N6-methyladenosine-modified long non-coding RNA KIF9-AS1 promotes stemness and sorafenib resistance in hepatocellular carcinoma by upregulating SHOX2 expression

Figure 1 The long noncoding RNA KIF9-AS1 was upregulated in hepatocellular carcinoma patients. Cancer tissues and adjacent non-cancerous tissues were collected from hepatocellular carcinoma patients. A: The expression of KIF9-AS1 in hepatocellular carcinoma tissues and para-cancerous tissues was calculated via quantitative reverse transcription polymerase chain reaction; B: The overall survival rate was analyzed via Kaplan-Meier analysis, n = 20. The data are shown as the means ± SDs. ^aP < 0.05, ^bP < 0.01. HCC: Hepatocellular carcinoma.

Figure 2 Depletion of the long noncoding RNA KIF9-AS1 suppressed the stemness characteristics and sorafenib resistance of hepatocellular carcinoma cells. Huh-7 cells were transfected with sh-KIF9-AS1 or sh-NC. A: The expression of KIF9-AS1 was measured via quantitative reverse transcription polymerase chain reaction; B: The viability of Huh-7 cells was assessed with a Cell Counting Kit-8 assay; C: The sphere formation ability of Huh-7 cells was examined; D: The protein levels of CD44, CD133 and epithelial cell adhesion molecule in Huh-7 cells were quantified via western blotting. Huh-7/R cells were transfected with sh-KIF9-AS1 or sh-NC; E: The IC₅₀ values in Huh-7 and Huh-7/R cells were calculated via a Cell Counting Kit-8 assay; F: The expression of KIF9-AS1 was measured via quantitative reverse transcription polymerase chain reaction; G and H: Cell proliferation and cell apoptosis were evaluated via colony formation assays and flow cytometry, respectively. The data are shown as the means ± SDs (n = 3). ^aP < 0.05, ^bP < 0.01. Each experiment was repeated three times. EpCAM: Epithelial cell adhesion molecule.

Figure 3 Methyltransferase-like 3 stabilizes and upregulates long noncoding RNA KIF9-AS1 expression in an N6-methyladenosine-insulin-like growth factor 2 mRNA-binding protein 1-dependent manner. A: The N6-methyladenosine (m6A) content in hepatocellular carcinoma tissues and para-cancerous tissues was examined using a commercial kit (n = 20); B: m6A levels of KIF9-AS1 in Huh-7 and Huh-7/R cells were examined via methylated RNA immunoprecipitation-quantitative polymerase chain reaction; C: Methyltransferase-like 3 (METTL3) protein expression in Huh-7 and Huh-7/R cells was quantified via western blotting; D: METTL3 protein expression in Huh-7 and Huh-7/R cells transfected with sh-METTL3 or sh-NC was quantified via western blotting; E: The m6A level of KIF9-AS1 was calculated via methylated RNA immunoprecipitation-quantitative polymerase chain reaction; F: The insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) protein level was evaluated via western blotting; G: The interaction between IGF2BP1 and KIF9-AS1 was verified via an RNA immunoprecipitation assay; H: The protein expression of IGF2BP1 was assessed via western blotting; I: After blocking the synthesis of new RNA with actinomycin D (5 μg/mL) for 0, 3, and 6 hours, the expression of KIF9-AS1 was evaluated via quantitative reverse transcription polymerase chain reaction. The data are shown as the means ± SDs (n = 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. Each experiment was repeated three times. m6A: N6-methyladenosine; METTL3: Methyltransferase-like 3; IGF2BP1: Insulin-like growth factor 2 mRNA-binding protein 1.

Figure 4 The long noncoding RNA KIF9-AS1 increases the stability and expression of short stature homeobox 2 through ubiquitin-specific peptidase 1-mediated short stature homeobox 2 deubiquitination. A: The expression of ubiquitin-specific peptidase 1 (USP1) and short stature homeobox 2 (SHOX2) in hepatocellular carcinoma tissues and para-cancerous tissues was evaluated via quantitative reverse transcription polymerase chain reaction (n = 20); B: Image of immunohistochemical staining of SHOX2 in tumor tissues and para-cancerous tissues; C: Correlation analysis between KIF9-AS1 and USP1 and between USP1 and SHOX2; D: The protein levels of USP1 and SHOX2 in Huh-7 and Huh-7/R cells were assessed via western blotting; E: The interaction between KIF9-AS1 and USP1 was verified via an RNA immunoprecipitation assay; F: The interaction between USP1 and SHOX2 was confirmed via a coimmunoprecipitation assay; G: The ubiquitination levels of SHOX2 in Huh-7 and Huh-7/R cells were determined via immunoprecipitation and western blotting; H: After cycloheximide treatment for 0, 3, 6, or 12 hours, the protein level of SHOX2 was evaluated via western blotting. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. Each experiment was repeated three times. USP1: Ubiquitin-specific peptidase 1; SHOX2: Short stature homeobox 2; HCC: Hepatocellular carcinoma; IgG: Immunoglobulin G.

Figure 5 The long noncoding RNA KIF9-AS1 enhances the stability and expression of short stature homeobox 2 through ubiquitin-specific peptidase 1-mediated short stature homeobox 2 deubiquitination. A: The ubiquitination levels of short stature homeobox 2 in Huh-7 and Huh-7/R cells were determined via immunoprecipitation and western blotting; B: Western blotting was conducted to analyze the short stature homeobox 2 protein. ^aP < 0.05, ^bP < 0.01. Each experiment was repeated three times. USP1: Ubiquitin-specific peptidase 1; SHOX2: Short stature homeobox 2; CHX: Cyclohexamide.

Figure 6 The long noncoding RNA KIF9-AS1 increases the stemness and sorafenib resistance of hepatocellular carcinoma cells through short stature homeobox 2. Huh-7 cells were transfected with sh-NC, sh-KIF9-AS1, sh-KIF9-AS1 + OE-NC or sh-KIF9-AS1 + OE-short stature homeobox 2. A: Short stature homeobox 2 (SHOX2) protein expression was quantified via western blotting; B: Cell viability was assessed via a Cell Counting Kit-8 assay; C: The sphere formation ability of Huh-7 cells was examined; D: The protein levels of the stemness markers CD44, CD133 and epithelial cell adhesion molecule in Huh-7 cells were evaluated via western blotting. Huh-7/R cells were transfected with sh-NC, sh-KIF9-AS1, sh-KIF9-AS1 + OE-NC or sh-KIF9-AS1 + OE-SHOX2; E: SHOX2 expression was measured via western blotting; F: The IC₅₀ values were determined via a Cell Counting Kit-8 assay; G: A colony formation assay was performed to evaluate cell proliferation; H: A flow cytometry assay was conducted to detect cell apoptosis. ^aP < 0.05, ^bP < 0.01. Each experiment was repeated three times. SHOX2: Short stature homeobox 2; EpCAM: Epithelial cell adhesion molecule.

Figure 7 Knockdown of the long noncoding RNA KIF9-AS1 inhibited sorafenib resistance in hepatocellular carcinoma xenograft model mice. Huh-7/R cells transfected with sh-KIF9-AS1 or sh-NC were subcutaneously transplanted into BALB/c nude mice to establish a mouse model of xenograft tumor growth. Sorafenib was administered orally twice a week at a dosage of 30 mg/kg from days 7-14 and 21-28. A-C: The tumor volume and weight of the mice were recorded; D: Images of immunohistochemical staining for Ki67 in tumor tissues; E: Short stature homeobox 2, CD44, CD133 and epithelial cell adhesion molecule expression levels were assessed via western blotting. n = 6. ^bP < 0.01, ^cP < 0.001. EpCAM: Epithelial cell adhesion molecule.

Figure 8 The mechanism by which the m6A-modified long noncoding RNA KIF9-AS1 promotes stemness and sorafenib resistance in hepatocellular carcinoma through ubiquitin-specific peptidase 1-mediated deubiquitination of short stature homeobox 2. HCC: Hepatocellular carcinoma; USP1: Ubiquitin-specific peptidase 1; SHOX2: Short-stature homeobox 2; METTL3: Methyltransferase-like 3; IGF2BP1: Insulin-like growth factor 2 mRNA binding protein 1; m6A: N6-methyladenosine; Ub: Ubiquitin.