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  • br Results br LncRNA TONSL AS

    2019-10-22


    3. Results
    3.1. LncRNA TONSL-AS1 is downregulated in gastric cancer tissues and cells
    To acquire the transcriptional profile of lncRNAs in gastric cancer, paired gastric cancer tissues and normal tissues (n = 3) were analyzed using ArrayStar lncRNA microarrays. We identified seven candidate lncRNAs (Fig. 1A) that were deregulated significantly between cancer and paired normal tissues. Some of these lncRNAs have previously been investigated in other cancer types, but no published studies have fo-cused on TONSL-AS1.
    We examined TONSL-AS1 expression in 71 paired clinical gastric cancer and normal tissues. TONSL-AS1 was significantly downregulated in cancer tissues compared with paired normal tissues (Fig. 1B). Then,
    Fig. 1. TONSL-AS1 is downregulated in gastric cancer tissues and cells.A. Representative result of gastric cancer lncRNA microarray analysis. N, Normal tissue; Ca, cancer tissue. B. In total, 71 paired gastric cancer tissues and normal tissues shows significantly decreased TONSL-AS1 in cancer tissues. C. Evaluation of TONSL-AS1 in different gastric cells. *P < 0.05, **P < 0.01. D. RNC-RNA from SGC-7901 Actinomycin D was extracted and reverse-transcribed using oligo-dT primers as indicated. Specific primers for TONSL-AS1 were analyzed by using qPCR. E. Specific antisense probes for Actinomycin D TONSL-AS1 were used to detect TONSL-AS1 by fluorescence in situ hybridization in SGC-7901 cells.
    we compared the TONSL-AS1 expression levels in immortalized gastric epithelial cell line (GES-1) and gastric cancer cells (SGC-7901, MGC-803, NCI-N87 and BGC-823). SGC-7901 and MGC-803 had the lowest TONSL-AS1 expression levels among all cells (Fig. 1C). Thus, we used SGC-7901 and MGC-803 to investigate the role of TONSL-AS1 in gastric cancer pathogenesis. To test the coding ability of TONSL-AS1, we analyze the coding potential score of TONSL-AS1 by Coding Potential Calculator (http://cpc.cbi.pku.edu.cn). The score (−0.209) indicated that the coding ability of TONSL-AS1 is weak. Next, we performed qPCR on the translating RNAs (RNC-RNAs) from SGC-7901 cells to further show that TONSL-AS1 has no translating ability. RNC-RNAs were reverse-transcribed using oligo-dT (for mRNAs). Total RNAs were reverse-transcribed by random primers as a positive control. As shown in Fig. 1D, TONSL-AS1-specific PCR products were not amplified from cDNA reverse-transcribed from RNC-RNAs with random primers, in-dicating that TONSL-AS1 is not bound to the ribosomal nascent chain complex and is not undergoing translation. Then we examined the TONSL-AS1 localization by fluorescence in situ hybridization. As shown in Fig. 1E, TONSL-AS1 was most localized in cell nucleus.
    3.2. LncRNA TONSL-AS1 inhibits proliferation of gastric cancer cells in vitro
    The patients’ clinicopathologic characteristics of TONSL-AS1 are detailed in Table 1. It revealed that TONSL-AS1 significantly correlated with lymph node metastasis. Then we overexpressed TONSL-AS1 in SGC-7901 and MGC-803 cells (Fig. 2A). TONSL-AS1 overexpression resulted in a significant decrease cell viability in SGC-7901 and MGC-803 by the CCK-8 assay (Fig. 2B). The colony formation assay also showed that TONSL-AS1 significantly attenuated the colony numbers of SGC-7901 and MGC-803 cells (Fig. 2C and D). Moreover, TONSL-AS1 
    Table 1
    Correlation between TONSL-AS1 expression and clinicopathologic character-istics of gastric cancer patients.
    Characteristics
    TONSL-AS1
    Cases High Low χ2 P
    TNM stage(AJCC) High 11 3 8
    Lymph node metastasis T3/T4 7 4 3
    inhibited SGC-7901 and MGC-803 anchorage dependent cell growth (Fig. 2E and F). These results indicate that TONSL-AS1 is negative for the cell growth in vitro.
    Fig. 2. TONSL-AS1 inhibits gastric cell proliferation and anchorage dependent cell growth.A. TONSL-AS1 expression level was validated by qPCR. SGC-7901 and MGC-803 cells were transfected with TONSL-AS1, the cells were subjected to RT-qPCR, Actin serves as an internal control for normalization.B. TONSL-AS1 inhibited cell proliferation. SGC-7901 and MGC-803 cells were transfected with TONSL-AS1 or the control empty vector, then examined with CCK-8 assay on days 1, 2, 3, 4 and