LncRNA NDRG1 aggravates progression and EMT of osteosarcoma by sponging miR-96-5p through the PI3K/AKT pathway

doi:10.21203/rs.3.rs-1358250/v1

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Research Article

LncRNA NDRG1 aggravates progression and EMT of osteosarcoma by sponging miR-96-5p through the PI3K/AKT pathway

https://doi.org/10.21203/rs.3.rs-1358250/v1

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Background

Osteosarcoma (OS) is the most common primary malignant bone tumors in adolescents. Large numbers of studies have focused on the long non-coding RNA (lncRNA) that plays essential roles in the progression of osteosarcoma. Nevertheless, the functions and underlying mechanisms of LncRNA NDRG1 in osteosarcoma remain unknown.

Methods

By ways of RNA sequencing, lncRNAs which expressed differentially between osteosarcoma and adjacent tissues were identified. Through in vitro and in vivo functional experiments, the effect of LncRNA NDRG1 in osteosarcoma growth and metastasis were investigated. LncRNA NDRG1 specific binding with miR-96-5p was verified through bioinformatic analysis and luciferase reporter assay. Regulation relationship between Lnc NDRG1 and miR-96-5p was further evaluated by the rescue experiments. Additionally, the changes in the expression of epithelial-mesenchymal transition (EMT) and the PI3K/AKT pathway molecular markers were verified by Western blot.

Results

LncRNA NDRG1 was up-regulated in osteosarcoma cell lines and tissues and the expression of LncRNA NDRG1 was correlated with the overall survival of osteosarcoma patients. LncRNA NDRG1 aggravated the progression of osteosarcoma both in vitro and in vivo. Mechanistically, LncRNA NDRG1 was found to promote progression and epithelial-to-mesenchymal transition (EMT) of osteosarcoma by sponging miR-96-5p through the PI3K/AKT pathway.

Conclusions

LncRNA NDRG1 aggravates the proliferation, metastasis and EMT of osteosarcoma by sponging miR-96-5p through the PI3K/AKT pathway. Therefore, LncRNA NDRG1 could act as a prognostic marker and a therapeutic target for osteosarcoma in the future.

Osteosarcoma

LncRNA NDRG1

miR-96-5p

proliferation

migration

EMT

PI3K/AKT pathway

Osteosarcoma (OS) is the most common malignant bone tumor which accounts for approximately 56% of bone sarcomas and typically affects children, adolescents and young adults[1, 2]. Such bone tumor is associated with highly aggressive and unfavorable prognoses[3]. Current therapies for osteosarcoma incorporate neoadjuvant chemotherapy, definite surgery and adjuvant chemotherapy[4]. With the advance of the treatment to osteosarcoma, the 5-year survival rate among patients with localized osteosarcoma is approximately 60% but is only 20% among patients with metastases or recurrent disease[5]. Therefore, it is important to clarify the molecular mechanism underlying OS progression so as to provide effective treatment for OS patients in the future[6].

Long non-coding RNAs (lncRNAs) are defined as transcripts of more than 200 nucleotides with no potential of coding protein. Many published articles reveal that lncRNAs play essential roles in the regulation of cell proliferation, migration, invasion, apoptosis, drug resistance and chromatin remodeling[7–10]. Moreover, lncRNAs could act as competing endogenous RNAs (ceRNAs) that sponge microRNAs (miRNAs) to regulate the expression of down-stream messenger RNA (mRNA)[11]. Over the last few years, lncRNAs have gradually been recognized to play crucial roles in the progression of OS as well. For example, lncRNA DANCR was recognized as a competitive endogenous RNA which promotes ROCK1-mediated proliferation and metastasis by decoying of miR-335-5p and miR-1972 in osteosarcoma[12]. LncRNA SNHG16 aggravated osteosarcoma progression by acting as the ceRNA of miR-1285-3p[6].

MicroRNAs (miRNAs) are considered as short noncoding RNAs with the length of approximately 23 nucleotides[13]. MiRNAs play important roles in regulating genes expression through directly repressing translation of mRNA. Increasing research revealed that miRNAs are dynamic, exhibiting different expression level in different tissue and diseases, and closely related to the initiation and progression of osteosarcoma[14]. Nowadays, many studies validated that lncRNAs could regulate initiation, proliferation, and metastasis of malignant tumors by sponging miRNAs.

In our research, we exhibited a newly screened lncRNA in osteosarcoma: LncRNA NDRG1 which plays an important role in the progression of osteosarcoma. By reverse transcription-quantitative PCR (RT-qPCR), we found that LncRNA NDRG1 was up-expressed significantly in osteosarcoma tissue and osteosarcoma cell lines (MG63, U2OS, HOS and 143B), and the high expression of Lnc NDRG1 were associated with poor prognosis of osteosarcoma patients. Subsequently, functional experiments including CCK8, EdU, transwell assay and wound healing assay showed that LncRNA NDRG1 aggravated proliferation and migration of osteosarcoma in vitro. Animal experiments showed that Lnc NDRG1 promotes OS proliferation and metestasis in vivo. Mechanistically, LncRNA NDRG1 was shown to aggravate progression and epithelial-to-mesenchymal transition (EMT) of osteosarcoma by sponging miR-96-5p through the PI3K/AKT pathway. Our study shed lights onto the design of novel therapeutic targets to treat osteosarcoma.

Specimens Collection

A total of 18 osteosarcoma patients were included into the study. OS tissues and paired adjacent normal tissues were obtained from Jinling Hospital (Nanjing, China) from January 2018 to January 2020. The study was reviewed and approved by the Ethics Committee of Jinling Hospital (Nanjing, China). And all the patients provided informed written consent authorizing the use of specimens for the intended research. All resected specimens were stored at − 80 °C prior to RNA extraction.

Cell culture

Human OS cell lines (MG63,U2OS, HOS and 143B) and human osteoblast cell line (hFOB 1.19) were obtained from the Cell Bank of the Chinese Academy of Sciences(Shanghai, China). All types of cell were cultured in DMEM (Hyclone; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.) , 100 U/ml of penicillin (Life Technologies) and 100 μg/ml streptomycin (Life Technologies) at 37 °C in 5% CO2 and 95% air.

Cell transfection

Three different small interfering (si)RNAs against Lnc NDRG1 and miR-96-5p mimics, mimics NC, miR-96-5p inhibitor and inhibitor NC were designed and synthesized by Ribobio(Guangzhou, China), and transfected into MG63 and 143B cells using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were collected 48h after transfection, and the knockdown or over-expression efficiency was detected by reverse transcription-quantitative PCR (RT-qPCR). The lentivirus-containing short hairpin RNA(shRNA) targeting Lnc NDRG1 was purchased from GenePharma(Shanghai, China), the shRNA was transfected into 143B cell line, after transfecting for 48h, the cells were selected with puromycin(2μg/mL) for 2 weeks to construct stable Lnc NDRG1 knock down cell line.

RNA extraction and RT-qPCR

Total RNA was extracted from the cultured cells and tissues using Trizol Reagent (Invitrogen, CA, USA) according to the manufacturer’s instruction. For the quantification of miRNA, 0.2 μg of total RNA obtained from cultured cells or tissues was reverse-transcribed to cDNA using AMV reverse transcriptase (TaKaRa, Dalian, China). For the quantification of lncRNA and mRNA, 1 μg of total RNA was reverse-transcribed to cDNA using Oligo d(T) primer (TaKaRa, Dalian, China), the real-time PCR analyses were performed using SYBR Green dye(Invitrogen) with LightCycler96 System (Roche, IN, USA). All reactions were run in triplicate. After the reactions were complete, the cycle threshold (CT) values were determined with the LightCycler96 software. A comparative CT method was used to compare each condition to the control reactions. In miRNA RT-PCR reaction, U6 was used as an internal control; in lncRNA RT-PCR reaction, GAPDH was used as an internal control, and the relative level was calculated with the equation 2⁻^ΔΔCT.

Nucleus and cytoplasm extraction

Nuclear and cytoplasmic fractions were isolated with the reagents in a PARIS™ kit (AM1556, Thermo Fisher Scientific, Waltham, USA). MG63 and 143B cells were lysed in Cell Fraction Buffer on ice for 10 min. Subsequently, after centrifugation at 500 g for 3 min at 4 °C, the supernatant was collected as the cytoplasmic fraction. Then, the pelleted nuclei were washed with Cell Fraction Buffer and used as the nuclear fraction.

Cell viability assay

The proliferation capability of OS cells (MG63 and 143B) were assessed by cell counting kit-8 assay (CCK-8) according to the manufacturer’s instruction. The transfected cells were seeded into 96-well plates (5 × 10^³ cells/well). At 12, 24, 36, 48 and 60h after transfection, 10 μl of CCK-8 reagent was added to the test well and incubated for 2 h at 37 °C away from light. The absorbance was measured at a wavelength of 450 nm.

EdU assay

5-Ethynyl-2’-deoxyuridine(EdU) assay was conducted with Cell-LightTM EdU Apollo567 kit (Guangzhou Ruibo Biotechnology). The siRNA or mimics or inhibitor were transfected into MG63 and 143B cells. After transfection for 24 hours, MG63 and 143B cells were seeded in 48-well plates(Corning). When the density of cells turned to 80%, reagent A was added to the medium and cells were cultured for another 4 hours. Then, according to the manufacturer's instruction, the cells were stained. When the staining was done, photos were taken under the microscope (EVOS M7000; Thermo Fisher) at 100×.

Wound healing assay

Wound healing assays were performed to evaluate the migration ability of osteosarcoma cells. After transfection for 24h, MG63 and 143B cells were seeded into six-well plates (8 × 10^⁵ cells/well). After seeding for 48h, pipette tips (200 μl) were used to scrape a straight scratch in the confluent cell layer and then cultured in serum-free medium. After washing the cells with PBS to remove cellular fragments, each wound was imaged at 0 and 24 h under the microscope (EVOS M7000; Thermo Fisher) at 100×. Cell migration was quantified by measuring the relative wound areas with ImageJ.

Transwell assay

For the transwell assay, after transfection for 24h, cells suspended in serum-free DMEM medium were seeded into the upper transwell chamber(Corning). In the lower chamber, DMEM containing 20% FBS was filled in the well. After culturing for 36 hours, the filters were fixed in methanol and stained with 0.1% crystal violet. The upper cells of the filters were gently abraded, and the lower cells migrated across the filters were imaged and counted under the microscope(EVOS M7000; Thermo Fisher) at 100×. The numbers of migrated cells were counted and calculated by ImageJ (NIH).

Luciferase reporter assay

Wild-type and mutant Lnc NDRG1 fragments were constructed and inserted into the luciferase reporter plasmid(GenePharma, Shanghai, China). Osteosarcoma cells were seeded in 24-well plates and co-transfected with 0.2 μg of firefly luciferase reporter plasmid, 0.2 μg of β-galactosidase (β-gal) expression plasmid (Ambion), and equal amounts (50 pmol) of miR-96-5p mimics, miR-96-5p inhibitor or the negative control RNAs with Lipofectamine 3000 (Invitrogen). The β-gal plasmid was used as the transfection efficiency control. The cells were assayed using a luciferase assay kit 24 h post-transfection (Promega, Madison, WI, USA).

Western Blot

The total proteins were extracted through radio-immunoprecipitation assay(RIPA) (Beyotime, Shanghai, China) supplemented with PMSF. The extracted protein lysates were separated in 10% SDS-polyacryl-amide gel electrophoresis(SDS-PAGE) and transferred to 0.22 μm PVDF membranes (Millipore, Massachusetts, USA). The membrane was sealed with 5% skim milk and then incubated with primary antibodies at 4°C overnight. Then the membrane was washed 3 times with 1× TBST for 20 min each. Secondary antibodies were incubated for 1 h at room temperature followed by another 3 times of 10-min wash with 1× TBST. After that, the membrane was incubated with ECL substrate (Thermo Fisher, CA, USA) according to the manufacturer’s instructions and the bands were detected with the SuperSignal West Pico chemiluminescence substrate (Pierce, Thermo Scientific). The protein bands were analyzed with ImageJ.

Animal experiments

Male BALB/c nude mice(4 weeks old) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China), and randomly divided into two groups (n=5, respectively). 143B cells transfected with a lentiviral vector sh-Lnc NDRG1 or sh-NC were subcutaneously injected into armpit of nude mice (1 × 10⁷ cells/mice), respectively. The volume of the tumors was measured every week after implantation. The tumor volume was calculated according to the formula: tumor volume [mm³] = (length [mm]) × (width [mm])² × 0.52. The mice were sacrificed after 28 days. The xenograft tumors were excised and weighed. Part of the tumors were used for total RNA extraction, and the remains were fixed in 4% paraformaldehyde for 24 h and then processed for hematoxylin and eosin (H&E) staining as well as immunohistochemical staining for Ki67. For the metastasis assay, 143B cells (1 × 10^⁵) were injected into the tail veins of mice (three mice per group). Lung metastasis was monitored with a Xenogen IVIS Spectrum Imaging System (PerkinElmer, USA). After 8 weeks, the lungs of mice were excised under anaesthesia, and the number of lung metastatic nodules were counted and validated using haematoxylin and eosin (HE)-stained sections by microscopy.

Immunohistochemistry (IHC)

Immunohistochemistry (IHC) was performed according to the manufacturer’s constructions. After the samples were deparaffinized with xylene and rehydrated with ethanol, the samples were incubated with 3% H₂O₂ for 5 min to block endogenous peroxidase activity. Then, antigen retrieval was performed by incubating the samples with sodium citrate buffer (pH 6.0) for 20 min at 95 °C, after which the samples were blocked with 5% normal goat serum for 10 min at 20 °C. Subsequently, the sections were incubated with polyclonal antibodies against Ki67(1:500, Servicebio, Wuhan, China) at 4 °C overnight and then incubated with secondary antibodies(1:200, Servicebio, Wuhan, China). The image were captured by Olympus FSX100 microscope (Olympus,Japan).

Statistical analysis

All date were expressed as mean ± SD. Statistical analyses were performed using Prism software (GraphPad Software 8), and consisted of analysis of variance followed by Student’s t-test when comparing two experimental groups. One-way ANOVA analysis was used to compare the differences among groups. Overall survival (OS) rates were determined using the Kaplan-Meier method. All experiments were triplicated, and p < 0.05 was considered significant.

Lnc NDRG1 is up-expressed in osteosarcoma cell lines and tissues, and is associated with poor prognosis

To identify lncRNAs that are essential to osteosarcoma progression, RNA-seq was performed for 5 pairs of OS and adjacent normal tissues(H1812143). And we present the results of transcripts which the fold change>2.5 with heatmap according to our RNA-seq date(Fig.1A). Then we selected Lnc NDRG1(ENST00000521414) as the targert lncRNA to investigate the biological role of lncRNAs in osteosarcoma. Firstly, we investigated the relative expression of Lnc NDRG1 in osteosarcoma cell lines and tissues. We found that Lnc NDRG1 was significantly up-regulated in osteosarcoma cell lines(MG63, U2OS, HOS and 143B) compared with normal control cell line(hFOB) and was significantly up-regulated in osteosarcoma tissues compared with adjacent normal tissues as well(Fig.1B-C). Kaplan-Meier analysis demonstrated that high expression of Lnc NDRG1 were associated with poor prognosis of osteosarcoma patients(Fig.1D).

Lnc NDRG1 aggravates the proliferation, migration and EMT of osteosarcoma cells in vitro

In order to investigate the function of Lnc NDRG1 in the progression of OS, Lnc NDRG1 siRNA or negative control were transfected into MG63 and 143B cells. The expression of Lnc NDRG1 was detected by RT-qPCR after transfection for 48h. Lnc NDRG1 siRNA transfection resulted in knocking down of Lnc NDRG1 significantly in OS cells(Fig.2A). Then, the effect of Lnc NDRG1 on OS proliferation was examined by CCK8 and EdU assay. In MG63 and 143B cell lines, knocking down Lnc NDRG1 led to decreased cell proliferation ability compared with negative control (NC) transfection group(Fig.2B-D). In transwell and wound healing assays, we found that Lnc NDRG1 knocking down reduced the migration ability of OS cells(Fig.2E-H). Epithelial to mesenchymal transition (EMT) plays an important role in cancer progression, especially in cancer metastasis[15]. Many previous articles revealed that EMT participates in osteosarcoma progression ae well[16-18]. Therefore, we explored the effect of Lnc NDRG1 on the EMT of osteosarcoma cells. We detected EMT-related genes E-cadherin, N-cadherin and Vimentin in MG63 and 143B cells by Western blot. And we found that the expression of E-cadherin in si-Lnc NDRG1 group was significantly increased compared with si-NC group. In contrast, N-cadherin and Vimentin were decreased, such results suggest that Lnc NDRG1 could promote EMT in osteosarcoma cells(Fig.2I-J). All in all, these data indicate that Lnc NDRG1 aggravates proliferation, migration and EMT of osteosarcoma cells in vitro.

Lnc NDRG1 functions as a sponge of miR-96-5p

It has already been reported that lncRNAs may act as ceRNAs through binding to miRNA to regulate the expression of down-stream genes[19]. So, we conducted the nuclear-cytoplasmic fractionation assay firstly, the results indicated that Lnc NDRG1 was primarily localized in the cytoplasm of MG63 and 143B cell lines(Fig.3A) which means Lnc NDRG1 may exert functions through ceRNA mechanism. Then, to determine the miRNA which interacts with Lnc NDRG1, we selected two miRNA target prediction databases (miRcode, miRDB) and identified 6 candidate miRNAs(miR-133a-3p, miR-96-5p, miR-1297, miR-34b-5p, miR-449c-5p, miR-182-5p) that may bind to Lnc NDRG1 (Fig.3B)[20, 21]. After that, we detected the expression of such miRNAs in MG63 and 143B cell lines which transfected with Lnc NDRG1 siRNA or negative control respectively. The results showed that miRNA-96-5p was significantly up-regulated in Lnc NDRG1 siRNA group compared with negative control group (Fig.3C). Therefore, Lnc NDRG1 may sponge miRNA-96-5p to exert functions. Finally, we conducted the luciferase reporter assay and the results indicated that miRNA-96-5p binds to Lnc NDRG1 directly (Fig.3D-E). Then, we put our emphasize on miRNA-96-5p. RT-qPCR indicated that miRNA-96-5p was low-expressed in osteosarcoma cell lines and tissues(Fig.3F-G).

MiR-96-5p suppresses cell proliferation, migration and EMT in vitro

To investigate the biological function of miR-96-5p, miRNA mimics or inhibitor of miR-96-5p were transfected into MG63 amd 143B cell lines. The expression of miR-96-5p was obviously increased in mimics group, and correspondingly decreased in inhibitor group (Fig.4A). The ability of proliferation and migration of MG63 and 143B cells were obviously weakened after overexpression of miR-96-5p, while it was enhanced after miR-96-5p knocking down (Fig.4B-I). Then, we explored EMT-related genes expression in MG63 and 143B cells which transfected with miR-96-5p mimics or inhibitor. The results showed that the expression of E-cadherin in MG63 and 143B cells transfected with miR-96-5p mimics was significantly increased compared with the mimics NC group; while the expression of N-cadherin and Vimentin were decreased. The expression of E-cadherin in MG63 and 143B cells transfected with miR-96-5p inhibitor was significantly decreased compared with the inhibitor NC group and the expression of N-cadherin and Vimentin were increased(Fig.4J-K). Our results indicated that miR-96-5p acts as the role of tumor supressor in osteosarcoma, which could repress osteosarcoma progression by supressing proliferation, migration and EMT. The results were corresponded to previous research[22, 23].

MiR-96-5p reverses the oncogenic effects of Lnc NDRG1 in osteosarcoma cells

Then, we performed the rescue experiments to illuminate whether Lnc NDRG1 exerts function by sponging miR-96-5p. MG63 and 143B cells were cotransfected with miR-96-5p inhibitor and Lnc NDRG1 siRNA. The results indicated that the transfection with miR-96-5p inhibitor significantly promoted the proliferation and migration of MG63 and 143B cell lines, and reversed the suppressive effects which induced by transfecting with Lnc NDRG1 siRNA in CCK-8, EdU, transwell and wound healing assays(Fig.5A-G). The effects of Lnc NDRG1 in the EMT of osteosarcoma cells were reversed by miR-96-5p as well(Fig.5H-I). Our results demonstrate that miR-96-5p reverses the oncogenic effects of Lnc NDRG1 in osteosarcoma cells.

Lnc NDRG1 aggravates osteosarcoma proliferation and metastasis in vivo

In order to investigate the role of Lnc NDRG1 in tumor proliferation in vivo, 143B cells were stably transduced with lentivirus sh-Lnc NDRG1 and lentivirus sh-NC respectively. Then, the cells were subcutaneously injected into the armpit of BALB/c nude mice. Four weeks later, the mice were sacrificed and we found that the expression of Lnc NDRG1 in sh-Lnc NDRG1 group was down regulated compared with sh-NC group(Fig.6A-B). Tumor mass and volume in the Lnc NDRG1 knocking down group were significantly decreased compared with the control group(Fig.6C-D). In addition, IHC staining revealed decreased Ki-67 expression in Lnc NDRG1 knockdown group(Fig.6E). Furthermore, to determine whether Lnc NDRG1 could promote OS metastasis in vivo, 143B cells transduced with lentivirus luciferase were injected into the tail veins of male nude mice. The fluorescence intensity in the lung in the sh-Lnc NDRG1 group were significantly lower than the sh-NC group (Fig.6F). Moreover, the number of metastatic nodules in the lung in the sh-Lnc NDRG1 group was notably reduced than the sh-NC group(Fig. 6G-I). Such results suggest that Lnc NDRG1 could aggravate osteosarcoma proliferation and metastasis in vivo.

Lnc NDRG1/miR-96-5p aggravates osteosarcoma progression through the PI3K/AKT pathway

To further explore the downstream signal pathways of Lnc NDRG1/miR-96-5p axis, we firstly predicted the downstream genes of miR-96-5p through Targetscan website[24]. Then we predicted the downstream signal pathways of such genes by KEGG pathway enrichment analysis(Fig.7A). As we all know that the PI3K/AKT signaling is one of the most frequently dysregulated signaling pathways observed in cancer that plays crucial roles in tumor initiation, progression and therapy responses[25]. So we selected the PI3K/AKT signal pathway for further study conbined with our KEGG analysis results. After that, we detected the expression of the genes in the PI3K/AKT pathway by Western blot. And we found that the expression of phosphorylated PI3K (p-PI3K) and phosphorylated AKT(p-AKT) were down-regulated obviously in si-Lnc NDRG1 group compared with si-NC group while the expression of PI3K and AKT did not exhibit significant difference(Fig.7B-C). Then, we explored such genes expression in MG63 and 143B cells which transfected with miR-96-5p mimics or inhibitor. We found that the expression of p-PI3K and p-AKT in MG63 and 143B cells transfected with miR-96-5p mimics was significantly down-regulated compared with the mimics NC group; the expression of p-PI3K and p-AKT in MG63 and 143B cells transfected with miR-96-5p inhibitor was significantly up-regulated compared with the inhibitor NC group. No significant differences in the expression of PI3K and AKT were seen(Fig.7D-E). Finally, the effects of Lnc NDRG1 in the PI3K/AKT pathway of osteosarcoma cells were reversed by miR-96-5p through resecue experiments(Fig.7F-G).

Nowadays, large number of lncRNAs have been discovered and more evidence indicates that lncRNAs play important roles in the development and progression of multiple cancers[26–28]. As to osteosarcoma, there are many published articles demonstrating that lncRNAs participate in the development of OS by regulating cell proliferation, migration, metastasis, apoptosis and chemoresistance[6, 7, 16, 29, 30]. For instance, LncRNA MALAT1 promotes tumor angiogenesis by regulating microRNA-150-5p/VEGFA signaling in osteosarcoma[31]; LncRNA SNHG16 promotes epithelial-mesenchymal transition by upregulating ITGA6 through miR-488 inhibition in osteosarcoma[32]; LncRNA HCG18 promotes osteosarcoma growth by enhanced aerobic glycolysis via the miR-365a-3p/PGK1 axis[33] and so on.

In this article, we selected Lnc NDRG1 for further study from our own RNA-seq results(H1812143). LncRNA NDRG1 is a new lncRNA that has not been reported previously. We proved that Lnc NDRG1 was up-expressed obviously in osteosarcoma cell lines and osteosarcoma tissues. Then we found that high expression levels of Lnc NDRG1 were associated with poor prognosis of osteosarcoma patients in TCGA datebase. So we supposed that Lnc NDRG1 may play an important role in the progression of osteosarcoma. To verify our thought, we firstly knocked down Lnc NDRG1 via siRNA; then the proliferation, migration and EMT ability was examined. We found that knocking down Lnc NDRG1 results in low proliferation, migration and EMT activity in both MG63 and 143B cell lines. Moreover, we further demonstrated that Lnc NDRG1 could aggravate proliferation and metastasis of osteosarcoma in vivo. In the end, we found that Lnc NDRG1/miR-96-5p axis exerts functions through the PI3K/AKT pathway. Such results revealed that Lnc NDRG1 may take part in the development of osteosarcoma and could be a potential therapeutic target in the future.

Previous studies have proved that the possible mechanisms of lncRNAs was closely associated with the distribution of lncRNAs in cells[34]. So we conducted the nuclear-cytoplasmic fractionation assay, the results indicated that Lnc NDRG1 was primarily localized in the cytoplasm of MG63 and 143B cell lines, which indicated that our lncRNA might function as a miRNA sponge(ceRNA). Therefore, we predicted the down-stream miRNAs through miRNA target prediction databases and demonstrated the binding sites between Lnc NDRG1 and miR-96-5p via the luciferase assay. Our results suggested that Lnc NDRG1 may exert functions by sponging miR-96-5p. Nevertheless, the function of miR-96-5p in osteosarcoma remains uncertain. Previous studies have demonstrated that miR-96-5p represses breast cancer proliferation and invasion through Wnt/β-catenin signaling via targeting CTNND1[35]; miR-96-5p regulated TGF-β/SMAD signaling pathway and suppressed endometrial cell viability and migration via targeting TGFBR1[36]. Our study indicated that miR-96-5p was significantly decreased in OS cell lines and OS tissues. Furthermore, our study demonstrated that miR-96-5p could inhibit OS cell proliferation, migration and EMT in vitro. The results implied that miR-96-5p may play a tumor supressor role in osteosarcoma which were consistent with previous study[23].

Overall, our study first demonstrated that the expression of lncRNA NDRG1 is upregulated in OS tissues and cell lines. High expression levels of lncRNA NDRG1 are associated with poor clinical outcomes of OS patients. Furthermore, our results indicated that Lnc NDRG1 aggravates OS cells proliferation, migration and EMT in vitro by sponging miR-96-5p through the PI3K/AKT pathway and aggravates OS progression in vivo. These results validated the interaction between lncRNA NDRG1 and miR-96-5p, such interaction could be a novel pathway in osteosarcoma which could provide a promising therapeutic target for the treatment of OS patients in the future. By the way, miRNA generally pairs to mRNA to regulate the transcription of target genes, and it is a limitation that the we did not study the target gene regulated by miR-96-5p, and we plan to conduct the related research in the future.

In summary, lncRNA NDRG1 could act as a ceRNA of miR-96-5p, thus aggravated the progression and EMT of osteosarcoma through the PI3K/AKT pathway. Therefore, Lnc NDRG1 may serve as a prognostic marker and a therapeutic target for osteosarcoma in the future.

LncRNA

Long non-coding RNA

NDRG1

N-myc downstream regulated 1

osteosarcoma

miRNA

microRNA

EMT

epithelial-to-mesenchymal transition

RT-Qpcr

reverse transcription quantitative polymerase chain reaction

ceRNA

competing endogenous RNA

CCK8

Cell Counting Kit-8

EdU

5-Ethynyl-2’-deoxyuridine

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

shRNA

Short hairpin RNA

IHC

Immunohistochemistry

Acknowledgements

We thank the support of Jiangsu Engineering Research Center for microRNA Biology and Biotechnology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University all the time.

Ethics approval and consent to participate

All methods were carried out in accordance with relevant guidelines and regulations. All methods of animal experiments were reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) and was approved by the Ethics Committee of Jinling Hospital (Nanjing, China). All of the informed consent was obtained from the subjects and/or their legal guardian(s) and was approved by the Ethics Committee of Jinling Hospital (Nanjing, China).

Funding

This work supported by grants from health planning committee youth project of Wuxi. Grant Number: Q201924

Availability of data and materials

The datasets generated and/or analyzed during the current study are available in the GEO repository. (https://www.ncbi.nlm.nih.gov/gds/).

Author Contribution Statement

GZ and ZW designed this research. ZW, JZ and QH contribute equally to this work. ZW, LY and ZL carried out most experiments in this work and GZ drafted this manuscript. JH, HZ helped with the western bolt experiments. QT helped with the functional experiments. YZ, GF and JQ helped with the animal experiment. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Author details

¹Nanjing Medical University, Hanzhong Road 104 Nanjing china.² Jinling Hospital, Department of Orthopaedics, Zhongshan Road 305 Nanjing China.³Nanjing University, Jinling Hospital, Department of Orthopaedics, Zhongshan Road 305 Nanjing China.⁴Southern Medical University, Jinling Hospital, Department of Orthopaedics, Zhongshan Road 305 Nanjing China.⁵Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital, Department of Neurosurgery, Hanzhong Road 104 Nanjing China.⁶Affiliated Yixing Hospital of Jiangsu University, Department of Digestion Medicine, Tongzhenguan Road 75 Yixing China.

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No competing interests reported.

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LncRNA NDRG1 aggravates progression and EMT of osteosarcoma by sponging miR-96-5p through the PI3K/AKT pathway

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