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LncRNA OIP5-AS1 loss-induced microRNA-410 accumulation regulates cell proliferation and apoptosis by targeting KLF10 via activating PTEN/PI3K/AKT pathway in multiple myeloma

Numerous studies confirmed that aberrant miRNAs expression contributes to multiple myeloma (MM) development and progression. However, the roles of specific miRNAs in MM remain to be investigated. In present study, we demonstrated that miR-410 expression was increased in MM newly diagnosed and relapsed tissues and cell lines. Clinical analysis revealed that miR-410 was positively correlated with advanced ISS stage. Moreover, high miR-410 expression in MM patients showed an obvious shorter overall survival and progression-free survival. Gain- and loss-of function experiments indicated that miR-410 promoted cell proliferation, cell cycle progression and apoptosis inhibition both in vitro and in vivo. Moreover, KLF10 was identified as a direct downstream target of miR-410 in MM cells, and mediated the functional influence of miR-410 in MM, resulting in PTEN/AKT activation. In clinical samples of MM, miR-410 inversely correlated with KLF10. Alteration of KLF10 expression or AKT inhibitor at least partially abolished the biological effects of miR-410 on MM cells. Furthermore, downregulated expression of lncRNA OIP5-AS1 was inversely correlated with miR-410 expression in MM tissues. LncRNA OIP5-AS1 could modulate the miR-410 expression and regulate its target KLF10/PTEN/AKT-mediated cellular behaviors. Taken together, this research supports the first evidence that lncRNA OIP5-AS1 loss-induced miR-410 accumulation facilitates cell proliferation, cycle progression and apoptosis inhibition by targeting KLF10 via activating PTEN/PI3K/AKT pathway in MM.

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by aberrant infiltration and accumulation of malignant plasma cells within the bone marrow (BM). It is the second most common hematologic neoplasms with dismal prognosis despite advanced progress in diagnosis and treatment, including immunomodulatory medicine, proteasome inhibitors and autologous stem cell transplantation. [1][2][3][4][5] However, the detailed potential mechanisms for the development and progression of MM remain unclear. Therefore, it is urgent to elucidate the molecular mechanisms of MM and identify new prognostic biomarker to provide potential therapeutic targets for MM patients.
MicroRNAs (miRNAs), a class of endogenous evolutionarily conserved non-coding single-stranded small RNAs, act as post-transcriptional regulator of gene expression in cancer initiation, development and progression by interacting with complementary sequences within the 3′-untranslated region (UTR) of target mRNA to induce mRNA degradation or translational repression. [6][7][8][9] Increasing evidence confirm that aberrant miRNAs play critical roles in multiple biological processes in MM, [10][11][12] including cell proliferation, apoptosis, drug-resistance, metastasis and stem cell renewal and have been identified as promising therapeutic and prognostic biomarkers in MM diagnosis and treatment.
MiR-410, a novel cancer-related microRNA, has been found to be dysregulated in cancers. [13][14][15] Li et al demonstrated that miR-410 promotes cell proliferation by targeting BRD7 in nonsmall cell lung cancer (NSCLC). 16 In prostate cancer, miR-410 could be served as a potential serum biomarker for the diagnosis. 17 MiR-410 acts as oncogene in NSCLC through downregulating SLC34A2 via activating Wnt/β-catenin pathway. 18 These reports identified miR-410 as an oncogene. However, miR-410 functions as a tumor suppressor by targeting angiotensin II type 1 receptor in pancreatic cancer. 19 MiR-410 suppressed migration and invasion by targeting MDM2 in gastric cancer. 20 These studies revealed that miR-410 was a tumor suppressor. Therefore, the functional roles of miR-410 in human cancers are cancertype specific. Nevertheless, the functional importance of miR-410 and the molecular mechanisms in MM are still unclear.
In present study, we investigated the expression and biological role of miR-410 in MM progression. Our results showed that miR-410 was significantly upregulated in MM samples and cells for the first time. Its overexpression was associated with poor prognosis of MM patients. Gain-and loss-of-function experiment revealed that miR-410 promoted proliferation, cell cycle and apoptosis resistance of MM cells in vitro and in vivo. Notably, Krüppel-like factor 10 (KLF10) was identified as direct target of miR-410, resulting in activation of AKT signaling in cell proliferation and apoptosis. MiR-410 was inversely regulated by lncRNA OIP5-AS1. These results showed a novel role for miR-410 in predicting of prognosis and promoting tumor growth of human MM.
Results miR-410 was significantly increased in MM samples and cells. To investigate the potential role of miR-410 in MM, we first performed qRT-PCR to determine the miR-410 expression in 97 newly diagnosed MM samples and 14 healthy donors' tissues. The data revealed that the mean level of miR-410 in MM tissues was significantly higher than that in normal plasma cells (Po0.01, Figure 1a). Moreover, we also observed that relapsed patients have higher miR-410 compared newly diagnosed patients (Po0.01, Figure 1a). Consistently, the expression of miR-410 in MM patients was also associated with ISS stage. The data revealed that the levels of miR-410 were increased at advanced stages (Po0.05, Figure 1b). Furthermore, we assessed miR-410 expression in MM cell lines and normal bone marrow-derived plasma cells (nPCs). All MM cell lines (RPMI-8266, U266 and NCI-H929) exhibited high expression as compared to nPCs (Po0.05, Figure 1c). These results indicated that miR-410 may be involved in the development of MM.
Increased miR-410 is associated with poor survival in newly diagnosed MM patients. Since we have confirmed the correlation between high miR-410 expression and MM progression, we next explored the potential roles of miR-410 in MM patients' clinical outcome. We determined 0.33 (mean level of miR-410) as a cutoff value for the expression level of miR-410. The expression of miR-410 was considered as either low or high. Kaplan-Meier survival curves suggest that patients with high miR-410 expression had remarkable shorter overall survival (OS, P = 0.0001, Figure 2a) and progression-free survival (PFS, P = 0.0002, Figure 2b) in newly diagnosed MM patients. These data suggest that miR-410 could be identified as a potential biomarker for the prognosis outcome of MM patients.
miR-410 promotes cell proliferation, cell cycle progression and inhibits apoptosis in MM cells in vitro and in vivo. To investigate the biological significance of miR-410 in MM, we stably overexpressing miR-410 in NCI-H929 cells by lentivirus system and stably knockdown miR-410 in RPMI-8266 cells which contained different endogenous miR-410 levels. As measured by qRT-PCR, we confirmed that miR-410 was effectively upregulated in NCI-H929 or downregulated in RPMI-8266 cells (Po0.01, Figure 3a). First, we assessed cell growth by CCK8 assays and found that miR-410 overexpression increased cell proliferation (Po0.05, Figure 3b). Next, as determined by flow cytometric analysis, the ectopic-expression of miR-410 promoted cell cycle transition from G1 to S phase (Po0.05, Figure 3c) and apoptosis resistance (Po0.05, Figure 3d). Furthermore, western blot confirmed that upregulated miR-410 markedly increased cycle-related protein, Cyclin D1 and apoptosis inhibition protein Bcl-2, while inhibited cycle inhibitor p27 and pro-apoptosis protein Bax (Po0.05, Figure  To further confirm the in vitro functional significance on MM cells, we establish the subcutaneous tumor model and the tumor growth curves revealed that miR-410 overexpression significantly promoted the tumor growth, while miR-410 knockdown retarded the tumor growth of MM cells in mice (Po0.05, Figure 4a). Next, we performed immunohistochemistry for Ki67 and TUNEL assays in the xenografted tissues. As   miR-410 expression was inversely correlated with KLF10 in MM tissues. To further investigate the relationship between miR-410 and KLF10 in vivo, we examined the mRNA and protein expression in diverse miR-410 expression groups. Results showed that KLF10 mRNA and protein levels were significantly lower in high miR-410 group than that in low miR-410 group in MM tissues (Po0.05, Figures 6a  and b). In addition, we demonstrated that the mRNA level of KLF10 in the MM tissues was inversely correlated with miR-410 expression (R 2 = 0.7150, Po0.0001, Figure 6c). In conclusion, these data suggest that KLF10 was a direct downstream target of miR-410 in MM.
Alterations of KLF10 expression partially rescued the miR-410-induced biological effects on MM cells. To further confirm that KLF10 exerted its biological function as the target of miR-410, we restored KLF10 expression by overexpression construct plasmid in miR-410-overexpressing NCI-H929 cells (Po0.05, Figure 7a). Our data showed that miR-410 is negatively regulated by lncRNA OIP5-AS1 in MM cells. To investigate the reason for miR-410 was increased in MM, we predicted a target by Starbase 2.0 and found lncRNA OIP5-AS1 is a molecular sponge that modulates miR-410. Next, our data revealed the levels of OIP5-AS1 in MM tissues were notably lower than those in healthy donors (Po0.05, Figure 9a). Spearman correlation analysis revealed that the levels of OIP5-AS1 were inversely

Discussion
Increasing evidence has demonstrated that abnormal miRNAs play critical role in the cancer initiation, development and progression of MM, which has been emphasized as valuable diagnostic and prognostic biomarker and attractive therapeutic targets of MM. However, the clinical significance and molecular mechanisms of specific miRNA are still needed to be investigated. Here, we demonstrated for the first time that the expression of miR-410 was significantly upregulated in newly diagnosed and relapsed patients than healthy donors. Moreover, our data suggest that increased miR-410 expression was positively associated with advanced ISS stage. In addition, we provided the first evidence that overexpression of miR-410 conferred an obvious poor prognosis of MM patients. Therefore, these data confirmed Previous studies reported that miR-410 is overexpressed in liver and colorectal tumors and enhances tumor cell growth by silencing FHL1 via a methylation-related direct/indirect mechanism. 21 MiR-410 induces stemness by inhibiting Gsk3β but upregulating β-catenin in non-small cells lung cancer. 22 However, miR-410 regulates MET to influence the proliferation and invasion of glioma. 23 MiR-410 suppresses cell proliferation and invasion of osteosarcoma by targeting VEGF. 24 Therefore, the expression status and role of miR-410 in human cancers is a controversial topic. In the present study, we disclosed the biological function of miR-410 in MM. We demonstrated that miR-410 overexpression promoted cell proliferation, cell cycle progression and apoptosis inhibition of NCI-H929 cells, while miR-410 knockdown facilitated these cellular behaviors of RPMI-8266 cells in vitro. In addition, miR-410 overexpression promoted, while miR-410 knockdown inhibited tumor growth in subcutaneous tumor model. These data revealed that miR-410 functions as an oncogene by regulating proliferation, cell cycle and apoptosis in MM cells.
KLF10, originally called TGF-β inducible gene 1 (TIEG1), has been found to be downregulated in human cancers and inhibits growth, radio-sensitivity and metastasis of cancer cells. [25][26][27][28] Here, we confirmed that KLF10 was a direct downstream target of miR-410 based on the following reasons: firstly, luciferase activity assays indicated that miR-410 could bind with the 3′-UTR of KLF10. Next, we found miR-410 inversely regulated KLF10 abundance in MM cells and an inverse correlation between miR-410 and KLF10 expression was observed in MM tissues. Moreover, KLF10 restoration abrogated the effects of miR-410 on the proliferation, cell cycle and apoptosis of MM cells. Previous studies confirmed that KLF10 could regulate the PTEN/AKT signaling pathway. 29,30 Here, we disclosed that miR-410 promoted Previous studies reported that miR-410 is regulated by lncRNA expression. 31 Here, we identified lncRNA OIP5-AS1 was downregulated in MM tissues compare to healthy donors, and was inversely correlated miR-410 expression in MM tissues. Next, we demonstrated that OIP5-AS1 inversely regulated miR-410 expression and promotes KLF10-mediated PTEN/ AKT signaling in MM cells. Taken together, the OIP5-AS1-miR-410-KLF10/PTEN/AKT signaling axis probably exerts key functions in the cell proliferation, cell cycle progression and apoptosis inhibition of MM and may represent a therapeutic target for MM patients.
In conclusion, we show that miR-410 acts as an oncogene in MM. Firstly, our results demonstrate that miR-410 expression was upregulated in MM tissues and cell lines. Then, our clinical data suggest that miR-410 may be used as a novel prognostic marker for MM patients. Moreover, loss of lncRNA OIP5-AS1 induced miR-410 accumulation facilitates cell proliferation, cell cycle progression and apoptosis inhibition via targeting KLF10/PTEN/AKT signaling in MM cells. Taken together, our results verify that miR-410 may be served as a potential target for cancer therapeutics in MM. Quantitative reverse transcriptase PCR. Total RNA from MM tissues and cells was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. cDNA was reverse-transcribed from 2 μg total RNA using a Reverse Transcription Kit (Takara, Biochemical, Tokyo, Japan). cDNA was then amplified with a SYBR Premix Ex Taq II (Perfect Real-Time) kit (Takara). The gene expression levels were calculated using the delta-delta Ct method with U6 or GAPDH as an internal control. Hsa-miR-410 primer (HmiRQP0485), snRNA U6 qPCR Primer (HmiRQP9001), KLF10 (HQP018084) and GAPDH (HQP006940) were purchased from Genecopoeia (Guangzhou, China).
LncRNA OIP5-AS1 vectors, including OIP5-AS1 expression vector, the empty vector, OIP5-AS1 siRNA and control were synthesized and purchased from by Shanghai Genepharm Co. Ltd (Shanghai, China). The KLF10 overexpression plasmid and specific siRNA against KLF10 and a scramble siRNA were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Cells were seeded at a concentration  For cell cycle assay, the cells were seeded in 6-well plates at 2 × 10 5 /well. 48 h after transfection, the cells were fixed in 70% ethanol at 4°C for 24 h and stained with 50 μg/ml propidium iodide (Keygen, Nanjing, China). An Annexin-V-Fluos Staining kit (Roche, Basel, Switzerland) was used to analyze apoptosis levels.
Luciferase reporter assay. The 3′-UTR sequence of KLF10 predicted to interact with miR-410, together with a corresponding mutated sequence within the predicted target sites, were synthesized and inserted into the pmiR-GLO called wt KLF10 3′-UTR and mt-KLF10 3′-UTR. Subsequently, HEK293 cells that were plated into 24-well plate and were transfected with miR-410 inhibitor or negative control. Cells were co-transfected with the wild-type or mutant 3′-UTR of KLF10 vector using the Lipofectamine 2000 reagent (Invitrogen). After 48 h, cells were harvested and measured according to the manufacturer's instructions (Dual-Luciferase Assay System; Promega). pRL-TK expressing Renilla luciferase was cotransfected as an internal control to correct the differences in both transfection and harvest efficiencies.
In vivo experiments. Four-to-six-week-old female BALB/c nude mice (Centre of Laboratory Animals, The Medical College of Xi'an Jiaotong University, Xi'an, China) were used to establish the nude mouse xenograft model. NCI-H929 (5 × 10 6 ) cells that were transduced with miR-410 or miR-control vectors or RPMI-8266 cells with anti-miR-410 were mixed in 150 μl of Matrigel and were inoculated subcutaneously into the flank of nude mice. The tumor volume for each mouse was determined by measuring two of its dimensions and then calculated as tumor volume = length × width × width/2. After 3 weeks, the mice were sacrificed by cervical dislocation under anesthesia with ether and the xenograft tumor tissue was explanted for examination. Animal protocols were approved by the Institutional Animal Care and Use Committee of Xi'an Jiaotong University.
Statistical analysis. Data are presented as the mean ± SD and performed at least three independent replicates. SPSS software, 16.0 (SPSS, Inc., Chicago, IL, USA) and Graphpad Prism 6.0 (San Diago, CA, USA) were used for a two-tailed Student's t-test, Pearson's correlation analysis, Kaplan-Meier method and the log-rank test to evaluate the statistical significance. Differences were defined as Po0.05.