Differentially expressed circRNAs and circRNA/miRNA/mRNA
regulatory network
Additionally, we have also identified the differentially expressed
circRNAs between OS and corresponding adjacent nontumor tissue samples.
Compared with nontumor samples, there were totally
1088 significantly
differentially expressed circRNAs in
OS samples, including 1052 upregulated circRNAs and 36 downregulated
circRNAs (Figure 1a). The expression levels of differentially expressed
circRNAs were significantly different
(Figure 1b).
Furthermore, we have estimated the circRNA/miRNA interactions using
miRanda and TargetScan. After the cross analysis of predicted results of
two software, there were 36 differentially expressed circRNAs and their
corresponding miRNAs in regulatory network
(Supplementary Figure 2a). Among
them, circ_0001722 with small
expression standard deviations in three OS samples and three nontumor
samples (standard deviation values were 3.1 and 0.7, respectively) was
selected for subsequent analysis. Regarding circ_0001722, there were 11
overlapped regulatory pairs between miRanda and TargetScan
(Supplementary Figure 2b), including miR-365b-5p, miR-3122, miR-211-5p,
miR-208b-5p, miR-3913-5p, miR-204-5p, miR-365a-5p, miR-5006-3p,
miR-208a-5p, miR-3137 and miR-6875-3p. Then, the target genes of these
11 miRNAs were also estimated utilizing miRTarBase database. We found
that miR-204-5p and its corresponding 76 target genes, comprising RUNX2,
were supported by most experimental
validation (469 experimental results). The
circRNA/miRNA/mRNA regulatory
network, based on circ_0001722, 11 miRNAs, and target mRNAs, was shown
in Figure 1c.
Expression of circ_0001722 is significantly upregulated inOS tissues and cell
lines
Firstly, we determined whether
circ_0001722 is a closed circular
RNA that is resistant to RNase R digestion. We investigated its
expression level in OS tissues and cell lines. Our result showed that
RNase R digestion could decrease the RNA level of linear GAPDH, but
could not affect the level of circ_0001722 significantly (Figure 1d),
indicating that circ_0001722 was resistant to RNase R digestion. To
confirm circ_0001722 is a closed circRNA, we ran PCR amplification of
circ_0001722 with specific divergent primers. PCR products amplified by
divergent primers were detected by Sanger sequencing to confirm the
circular structure of circ_0001722. Our data showed that the back
splicing junction sites of circ_0001722 were consistent with the
sequence from circBase (Figure 1e). Then, we measured the expression
level of circ_0001722 in a series of 20 surgically removed fresh OS
tissues and their corresponding adjacent nontumor tissues by qRT-PCR.
Comparing with nontumor tissues, the expression level of circ_0001722
in OS tissues was significantly upregulated (P <0.01;
Figure 1f). Similarly, the expression of circ_0001722 in OS cell lines
(MG63 and U2OS) was significantly higher than in normal human fetal
osteoblastic cell line (hFOB 1.19)
(P <0.01; Figure 1g).
Downregulation ofcirc_0001722 suppresses
cell proliferation of human OS cells in vitro and in
vivo.
We noticed that the expression of circ_0001722 was higher in MG63 cells
and U2OS cells. Based on the observation, exogenous shRNA
(sh-circ_0001722) was used to knock down circ_0001722 expression in
two OS cell lines (Figure 2a). Based on the high silencing efficiency,
we carried out cell proliferation assay and matrigel invasion assay. The
results showed that the viability level of MG63 cells and U2OS cells
transfected with sh-circ_0001722 was lower than that of the cells
transfected with sh-Mock (Figure 2b). Consistently, sh-circ_0001722
significantly decreased the number of OS cells invaded through the
matrigel. Quantitative analysis of cell numbers revealed that, in the
negative control (sh-Mock) and blank control groups, the number of cells
invaded through the matrigel was almost 5 times higher than
sh-circ_0001722 group (figure 2c). The above experiments verified to
some extent that silencing circ_0001722 can inhibit the proliferation
and invasion of OS cells in vitro . To investigate whether
circ_0001722 regulates the tumorigenesis of OS in vivo , we
established OS xenograft mouse models. MG63 cells transfected with
sh-circ_0001722 or sh-Mock were inoculated subcutaneously in the right
flank of athymic nude mice (5 × 106 cells per mouse, 5
mice per group). After 4 weeks, all experimental mice were euthanized
and tumor tissues were collected (Figure 2d). Comparing with sh-Mock
group, smaller tumor volume and lower tumor weight were observed in
sh-circ_0001722 group. The same conclusion was reached with thein vitro experimental results. Downregulation of circ_0001722
can suppress tumorigenesis of OS cells in vivo . In addition,
compare with the control group, knocking down of circ_0001722 led to
the decrease of Ki67 expression, which implied the cell proliferation of
sh-circ_0001722 group is slower than sh-Mock group. Taken altogether,
downregulation of circ_0001722 can restrain the growth of OS cellsin vitro and in vivo .