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. 2016 Aug 30;7(35):57099-57116.
doi: 10.18632/onco_target.10981.

MiRNA-22 inhibits oncogene galectin-1 in hepatocellular carcinoma

Yu You  1 , Jia-Xin Tan  1 , Hai-Su Dai  1 , Hao-Wei Chen  1 , Xue-Jun Xu  1 , Ai-Gang Yang  1 , Yu-Jun Zhang  1 , Lian-Hua Bai  1 , Ping Bie  1
Affiliations

MiRNA-22 inhibits oncogene galectin-1 in hepatocellular carcinoma

Yu You et al. Onco_target. .

Abstract

Hepatic stellate cells (HSCs) induce immune privilege and promote hepatocellular carcinoma (HCC) by suppressing the immune system. On the other hand, galectin-1 and miRNA-22 (miR-22) are dysregulated in HCC and serve as prognostic indicators for patients. In this study, therefore, we measured galectin-1 and miR-22 expression in HSCs isolated from HCC tissues (Ca-HSCs), and in normal liver tissues (N-HSCs) as a control. We also investigated the apoptosis rate among T cells and the production of cytokines (IFN-γ and IL-10) in HSCs co-cultured with T cells. And we used immunohistochemical staining to tested for correlation between galectin-1 expression, CD3 expression and clinicopathological features in 162 HCC patients. Our results showed that galectin-1 expression was much higher in Ca-HSCs than in N-HSCs. Overexpression of galectin-1 promoted HSC-induced T cell apoptosis and cytokine production (IFN-γ and IL-10), while miR-22 expression inhibited it. Galectin-1 expression correlated negatively with miR-22 expression in HSCs. High galectin-1 and low CD3 expression levels were associated with poor prognosis in HCC patients. These results suggest that the immunosuppressive microenvironment promoted by HSC-derived galectin-1 in HCC can be inhibited by miR-22. Galectin-1 and miR-22 could potentially serve as prognostic markers and therapeutic _targets in HCC.

Keywords: galectin-1; hepatic stellate cells; hepatocellular carcinoma; miRNA-22.

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Conflict of interest statement

No conflicts of interest to declare.

Figures

Figure 1
Figure 1. Activated α-SMA-positive HSCs could induce CD3+ T cells apoptosis
Primary N-HSCs (HSCs isolated from normal liver tissues) were analysed for A-B. α-SMA expression by immunofluorescence and immunohistochemical assays and for C-D. the ability to induce apoptosis in T cells by co-culture assays. For the co-culture assays, CD3+ T cells were co-cultured with quiescent HSCs (pre-cultured for 2 days) or activated HSCs (pre-cultured for 7 days) at different ratios (HSC:T = 1:5, 1:10 and 1:20). The data for each group are representative of 4 independent experiments representing 4 different liver samples and repeated 3 times. T cells were cultured alone as a negative control. After the cells were co-cultured for 48 hours, T cell apoptosis was measured by flow cytometry (annexin V-FITC apoptosis detection). All the groups with activated HSCs showed significantly higher apoptosis rates for T cells alone than for groups with quiescent HSCs. Among the groups with activated HSCs, the ratios 1:5 and 1:10 showed the highest T cell apoptosis rate. Data are shown as the means (± SD) of triplicates (n = 4). *P < 0.05, **P < 0.01.
Figure 2
Figure 2. The expression of galectin-1 in human HSCs
Primary HSCs isolated from normal liver tissues were tested for galectin-1 expression by A. RT-qPCR, B. western blot (line 1 for quiescent HSCs, line 2 for K562 cell line and line 3 for activated HSCs, 20μg per lane), C. immunohistochemical assays for activated HSCs, and D. ELISA (20,000 HSCs per well). In A. and B., the K562 cell line was used as positive control for galectin-1 mRNA and protein expression. Data are shown as the means (± SD) of triplicates (n = 4).
Figure 3
Figure 3. Knockdown and overexpression of galectin-1 in N-HSCs
After the cells were transfected, qPCR (n = 3), western blot (30μg per lane, n = 3) and ELISA (20,000 HSCs per well, n = 4) were used to analyse galectin-1 knockdown A. and overexpression B. in HSCs. Data are shown as the means (± SD) of triplicates. *P < 0.05, **P < 0.01. NC, negative control group; Scr, non-_targeting scrambled sequence group; sh, small hairpin RNA sequence transfection group; pcDNA3.1, negative control group; Over, galectin-1 overexpression group.
Figure 4
Figure 4. The expression of galectin-1 in HSCs promotes HSC-induced T cell apoptosis and Th1/Th2 cytokine balance skewing
A. Flow cytometry (annexin V-FITC apoptosis detection) analyses to detect T cell apoptosis in CD3+ T cells, cultured alone or co-cultured with HSCs subjected to different pre-treatments (cell transfection for galectin-1 knockdown and overexpression: sh-3 group versus Scr group; Over group versus pcDNA3.1 group) for 48 hours at a ratio of 10:1 (T:HSC), B. ELISA showing the levels of cytokines (IFN-γ and IL-10) in the supernatant. Data are shown as the means (± SD) of triplicates (n = 7). *P < 0.05. NC, negative control group; Scr, non-_targeting scrambled sequence group; sh, small hairpin RNA sequence transfection group; pcDNA3.1, negative control group; Over, galectin-1 overexpression group; No HSCs, T cells cultured alone.
Figure 5
Figure 5. Ca-HSCs showed a higher galectin-1 expression and a stronger immunosuppressive capacity than N-HSCs
Expression of galectin-1 by A. RT-qPCR, B. western blot (30μg per lane), and C. ELISA (20,000 HSCs per well) (n = 4) in primary HSCs isolated from normal liver tissues (N-HSCs) or HCC tissues (Ca-HSCs). D-E. T cell apoptosis and ELISA analyses of CD3+ T cells co-cultured with N-HSCs (isolated from normal liver samples) or Ca-HSCs (isolated from HCC samples). F. Levels of cytokines (IFN-γ and IL-10) in the supernatant (n = 6) measured by flow cytometry (annexin V-FITC apoptosis detection). Data are shown as the means (± SD) of triplicates. *P < 0.05, **P < 0.01.
Figure 6
Figure 6. Negative regulation relationship between galectin-1 and miR-22 expression in HSCs
Expression of A. galectin-1 mRNA, B. miR-22 expression, and C. their relationship, in N-HSCs (n = 12) and Ca-HSCs (n = 12) measured by qPCR. D. Genetic constructs: 3′-UTR of galectin-1 cloned downstream of a luciferase reporter gene (wt-galectin-1), and its mutant version (mut-galectin-1). E. Luciferase activity detected by a dual-luciferase reporter assay in HSCs co-transfected for 48 hours with wt-galectin-1/mut-galectin-1 vector and miR-22 expression plasmid/negative control (mimic NC; n = 3 for each group). F. MiR-22 expression measured by qPCR in N-HSCs transfected with hsa-miR-22 mimic (n = 4). G. Expression of galectin-1 in HSCs measured by western blot (30μg per lane) and H. ELISA (20,000 HSCs per well). Data are shown as the means (± SD) of triplicates. *P < 0.05, **P < 0.01. Wt-galectin-1, luciferase reporter gene was cloned with wild type galectin-1; mut-galectin-1, mutant version of galectin-1; mimic NC, mimic negative control; hsa-miR-22, Homo sapiens miRNA-22.
Figure 7
Figure 7. Role of miR-22 in HSC-derived T cell apoptosis and Th cytokine balance skewing
T cell apoptosis and cytokine (IFN-γ and IL-10) levels in the supernatant of 4 groups of HSCs isolated from normal liver samples and co-cultured with CD3+ T cells for 48 hours at a ratio of 1:10 (HSC:T) (n = 6 for each group), measured by A. flow cytometry and B. Elisa. Each of the 4 groups was subjected to different pre-treatments: transfection with 1) negative control mimic for miR-22 (mimic NC), 2) miRNA-22 of homo sapiens type mimic (hsa-miRNA-22 mimic), 3) hsa-miRNA-22 mimic plus galectin-1 overexpression plasmid (hsa-miRNA-22 mimic + Over), and 4) hsa-miRNA-22 mimic plus negative control for galectin-1 overexpression (hsa-miRNA-22 mimic + pcDNA3.1) Data are shown as the means (± SD) of triplicates. N.S. for P > 0.05; *P < 0.05.
Figure 8
Figure 8. Immunofluorescent labelling for galectin-1 and α-SMA in liver samples
A1-A4. α-SMA and galectin-1 levels in normal liver samples (n = 12) and B1-B4. HCC (n = 31) by immunofluorescence. C. Levels of HSC-derived galectin-1 assessed by evaluating the number of galectin-1-positive hepatic stellate cells (both positive for galectin-1 and α-SMA) over the total number of HSCs (only positive for α-SMA). Data are shown as the means (± SD). Magnification: ×100. *P < 0.05.
Figure 9
Figure 9. Expression of galectin-1, α-SMA and CD3 in liver samples
A. Galectin-1, α-SMA, and CD3 levels in HCC liver samples (n = 162) and normal liver samples (n = 12) (paraffin-embedded specimens) measured by IHC, scored and grouped into low expression and high expression groups. B. Galectin-1 and CD3 levels in HCC and normal liver samples analysed using the χ2 test. Magnification: ×100. N.S. for P > 0.05; *P < 0.05.
Figure 10
Figure 10. Correlation between miR-22 expression, galectin-1 expression, CD3 expression and the clinicopathological features of HCC
A. IHC analysis to measure expression of α-SMA and galectin-1 in consecutive sections of HCC samples (n=53). B-D. Galectin-1 expression in different TNM stages (TNM I-IV: A1-A4.) and CD3 expression in different TNM stages (TNM I-IV: B1-B4.) measured by IHC. The correlations between galectin-1 expression, CD3 expression, and TNM stage of patients with HCC were analysed using the χ2 test (TNM I, n = 13; TNM II, n = 55; TNM III, n = 58; TNM IV, n = 36). C-E. Expression of C1-C2. galectin-1 and D1-D2. CD3 in Ca-HSCs isolated from HCC patient liver samples, separated into low (n = 13) and high (n = 13) miR-22 expression groups, according to the median miR-22 expression. Magnification: ×200 for CD3 staining (B1-B4, B1-B2), ×100 for galectin-1 staining (a1-a4, c1, c2). *P < 0.05.
Figure 11
Figure 11. Kaplan-Meier curve of overall survival based on galectin-1 and CD3 expression in 162 HCC patients
HCC patients were grouped into high (n = 105) and low (n = 57) galectin-1 expression groups, or high (n = 68) and low (n = 94) CD3 expression groups. The correlation between A. galectin-1 or B. CD3 expression and survival was analysed using the Kaplan-Meier method. The P-values were determined using the log-rank test.
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