect of HAS-2 antisense oligonucleotide treatment on HAS-2 or HAS-3 mRNA expression by MG-63 cells
Aliquots of total RNA (0.125 μg) derived from MG-63 cells cultures treated with 2 μM HAS-2 antisense or control oligonucleotides for varying times (16 and 40 hours after addition of oligonucleotides is shown) were reverse-transcribed and PCR co-amplified for 26 cycles with HAS-2 or HAS-3 mimics and HAS-2 or HAS-3 specific primers, respectively. The products were separated on 1.5% agarose gel and visualized by SYBR Green I staining. The stained products were scanned and quantified using a fluoroimaging system. The slower migrating bands in all lanes are the mimics. Panels A and B depict competitive RT-PCR amplification of total RNA isolated from MG-63 cultures treated with HAS-2 antisense oligonucleotides for 16 hours; 40 hour cultures in panels C and D. HAS-2 primers and mimics are shown in panels A and C; HAS-3 primers and mimics in panels B and D. Lanes 1–4 in each panel represent amplification of RNA isolated from HAS-2 antisense oligonucleotide-treated cultures; lanes 5–8, HAS-2 sense oligonucleotides treated cultures. For HAS-2 PCR analyses, lanes 1–4 and 5–8 represent samples co-amplified with 50, 25, 12.5, and 6.25 attomol of HAS-2 DNA mimic, respectively. For HAS-3 PCR analyses, lanes 1–4 and 5–8 represent samples co-amplified with 1, 0.5, 0.25, and 0.125 attomol of HAS-3 DNA mimic, respectively. HAS-2 target, 409 bp; mimic, 523 bp. HAS-3 target, 331 bp; mimic, 421 bp. Std represents 100 base pair DNA ladder markers.
Figure 3
Figure 3. Hyaluronan accumulation by MG-63 cells during proliferative and confluent growth
MG-63 cells cultured in monolayer were fixed in 2% paraformaldehyde and stained for HA using a biotinylated HABP followed by streptavidin peroxidase. Low density cultures in proliferative phase of growth are shown in Panel A; confluent cultures in Panel C. Nuclear staining with DAPI of the same cells shown in panels A and C are shown in panels B and D, respectively. The arrows shown in panel A depict cytoplasmic HABP-positive staining. All cells were photographed and printed at the same magnification. (original magnification: 200x)
Figure 4
Figure 4. The effect of HAS-2 antisense oligonucleotides on HA retention in MG-63 cells
MG-63 cells were fixed in 2% paraformaldehyde and stained for HA with biotinylated HABP 12 hours (panels A and B) or 24 hour (panels C and D) following the initial addition of HAS-2 antisense (panels A and C) or sense (panels B and D) oligonucleotides. Fixed erythrocytes were applied to cultures of MG-63 cells 16 hours (panels E and F) or 40 hours (panels G and H) following the initial addition of HAS-2 antisense (panels E and G) or sense (panels F and H) oligonucleotides. All cells were photographed and printed at the same magnification. (original magnification: 200x).
Figure 5
Figure 5. The effect of HAS-2 oligonucleotides on MG-63 cell proliferation
MG-63 cells incubated for 24 and 48 hours after initial addition of antisense or sense oligonucleotides were analyzed for changes in cell proliferation by MTT assay. Bars represent the average ± s.d. of absorbance values at 570nm. Data was averaged from triplicate expreriments. Differences between antisense (open bars) and sense (shaded bars) treatments exhibited statistical significance, p=0.012 and p=0.0002 for data obtained at both 24 and 48 hours post transfection, respectively.
Figure 6
Figure 6. The effect of HAS-2 antisense oligonucleotides on MG-63 cell motility and invasiveness
Following transfection with antisense or sense oligonucleotides, MG-63 cells were assayed for cell motility and invasiveness through the use of modified Boyden chamber assays without (A) or with matrigel (B) respectively, for an additional 12 and 24 hours. The cells were then fixed with 2% paraformaldehyde and visualized with hematoxylin staining. The number of cells on lower surface of the membrane was calculated in 4 randomly selected high power fields of view. Changes in the number (average ± s.d.) of migrating cells after 12 and 24 hours of migration are summarized in panel A. Differences between antisense and sense treatments exhibited statistical significance for data obtained following 12 hours (p=0.0066) and 24 hours (p=0.00065) of migration. Changes in the number (average ± s.d.) of invading cells are summarized in panel B. Differences between antisense (open bars) and sense (shaded bars) treatments exhibited statistical significance with a p=0.013 for data obtained 24 hours post transfection.

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