MiR-101 targets USP22 to inhibit the tumorigenesis of papillary thyroid carcinoma

Abstract

Increasing evidence suggests that microRNA-101 (miR-101) is involved in the progression of various human cancers, including papillary thyroid carcinoma (PTC). However, the biological functions of miR-101 and underlying molecular mechanisms in PTC remain largely unknown. In this study, we demonstrated that miR-101 underexpression in PTC tissue was associated with lymph node metastasis and poor prognosis of PTC patients. MiR-101 reduced PTC cell proliferation, apoptosis resistance, and invasion. Ubiquitin-specific protease 22 (USP22) was confirmed as a direct target of miR-101. USP22 restoration attenuated the inhibitory effects of miR-101 on PTC malignant traits in vitro. In vivo, miR-101 overexpression or USP22 depletion reduced the tumorigenesis of PTC. Overall, our findings provide new insight into the mechanism of PTC inhibition by miR-101, suggesting the potential of miR-101 as a therapeutic target in PTC patients.

Keywords: MicroRNA-101; papillary thyroid carcinoma; tumorigenesis; ubiquitin-specific protease 22.

Figure 1

The levels of miR-101 and USP22 in PTC tissues and cell lines and the association between miR-101 expression and clinicopathological characteristics of PTC. (A) qPCR analysis of miR-101 expression in PTC (T) and the adjacent non-cancerous (N) tissues (n = 20). U6 was used as the endogenous control. (B) Kaplan-Meier survival curves of 60 PTC patients divided by miR-101 levels. (C) The association between miR-101 expression and lymph node metastasis (n = 120). (D) qPCR analyses of USP22 mRNA levels in T and N tissues (n = 20). GAPDH was used as the internal control. (E) The inverse association of miR-101 level and USP22 expression in PTC patients. (F) Western blot analysis of USP22 protein expression in T and N tissues. β-actin was used as the internal control. qPCR (G) and Western blot (H) assays were performed to determine miR-101 and USP22 expression in five PTC cell lines (HTH83, TPC-1, K1, NIM-1 and B-CPAP) and a Nthy-ori 3-1 cell line. U6 and β-actin were used as the endogenous controls. All data are shown as means ± SD of three separate experiments. *P < 0.05, **P < 0.01, as compared with N group (A, D); **P < 0.01, as compared with Nthy-ori 3-1 group (G).

Figure 2

MiR-101 inhibited proliferation, apoptosis resistance, migration and invasion of PTC cells. K1 cells were transfected with miR-NC or miR-101 mimics. (A) MTT assay was performed to determine cell viability. (B) Representative photographs of the colony formation of K1 cells and related quantitative analysis. (C) EdU staining was performed and the percentages of EdU-positive cells were counted. Scale bar: 10 mm. The caspase-3 activity (D) and enrichment factor (E) were analyzed. Cell migration (F) and invasion (G) were determined using Transwell chambers, and the percentage of migrated and invaded cells was calculated. Scale bar: 10 mm. All data are shown as means ± SD of three separate experiments. *P < 0.05, **P < 0.01, as compared with miR-NC group.

Figure 3

Identification of USP22 as a direct target of miR-101. (A) The putative target genes of miR-101 were predicted using TargetScan, PicTar, and miRanda softwares. (B) The predicted binding sites of miR-101 in the 3’-UTR of USP22. (C) Dual-luciferase reporter assays were performed 24 h after co-transfection of K1 cells with miR-NC or miR-101 mimics and a pGL3 construct containing WT or MUT 3’-UTR of USP22. Data were normalized to those from cells co-transfected with miR-NC and pGL3 plasmid. The mRNA (D) and protein (E) levels of USP22 in K1 cells transfected with miR-NC or miR-101 mimics were measured by qPCR and Western blot assays. GAPDH and β-actin were used as internal controls, respectively. All data are shown as means ± SD of three separate experiments. **P < 0.01, as compared with miR-NC group.

Figure 4

USP22 restoration counteracted the suppressive effects of miR-101 on PTC cell proliferation and apoptosis resistance. K1 cells were transfected with miR-NC, miR-101 mimics, or miR-101 mimics + USP22-expressing plasmid. (A) Cell viability was measured by MTT assay. (B) The colony number was calculated. (C) The EdU-positive cells were counted to assess cell proliferation. Caspase-3 activity (D) and nucleosomal fragmentation (E) were determined. (F) The levels of USP22, cyclin D2, Rb, Bcl-2, Bax, cl-caspase-3, and caspase-3 were measured by western blot analysis. β-actin was used as endogenous control. All data are shown as means ± SD of three separate experiments. *P < 0.05, as compared with miR-NC group or miR-101 group; #P < 0.05, as compared with miR-101 group.

Figure 5

USP22 overexpression reduced the inhibitory effects of miR-101 on PTC cell migration and invasion. K1 cells were transfected with miR-NC, miR-101 mimics, or miR-101 mimics + USP22-expressing plasmid. Transwell assays were performed to detect cell migration (A) and invasion (B), and the number of migrated and invaded cells was calculated. (C) Expression of USP22 and invasion-related proteins containing E-cadherin, vimentin, and snail was measured by western blot analysis. β-actin was used as endogenous control. All data are shown as means ± SD of three separate experiments. *P < 0.05, as compared with miR-NC group; #P < 0.05, as compared with miR-101 group.

Figure 6

MiR-101 overexpression or USP22 knockdown suppressed tumor growth and metastasis and promoted apoptosis of PTC cells in vivo. SCID mice were injected subcutaneously or via their tail veins with K1-luc cells infected with a control lentivirus (Lenti-pGCsi or Lenti-pLKO.1) or a recombinant lentivirus expressing a miR-101 precursor (Lenti-pGCsi-miR-101) or shUSP22 (Lenti-shUSP22). A. In vivo luciferase imaging of the xenografts at 5 weeks after implanted with K1-luc cells. B. Representative gross photos of tumors after 12 weeks of implantation. C. Tumor volume was measured and calculated every two weeks. D. The numbers of metastatic foci in the lungs of mice from various groups at 8 weeks after tail vein injection. E. TUNEL assay was performed to detect the percentage of apoptotic cells. F. Representative results of western blot analyses of USP22, cyclin D2, Rb, E-cadherin, snail, cl-caspase-3, and caspase-3 in tumor tissues. β-actin was used as endogenous control. All data are shown as means ± SD of three separate experiments. *P < 0.05.