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Lncrna LINC00958 Promotes Tumor Progression Through Mir-4306/CEMIP Axis in Osteosarcoma

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Lncrna LINC00958 Promotes Tumor Progression Through Mir-4306/CEMIP Axis in Osteosarcoma European Review for Medical and Pharmacological Sciences 2021; 25: 3182-3199 LncRNA LINC00958 promotes tumor progression through miR-4306/CEMIP axis in osteosarcoma Y. ZHOU1, T. MU2 1Department of Orthopedics, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China 2Department of Stomatology, Emergency General Hospital, Beijing, China Abstract. – OBJECTIVE: To investigate the er gene assay verified that LINC00958 competi- mechanism by which LINC00958 affects osteo- tively bound miR-4306 and repressed its expres- sarcoma progression through miR-4306/CEMIP sion. Silencing of LINC00958 inhibited prolifera- axis. tion, cell cycle, metastasis, and invasion of os- PATIENTS AND METHODS: The microarray teosarcoma cells while inducing cellular apopto- data (GSE66673) for gene expression in osteo- sis. The introduction of miR-4306 inhibitors re- sarcoma cells were obtained from the Gene Ex- versed the tumor-suppressing effect of silenc- pression Omnibus (GEO) database, and differ- ing LINC00958. miR-4306 binds to CEMIP and entially expressed genes were analyzed by bio- suppressed its expression. Xenograft tumor ex- informatics tools. Real-time quantitative PCR periments and tumor metastasis assays in nude (RT-qPCR) was performed to detect the expres- mice demonstrated that silencing LINC00958 in- sion levels of LINC00958, miR-4306, and CEMIP hibited osteosarcoma cells’ growth and metas- in osteosarcoma tissues and cell lines. West- tasis while inhibiting miR-4306 reversed this ef- ern blot was performed to detect the expression fect. Kaplan-Meier analysis showed that high ex- levels of CEMIP. Subcellular fractionation analy- pression of LINC00958 was significantly associ- sis and RNA Fluorescence in situ hybridization ated with poor prognosis of osteosarcoma pa- (FISH) assay were performed to analyze the sub- tients. cellular localization of LINC00958. The target re- CONCLUSIONS: LINC0095 promotes tumor- lationship between LINC00958, CEMIP, and miR- igenesis and metastasis in osteosarcoma by 4306 was verified by public bioinformatics da- competitively inhibiting miR-4306 expression, tabase and dual-luciferase reporter assay. RNA leading to elevated expression of CEMIP. immunoprecipitation (RIP) assay was performed to detect LINC00958 and miR-4306 bound to Key Words: AGO2. The biological functions of LINC00958 LncRNA LINC00958, MiR-4306, CEMP, Osteosar- and miR-205 on proliferation, cell cycle, apop- coma. tosis, migration, and invasion of osteosarco- ma cells were evaluated by gain-of-function and loss-of-function experiments. Tumorigenic and metastatic abilities of cells in vivo were detect- Introduction ed by xenograft tumor experiments and tumor metastasis assays in nude mice. Correlation be- Osteosarcoma, also known as osteogenic sar- tween miR-4306 and LINC00958 or CEMIP ex- coma, is one of the most common primary ma- pression in osteosarcoma tissues was analyzed lignant tumors of bone, most often occurring using Pearson correlation analysis. Kaplan-Mei- in adolescents under the age of 20 or children1. er analysis was performed to assess the rela- tionship between LINC00958 expression and Nearly 60% of all osteosarcoma lesions occur overall survival of osteosarcoma patients. in the distal femur, proximal tibia, femur, and RESULTS: LINC00958 expression levels sig- pelvis2. Clinically, osteosarcoma is highly ma- nificantly increased in osteosarcoma tissues lignant, rapidly growing, highly aggressive, and while miR-4306 expression levels significant- early metastatic3,4. Osteosarcoma has the highest ly decreased, and the expression of these two mortality and morbidity rate among all types of genes was negatively correlated. Subcellu- 5 lar fractionation analysis and RNA FISH assay bone cancer, especially in adolescents . Due to demonstrated that LINC00958 was mainly local- the lack of specificity of clinical symptoms in ized in the cytoplasm. Dual-luciferase report- the early stage of osteosarcoma, the prognosis of 3182 Corresponding Author: Yu Zhou, MD; e-mail: [email protected] LncRNA LINC00958 promotes tumor progression through miR-4306/CEMIP axis in osteosarcoma patients is poor, with a 5-year survival rate of 52- untranslated region (3’UTR) of target mRNAs, 56% and a high rate of pulmonary metastasis and resulting in degradation or translational repres- recurrence after treatment6,7. Therefore, strength- sion of target mRNAs and negative regulation of ening research on the pathogenesis of osteosarco- gene expression at the post-transcriptional lev- ma can provide an important basis and reference el18,19. Some studies indicate that the interaction for clinical diagnosis and treatment. between lncRNA and miRNA plays a critical Long non-coding RNA (LncRNA) is a class of role in tumor development. Wang et al20 found nucleotide fragments whose transcripts are longer that miR-335 expression was downregulated in than 200 nt without coding capacity, which plays osteosarcoma tissues and cell lines and func- a biological role in embryonic development, cell tioned as a tumor suppressor gene. Upregulation growth and disease development through splic- of miR-335 can inhibit migration and invasion of ing regulation, chromatin remodeling and RNA osteosarcoma cells by targeting Rho-associated degradation8-10. Some studies11-13 have shown that coiled-coil protein kinase 1 (ROCK1). Accumu- lncRNAs are often aberrantly expressed in hu- lating evidence suggests that miRNAs regulate man tumors and are involved in tumor initiation, a variety of osteosarcoma cell functions, includ- progression, and metastasis. With the in-depth ing proliferation, differentiation, metastasis, and study of lncRNAs, a large amount of evidence apoptosis. has revealed that the upregulation or downregula- The ceRNA regulatory network is a recently tion of lncRNAs is closely related to mechanisms discovered novel mechanism by which RNAs such as the development of osteosarcoma14. In os- interact with each other in vivo and can regulate teosarcoma cells, elevated expression of lncRNA the action of coding genes, which also extends tends to promote cell proliferation. Liu et al15 the previous understanding of the large number found that LINC01296 expression was elevated of non-coding RNAs in vivo21,22. Numerous in- in osteosarcoma, while silencing LINC01296 ex- vestigations23,24 have demonstrated that ceRNA pression inhibited osteosarcoma cell proliferation networks are widespread in different tumors, and and induced apoptosis. It was also found that the ceRNAs may have an impact on tumor develop- expression level of cyclin D1 was positively cor- ment and prognosis, which could be an indicator related with LINC01296 expression in osteosar- for early diagnosis, prognostic evaluation, and a coma tissues, which partly explained the pro-tu- target for cancer therapy. In this study, we iden- morigenic effect of LINC01296 in osteosarcoma. tified a long non-coding RNA LINC00958 sig- Recent studies have shown that lncRNAs also nificantly overexpressed in osteosarcoma tissues play an important role in tumor invasion and me- and cells and investigated its biological function tastasis. According to Ma et al16, the expression and mechanism on promoting cell metastasis and of FOXF1 and its antisense transcript FOXF1- invasion in osteosarcoma cells. We found that AS1 (lncRNA) were significantly upregulated in LINC00958 acts as a ceRNA for miR-4306 and osteosarcoma tissues and cell lines, which were regulates metastasis and invasion of osteosarco- associated with poor prognosis in osteosarco- ma cells via CEM P. ma patients. Furthermore, silencing FOXF1-AS1 and FOXF1 significantly inhibited osteosarcoma cell migration and invasion in vitro and in vi- Patients and Methods vo by decreasing the expression of MMP2 and MMP9, whereas enhancing the expression of Bioinformatics Analysis FOXF1-AS1 had the opposite effect. In this study, The microarray data (GSE66673) for gene ex- LINC00958 was found to be abnormally overex- pression in osteosarcoma cell was obtained from pressed in highly metastatic osteosarcoma cells the GEO database (https://www.ncbi.nlm.nih. in the GSE38666 dataset deposited in the GEO gov/geo/), and the parental osteosarcoma cells database, which prompted us to further define its SAOS-2 and SAOS-2-derived LM5 cells with functional role in the mechanism of osteosarcoma increased metastatic potential were selected from metastasis. this microarray study. The parental osteosarcoma MicroRNAs (miRNAs) are small non-coding cells were used as controls for differential gene RNAs composed of 19-25 nucleotides17. miR- expression analysis. Then, background correction NAs affect cell proliferation, metastasis, dif- and standardized preprocessing were performed ferentiation and apoptosis through complete or using the affy package in the R language, and incomplete complementary binding to the 3’ “limma” package was used for screening dif- 3183 Y. Zhou, T. Mu ferentially expressed genes with the screening Cell culture: The human normal osteoblast cell threshold of | log2FolfChange | > 2.0 and adj.P.Val line hFOB 1.19, osteosarcoma cell lines HOS, (the corrected p-value) < 0.0525,26. The “pheat- MG-63, U2OS, 143B, and human embryonic map” package was used to construct the differ- kidney cells HEK-293T were purchased from ential gene expression heatmap. Next, the down- the American Type Culture Collection (ATCC; stream miRNAs of LINC00958 were predicted Manassas, VA, USA). The cells were cultured in using the RNA22 database (https://cm.jefferson. Dulbecco’s Modified Eagle’s Medium
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