The miR-34a-5p promotes the multi-chemoresistance of osteosarcoma via repression of the AGTR1 gene

The two cell lines (SJSA-1 (ATCC NO. CRL-2098) [24] and G-292 (ATCC NO. CRL-1423) [25] used in this study) were purchased from ATCC. The cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) implemented with 10% fetal bovine serum and 1% glutamine at 37 °C in 5% CO₂.

RNA-seq analysis was performed by BGI-Tech (Shenzhen, China). Sample preparation and data analysis were done as reported previously [26].

All the sequences including the antagomiR, mimic, siRNA, the scramble sequence (negative control, NC) were supplied by Guangzhou Ribobio, China. The expression constructs for AGTR1 (EX-A0417-M98-5) fused with a GFP tag were supplied by Guangzhou Fulengen (Guangzhou, China). The transfection method mentioned above was performed according to the manufacturer’s instruction. The partial sequences used in this study are as follows:

Clinical grades of the following drugs were used, Dox (Haizheng, Zhejiang, China); Etop (Hengrui, Jiangsu, China); Carb: carboplatin (Qilu, Shandong, China) and CDDP (Haosen, Jiangsu, China) [5, 27, 28]. The method of MTT assay has been described in our previous report [26].

The annexin V-FITC/propidium iodide (PI) staining assay was used to detect the apoptosis of G-292 cells transfected with either 5PM, si-AGTR1 or their corresponding NC. Cells growing to the logarithmic growth phase were harvested and rinsed after washing with cold PBS. Then, FITC-labeled enhanced annexinV (3 μl) and propidium iodide (3 μl, 20 μg/ml) were added to the cell suspension (100 μl) for labeling (Vazyme, China). After incubation in the dark for 15 min at room temperature, the samples were diluted with 50 μl PBS. Apoptotic cells were then evaluated by gating PI and Annexin V-positive cells on a FACSCalibur instrument. The results were analyzed according to the manufacturer’s instructions. The experiments were performed at least three times independently, and a representative is shown.

A luciferase reporter assay was performed to test the binding of miR-34a-5p to AGTR1. The detailed methods were described previously [29]. The full-length AGTR1 3’-untranslated region (UTR, 894 bp) containing the target sequence of miR-34a-5p was inserted into the pGL3 -reporter plasmid to construct pGL3-luc-AGTR1 WT and pGL3-luc-AGTR1 Mut. Cells were seeded into 96-well plates at approximately 1x10⁴ cells per well. Then the cells were transfected with a mixture of pGL3-luc-AGTR1 WT or Mut (50 ng), Renilla (5 ng), mimic or NC nucleotides (5 pmol) using the riboFECT CP transfection kit according to the manufacturer’s instruction. After transfection in twenty-four hours, the cells were assayed by the Dual-Luciferase Reporter Assay System (Promega) using a Promega GloMax 20/20 luminometer. The relative luciferase activities of the UTR construct and pathway reporter constructs were analyzed as reported previously [5].

The total RNA was extracted from the cells using Trizol (Tiangen, China) according to the manufacturer’s instructions. The mRNAs were analyzed as previously reported [29]. The sequences of primers and probes used for the qRT-PCR analysis are as follows:

To detect and quantify the expression of miR-34a-5p, Total RNA was reverse transcribed using a Bulge-Loop™ miRNA qRT-PCR Primer Set (Ribobio) and quantified by SYBR Green-based real-time PCR analysis. The Ct values of the target miRs were normalized to the Ct values of U6 RNA before quantification using the 2^−ΔΔ Ct method.

Cells were lysed with a lysis buffer [29]. Anti-AGTR1 (25343-1-AP) was purchased from San Ying Biotechnology, China. The target proteins were then detected with anti-rabbit IgG peroxidase-conjugated antibody (SA00001-2; San Ying Biotechnology, China). The target bands were detected by an enhanced chemiluminescence reaction (Pierce), and the relative density (level) of proteins over the GAPDH (10494-1-AP; San Ying Biotechnology, China) band was quantified with the Gel-Pro Analyzer.

The xenograft model on nude mice was generated and analyzed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The analysis was performed as previously reported [29]. The expression of AGTR1 protein was measured using immunochemical analysis. Antigens were retrieved by pretreating dewaxed sections and processed with the Super Sensitive Link-Labeled Detection System (Biogenex, Menarini, Florence, Italy). Pictures were taken using a LEICA DM 4000B microscope. The animal study proposal was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Science and Technology of China. All of the mouse experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People’s Republic of China.

Apoptosis assays, cell viability, quantitative RT-PCR, and luciferase reporter assays were performed in triplicate, the data are presented as the means, and the error bars indicate the S.D. Excel was used to process the data. The differences were considered statistically significant at p < 0.05 using Student’s t -test.

Our previous result suggested that G-292 and SJSA-1 cell lines are the multi-chemosensitive and multi-drug resistant OS cell lines, respectively [29]. Indeed, the IC₅₀ profiling experiments against the following four drugs: Doxorubicin (Dox), Etoposide (Etop), Cisplatin (CDDP), Carboplatin (Carb) demonstrated that SJSA-1 cells is more resistant against all the four drugs. The chemoresistance index of the SJSA-1 cells is 20.32, which is drastically higher than that of the G-292 cells (Fig. 1a). To find the mechanistic insights that govern the multi-chemoresistance of OS cells, we performed an RNA-seq-based miR-omic analysis of G-292 and SJSA-1 cells, and several related miRNAs were selected based on a reference survey (Additional file 1: Figure S1). MiR-34a-5p was selected as our target, which correlated well with the probe and RNA-seq analyses. The miR-34a-5p expression is 3.41-fold higher in the SJSA-1 cells than in the G-292 cells by miR-omic and 186.83-fold higher by qRT-PCR analysis (Fig. 1b and c).

A given miRNA usually suppresses the expression of various target genes and thus regulates related pathways. We thus proposed the target genes of miR-34a-5p based on the following websites: TargetScan (http://​www.​targetscan.​org/​), miRDB (http://​mirdb.​org/​miRDB/​) and microRNA.org (http://​www.​microrna.​org/​microrna/​getMirnaForm.​do). We subsequently compared the expression pattern of shared predicted mRNAs between G-292 and SJSA-1 cells by the RNA-seq based miR-omic analysis. Dozens of genes have been found that differentially expressed in the two cell lines. Among them, the AGTR1 gene is one of the most significantly differentiated genes that negatively correlate with miR-34a-5p expression (Additional file 1: Figures S1, Additional file 2: Figures S2 and Additional file 3: Figures S3. Consequently, the expression level of AGTR1 was higher in G-292 than SJSA-1 at both mRNA (RNA-seq based miR-omic: 490.16:1, and qRT-PCR analysis: 28.49:1) and protein level (western blot: 3.21:1) (Figs. 2a b and 2c). The lower expression of AGTR1 in multi-chemoresistant cells SJSA-1 suggests that AGTR1 is a negative regulator of OS multi-chemoresistance.

The miR-34a-5p level was dramatically higher in SJSA-1 cells than G-292 cells. We found that AGTR1 negatively correlates with the level of miR-34a-5p. To check whether AGTR1 is one of the authentic targets of miR-34a-5p, we detected the AGTR1 level in the miR-34a-5p mimic transfected G-292 and the antagomiR transfected SJSA-1 cells versus the NC (scramble sequence control) transfected. The transfection of miR-34a-5p mimic in G-292 cells increased its expression to about 21-fold, whereas the transfection of miR-34a-5p antagomiR in SJSA-1 significantly decreased its level to 38% (Fig. 3a and b). In agreement with the changes of the miR-34a-5p level, a miR-34a-5p mimic transfection decreased the AGTR1 mRNA to 12% (Fig. 3c) and protein to nearly 79% (Fig. 3e) compared to that in the NC transfected G-292 cells. By contrast, miR-34a-5p antagomiR transfection increased the mRNA level of AGTR1 by 1.97 folds (Fig. 3d) and the protein level by 1.46 folds in SJSA-1cells (Fig. 3e).

To further confirm whether AGTR1 is a direct target of miR-34a-5p, we cloned the wild-type AGTR1 gene at the downstream of the Renilla luciferase gene in pGL3-control vector (Promega) to create pGL3-AGTR1 UTR WT or pGL3-AGTR1 UTR Mut (Fig. 3f). The constructs pGL3-AGTR1 UTR WT or pGL3-AGTR1 UTR Mut and pGL3 enhancer control were transfected into G-292 and SJSA-1 cells respectively, to determine the function of miR-34a-5p in different OS cells. The pGL3-AGTR1-UTR WT gave the relative luciferase activities of 0.84 and 0.62 in SJSA-1 and G-292 cells, respectively (Fig. 3g). The transfection of miR-34a-5p-mimic into G-292 cells significantly brought down the luciferase activity of pGL3-AGTR1-UTR WT construct, whereas the control cells showed almost the same activity upon the transfection of miR-34a-5p-mimic (Fig. 3g). Meanwhile, the transfection of miR-34a-5p-antagomiR into SJSA-1 cells raised the luciferase activity of pGL3-AGTR1-UTR WT construct (Fig. 3g). Furthermore, the mutation of the 3'-UTR showed similar effect as the wild type with the transfection miR-34a-5p-antagomiR into SJSA-1 cells. By contrast, the comparable luciferase activity was detected in the pGL3-AGTR1-UTR Mut with the transfection of miR-34a-5p-mimic into G-292 cells, suggesting that miR-34a-5p indeed targets the 3’-UTR region of AGTR1 (Fig. 3g). Getting together, AGTR1 is indeed, a direct target of miR-34a-5p and may dedicate the miR-34a-5p’s promoting effect on the OS drug resistance.

To investigate the role of AGTR1 in the OS chemoresistance, we first transfected si-AGTR1 into G-292 cells and tested the level of AGTR1. The transfection of si-AGTR1 indeed decreased the level of AGTR1 at both mRNA (0.78:1) and protein level (0.45:1), compared to the control cells (Fig. 4a and b). A similar effect was also found with the transfection of miR-34a-5p-mimic into G-292 cells. We then compared the cell apoptosis triggered by an IC₅₀ dosed drug in the miR-34a-5p mimic or si-AGTR1 transfected G-292 cells. The transfection of miR-34a-5p mimic or si-AGTR1 in G-292 cells increased the chemoresistance to some extent against the following four drugs: Dox, Etop, CDDP, Carb (Fig. 4c). Afterwards, we increased the level of AGTR1 by transfection of miR-34a-5p antagomiR or overexpression of AGTR1 in SJSA-1 cells. In agreement with the elevated level of AGTR1 in both mRNA and protein levels (Fig. 4d and e), the cell survival rate was slightly decreased for all the four drugs, except for Carb (Fig. 4f). The results correlate well with the negative regulation of AGTR1 in the multi-drug resistance of OS cells. In accordance with its negative effect on drug resistance, a siRNA-mediated AGTR1 repression reduced the apoptotic cells from 16.2 to 14.2%, indicating an elevated cell survival rate upon the addition of si-AGTR1 into G-292 cells (Fig. 4g, h and i). A similar effect was also found in the miR-34a-5p-mimic transfected G-292 cells (Fig. 4g, h and i). Taken together, The AGTR1 gene does contribute a great deal to the miR-34a-5p’s promoting effect on the OS drug resistance.

Recently, miR-34a-5p was shown to promote Dox chemoresistance of OS in tumor xenografts of nude mice by repressing its target gene CD117 [29]. In this study, we semi-quantified the levels of AGTR1 protein by immuno-histological analysis in the same section of mice tumor tissues that were injected with either Dox or PBS. The intratumoral injection of miR-34a-5p’s agomiR into G-292 decreased AGTR1 expression. By contrast, the injection of miR-34a-5p’s antagomiR into SJSA-1 increased AGTR1 expression in Dox- or PBS-treated mice (Fig. 5). The results further confirmed that miR-34a-5p has a significant positive effect on both the growth and chemoresistance of OS cells in vitro and cell-derived tumor xenografts in nude mice (Additional file 4: Figures S4).