The Clinical Prognostic Value And Potential Regulatory Mechanism Of Long Non-coding RNA MALAT1 In Osteosarcoma

Objective:To detect the expression of MALAT1 in primary osteosarcoma and noncancerous tissues, in serum of osteosarcoma patients and healthy controls. The expression level of MALAT1 and clinicopathologic factors were evaluated and the clinical prognostic value was also explored. Moreover, we also investigated the underlying functional role of MALAT1 in osteosarcoma cell migration, invasion and proliferation, and the regulatory mechanism on E-cadherin and miR-205 expression.Method:1. Sixty-six primary osteosarcoma tissues and adjacent noncancerous tissues from osteosarcoma patients were collected..Besides,45 serum samples from osteosarcoma patients and 30 serum samples from healthy individuals were also collected and then centrifuged with 3000 r/min for 10min. The collected serum must be determined immediately, stored at 4? within one week, or-20? or-80? for long-time detection (more than 1 week). Quantitative real-time PCR (RT-qPCR) was used to detect the expression of MALAT1, EZH2 mRNA, E-cadherin mRNA and miR-205 expression levels. All patients received a standard follow-up with computed tomography of the abdomen after surgery. Written informed consent was obtained from all patients according to the guidelines approved by the Ethics Committee of the Second SW1353pital of Shandong University.2. Receiver operator characteristic (ROC) curves were drawn to determine the diagnostic value of MALAT1, and survival curves of osteosarcoma patients were estimated via the Kaplan-Meier method and the difference in survival curves was estimated using log-rank testing.3. The human osteosarcoma cell lines U2OS, MG63, SAOS-2 and SOSP-9607 were used in this study for cell functional assay. Besides, one normal osteoblast cell line hFOB was used as the control. The above cell lines were transfected with siRNAs with Lipofectamine 2000 in reduced serum medium according to the manufacturer's instructions and final concentration of siRNAs was 100 nM. Knock down effect of si-MALAT1 was examined by RT-qPCR using RNA extracted 48 hours after transfection. The siRNA sequences of lncRNA-MALAT1, EZH2 and negative control used in this study are listed in Table 1.4. CCK8 assay was used to detect the osteosarcoma cell proliferation; cell migration and invasion were assessed with Boyden chambers or modified Boyden chambers according to the protocol of the manufacturer; RIP and ChIP experiments were performed to investigate whether ribonucleoprotein (RNP) complex contained LncRNA MALAT1 and E-cadherin promoter, and its potential binding protein (EZH2) in osteosarcoma cells; western blot or immunofluorescence was used to detect the relative expression of proteins.Results:1. The expression of MALAT1 in osteosarcoma cells was significantly higher when compared with the normal osteoblast cell line hFOB. Similarly, the expression of MALAT1 in 66 primary osteosarcoma tissues were also significantly increased compared with the noncancerous tissues, and the osteosarcoma tissues in 60.6%(40 of 66) of cases had at least two-fold higher expression of MALAT1. Moreover, the expression of MALAT1 was significantly higher in pT (pT3+pT4), pN (+), pM (+), and high stage(3+4) patients compared to lower stage(l+2) patients. Kaplan-Meier survival analysis indicated that high MALAT1 expression was associated with shorter overall survival in osteosarcoma patients.2. RT-qPCR showed that MALAT1 expression in osteosarcoma tissues was negatively correlated with E-cadherin mRNA expression level. Transwell assay indicated that MALAT1 promoted the migratory and invasive capacity of osteosarcoma cells. Moreover, MALAT1 also suppressed the E-cadherin protein expression while promoted Vimentin protein expression in osteosarcoma. CCK8 assay was used to evaluate the cell proliferation capacity and the results found that MALAT1 knockdown could sufficiently suppressed the cell viability of osteosarcoma cells.3. RT-qPCR indicated that EZH2 mRNA was significantly elevated in primary osteosarcoma tissues and cell lines. The spearmen correlation test showed that MALAT1 expression was significantly positively correlated with EZH2 mRNA expression. RIP assay revealed a significant enrichment of MALAT1 with EZH2 antibody compared with the non-specific IgG antibody. Moreover, the region containing the nucleotides 7501-8708 at the 3'end of MALAT1 interacted most strongly with EZH2.4. E-cadherin expression was negatively regulated by EZH2 in osteosarcoma. Besides, the ChIP assay showed that the histone associated DNAs that were immunoprecipitated with antibody against EZH2 and H3K27-me3 were individually amplified with primers sets covering the E-Cadherin gene promoter regions, and the interaction of EZH2 and H3K27me3 in the E-cadherin promoter is specifically mediated by MALAT1. Furthermore, si-EZH2 reversed the MALAT1 induced cell metastasis and EMT process.5. miR-205 expression was significantly lower in osteosarcoma cell lines. Knock down of MALAT1 significantly increased miR-205 expression in osteosarcoma cells, however after overexpression of miR-205, MALAT1 expression was significantly decreased. Importantly, the promoted cell growth induced by MALAT1 was significantly abrogated by miR-205 in osteosarcoma cells, indicating that miR-205 mediated the effect of MALAT1 on osteosarcoma cell growth.Conclusion: MALATl was highly expressed in osteosarcoma tissues and was correlated with tumor metastasis and growth. Patients with high MALAT1 expression was associated with poor survival, which indicated that MALAT1 might serve as a prognostic factor in osteosarcoma. The subsequent mechanistic experiments showed that MALAT1 could promote the osteosarcoma cell EMT, metastasis and cell proliferation. EZH2 is highly expressed and associated with 3'end region of MALAT1 in CRC, and this association suppressed the expression of E-cadhrin. Finally, the interaction between MALAT1 and miR-218 was observed, which further indicated the regulatory mechanism of MALAT1 on osteosarcoma cell proliferation. In a word, this study illuminates the prognostic role of MALAT1 in osteosarcoma patients and further demonstrates how high MALAT1 expression confers a tumor-promoter role in osteosarcoma. Thus, MALAT1 inhibition may represent a promising prognostic and therapeutic option for preventing osteosarcoma progression.