| BackgroundChondrosarcoma (CHS) is a major malignancy of bone apart from osteosarcoma, but the molecular pathophysiology of chondrosarcomas has not been fully understood. Pathologically, there are four kinds of chondrosarcoma: conventional, dedifferentiated, mesenchymal, and clear cell chondrosarcomas. As the approximate 85% of all chondrosarcomas, conventional chondrosarcomas have been found to be highly resistant to chemo- and radiotherapy. At present, surgical resection is still the main therapeutic method for chondrosarcoma. However, disability and functional defect caused by surgery might result in serious burden of life and the psychological shadow for patients. Thus, it is urgent to seek new treatments to get better prognosis for chondrosarcoma patients. With the rapid development of molecular biology techniques, more researches are focused on the molecular-targeted treatment, which also provides a new and effective way for the treatment of chondrosarcoma. Therefore, it makes a big difference to find new molecular targets for chondrosarcoma treatment.Metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) is a long non-coding RNA ubiquity expressed in various tissues and highly conserved in mammals. With respect to its function, MALAT-1 participates in the regulation of gene expressions, alternative splicing and cell cycle. MALAT-1 was originally found to be overexpressed in patients at high risk for metastasis of non-small cell lung tumors (NSCLC). Subsequently, various studies showed that MALAT-1 was overexpressed in several cancers, such as colon, prostate, breast and oral squamous cell carcinoma. These findings indicated the oncogenic function of MALAT-1. However, the role of MALAT-1 in chondrosarcoma is poorly understood.Various pro-survival pathways are known to play important roles in biological processes. Among them, the Notch signaling pathway is increasingly being studied as a novel mechanism for tumorigenesis. Interestingly, Notch can serve as the apparently opposite functions in tumor development, such as an oncogene or a tumor suppressor, a repressor or inducer of terminal differentiation. To date, four Notch receptors with Notch 1-4 and five corresponding ligands including Delta-like-1, Delta-like-3, Delta-like-4, Jagged-1, and Jagged-2 were identified in mammals. Overexpression of the Notch receptors and/or their ligands has now been identified in multiple cancers, including breast, brain, prostate and ovarian. Recently, Notch signaling pathway was found to be involved in the apoptosis of osteosarcoma cells. According to the functional role of Notch signaling during cartilage development, Notch-1 is supposed to be a marker for chondrogenic progenitor cells.In the present study, we examined the differential expression of MALAT-1 in chondrosarcoma tissues and cells with fluorescent quantitative method. Then, the effect of MALAT-1 on the proliferation of chondrosarcoma cells were investigated through MALAT-1 overexpression or MALAT-1 knockdown. Finally, we found that MALAT-1 promoted the proliferation of chondrosarcoma cell via activating Notch-1 signaling pathway. Our findings elucidated the important role of MALAT-1 in chondrosarcoma and the corresponding molecular mechanism, which put MALAT-1 and Notch-1 forward as valid therapeutic targets to be further tested.Materials and Method1. Clinical samplesOur study consisted of 20 chondrosarcoma tissue samples and 20 adjacent noncancerous tissue taken from the Subei People’s Hospital. The pathological types of chondrosarcoma were conventional chondrosarcoma in 17 cases and myxoid chondrosarcoma in 3 cases. Histologically, they were grade II-III. Among them, lymph node metastasis occurred in 4 cases. All participants consented to molecular analyses, and the protocols were approved by the local ethics committee. All tissue specimens were immediately frozen in liquid nitrogen after surgery and stored at-80℃ until RNA isolation, and diagnosis of chondrosarcoma was verified by pathologists.2. Cell cultureChondrosarcoma cell lines CH2879, L835, JJ012 and cartilage cells TC28a2 were cultured in RPMI1640 (Invitrogen Life-Technologies, Scotland, UK) supplemented with 1% penicillin/streptomycin (P/S) (100 U/ml), and 10% heat-inactivated fetal calf serum (Invitrogen Life-Technologies, Scotland, UK). Cells were grown at 37℃ in a humidified incubator with 5% CO2.3. Vector construction(1) pcDNA-MALAT-1:According to the MALAT-1 sequence, single stranded oligo was designed and synthetized for MALAT-1. The polymerase chain reaction (PCR) product of MALAT-1 gene was inserted into T vector, and then transferred to the bacteria. After extracting the integrating T vector, gene sequencing was performed to confirm the correction of sequence. The corrected fragment was cloned into pcDNA3.1 vector and the vector pcDNA-MALAT-1 for overexpression of MALAT-1 was obtained.(2) pcDNA-Notch-1:The approach for constructing pcDNA-Notch-1 to overexpress Notch-1 was similar to the construction of pcDNA-MALAT-1.4. siRNA interference sequenceAccording to the sequence of MALAT-1 gene and Notch-1 gene, siRNA interference sequence for MALAT-1 and Notch-1 were designed and synthetized.5. Cell transfectionJJ012 were transfected with siRNA (GenePharma, Shanghai, China) against MALAT-1 to knock down MALAT-1 or pcDNA-Notch-1 to overexpress Notch-1 using Lipofectamine2000 transfection reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. CH2879 cells were transfected with siRNA (GenePharma, Shanghai, China) against Notch-1 to knock down Notch-1 or pcDNA-MALAT-1 to overexpress MALAT-1 using Lipofectamine2000 transfection reagent. siRNA-control and pcDNA were used as a negative control.6. Quantitative real-time PCRThe total RNA from treated cells was isolated by Trizol (Invitrogen) and purified by RNeasy Mini Kit and RNase-free DNase Set (Qiagen) according to the manufacturer’s protocols. First strand cDNA was synthesized with the PrimeScriptTM RT Kit (Takara Biotechnology Co, Dalian, China). MALAT-1 and Notch-1 expression were detected by both semi-quantitative polymerase chain reaction (PCR) and quantitative qPCR using PrimeScriptTM PCR Master Mix (Takara Biotechnology Co, Dalian, China) and an ABI 7500 Real-Time PCR system. GAPDH was used as an internal control. The assay was run in triplicate for each sample.2-ΔΔCt method was used to calculate the relative expression of target gene.7. Western blottingWestern blotting assay was performed to examine the protein levels of Notch-1, Hes-1, Hey-1 and Hey-2. Proteins were separated by SDS-PAGE and electroblotted to polyvinylidene difluoride membranes (Millipore) according to manufacturer’s manual. The membranes were incubated with the primary antibodies of Notch-1, Hes-1, Hey-1 and Hey-2and detected by chemiluminescence. β-actin was as the control. Quantity one software was used to detect the iniensity of protein fragment.8. Cell viability assayJJ012 and CH2879 cells were seeded in 96-well plates and transfected with si-MALAT-1 or pcDNA-MALAT-1, respectively. Then,10 μL of 3-(4,5-dimethylthiazolyl-2) 2,5-diphenyltetrazolium bromide (MTT,5 mg/mL, Sigma-Aldrich) was added to each well and incubated for 4 h at 37 ℃. After 4 hours, 150 μL ofDMSO was added to each well and optical density was measured at a wavelength of 570 nm.9. RNA pull-down assayRNA pull-down assay was performed to detect the binding of MALAT-1 and Notch-1. Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase were used for in vitro transcription of biotin-labelled MALAT-1 and biotin-labelled MALAT-1 antisense, the products of which were purified with RNeasy Mini Kit (Roche). RIP buffer was utilized to prepare the cell lysate and then the cell lysate were incubated with the biotinylated RNA biotin-labeled RNAs at 4℃, following adding the washed streptavidin agarose beads to each binding reaction for incubating for 1 h at room temperature. Beads were washed five times and the retrieved proteins were run on SDS-PAGE gels for western blot.10. Cells were treated with cycloheximideCells were treated with 5 μg/ml of cycloheximide and collected at the treatment of 3 h,6 h and 9h. Western blotting was performed to detect the level of Notch-1.11. Animal experimentCultured JJ012 cells which were transfected with si-MALAT-1 or si-control or CH2879 cells which were transfected with pcDNA-MALAT-1 or pcDNA were suspended in 0.2 mL PBS and then injected into the right flank of mice (6 mice per group). The tumor size was measured in two orthogonal directions using calipers, and the tumor volume (mm3) was estimated using the equation length × (width)2 × 0.5.12. Statistical analysisAll statistical analyses were performed using SPSS 13.0 software (Chicago, IL, United States). The results of PCR were analyzed by one-way ANOVA. The differences among means were statistically analyzed by a t-test. P<0.05 was considered statistically significant.Results1. The effect of MALAT-1 on the proliferation of chondrosarcoma cellsAccording to the results of quantitative real-time PCR, we found that the mRNA level of MALAT-1 was increased by 3.55 times in the chondrosarcoma tissues compared to that in the controls. In addition, the mRNA level of MALAT-1 in chondrosarcoma cells CH2879, L835, JJ012 was higher than that in control cellsTC28a2. Moreover, the mRNA level of MALAT-1 was the highest in JJ012 among the three kinds of chondrosarcoma cells, which was 4.05 times to that in control cells. While, CH2879 cells expressed the lowest level of MALAT-1. These results indicated that abnormal expression of MALAT-1 was observed in chondrosarcoma tissues and cells, suggesting the important roles of MALAT-1 for the occurrence and development of chondrosarcoma.To further examine the important role of MALAT-1 for chondrosarcoma, the effect of MALAT-1 on the proliferation of chondrosarcoma cells was investigated. CH2879 cells were transfected with pcDNA-MALAT-1, which increased the level of MALAT-1 by 10.02 rates. It has been shown that the cell viability of CH2879 cells transfected with pcDNA-MALAT-1 were greatly enhanced by 94% compared with that of CH2879 cells transfected with pcDNA. Next, JJ012 cells were transfected with si-MALAT-1 to obtain MALAT-1 knockdown, and the cell viability of JJ012 cells were detected. The mRNA level of MALAT-1 was significantly decreased by si-MALAT-1, which also greatly suppressed the cell viability of JJ012 cells to 51%. These data indicated that the proliferation of chondrosarcoma cells was promoted by MALAT-1 overexpression and was decreased by MALAT-1 knockdown.2. The expression of Notch-1 signaling pathway in chondrosarcoma tissues and cellsTo investigate theexpression of Notch-1 signaling pathway in chondrosarcoma tissues, we examined the protein level of Notch-1, Hes-1, Hey-1, Hey-2 in the chondrosarcoma tissues and the control tissues. The protein level of Notch-1 was enhanced in the chondrosarcoma tissues, as well as that of its target proteins Hes-1, Hey-1, Hey-2. Then we extracted the protein from chondrosarcoma cells CH2879, L835, JJ012 and the control cell TC28a2. It has been shown that the protein level of Notch-1 as well as Hes-1, Hey-1, Hey-2 were significantly enhanced in chondrosarcoma cells. The protein level of Notch-1 was the highest in JJ012 cell and was the lowest in CH2879 cell. These results indicated the highexpression of Notch-1 in chondrosarcoma tissues and cells, suggesting the important roles of Notch-1 for the occurrence and development of chondrosarcoma.3. MALAT-1 promotes the proliferation of chondrosarcoma cell via activating Notch-1 signaling pathwayTo investigate the regulated effect of MALAT-1 on the Notch-1 signaling pathway, JJ012 cells were transfected with si-MALAT-1 or si-control, then the mRNA level of Notch-1 and the protein level of Notch-1 and Hes-1, Hey-1, Hey-2 were detected. si-MALAT-1 exhibited no significant effect on the mRNA level of Notch-1. While the protein level of Notch-1 and Hes-1, Hey-1, Hey-2 were reduced by si-MALAT-1. Moreover, the expression of Notch-1 signaling pathway was examined in CH2879 cells transfected with pcDNA-MALAT-1. Overexpression of MALAT-1 enhanced the protein level of Notch-1 and Hes-1, Hey-1, Hey-2. To futher detect the effect of MALAT-1 on Notch-1,5 μg/ml of cycloheximide (CHX) was used to treat JJ012 which was transfected with si-MALAT-1 or si-control. The results of western blot indicated that si-MALAT-1 further accelerated the degradation of Notch-1 induced by CHX.Biotin-RNA pull-down assay was a good approach to examine the interaction between RNA and protein. To further elucidate the relationship between MALAT-1 and Notch-1, RNA pull-down assay was performed. Biotin-MALAT-1 or biotin-MALAT-1 antisense was incubated with cell extracts, targeted with streptavidin beads, and washed, and the associated proteins were resolved on a gel. The results indicated that MALAT-1 could bind to Notch-1.These results indicated that MALAT-1 could activate the Notch-1 signaling pathway at post-transcriptional level.According to the above experiments, we knew that MALAT-1 could promote the proliferation of chondrosarcoma cells. Here, we investigated the effect of Notch-1 on the proliferation of chondrosarcoma cells regulated by MALAT-1. JJ012 cells were divided into four groups:(1) si-control; (2) si-MALAT-1; (3) si-MALAT-1+pcDNA; (4) si-MALAT-1+pcDNA-Notch-1. The results of MTT assay indicated that Notch-1 overexpression reversed the decreasing of cell viability induced by si-MALAT-1. Furthermore, we found that Notch-1 knockdown reversed the effect of pcDNA-MALAT-1 on the proliferation of chondrosarcoma cells. These data indicated that MALAT-1 promoted the proliferation of chondrosarcoma cell via activating Notch-1 signaling pathway.4. MALAT-1 promotes the tumor growth in a subcutaneous chondrosarcoma cells xenograft modelTo determine the effect of MALAT-1 in vivo, we evaluated its effect in a nude mouse xenograft model of JJ012 cells or CH2879 cells. JJ012 cells transfected with si-MALAT-1 or si-control were subcutaneously inoculated into the nude mice (6 mice per group). Then, the tumor volume was measured. si-MALAT-1 inhibited tumor growth significantly after inoculation for 28 days. CH2879 cellstransfected with pcDNA-MALAT-1 or pcDNA were subcutaneously inoculated into the nude mice (6 mice per group) and the tumor volume were measured. The tumor volume was increased in xenograft tumors of mice treated with CH2879 cellscontaining pcDNA-MALAT-1 compared with the control. Results in vivo further confirmed the promoted effect of MALAT-1 on the tumor growth.Conclusions1. High expression of MALAT-1 was observed in the chondrosarcoma tissues and cells.2. MALAT-1 might contribute to the development of chondrosarcom. Practically speaking, MALAT-1 could promote the proliferation of chondrosarcoma cell.3. High expression of Notch-1 signaling pathway was observed in chondrosarcoma tissues and cells.4. In chondrosarcoma cells, MALAT-1 could activate Notch-1 signaling pathway through binding to Notch-1.5. MALAT-1 promoted the proliferation of chondrosarcoma cell via activating Notch-1 signaling pathway.6. MALAT-1 overexpression promoted the tumor growthin a subcutaneous chondrosarcoma cells xenograft model. |
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