The stem–loop of miR-210 is located in an intron of a noncoding RNA, which is transcribed from AK123483 on chromosome 11p15.5 [
11]. MiR-210 is regulated by HIF-1α, HIF-2α and nuclear factor κB (NFκB) [
11,
13,
20]. HIF-1α directly binds to a hypoxia responsive element (HRE) on the proximal miR-210 promoter, located 400 bp upstream of the structure [
11]. NFκB p50 can physically interact with a conserved κB binding site and activate miR-210 promoter under hypoxia [
20].
MiR-210 expression is elevated in a variety of human solid tumors [
21,
22]. The role of miR-210 in tumorigenesis has been investigated in several reports. However, the results of these experiments are somewhat controversial. It has been reported that high levels of miR-210 were associated with disease recurrence and short overall survival in head and neck squamous cell carcinoma [
23] and display an inverse correlation with disease-free and overall patient survival in human breast cancer samples [
24]. In addition, miR-210 levels correlate with breast cancer aggressiveness and metastatic potential [
25]. However, genomic deletions of miR-210 in human epithelial ovarian cancer samples suggested these deletions as a possible trigger to tumorigenesis [
12]. Huang
et al. have demonstrated that stably expression of miR-210 in implanted tumor tissue could repress tumor growth in immunodeficient mice [
26]. Our preliminary findings suggested that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy showed an enhanced effect on hypoxic human hepatoma cells
in vitro[
17]. In the present study, in order to investigate the effect of knockdown of miR-210 in tumorigenesis in vivo, we subcutaneously implanted miR-210 downregulated human hepatoma SMMC-7721 cells into nude mice. We found that tumor growth was significantly delayed in SMMC-7721/Lv-anti-210 xenograft. To investigate the mechanism underlying the knockdown of miR-210 mediated tumor growth delay, we analyzed protein expression of HIF-1α gene and miR-210 targeted MNT, EFNA3 and AIFM3 genes in human hepatoma xenograft by Western blot. MNT represses Myc target genes by binding the E box DNA sequence (CANNTG) after forming heterodimers with Max [
27,
28]. MiR-210 could override hypoxia-induced cell cycle arrest by downregulating MNT [
13]. EFNA3 is an ephrin family member involving vascular development [
29]. Over-expression of EFNA3 significantly blocked the angiogenesis effect of miR-210 [
30]. AIFM3, a gene homologous to the apoptosis-inducing factor (AIF), is a direct target of miR-210 in human hepatoma cells [
17,
31]. AIFM3 increases cytochrome
c release and induces apoptosis in a caspase-dependent manner [
32]. Our preliminary studies showed that AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells, which suggest miR-210 downregulation mediate enhanced radiation induced apoptosis in hypoxic human hepatoma cells through AIFM3 gene at least in part [
17]. The Western blot results indicated that knockdown of miR-210 decreased HIF-1α protein and increased protein expression of MNT, EFNA3 and AIFM3 genes in human hepatoma xenograft. HIF-1α protein downregulation by knockdown of miR-210 might be due to destruction of a hypoxia-induced positive feedback loop, in which HIF-1α induce miR-210 expression in hypoxia, which represses glycerol-3-phosphate dehydrogenase 1-like (GPD1L), contributing to increase of HIF-1α protein stability [
33]. HIF-1α downregulation may inhibit proliferation, induce apoptosis, and enhance radiosensitivity in hypoxic cancer cells [
4,
5]. In immunohistochemical studies anti-sense miR-210 therapy group showed decreased Ki-67-positive cells and IMVD and increased TUNEL-positive cells compared with control and negative control vector group. These results suggest that knockdown of miR-210 may lead to tumor growth delay by cell proliferation and angiogenesis suppression and apoptosis enhancement.
In order to investigate the efficacy of knockdown of miR-210 in combination with radiotherapy in nude mice bearing human hepatoma SMMC-7721 cells, mice were monitored for tumor growth and survival after treatment as described in Materials and Methods. Results showed that the average tumor volume in combined therapy group reached 593 mm3 on day 30, only 33.46% of control group. In addition, survival durations were significantly longer in combined therapy group compared with control, radiotherapy or anti-sense miR-210 therapy group. These results suggest that knockdown of miR-210 in combination with radiotherapy is more effective than radiotherapy alone or anti-sense miR-210 therapy alone in suppressing tumor growth and extending survival duration. Analyzing associated mechanisms of the in vivo efficacy of combined therapy, we observed its inhibitory effects on cell proliferation (by Ki-67 staining) and tumor angiogenesis (by CD31 staining) and an enhancing effect on apoptosis (by TUNEL staining) in human hepatoma xenograft.