Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Mar 6;19:1073-1085.
doi: 10.1016/j.omtn.2019.12.043. Epub 2020 Jan 15.

miR-552 Regulates Liver Tumor-Initiating Cell Expansion and Sorafenib Resistance

Affiliations
Free PMC article

miR-552 Regulates Liver Tumor-Initiating Cell Expansion and Sorafenib Resistance

Tao Han et al. Mol Ther Nucleic Acids. .
Free PMC article

Abstract

MicroRNAs (miRNAs) are involved in tumorigenesis, progression, recurrence, and drug resistance of hepatocellular carcinoma (HCC). However, few miRNAs have been identified and entered clinical practice. Herein, we report that microRNA (miR)-552 is upregulated in HCC tissues and has an important function in liver tumor-initiating cells (T-ICs). Functional studies revealed that a forced expression of miR-552 promotes liver T-IC self-renewal and tumorigenesis. Conversely, miR-552 knockdown inhibits liver T-IC self-renewal and tumorigenesis. Mechanistically, miR-552 downregulates phosphatase and tensin homolog (PTEN) via its mRNA 3' UTR and activates protein kinase B (AKT) phosphorylation. Our clinical investigations elucidated the prognostic value of miR-552 in HCC patients. Furthermore, miR-552 expression determines the responses of hepatoma cells to sorafenib treatment. The analysis of patient cohorts and patient-derived xenografts (PDXs) further demonstrated that miR-552 may predict sorafenib benefits in HCC patients. In conclusion, our findings revealed the crucial role of the miR-552 in liver T-IC expansion and sorafenib response, rendering miR-552 an optimal target for the prevention and intervention in HCC.

Keywords: PTEN; hepatocellular carcinoma; miR-552; sorafenib; tumor-initiating cells.

Figures

Figure 1
Figure 1
miR-552 Is Upregulated in Liver T-ICs and Predicts Poor Prognosis (A) Real-time PCR analysis of miR-552 in MACS-sorted CD133+ primary HCC cells relative to negative cells (n = 3). (B) Real-time PCR analysis of miR-552 in MACS-sorted EpCAM+ primary HCC cells relative to negative cells (n = 3). (C) Real-time PCR analysis of miR-552 in primary HCC adherent and spheroids cells (n = 3). (D) Real-time PCR was performed to check the expression of miR-552 in a sorafenib-resistant HCC xenograft (n = 3). (E) Real-time PCR analysis of miR-552 in primary HCC tissues and recurrence HCC tissues (n = 10). (F) The expression of miR-552 in 110 pairs of HCC (cohort 1) and neighboring noncancerous tissues (normal) was checked by real-time PCR analysis. (G) The overall free survival time after surgery of the patients in cohort 1 was compared between the “miR-552 low” (n = 55) and “miR-552 high” (n = 55) groups. (H) The disease survival time after surgery of the patients in cohort 1 were compared between the “miR-552 low” (n = 55) and “miR-552 high” (n = 55) groups. Data are represented as mean ± SD; *p < 0.05; two-tailed Student’s t test.
Figure 2
Figure 2
miR-552 Is Required for the Maintenance of Liver T-ICs (A) Hepatoma cells were infected with the miR-552 sponge virus, and the stable infectants were determined by real-time PCR (n = 3). (B) The expression of liver T-IC surface markers was checked in miR-552 sponge and control hepatoma cells (n = 3). (C) The expression of stemness-associated transcription genes was checked in miR-552 sponge and control hepatoma cells (n = 3). (D) Flow cytometric analysis of the proportion of EpCAM+ cells in miR-552 knockdown and control hepatoma cells (n = 3). (E) The protein expression of EpCAM in miR-552 knockdown and control hepatoma cells was checked by western blot assay. (F) Spheres formation assay of miR-552 sponge and control hepatoma cells (n = 3). (G) The frequency of liver T-ICs in miR-552 sponge and control hepatoma cells was compared by in vitro-limiting dilution assay (n = 8). (H) The frequency of liver T-ICs in miR-552 sponge and control hepatoma cells was compared by in vivo-limiting dilution assay (n = 8). Data are represented as mean ± SD; *p < 0.05; two-tailed Student’s t test.
Figure 3
Figure 3
miR-552 Promotes the Expansion of Liver T-ICs (A) Hepatoma cells were infected with miR-552 mimic virus, and the sable infectants were checked by real-time PCR (n = 3). (B) The expression of liver T-IC surface markers was checked in miR-552 mimic and control hepatoma cells (n = 3). (C) The expression of stemness-associated transcription genes was checked in miR-552 mimic and control hepatoma cells (n = 3). (D) Flow cytometric analysis of the proportion of EpCAM+ cells in miR-552 overexpression and control hepatoma cells (n = 3). (E) The protein expression of EpCAM in miR-552 overexpression and control hepatoma cells was checked by western blot assay. (F) Spheres formation assay of miR-552 overexpression and control hepatoma cells (n = 3). (G) The frequency of liver T-ICs in miR-552 mimic and control hepatoma cells was compared by in vitro-limiting dilution assay (n = 8). (H) The frequency of liver T-ICs in miR-552 mimic and control hepatoma cells was compared by in vivo-limiting dilution assay (n = 8). Data are represented as mean ± SD; *p < 0.05; two-tailed Student’s t test.
Figure 4
Figure 4
PTEN Is a Direct Target of miR-552 in Liver T-ICs (A) The phosphorylation of STAT3, SMAD3, β-catenin, Shp2, p53, and PTEN in miR-552 mimic and control hepatoma cells was determined by western blot assay. (B) The mRNA expression of PTEN in miR-552 mimic and control hepatoma cells was checked by a real-time PCR assay (n = 3). (C) A potential target site for miR-552 in the 3′ UTR of human PTEN mRNA, as predicted by the program TargetScan. To disrupt the interaction between miR-552 and PTEN mRNA, the target site was mutated. (D) Luciferase reporter assays performed in miR-552 mimic and control cells transfected with wild-type or mutant PTEN 3′ UTR constructs (n = 3). (E) Spearman correlation analysis of the relationship between PTEN mRNA and miR-552 expression in 40 HCC specimens. (F) miR-552 mimic and control hepatoma cells were infected with the PTEN overexpression virus or control virus and subjected to a real-time PCR assay (n=3), the relative gene expression of CD133, EpCAM, CD90 and CD24 were analysis. (G) miR-552 mimic and control hepatoma cells were infected with the PTEN overexpression virus or control virus and subjected to a real-time PCR assay (n=3), the relative gene expression of SOX2, OCT4, Nanog, c-Myc, Bmi-1 and β-catenin were analysis. (H) miR-552 mimic and control hepatoma cells were infected with the PTEN-overexpressing virus or control virus, and the EpCAM+ hepatoma cells were checked by the flow cytometric assay (n = 3). (I) miR-552 mimic and control hepatoma cells were infected with the PTEN-overexpressing virus or control virus and subjected to spheroid formation (n = 3). (J) miR-552 mimic and control hepatoma cells were infected with the PTEN-overexpressing virus or control virus and were then injected subcutaneously into NOD-SCID mice. Tumors were observed over 2 months; n = 8 for each group. Data are represented as mean ± SD; *p < 0.05; two-tailed Student’s t test.
Figure 5
Figure 5
miR-552 Promotes Liver T-IC Expansion via the PTEN/AKT Pathway (A) The phosphorylation of AKT in miR-552 mimic and control hepatoma cells checked by western blot assay. (B) The phosphorylation of AKT in miR-552 sponge and control hepatoma cells checked by western blot assay. (C) miR-552 mimic and control hepatoma cells were infected with the PTEN-overexpressing virus or control virus and subjected to western blot assay. (D) miR-552 mimic and control hepatoma cells were treated with MK2206 (5 μM) or not and subjected to a real-time PCR assay (n=3), the relative gene expression of CD133, EpCAM, CD90 and CD24 were analysis. (E) miR-552 mimic and control hepatoma cells were treated with MK2206 (5 μM) or not and subjected to a real-time PCR assay (n=3), the relative gene expression of SOX2, OCT4, Nanog, c-Myc, Bmi-1 and β-catenin were analysis. (F) miR-552 mimic and control hepatoma cells were treated with MK2206 (5 μM) or not, and the EpCAM+ hepatoma cells were checked by the flow cytometric assay (n = 3). (G) miR-552 mimic and control hepatoma cells were treated with MK2206 (5 μM) or not and subjected to spheroid formation (n = 3). (H) In vivo-limiting dilution assay of indicated hepatoma cells. Tumors were observed over 2 months; n = 8 for each group. Data are represented as mean ± SD; *p < 0.05; two-tailed Student’s t test.
Figure 6
Figure 6
miR-552 Is Associated with the Sensitivity of Sorafenib in HCC Patients (A) miR-552 mimic and control hepatoma cells were treated with sorafenib (2 μM) for 7 days, and their colony formation was examined (n = 3). (B) miR-552 mimic and control hepatoma cells were treated with sorafenib (10 μM) for 48 h, and their apoptosis was checked by flow cytometry (n = 3). (C) miR-552 mimic and control hepatoma cells were treated with 10 μM sorafenib, as indicated, for 48 h. The protein of cleaved poly (ADP-ribose) polymerase (PARP) was determined by western blot. (D) miR-552 sponge and control hepatoma cells were treated with sorafenib (2 μM) for 7 days, and their colony formation was examined (n = 3). (E) miR-552 sponge and control hepatoma cells were treated with sorafenib (10 μM) for 48 h, and their apoptosis was checked by flow cytometry (n = 3). (F) miR-552 sponge and control hepatoma cells were treated with 10 μM sorafenib, as indicated, for 48 h. The protein of cleaved PARP was determined by western blot. (G) The overall survival of patients between miR-552-high (n = 40) or miR-552-low (n = 40) groups was evaluated by Kaplan-Meier analysis in HCC cohort 2. (H) The overall survival of patients between miR-552-high (n = 40) or miR-552-low (n = 40) groups was evaluated by Kaplan-Meier analysis in HCC cohort 3. (I) PDXs with high miR-552 levels in their primary tumors were treated with sorafenib (30 mg/kg body weight) or vehicle for 24 days (n = 6 for each group). The xenograft growth was monitored. (J) PDXs with low miR-552 levels in their primary tumors were treated with sorafenib (30 mg/kg body weight) or vehicle for 24 days (n = 6 for each group). The xenograft growth was monitored. Data are represented as mean ± SD; p < 0.05; two-tailed Student’s t test.

Similar articles

See all similar articles

References

    1. Forner A., Reig M., Bruix J. Hepatocellular carcinoma. Lancet. 2018;391:1301–1314. - PubMed
    1. Llovet J.M., Zucman-Rossi J., Pikarsky E., Sangro B., Schwartz M., Sherman M., Gores G. Hepatocellular carcinoma. Nat. Rev. Dis. Primers. 2016;2:16018. - PubMed
    1. Mo D.C., Jia R.R., Zhong J.H. Letter to the Editor: Hepatic Resection Compared to Chemoembolization in Intermediate- to Advanced-Stage Hepatocellular Carcinoma: A Comment For Moving Forward. Hepatology. 2019;70:446–447. - PubMed
    1. Han T., Xiang D.M., Sun W., Liu N., Sun H.L., Wen W., Shen W.F., Wang R.Y., Chen C., Wang X. PTPN11/Shp2 overexpression enhances liver cancer progression and predicts poor prognosis of patients. J. Hepatol. 2015;63:651–660. - PubMed
    1. Singh A.K., Arya R.K., Maheshwari S., Singh A., Meena S., Pandey P., Dormond O., Datta D. Tumor heterogeneity and cancer stem cell paradigm: updates in concept, controversies and clinical relevance. Int. J. Cancer. 2015;136:1991–2000. - PubMed
Feedback