The inhibition of microRNA‐326 by SP1/HDAC1 contributes to proliferation and metastasis of osteosarcoma through promoting SMO expression

Abstract Osteosarcoma (OS) is a malignant bone cancer lacking of effective treatment target when the metastasis occurred. This study investigated the implication of MicroRNA‐326 in OS proliferation and metastasis to provide the clue for the treatment of metastatic OS. This study knocked down SP1 in MG63 and 143B cells and then performed Microarray assay to find the expression of miRNAs that were influenced by SP1. MTT, EdU, wound‐healing and cell invasion assays were performed to evaluated cell proliferation and invasion. OS metastasis to lung was detected in a nude mice model. ChIP assay and DAPA were applied to determine the regulatory effect of SP1 and histone deacetylase 1 (HDAC) complex on miR‐326 expression. Human OS tissues showed lowly expressed miR‐326 but highly expressed Sp1 and HDAC. Sp1 recruited HDAC1 to miR‐326 gene promoter, which caused the histone deacetylation and subsequent transcriptional inhibition of miR‐326 gene. miR‐326 deficiency induced the stimulation of SMO/Hedgehog pathway and promoted the proliferation and invasion of 143B and MG63 cells as well as the growth and metastasis in nude mice. SP1/HDAC1 caused the transcriptional inhibition of miR‐326 gene by promoting histone deacetylation; miR‐326 deficiency conversely stimulated SMO/Hedgehog pathway that was responsible for the proliferation and metastasis of OS.

MicroRNAs (miRNAs) are classical small non-coding RNAs constituted by approximately 22 nucleotides. miRNAs primarily influence the stability of mRNA by interacting with the 3′UTR. 8 The interaction generally causes the rapid degradation of mRNA before it is translated to protein, therefore miRNAs are associated with the down-regulation of the targeted genes. 9 Though miRNAs play important role in the post-transcription of genes, expression of miR-NAs themselves is regulated by complex mechanisms, for example, epigenetic modifications. 10 Epigenetic modifications which includes DNA methylation, multiple modifications of histone (eg histone acetylation and methylation) and changes of chromatin structure.
Studies confirmed that epigenetic modifications play an important role in the modulation of miRNA expression. 11,12 The epigenetic modifications of miRNA during the initiation and development of cancers conversely affect various biological processes of tumour, such as the proliferation, metastasis and drug resistance. 13,14 Our preliminary study found that histone deacetylase 1 (HDAC1) and SP1 were up-regulated in OS tissues compared to the normal tissues. HDAC1 is a key enzyme responsible for histone deacetylase. [15][16][17] Although SP1 is a transcription factor, it has been found to help HDAC1-mediated epigenetic modification. This study knocked down SP1 in two OS cell lines, followed by the miRNA Microarray detection. We found that miR-326 was up-regulated in both cell lines after SP1 knockdown, suggesting that miR-326 was negatively regulated by SP1. This result was in agree with the down-regulation of miR-326 in OS tissues based on the data in GEO data set, given SP1 is highly expressed in OS. Interestingly, SP1, acting as a transcription factor, promotes gene expression in most cases, but SP1 negatively regulated miR-326. We hypothesized that SP1 probably recruited HDAC1 to miR-326 gene promoter, which caused the histone deacetylation and subsequent transcriptional inhibition of miR-326 gene. This study performed ChIP assay to identify the hypothesis. miR-326 was predicted to target SMO that promotes cancer proliferation and metastasis by stimulating Hedgehog signal. Therefore, down-regulated miR-326 is proposed to enhance the cancer-promoting effects of SMO/Hedgehog signal. This study aimed to determine the role of a regulatory axis Sp1/HDAC1/miR-326/SMO/Hedgehog in the proliferation and metastasis of OS.  Table 1.

| RNA extraction and qRT-PCR analyses
Total RNA was isolated from frozen tumour specimens and cell lines using the Trizol reagent (Takara). The first-strand cDNA was generated using the miRNA Q-PCR Detection Kit (GeneCopoeia #R0101L

| Transfection
The siRNAs specifically targeting HDAC1/2/3, SP1 and control siRNA (the sequences are depicted in Table 2
The experiment was repeated three times.  Complete medium was subsequently added to the lower chamber.

| Trans-well assays
Following culture for 24 hours, the upper membrane was stained with 0.1% crystal violet in 4% Paraformaldehyde (PFA). The invading cells were quantified by counting 10 random fields under a light microscope (E200; Nikon Corporation).

| Wound-healing assay
Wound-healing assay was used to further investigate cell migration.

| Tumour xenograft in nude mice
The animal experiment was approved by the Ethical Committee for

| Haematoxylin and eosin (HE) staining
The lung tissues were taken from the nude mice and fixed in 10% buffered formalin. Afterwards, the tissues were embedded in paraffin, sectioned at 5 μm and stained with HE. The slides were observed and evaluated under light microscopy for histological examination.

| Statistical analysis
Statistical analysis was performed with the Student unpaired t test.
Statistical analysis of the data was performed using SPSS18.0 software (SPSS). A P value of <.05 was considered significant.

| Up-regulated SP1 and increased activities of HDACs in OS tissues and cells
Results showed that both mRNA and protein levels of SP1 were up-regulated in OS tissues compared to the corresponding normal tissues (P < .01 or P < .001, Figure 1A,B). In addition, mRNA and protein levels of SP1 were higher in the OS cell lines, such as U2OS (P < .001), MG63 (P < .05 or P < .01) and 143B cells (P < .01 or P < .001), than in hFOB1. 19, a normal osteoblastic cell line ( Figure 1C,D). Activities of all HDAC1, HDAC2 and HDAC3 were increased in OS tissues compared to the corresponding normal tissues (P < .001, Figure 1E). HDAC1 activity was also increased in some OS cell lines with most profound increase in MG63 (P < .01) and 143B (P < .01) cells, in comparison to that in hFOB1.19 cells ( Figure 1F).
The degree of the increase of HDAC2 and HDAC3 activity was less than that of HDAC1 activity, though HDAC2 and HDAC3 activities were significantly increased in 143B and Saos2 cells (P < .05), respectively. Based on these data, MG63 and 143B cells were used in further study.

| The changes of miRNA expression profiles caused by SP1 knockdown
This study knocked down SP1 in MG63 and 143B cells and then performed Microarray assay to find the expression of miRNAs that were influenced by SP1 (Figure 2A,B). The data showed that expression of 18 miRNAs was increased in both MG63 and 143B cells after SP1 knockdown ( Figure 2C). There were 20 miRNAs whose expression were down-regulated in both MG63 and 143B cells after SP1 knockdown. Among the up-regulated miRNAs ( Figure 2D), miR-326 was predicted to target SMO during bioinformatics analysis (http://www. targe tscan.org/vert_72/). Since SMO-mediated Hedgehog signalling plays critical roles in tumour occurrence and progression, this study aimed to investigate the regulatory effect of SP1/miR-326 axis on Hedgehog signalling. miR-326 expression was down-regulated in OS tissues compared to the corresponding normal tissues (P < .01, Figure 2E). Moreover, in comparison to hFOB1.19, all Saos-2, U2OS, MG63 and 143B cells showed decreased expression of miRNA-326 (P < .05 or P < .01, Figure 2F).

| miRNA-326 expression was regulated by SP1 and HDAC1
Using an open online Jaspar database (http://jaspar.gener eg.net), bioinformatics analysis showed that SP1 was able to bind to the promoter of miRNA-326 gene ( Figure 3A). As indicated by PCR, SP1 knockdown conversely caused the up-regulation of miRNA-326 in MG63 and 143B cells (P < .01, Figure 3B). Similarly, HDAC1 knockdown also increased miRNA-326 expression in MG63 and 143B cells (P < .01, Figure 3C). However, the increase caused by depletion of HDAC2 and HDAC3 was not significant. Treatment with HDAC inhibitor, vorinostat, induced the up-regulation of miRNA-326, as well (P < .001). We performed ChIP assay to determine the interaction between SP1, HDAC1 and acetylated histone 3 proteins and the promoter of miRNA-326 gene. Both antibodies of HDAC1 and acetylated histone 3 proteins extracted the DNA sequence of miRNA-326 gene promoter (P < .001, Figure 3D).
Knockdown of SP1 decreased the DNA enrichment in HDAC1 protein complex (P < .01), but increased the DNA enrichment in acetylated histone 3 protein complex (P < .01). Furthermore, using ChIP assay, we found that the enrichment of miRNA-326 gene promoter is higher in OS tissues compared to the corresponding normal tissues (P < .01, Figure 3E). We also conducted DAPA to further determine the interaction between SP1 and HDAC1 proteins and miRNA-326 promoter oligonucleotides. SP1 and HDAC1 proteins were pulled down by the WT oligonucleotides, but almost not by MT oligonucleotides ( Figure 3F). SP1 knockdown notably decreased the abundance of HDAC1 protein pulled down by the WT oligonucleotides.

| SP1/miR-326 modulated proliferation, migration and invasion of OS cells
To The invasion and migration of MG63 and 143B cells were suppressed after SP1 knockdown and miR-326 overexpression (P < .05 or P < .01, Figure 5A,B). Knockdown of SP1 and miR-326 at the same time strengthened the invasion and migration of MG63 and 143B cells (P < .05). miR-326 knockdown alone also strengthened the invasion and migration of MG63 and 143B cells (P < .01).

| SP1/miR-326 modulated SMO/GLI1/MMP-9 signalling pathway
Using miRanda and Targetscan, bioinformatics analysis showed that oncogene SMO is a potential target of miR-326. We initially performed western blot to measure SMO expression in OS cell lines.
Dual luciferase reporter assay was conducted to explore whether SMO targeted miR-326 directly. Transfection of miR-326 mimics inhibited the WT luciferase activity (P < .01, Figure 6B), while had not effect on MT luciferase activity. The regulatory effect of SP1/ miR-326 axis on SMO/GLI1/MMP-9 signalling pathway was evaluated using western blot assay. SP1 knockdown and miR-326 overexpression reduced protein levels of SMO, GLI1 and MMP-9 (P < .01 or P < .001, Figure 6C). The reduction of SMO, GLI1 and MMP-9 protein levels resulted from SP1 knockdown was abolished by miR-326 knockdown.

| SP1/miR-326 modulated the proliferation and metastasis of 143B cells in nude mice
Transfection with SP1-siRNA vector decreased SP1 but increased miR-326 expression in 143B-cell-xenografted tumour, as indicated by the results form PCR assay (P < .001, Figure 7A). Treatment with miR-326 inhibitor (antagonist) abolished the increased of miR-326 expression caused by SP1 knockdown. SP1 knockdown inhibited 143B tumour growth in nude mice (P < .01, Figure 7B). However, the inhibition of tumour growth was abolished by the reduction of miR-326. As indicated by immunohistochemistry, SMO and Ki67 protein abundances were decreased with SP1 knockdown. Knockdown of both SP1 and miR-326 seemed to had no effect on SMO and Ki67 protein abundances ( Figure 7C). This study investigated the metastasis of 143B cells to lung tissues using HE staining assay. 143B tumour belongs to solid tumour. It is easy to distinguish a solid tumour from F I G U R E 1 Up-regulated SP1 and increased activities of HDACs in OS tissues and cells. SP1 expression was measured in 20 pairs of OS tissues and the matched adjacent normal tissues using RT-qPCR (A) and western blot assays (B). Figure B shows five representive blots. SP1 expression was measured in OS cell lines and a normal osteoblastic cell line using RT-qPCR (C) and western blot assays (D). U6 and GAPDH were employed as miRNA and mRNA internal control, respectively. miR-326 expression was normalized to U6 expression. Activities of HDAC1/2/3 was measured in 20 pairs of OS tissues and the matched adjacent normal tissues (E) as well as in OS cell lines and a normal osteoblastic cell line (F). T means tumour tissues, N means matched adjacent normal tissues. A, B and E: **P < .01, ***P < .001 vs normal tissues. C, D and F: *P < .05, **P < .01, ***P < .001 vs hFOB1.19 cells lung tissues in which there are full of pulmonary alveoli. In lung tissues, SP1 knockdown was associated to smaller size of 143B tumour ( Figure 7D), but knockdown of both SP1 and miR-326 dramatically boosted the growth of 143B tumour. Figure 7E shows the molecular mechanism by which SP1/miR-326 regulated the proliferation and metastasis of OS. The molecular mechanism was described and disused in following discuss section.

| D ISCUSS I ON
Transcription factor Sp1 has been extensively reported to increase expression of many oncogenes, thereby promoting the progression of OS and other types of tumour. 18,19 However, there are seldom studies investigating the transcriptional effect of Sp1 on miRNA.
Mounting evidence has indicated that many miRNAs play important role in tumour occurrence and development. [20][21][22]  F I G U R E 6 SP1/miR-326 modulated SMO/GLI1/MMP-9 signalling pathway. A, Western blot was conducted to measure SMO expression in OS cell lines and a normal osteoblastic cell line. B, Dual luciferase reporter assay was conducted to explore whether SMO targeted miR-326 directly. Transfection of miR-326 mimics inhibited the WT luciferase activity, while had not effect on MT luciferase activity. C, MG63 and 143B cells were transfected with siRNA-SP1, siRNA-SP1 together with miR-326 inhibitor, miR-326 mimics and negative control. Western blot was conducted to measure SMO, GLI1 and MMP-9 protein level. A, *P < .05, **P < .01, ***P < .001 vs hFOB1.19 cells. B and C, *P < .05, **P < .01, ***P < .001 vs WT group that did not undergo transfection F I G U R E 7 SP1/miR-326 modulated the proliferation and metastasis of 143B cells in nude mice. A, Transfection with SP1-siRNA vector decreased SP1 but increased miR-326 expression in 143B-cell-xenografted tumour, as indicated by the results form PCR assay. Treatment with miR-326 inhibitor (antagonist) abolished the increased of miR-326 expression caused by SP1 knockdown. B, SP1 knockdown inhibited 143B tumour growth in nude mice. However, the inhibition of tumour growth was abolished by the reduction of miR-326. C, As indicated by immunohistochemistry, SMO and Ki67 protein abundances were decreased with SP1 knockdown. D, In lung tissues, SP1 knockdown was associated to smaller size of 143B tumour, but knockdown of both SP1 and miR-326 dramatically boosted the growth of 143B tumour. E, The molecular mechanism by which SP1/miR-326 regulated the proliferation and metastasis of OS. Sp1 epigenetically down-regulated miR-326 by recruiting HDAC1. Down-regulated miR-326 caused the overactivation of Hedgehog signalling pathway, resulting in the rapid proliferation, suppressed apoptosis and enhanced metastasis of OS. **P < .01, ***P < .001 vs control group

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data generated/analyzed in the present study are available upon reasonable request from the corresponding author.