The lncRNA SEMA3B-AS1/HMGB1/FBXW7 Axis Mediates the Peritoneal Metastasis of Gastric Cancer by Regulating BGN Protein Ubiquitination

Figure 5

SEMA3B-AS1 might destabilize the BGN protein by regulating FBXW7, thus promoting the ubiquitination degradation of BGN and inhibiting GC. Data were shown as (); , , , and . (a) The expression of BGN protein in gastric cancer and adjacent normal tissues was analyzed by bioinformatics using GSE54129 data set. (b) The relative mRNA expression of BGN was identified by qRT-PCR assay in 50 pairs of GC tissues and adjacent normal tissues. (c) The representative IHC staining image for BGN in the paracancerous normal tissues, GC tissues, and GS PM tissues. (d) The relative mRNA expression of FBXW7 in five GC cell lines and normal stomach epithelial cell line (GES-1) was detected by qRT-PCR assay. (e) The overexpression efficiency of FBXW7 and negative control (NC) was detected in HGC-27 using qRT-PCR. (f) The knockdown efficiency of FBXW7 and negative control (NC) was detected in SGC-7901 using qRT-PCR. The mRNA expression levels of BGN were detected by qRT-PCR assay after FBXW7 (g) overexpression or (h) knockdown in GC cells (HGC-27 or SGC-7901). The protein expression levels of BGN were detected by Western blotting assay after FBXW7 (i) overexpression or (j) knockdown in GC cells (HGC-27 or SGC-7901). (k) The interaction between FBXW7 and BGN was identified in HGC-27 cells by coimmunoprecipitation (co-IP). SDS-PAGE separated the immunoprecipitates of input (20%) and BGN. Western blotting was performed to confirm the interaction between FBXW7 and BGN. (l) Cell groups were treated with the proteasome inhibitor MG-132 (50 μM) or the protein synthesis inhibitor cycloheximide CHX (100 μg/mL) at different time points (1 h, 4 h, 8 h, and 12 h), and the changes of BGN protein at different time points were detected by Western blotting assay. (m) HGC-27 or SGC-7901 cells were treated with MG-132 (50 μM) and transfected with HA-Ubi and simultaneously overexpression or knockdown FBXW7 as well as SEMA3B-AS1 overexpressed or knocked down, respectively, FBXW7 vector served as a negative control. Cell lysates were immunoprecipitated with anti-BGN antibody to identify ubiquitination of BGN with anti-HA antibodies using Western blotting assay. (n) The mRNA relative expression levels of SEMA3B-AS1 and BGN were obvious negative association in GC patients using Pearson’s correlation analysis (, ).

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