Inhibition of the SERPINB5/HSP90AA1 axis restrains the proliferation and invasion of rectal cancer

Figure 1 Serpin family B member 5 was prominently upregulated in rectal cancer. A: Gene Expression Profiling Interactive Analysis database was used to illustrate the relative serpin family B member 5 (SERPINB5) levels in rectal cancer tissues (left red) and normal tissues (right gray), ^aP < 0.01; B and C: Western blot analysis was conducted to assess SERPINB5 protein level in rectal cancer tissues and matched adjacent tissues (n = 27); D and E: SERPINB5 expression in the human normal colonic mucosa cell line FHC and rectal cancer cell lines (HR8348, SW480, SW837, and SW1463) was determined through western blot analysis. Data are presented as mean ± SEM. ^aP < 0.01. SERPINB5: Serpin family B member 5; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 2 Effects of serpin family B member 5 on SW480 cell proliferation, invasion, and apoptosis. SW480 cells were transfected with pcDNA-serpin family B member 5 (SERPINB5) or short-hairpin RNA targeting SERPINB5. A and B: SERPINB5 protein level in SW480 cells was measured through western blot analysis; C: SW480 cell proliferation was examined with Cell Counting Kit-8 assay; D and E: SW480 cell invasion was evaluated with Transwell assay; F and G: SW480 cell apoptosis was tested through flow cytometry. Data are presented as mean ± SEM. ^aP < 0.01. SERPINB5: Serpin family B member 5; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; sh: short-hairpin RNA; FITC: Fluorescein isothiocyanate; PI: Propidium iodide.

Figure 3 Serpin family B member 5 interacted with heat shock protein 90 alpha class A member 1 in SW480 cells. A: Potential interacting proteins of serpin family B member 5 (SERPINB5) were predicted by using the STRING tool (https://cn.string-db.org/); B and C: Co-immunoprecipitation assay verified that SERPINB5 interacted with heat shock protein 90 alpha class A member 1 (HSP90AA1); D: PcDNA-SERPINB5 and short-hairpin RNA targeting SERPINB5 were transfected into SW480 cells. HSP90AA1 protein level was tested through western blot analysis; E: Gene Expression Profiling Interactive Analysis database revealed the relative expression of HSP90AA1 in rectal cancer tissues (left red) and normal tissues (right gray), ^aP < 0.01; F: Western blot analysis was employed to measure the relative HSP90AA1 protein level in rectal cancer tissues and matched adjacent tissues (n = 27). Data are presented as mean ± SEM. ^aP < 0.01. IP: Immunoprecipitation; IgG: Immunoglobulin G; SERPINB5: Serpin family B member 5; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; HSP90AA1: Heat shock protein 90 alpha class A member 1; sh: short-hairpin RNA.

Figure 4 Effects of heat shock protein 90 alpha class A member 1 on SW480 cell proliferation, invasion, and apoptosis. PcDNA-heat shock protein 90 alpha class A member 1 (HSP90AA1) and short-hairpin RNA targeting HSP90AA1 were transfected into SW480 cells. A and B: HSP90AA1 protein level in SW480 cells was tested through western blot analysis; C: SW480 cell proliferation was evaluated through Cell Counting Kit-8 assay; D and E: SW480 cell invasion was detected with Transwell assay; F and G: SW480 cell apoptosis was tested with flow cytometry. Data are presented as mean ± SEM. ^aP < 0.01. HSP90AA1: Heat shock protein 90 alpha class A member 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; sh: short-hairpin RNA; FITC: Fluorescein isothiocyanate; PI: Propidium iodide.

Figure 5 Heat shock protein 90 alpha class A member 1 overexpression reversed the effects of serpin family B member 5 knockdown on SW480 cell progression. A and B: Western blot analysis was utilized to measure heat shock protein 90 alpha class A member 1 protein expression; C: SW480 cell proliferation was evaluated with Cell Counting Kit-8 assay; D and E: SW480 cell invasion was evaluated with Transwell assay; F and G: SW480 cell apoptosis was tested with flow cytometry. Data are presented as mean ± SEM. ^aP < 0.01. HSP90AA1: Heat shock protein 90 alpha class A member 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; sh: short-hairpin RNA; SERPINB5: Serpin family B member 5; FITC: Fluorescein isothiocyanate; PI: Propidium iodide.

Figure 6 Serpin family B member 5 knockdown retarded rectal cancer tumor growth in xenograft mice. A: Tumors in the short-hairpin RNA targeting NC and short-hairpin RNA targeting serpin family B member 5 groups; B: Tumor volume was tested every 7 days following implantation; C: Tumor weight was assessed on day 28 after euthanasia; D and E: Serpin family B member 5 and heat shock protein 90 alpha class A member 1 protein levels in tumor tissues were examined through western blot analysis; F and G: Ki67 protein levels in tumor tissues were tested by using immunohistochemistry staining. Data are presented as mean ± SEM. ^aP < 0.01. SERPINB5: Serpin family B member 5; sh: short-hairpin RNA; HSP90AA1: Heat shock protein 90 alpha class A member 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.