Endoplasmic reticulum stress promotes sorafenib resistance via miR-188-5p/hnRNPA2B1-mediated upregulation of PKM2 in hepatocellular carcinoma

Abstract

Emerging evidence has shown that endoplasmic reticulum (ER) stress promotes sorafenib resistance in hepatocellular carcinoma (HCC). However, the underlying mechanisms are poorly understood. The purpose of this study was to explore the mechanism by which ER stress promotes sorafenib resistance in HCC. We found that pyruvate kinase isoform M2 (PKM2) was highly expressed in human HCC tissues and co-related with worse clinicopathologic features and overall survival. Activation of ER stress positively correlated with PKM2 expression both in HCC tissue samples and tunicamycin (TM)-induced HCC cell lines. PKM2 knockdown increased sorafenib-induced apoptosis and decreased the ability of colony formation, while upregulation of PKM2 reverses this phenomenon. Furthermore, high-throughput sequencing identified that activation of ER stress significantly downregulated the expression of miR-188-5p in HCC cells. According to bioinformatics analysis and dual-luciferase assays, we further confirmed that hnRNPA2B1 is the target gene of miR-188-5p. Downregulating the expression of hnRNPA2B1 with siRNA could decrease the expression of PKM2 and enhance sorafenib-induced apoptosis in HepG2 cells. Our study demonstrated that ER stress could promote sorafenib resistance through upregulating PKM2 via miR-188-5p/hnRNPA2B1. Therefore, targeting the miR-188-5p/hnRNPA2B1/PKM2 pathway and ER stress may prove instrumental in overcoming sorafenib resistance in HCC treatment.

Keywords: PKM2; endoplasmic reticulum stress; hepatocellular carcinoma; hnRNPA2B1; miR-188-5p; sorafenib.

Graphical abstract

Figure 1

Overexpression of PKM2 correlates with worse clinicopathologic features in HCC patients (A) The protein level of PKM2 in human liver tissues and HCC tissues was detected by western blot. (B and C) Representative expression and immunohistochemistry score of PKM2 in HCC and adjacent tissue specimens (100× and 200× magnification). (D) The Kaplan-Meier method was applied in overall survival analysis between the PKM2 high-expression and PKM2 low-expression groups. (E and F) The expression levels of PKM2 and PKM1 in different HCC cells were analyzed by western blot. (G and H) Immunofluorescence analysis of the location and fluorescence intensity semiquantitative analysis of the expression of PKM2 and PKM1 in HepG2 and L02 cells (40× magnification). ∗∗p < 0.01 (n = 3).

Figure 2

Upregulation of PKM2 contributes to sorafenib resistance in HCC cells (A) MTT assay analysis of cell viability of different HCC cells when exposed to sorafenib (1.25, 2.5, 5.0, 10, 20, and 40 μM) for 24 h. (B, C, H, and I) Western blot analysis of the efficiency of PKM2 knockdown (B and C) and overexpression (H and I). (D, E, J, and K) Flow cytometry analysis of apoptosis rate in Huh7 cells treated with sorafenib (10 μM, 24 h) after transfecting with PKM2 siRNA or si-NC (siRNA-negative control) for 24 h (D and E) and in HepG2 cells treated with sorafenib (10 μM, 24 h) after transfecting with overexpressed plasmid GV230-PKM2 or GV230-control for 24 h (J and K). (F, G, L, and M) Colony formation assay analysis of cell ability in Huh7 cells and HepG2 cells. ∗∗p < 0.01 (n = 3).

Figure 3

Overexpressed PKM2 is closely related to activated ER stress in HCC (A and B) Expression levels of PERK, GRP78, IRE1α, and ATF6 in HCC tissue samples were examined by immunohistochemistry (IHC) analysis and semiquantitative analysis by IHC score (200× magnification). (C) Correlation analysis between PKM2 expression and ER stress-related protein expression. (D) Immunofluorescence analysis of the localization and expression of PKM2 with GRP78 in HepG2 and L02 cells (40× and 200× magnification, respectively).

Figure 4

Activated ER stress in HCC triggers the expression of PKM2 (A and B) PKM2 and PKM1 protein levels were detected by western blot in HepG2 cells exposed to gradient concentrations of TM for 24 h and TM at 3 μM for gradient time. (C) Immunofluorescence analysis of PKM2 expression after HepG2 cells treated with TM (3 μM, 24 h) (80× magnification). (D and E) Western blot analysis of the knockdown efficiency of 3 UPR pathways. (F and G) PKM2, IRE1α, ATF6, and PERK protein expressions were determined by western blot after HepG2 cells transfected with the most effective UPR pathways siRNA for 24 h. ns, no statistical difference. (H and I) Western blot analysis of PKM2 and ER stress-related protein expression when Huh7 cells were transfected with 3 PKM2 interfering sequences for 24 h. ∗∗p < 0.01 (n = 3).

Figure 5

Activation of ER stress regulates the expression of PKM2 through miR-188-5p in HCC (A) The heatmap of miRNAs expressed in con-HepG2 cells and TM-HepG2 cells (3 μM, 24 h). (B) qRT-PCR analysis of the levels of differentially expressed miRNAs after treating with TM (3 μM, 24 h). (C) Schematic diagrams of miRNAs found to be critical in regulating PKM2 expression. (D and E) expression levels of PKM2 were detected by western blot in HepG2 cells transfecting with miR-188-5p mimic, inhibitors, and their corresponding controls for 24 h. (F and G) Analysis of the apoptosis rate of HepG2 cells treated with sorafenib (10 μM, 24 h) after transfecting with miR-188-5p mimic or negative control for 24 h was performed by flow cytometry. ∗p < 0.05, ∗∗p < 0.01 (n = 3).

Figure 6

hnRNPA2B1 is a direct target of miR-188-5p (A) hnRNPA2B1 mRNAs contain a complementary 3′ UTR sequence that matches the seed sequence of miR-188-5p. (B) Quantitative PCR analysis of the levels of hnRNPA2B1 mRNA after HepG2 cells transfected with miR-188-5p mimic or negative control for 24 h. (C and D) Expression levels of hnRNPA2B1 were determined by western blot in HepG2 cells transfected with miR-188-5p mimic, inhibitors, and their corresponding controls for 24 h. (E) miR-188-5p downregulates the luciferase reaction intensity of plasmids, which consist of wild-type 3′ UTR of hnRNPA2B1. (F and G) Expression of hnRNPA2B1 in HCC specimens were semiquantitatively analyzed by IHC (100× and 200× magnification). ∗∗p < 0.01 (n = 3).

Figure 7

ER stress promotes sorafenib resistance via the miR-188-5p/hnRNPA2B1/PKM2 pathway in HCC (A and B) The protein levels of hnRNPA2B1 were determined by western blot in HepG2 cells exposed to different concentrations of TM for 24 h. (C–E) Representative western blotting results of PKM2 and hnRNPA2B1 when HepG2 cells were transfected with 3 hnRNPA2B1 interfering sequences for 24 h. (F–I) Expression of PKM2 and hnRNPA2B1 was detected by western blot in hnRNPA2B1 knockdown HepG2 cells transfected with miR-188-5p inhibitors/INC and in hnRNPA2B1 overexpression HepG2 cells transfected with miR-188-5p mimic/NC. (J) The Kaplan-Meier method was applied in overall survival analysis between the hnRNPA2B1 high-expression and hnRNPA2B1 low-expression groups. (K and L) The apoptosis rate of HepG2 cells treated with sorafenib (10 μM, 24 h) after transfecting with si-hnRNPA2B1 or negative control for 24 h were detected by flow cytometry analysis. ∗∗p < 0.01 (n = 3).