Archives

  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • 2021-03
  • 3X FLAG Peptide br The statistical results reveal that TONSL

    2019-10-22


    5. The statistical results reveal that TONSL-AS1 significantly reduce the 3X FLAG Peptide growth.C. Colony formation assay showed that TONSL-AS1 inhibits cell proliferation. The representative images were captured at 14 day after seeding 100 cells into a 12-well plate as described in the “Materials and Methods” section. Scar bar: 1 cm.D. Quantification of the results in C.E. Soft agar colony formation assay showed that TONSL-AS1 inhibits cells anchorage-independent cell growth. SGC-7901 and MGC- 803 cells were transduced with TONSL-AS1 or the empty vector, and the representative images were captured at 14 days after seeding 1 × 104 cells into a 6-well plate as described in the “Materials and Methods” section. Scar bar: 100 μm F. Quantification of the results in E. For B, D and F Mean ± SD were derived from n = 3 independent experiments. **P < 0.01, ***P < 0.001; two-tailed unpaired Student's t-test.
    3.3. TONSL-AS1 inhibits cell motility and tumorigenesis
    Next, we detected the effect of TONSL-AS1 on cell migration and invasion. The results indicated that TONSL-AS1 could significantly 
    inhibit cell motility (Fig. 3A–D). To further explore the function of TONSL-AS1 in tumor progression, we established cell lines stably ex-pressing TONSL-AS1 via a lentiviral infection in SGC-7901 and MGC-803 cells respectively. Then we injected the nude mice subcutaneously
    Fig. 3. TONSL-AS1 inhibits tumorigenesis in vivo.A. The representative image of cell migration and invasion assay, which were performed to determine the SGC-7901 cell motility of TONSL-AS1. Scar bar: 100 μm B. Quantification of the results in A.C. The representative image of cell migration and invasion assay, which were performed to determine the MGC-803 cell motility of TONSL-AS1. Scar bar: 100 μm.D. Quantification of the results in F.E. Tumorigenesis assay showed that TONSL-AS1 inhibits cell growth in vivo. SGC-7901 and MGC-803 cells were transduced with TONSL-AS1 or the empty vector. Cells were injected subcutaneously to the mice as described in the “Materials and Methods” section.F. Tumor weights were measured and analyzed, TONSL-AS1 significantly reduces the tumor weight compared to the control group.G. Tumor volume were calculated every week, TONSL-AS1 significantly reduces the tumor volume compared to the control group. For B and D, Mean ± SD were derived from n = 3 independent experiments. For F and G, Mean ± SD were derived from n = 2 independent experiments. **P < 0.01,
    with SGC-7901 and MGC-803 cells that stably overexpressing TONSL-AS1 or vector control. Consistent with the in vitro results, two weeks after injection, tumor growth in the TONSL-AS1 overexpressing group was significantly decreased compared to the vector control group 3X FLAG Peptide (Fig. 3E). Both the tumor size and weight were substantially reduced in the TONSL-AS1-overexpressing group compared with the control group (Fig. 3F and G). Taken together, our data demonstrated that TONSL-AS1 played a crucial role in gastric cancer tumor growth both in vitro and in vivo. 
    TONSL-AS1 is on the reverse strand of the 8th human chromosome, we explored the neighboring protein-coding genes around the TONSL-AS1 gene locus on the genome. The positional relationship between TONSL-AS1 and TONSL were represent in Fig. 4A. We picked up four potential proteins (TONSL, VPS28, CYHR1 and KIFC2) for testing after inducing TONSL-AS1 in SGC-7901 and MGC-803 cells, then we ex-amined the expression of these relative gene via RT-qPCR (Fig. 4B). Compared with three others, the expression of TONSL was significantly increased (Fig. 4B). Western blotting confirmed that TONSL-AS1 in-creased the TONSL protein level in SGC-7901 and MGC-803 (Fig. 4C).
    Fig. 4. TONSL-AS1 increases TONSL expression.A. The relative expression level of four nearby genes of TONSL-AS1 were examined by qRT-PCR in TONSL-AS1 overexpressing cells. Actin was used as an internal control for normalization.B. The schematic model of the genomic relationship between TONSL-AS1 and TONSL was shown. TONSL, on the chromosome plus strand, is located about 120 bp downstream from TONSL-AS1 transcription start site.C. The western blotting results revealed that TONSL-AS1 increased the TONSL protein level in SGC-7901 and MGC-803 cells.D. The western blotting results revealed that the TONSL protein levels were decreased in SGC-7901 and MGC-803 cells compared to the normal gastric cell HPDE-6.E. TONSL-AS1 and TONSL exhibited the positive expression relationship in 140 gastric cancer tissues.F. Dual luciferase reporter assay indicated that TONSL-AS1 significantly activates the TONSL promoter activity. For F Mean ± SD were derived from n = 3 independent experiments. ***P < 0.001; two-tailed unpaired Student's t-test.