Inhibition of USP22 by miR-200b-5p represses gastric cancer cell proliferation and migration by targeting the NF-κB signaling pathway

Abstract

Gastric cancer (GC) is an aggressive tumor type with an intricate pathogenesis and limited therapeutic options. Ubiquitin-specific protease 22 (USP22) is a protein implicated in cell proliferation, metastasis, and tumorigenesis. However, the regulatory mechanisms governing USP22 in GC are still not fully understood. In this study, we perform bioinformatics analysis to identify conserved miRNA recognition sites for miR-200b-5p within the 3'UTR of USP22. Validation via luciferase reporter assay confirms the transcriptional regulation of USP22 by miR-200b-5p. Overexpression of miR-200b-5p markedly inhibits the proliferation and migration of GC cells in vitro and suppresses tumor growth in vivo. Conversely, ectopic expression of USP22 reversed this effect by modulating the NF-κB signaling pathway. Additionally, qPCR analysis reveals an inverse correlation between the miR-200b-5p level and USP22 expression in GC. Collectively, our findings indicate that miR-200b-5p-mediated inhibition of USP22 attenuates cell proliferation by targeting the NF-κB signaling pathway in GC, suggesting that miR-200b-5p and USP22 could serve as potential diagnostic or therapeutic targets for gastric cancer and other related human diseases.

Keywords: NF-κB; USP22; cell proliferation; gastric neoplasm; miR-200b-5p.

Figure 1

USP22 is a direct target of miR-200b-5p in GC cells (A) Mutant (Mut) 3′-untranslated region (3′UTR) USP22 sites and probable miR-200b-5p binding sites. Underlined is the site of mutation. WT stands for “wild type”, whereas MT stands for “mutant type”. (B,C) Relative luciferase activity in 293T (B) or SGC7901 (C) cells cotransfected with the WT/MT-USP22-3′UTR reporter plasmid and the miR-200b-5p mimic or miR-NC. (D,E) USP22 mRNA and protein expressions in SGC7901 and BGC823 cells transfected with miR-NC, the miR-200b-5p mimic, or the miR-200b-5p inhibitor. GAPDH was used as an internal control. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

miR-200b-5p inhibits GC cell proliferation in vitro (A) The expression level of miR-200b-5p determined by qRT-PCR after SGC7901 and BGC823 cells were transfected with the miR-200b-5p mimic or control miRNA (miR-NC). (B,C) Cell viability of SGC7901 and BGC823 cells transfected with the miR-200b-5p mimic or miR-NC was measured via CCK-8 assay. (D) Statistical analysis of the outcomes of the 72-h CCK-8 assay. (E) Colony formation assay was used to assess cell proliferation. (F) Statistical analysis of (E). The error bars show the standard deviation, and the results are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Overexpression of USP22 ablates the inhibitory effects of miR-200b-5p in GC cells (A) qPCR results showing the expression of USP22 in pLV-USP22-infected SGC7901 and BGC823 cells. (B,C) SGC7901 and BGC823 cell proliferation was assessed via the CCK-8 assay. The miR-200b-5p-mediated inhibition of cell proliferation was reversed by the ectopic expression of USP22. The findings of the statistical analysis of the CCK-8 assay are displayed on the right. (D) Colony formation assessment in SGC7901 and BGC823 cells. Ectopic expression of USP22 reversed the miR-200b-5p-mediated inhibition of cell proliferation. The statistical analysis results are displayed on the right. (E) Cell migration experiment. The expression of USP22 reversed the miR-200b-5p-mediated inhibition of SGC7901 and BGC823 cell migration. *P < 0.05, **P < 0.01.

Figure 4

miR-200b-5p represses USP22 by inhibiting the NF-κB pathway (A) SGC7901 cells were transfected with miR-200b-5p and treated with or without TNF for 48 h; then, luciferase reporter assays were performed. (B) The miR-200b-5p-mediated inhibition of NF-κB reporter gene activity in SGC7901 cells was reversed by the ectopic expression of USP22. (C) Western blot analysis showing the expression of USP22 in (B). (D,E) USP22 mRNA and protein levels in shUSP22-infected cells were measured via qPCR (D) and western blot analysis (E). (F) The miR-200b-5p-induced inhibition of NF-κB reporter gene activity in SGC7901 cells was amplified by USP22 knockdown as shown by luciferase reporter assay. *P < 0.05, **P < 0.01.

Figure 5

miR-200b-5p suppresses tumorigenicity in mice (A,B) Tumor growth in nude mice. (C) The weights of the SGC7901-miR-200b-5 tumors were significantly lower than the weights of the control tumors at 30 days following injection. (D) Statistical analysis of the tumor volume and weight in each group. (E) miR-200b-5p expression and USP22 mRNA levels in SGC7901 xenograft tumors as determined by qPCR. n = 5. **P < 0.01.

Figure 6

Inverse correlation between miR-200b-5p expression and USP22 mRNA expression in GC clinical samples and cell lines (A,B) qPCR was used to evaluate miR-200b-5p (A) and USP22 (B) mRNA expressions in GC tissues and nearby normal tissues. (C) Pearson correlation analysis revealed that the expression levels of USP22 and miR-200b-5p were correlated in GC tissues. n = 30. (D,E) The levels of miR-200b-5p and USP22 mRNA expression were determined in five different GC cell lines (GES, SGC7901, BGC823, MGC803, and HGC27) via qPCR.

Figure 7

A possible schematic diagram and molecular mechanism by which miR-200b-5p regulates cell proliferation via USP22