Targeting STK26 and ATG4B: miR-22-3p as a modulator of autophagy and tumor progression in HCC

Abstract

Drug-induced protective autophagy significantly affects the efficacy of anticancer therapies. Enhancing tumor cell sensitivity to treatment by inhibiting autophagy is essential for effective cancer therapy. Our study, analyzing data from The Cancer Genome Atlas (TCGA) public database, HCC cell lines, and liver cancer tissue samples, found that miR-22-3p is expressed at low levels in HCC and is significantly associated with clinicopathological features and patient prognosis. Functional assays and xenograft models demonstrated that miR-22-3p suppresses HCC progression. Moreover, Western blot analysis and the LC3B double reporter (mRFP1-EGFP-LC3B) confirmed that miR-22-3p inhibits autophagy in HCC cells. Further investigation identified Sterile 20-like kinase 26 (STK26) and Autophagy Related 4B Cysteine Peptidase (ATG4B) as targets of miR-22-3p. STK26, which is overexpressed in HCC, promotes malignant characteristics such as proliferation, migration, and invasion. Additionally, STK26 facilitates autophagy in HCC by phosphorylating ATG4B at serine 383. miR-22-3p inhibits autophagy by targeting STK26 and ATG4B, thus preventing the phosphorylation of ATG4B at serine 383. Sorafenib treatment increases the levels and phosphorylation of STK26 and ATG4B, inducing protective autophagy. The combination of miR-22-3p with sorafenib demonstrated enhanced antitumor effects both in vitro and in vivo. In conclusion, our findings suggest that miR-22-3p inhibits HCC progression by regulating the expression of STK26 and ATG4B, potentially through autophagy inhibition, thereby increasing sensitivity to sorafenib treatment. This offers a new therapeutic approach for effective HCC.

Keywords: Autophagy; Hepatocellular carcinoma; Sorafenib; Sterile 20-like kinase 26; miR-22-3p.

Fig. 1

miR-22-3p is downregulated in hepatocellular carcinoma and associated with poor prognosis. (A) Analysis using the TCGA database revealed that miR-22-3p expression is significantly reduced in breast cancer, cholangiocarcinoma, hepatocellular carcinoma, pheochromocytoma, and paraganglioma. (B) miR-22-3p expression is significantly downregulated in hepatocellular carcinoma. (C) Paired analysis showed that miR-22-3p expression is significantly lower in hepatocellular carcinoma tissues compared to corresponding normal tissues. (D) Survival curve analysis indicated that patients with low miR-22-3p expression have significantly shorter overall survival (OS). (E) Real-time PCR analysis of miR-22-3p expression in the normal liver cell line THLE-2 and five hepatocellular carcinoma cell lines. (F) Real-time PCR analysis of STK26 expression in hepatocellular carcinoma tumor tissues and adjacent non-tumor tissues. (G) Real-time PCR analysis of miR-22-3p expression in HCCLM3 and SNU398 cells following transfection with miR-22-3p mimic. Data are presented as mean ± standard deviation of three independent experiments. Significant differences compared to the control group: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2

miR-22-3p inhibits HCC cell proliferation and autophagy in vitro. (A) EDU assay assessing proliferation of HCCLM3 and SNU398 cells following transfection with miR-22-3p mimic and inhibitor. (B) Scratch assay evaluating the effect of miR-22-3p on HCC cell migration. (C) Transwell assay analyzing the impact of miR-22-3p on HCC cell invasion. (D) Western blot analysis of autophagy-related proteins LC3B, P62, and STK26, ATG4B, and p-ATG4B (Ser383) in cells transfected with miR-22-3p mimic and inhibitor. (E) After transfecting HCCLM3 cells with miR-22-3p, autophagic flux was assessed using the LC3B dual-reporter system (mRFP1-EGFP-LC3B). Yellow puncta indicate autophagosomes, while red puncta represent the fusion of autophagosomes with lysosomes. Data are presented as mean ± standard deviation of three independent experiments. Significant differences compared to the control group: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3

miR-22-3p targets and regulates STK26 and ATG4B. (A) Bioinformatic prediction using miRanda and TargetScan identified miR-22-3p as the only miRNA targeting both STK26 and ATG4B. (B) miR-22-3p shares common binding sites in the 3′UTR of STK26 and ATG4B. (C) Analysis of the TCGA database showed a significant negative correlation between miR-22-3p and the expression of STK26 and ATG4B, while STK26 and ATG4B expression was positively correlated. (D) Wild-type and mutant dual-luciferase reporter gene plasmids were constructed for STK26-3′UTR and ATG4B-3′UTR. Transfection with miR-22-3p mimic significantly reduced the fluorescence intensity of the wild-type STK26-3′UTR and ATG4B-3′UTR. Data are presented as mean ± standard deviation of three independent experiments. Significant differences compared to the control group: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4

STK26 is highly expressed in hepatocellular carcinoma and closely associated with poor prognosis. (A) TCGA database analysis revealed that STK26 is upregulated in various cancers, including breast cancer, colorectal adenocarcinoma, cholangiocarcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, lung squamous cell carcinoma, and gastric adenocarcinoma. (B) STK26 expression is significantly higher in hepatocellular carcinoma tissues compared to non-tumor tissues. (C) Paired tumor samples from hepatocellular carcinoma patients showed significant upregulation of STK26. (D) Kaplan-Meier survival analysis indicated that high STK26 expression is associated with significantly shorter overall survival in hepatocellular carcinoma patients. (E) UALCAN database analysis showed changes in STK26 promoter methylation levels. (F) Western blot analysis of STK26 expression in THLE-2, HUH-7, HCCLM3, SNU398, and Hep3B cell lines. (G) Immunohistochemistry (IHC) analysis of STK26 expression in hepatocellular carcinoma and adjacent non-tumor tissues. Data are presented as mean ± standard deviation of three independent experiments. Significant differences compared to the control group: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5

STK26 promotes hepatocellular carcinoma cell proliferation, migration, and invasion. (A) Knockdown and overexpression of STK26 were performed in HCCLM3 and SNU398 cells using three different sh-STK26 and pcDNA3.1-STK26 plasmids, with Western blot analysis confirming knockdown and overexpression efficiency. (B) EDU assay assessing cell proliferation, (C) Scratch healing assay evaluating cell migration, (D) Transwell invasion assay assessing the impact of STK26 on hepatocellular carcinoma cell invasion. Data are presented as mean ± standard deviation of three independent experiments. Significant differences compared to the control group: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 6

STK26 promotes hepatocellular carcinoma growth and autophagy in vivo. Using STK26 knockdown HCCLM3 cells in a nude mouse xenograft model: (A) Photographs of tumor tissues, (B) Tumor growth curves, (C) Tumor weight records, (D) Immunohistochemistry (IHC) analysis of STK26 and Ki-67 protein expression in tumor tissues. (E) Western blot analysis of autophagy-related proteins LC3B, P62, STK26, ATG4B, and p-ATG4B (Ser383). (F)Autophagic flux was assessed in HCCLM3 cells using the LC3B dual-reporter system (mRFP1-EGFP-LC3B). Data are presented as mean ± standard deviation of three independent experiments. Significant differences compared to the control group: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 7

miR-22-3p regulates STK26 to inhibit cell proliferation, migration, and invasion. (A) EDU assay assessing cell proliferation, (B) Scratch assay evaluating cell migration, (C) Transwell chamber assay analyzing cell invasion. (D) Western blot analysis of STK26, P62, p-ATG4B, GAPDH, and LC3II/I protein expression. (E) Evaluation of autophagic flux in HCC cells using the LC3B double reporter (mRFP1-EGFP-LC3B). All data are presented as mean ± standard deviation of three independent experiments. Significant differences compared to the control group: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig. 8

miR-22-3p enhances sorafenib-induced hepatocellular carcinoma cell death by regulating autophagy. (A) Western blot analysis of the effects of different concentrations of sorafenib (0-20μM) on the expression of STK26, ATG4B, p-ATG4B, autophagy marker protein LC3B, and P62, along with an evaluation of autophagic flux using the LC3B double reporter (mRFP1-EGFP-LC3B) (B). (C) Flow cytometry analysis of the effects of miR-22-3p and sorafenib on hepatocellular carcinoma cell apoptosis.In vivo experiments in nude mice injected with HCCLM3 cells stably expressing miR-22-3p and treated with sorafenib: (D) Photographs of tumor tissues, (E) Tumor growth curves and weight records, (F) Immunohistochemistry (IHC) analysis of STK26, ATG4B, and Ki-67 expression in tumor tissues. All data are presented as mean ± standard deviation of three independent experiments. Significant differences compared to the control group: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.