Microrna-1294 Targets HOXA9 and Has a Tumor Suppressive Role in Osteosarcoma
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European Review for Medical and Pharmacological Sciences 2018; 22: 8582-8588 MicroRNA-1294 targets HOXA9 and has a tumor suppressive role in osteosarcoma Z.-F. ZHANG1, G.-R. LI2, C.-N. CAO3, Q. XU1, G.-D. WANG1, X.-F. JIANG1 1Department of Joint Surgery, Yantai Yuhuangding Hospital, Yantai, Zhifu District, Yantai, Shandong, P. R. China 2Department of Spinal Surgery, Yantai Yuhuangding Hospital, Yantai, Zhifu District, Yantai, Shandong, P. R. China 3Department of Hyperbaric Oxygen Therapy, Yantai Yuhuangding Hospital, Yantai, Zhifu District, Yantai, Shandong, P. R. China Zuofu Zhang and Guangrun Li contributed equally to this work Abstract. – OBJECTIVE: MicroRNA-1294 cer type worldwide1,2. OS has a high potential for (miR-1294) was reported to act as a tumor sup- metastasis that results in the unsatisfactory OS pressor in several cancers. However, the biolog- prognosis despite the great achievements in the ical function of miR-1294 in osteosarcoma (OS) treatment measures in recent decades3-5. Hence, has not been investigated. We, therefore, inves- tigated the clinical significance and underlying there is an urgent requirement to deeply investi- mechanisms of miR-1294 in OS. gate the abnormally expressed molecules related PATIENTS AND METHODS: Quantitative Re- to the metastasis of OS to provide novel sugge- al-Time-Polymerase Chain Reaction (qRT-PCR) stions to develop anti-cancer therapeutic strate- was conducted to detect the levels of miR-1294. gies. Extensive studies have revealed the initia- Targets of miR-1294 were validated by luciferase tion and progression of OS is associated with the reporter assay and Western blot. In vitro functional abnormal expression of tumor suppressor genes assays were performed to investigate the effects 5,6 of miR-217 on cell proliferation and invasion. or oncogenes . MicroRNAs (miRs) are a family RESULTS: We found miR-1294 was downregu- of non-coding RNAs and have dual functions in lated in OS tissues and cell lines. Downregulation tumors: namely tumor suppressive role and onco- of miR-1294 has a significant negative impact on genic role7,8. MiRs are reported to exert its bio- the overall survival of OS patients. Overexpres- logical roles in tumors through complementary sion of miR-1294 suppresses OS cell prolifera- binding to the 3’-untranslated region (3’-UTR) of tion and invasion in vitro. Then, luciferase report- 9 er assay validated Homeobox A9 (HOXA9) was a targeted genes . Involvement of miRs in the pro- downstream target of miR-1294. Expression pat- gression and metastasis of OS has also received 10 terns of miR-1294 were inversely correlated with considerable attentions . For example, miR-34a- HOXA9 in OS tissues, strengthening the find- 5p expression was found elevated in OS tissues ings from the luciferase reporter assay. Further and negatively regulate angiotensin II type 1 re- functional assays revealed that overexpression ceptor (AGTR1) expression to affect the chemore- of HOXA9 could reverse the inhibition effects of sistance of OS11. Li et al12 revealed that miR-202- miR-1294 on cell proliferation and invasion. CONCLUSIONS: These results suggested 5p downregulation induced OS cell migration miR-1294 functions as a tumor suppressor in OS and invasion inhibition, showing a potential to progression by targeting HOXA9. develop miR-202-5p as a treatment target for OS. However, the mechanism of action of miRNAs in Key Words: OS is not understood clearly. HOXA cluster be- MiR-1294, Osteosarcoma, HOXA9, Tumor suppressor. longs to the Homeobox (HOX) gene family and is reported to play a crucial role in regulating cell differentiation13. Hence, it is reasonable to deduce Introduction that the dysregulation of these genes has the po- tential to regulate cancer progression14. HOXA9 Osteosarcoma (OS) often occurred in children protein has been reported to function as a tumor and adolescents and is one of the most lethal can- suppressor or oncogene in a context-dependent 8582 Corresponding Author: Xiaofeng Jiang, MD; e-mail: [email protected] MicroRNA-1294 targets HOXA9 and has a tumor suppressive role in osteosarcoma manner15-17. HOXA9 can inhibit HIF-1α-mediated bicinchoninic acid (BCA) protein determination glycolysis to suppress the progression of cutane- kit (Beyotime) and separated with 10% sodium ous squamous cell carcinoma15. Recently, HOXA9 dodecyl sulphate-polyacrylamide gel electropho- was found overexpressed in gastric cancer, impli- resis (SDS-PAGE) (30 µg). Subsequently, protein cating poor prognosis of these patients16. Notably, samples were transferred to polyvinylidene diflu- Zhang et al17 showed HOXA9 was directly regu- oride (PVDF) membrane (Beyotime) and blocked lated by miR-182 to regulate OS cell behaviors. with fat-free milk. After washed with TBST, However, more researches are still needed to reve- membranes were cultured with mouse monoclo- al the exact mechanism of HOXA9 in OS. In this nal antibodies (anti-HOXA9: ab51236; anti-GAP- study, we demonstrated the aberrantly expressed DH: ab8245; Abcam, Cambridge, MA, USA) at status of miR-1294 in OS specimens and cell li- 4°C for overnight and horseradish peroxidase nes. Next, the dual-luciferase reporter assay was (HRP) conjugated goat-anti-mouse secondary an- conducted to validate HOXA9 as a direct target tibody (ab6789; Abcam, Cambridge, MA, USA) of miR-1294. Functional studies were utilized to at room temperature for 1 h. Protein signals were investigate the roles of miR-1294 in OS. detected using enhanced chemiluminescent re- agent (Beyotime, Shanghai, China) and analyzed with ImageJ 1.41 software (National Institutes of Materials and Methods Health, Bethesda, MD, USA). Cell Culture and Collection of Tissues Total RNA Isolation From Tissues Normal human osteoblastic cell line hFOB 1.19 and Cell Lines and OS cell lines (MG-63 and HOS) used in this RNA samples were extracted by the TRIzol study were purchased from the American Type reagent (Invitrogen, Carlsbad, CA, USA) in line Culture Collection (ATCC, Manassas, VA, USA). with the supplier’s recommendations. RNA con- These cell lines were maintained with Dulbecco’s centration was measured by Nanodrop ND-2000 Modified Eagle Medium (DMEM, Invitrogen, spectrophotometer (Thermo Fisher Scientific, Carlsbad, CA, USA) containing 10% fetal bovine Inc., Waltham, MA, USA). serum (FBS, Invitrogen, Carlsbad, CA, USA) in a 37°C incubator containing 5% CO2. Sixty-four OS Quantitative Reverse Transcription PCR tissues and matched normal tissues were obtained (qRT-PCR) Analysis from Yantai Yuhuangding Hospital and stored Extracted RNA samples were reverse tran- at -80°C until further usage. The study protocol scribed into cDNA using PrimeScript™ RT Mas- was approved by the Ethics Committee of Yantai ter Mix (TaKaRa, Dalian, China). qRT-PCR was Yuhuangding Hospital. These patients received conducted using SYBR® Premix Ex Taq™ (TaKa- treatment between October 2011 and December Ra, Dalian, China) at Applied Biosystems Ste- 2012 and written informed consent was obtained pOne Plus Real Time-PCR System (Thermo Fish- from all the participated patients. er Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s instructions. Primer se- Cell Transfection quences for miR-1294 were 5’-ACACTCCAGCT- MiR-1294 mimic, inhibitor, HOXA9 construct GGGTGTGAGGTTGGCATTG-3’ (forward) were synthesized by GenePharm (Shanghai, Chi- and 5’-CTCAACTGGTGTCGTGGAGTCGG- na). The corresponding negative controls (NC) CAATTCAGTTGAGAGACAACA-3’ (reverse). were also purchased from GenePharm. Cell tran- Primer sequences for U6 snRNA were 5’-CTC- sfection was conducted using Lipofectamine 2000 GCTTCGGCAGCACA-3’ (forward) and 5’-AAC- (Thermo Fisher Scientific, Waltham, MA, USA) GCTTCACGAATTTGCGT-3’ (reverse). Relative according to the manufacturer’s instructions. expression levels were calculated using the com- After transfection for 48 h, cells were collected parative threshold cycle (Ct) method. All experi- for following experiments. ments were conducted in triplicate. Western Blot Analysis Luciferase Reporter Assay Protein samples were extracted by radioimmu- The wild-type (wt) or mutant (mut) sequence of noprecipitation assay (RIPA) buffer containing HOXA9 3’-UTR were cloned into pGL3-M vec- protease inhibitors (Beyotime, Shanghai, Chi- tor (Promega, Madison, WI, USA) and designated na). Protein concentration was detected using as HOXA9-3’-UTR-wt or HOXA9-3’-UTR-mut. 8583 Z.-F. Zhang, G.-R. Li, C.-N. Cao, Q. Xu, G.-D. Wang, X.-F. Jiang Cells were co-transfected with HOXA9-3’-UTR- 1294 might be a regulator of HOXA9 as they can wt or HOXA9-3’-UTR-mut and miR-1294 mim- perfectly bind with each other (Figure 1A). Lucif- ic or NC using Lipofectamine 2000. Luciferase erase activity assay showed that miR-1294 mimic activity was detected by dual-luciferase reporter significantly reduced the luciferase activity of assay system (Promega, Madison, WI, USA) after OS cells transfected with HOXA9-3’-UTR-wt 48 h transfection. but not HOXA9-3’-UTR-mut (Figure 1B). West- ern blot analysis results showed that miR-1294 Cell Proliferation Assay mimic markedly decreased HOXA9 protein Cell counting kit-8 (CCK-8) assay was con- expression in OS cells (Figure 1C). Correlation ducted to measure cell proliferation. Cells were analysis showed that miR-1294 and HOXA9 ex- plated in 96-well plate at the density of 5 × 103 pression in OS tumor tissues was inversely cor- cells/well and cultured for 0 h, 24 h, 48 h, and 72 related (Figure 1D). h. Subsequently, 10 μl CCK-8 reagent (Beyotime, Shanghai, China) was added to each well and cul- MiR-1294 Was Downregulated in OS tured for an additional 2 h. After that, the optical Tissues and Cell Lines density of each well was detected using micro- We further investigated the expression status plate reader (BioTek, Winooski, VT, USA) at 450 of miR-1294 in both OS tissues and cell lines us- nm. All experiments were conducted in triplicate. ing qRT-PCR. The results revealed that miR-1294 was frequently downregulated in OS tissues com- Cell Invasion Assay pared with their matched normal tissues (Figure Transwell invasion assay was performed to 2A). Then, miR-1294 expression in OS cell lines measure cell invasion.