Citation

  • Authors: Guan J. et al.
  • Year: 2022
  • Journal: Evidence-Based Complementary and Alternative Medicine 2022
  • Applications: in vitro / DNA, shRNA plasmid / PEIpro
  • Cell types:
    1. Name: HEK-293T
      Description: Human embryonic kidney Fibroblast
      Known as: HEK293T, 293T
    2. Name: SKOV3

Method

Lentiviral vectors pLKO.1, psPAX2, and pMD2.G were purchased from BioFeng. The GOLM1 full-length coding sequence was cloned from SKOV3 genome into pcDNA3.1 vector. pCDH-EF1-Luc-T2A-tdTomato was purchased from Youbio for bioluminescence imaging in vivo. PEIpro was used for transfection and lentivirus package according to the manufacturer’s instructions (Polyplus). qPCR analysis of GOLM1 and B7-H3 expression in SKOV3 cell line with transient exogenous expressed GOLM1 using PEIpro reagent. Next, we transiently induced exogenous GOLM1 expression plasmid into SKOV3 cell lines. Surprisingly, we found that exogenous GOLM1 expression increased B7-H3 mRNA expression about 3-fold.

Abstract

Introduction. This study was aimed at exploring whether the Golgi membrane protein 1 (GOLM1) enhanced ovarian cancer metastasis through B7-H3-dependent way. Methods. We collected the ovarian cancer patient samples from available databases including GEPIA, starBase, and Protein Altas that have GOLM1 and B7-H3 mRNA and protein expression. Ovarian cancer cell line SKOV3 was purchased. Knockdown GOLM1 and B7-H3 cell lines were obtained through introducing shRNAs by lentivirus package system, while GOLM1 or B7-H3 overexpression cell line was obtained by introducing GOLM1 full-length gene. Furthermore, wound-healing assay and Transwell assay were performed to assess tumor invasion and metastasis abilities; related proteins’ expression was quantitated by western blotting, ELISA, and flow cytometry assay. The protein interaction was quantified by co-immunoprecipitation. Results. GOLM1 has the correlative expression pattern with B7-H3 in ovarian cancer through patient sample databases (R = 0.421). GOLM1 knockdown had minimal impact on B7-H3 mRNA synthesis, while downregulated B7-H3 protein expression on tumor membrane and soluble B7-H3 (sB7-H3) level () through physical interaction, GOLM1 knockdown, significantly reduce tumor invasion and metastasis in vitro (). Moreover, exogenous sB7-H3 significantly rescued this inhibitory effect. Both GOLM1 and B7-H3 knockdown restrained tumor growth and metastasis in immunodeficient mice and prolonged the survival rate. Conclusions. GOLM1 acts as an initial oncogenic driving gene by promoting ovarian cancer invasion and metastasis through modulating B7-H3 protein maturation and secretion.

Pubmed