MiR-125b attenuates human hepatocellular carcinoma malignancy through targeting SIRT6 - PubMed (original) (raw)
. 2018 Jun 1;8(6):993-1007.
eCollection 2018.
Yuxia Yang 1, Minghui Liu 1, Boya Liu 1, Xin Yang 1, Miao Yu 1, Hao Qi 1, Mengmeng Ren 1, Zhe Wang 1, Junhua Zou 1, Feng Li 2, Xiaojuan Du 3, Hongquan Zhang 2, Jianyuan Luo 1
Affiliations
- PMID: 30034937
- PMCID: PMC6048406
MiR-125b attenuates human hepatocellular carcinoma malignancy through targeting SIRT6
Shi Song et al. Am J Cancer Res. 2018.
Abstract
Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancers. It has been demonstrated that aberrant expression of miRNAs plays an important role in HCC development. Here, we observed decreased miR-125b expression status in tumor samples from HCC patients, and the five years survival rate of HCC patients with low miR-125b expression is poor. By using bioinformatics prediction tools combining with luciferase reporter assay, we identified that miR-125b can suppress the expression of SIRT6 by directly targeting the seed-matching region of its 3'UTR. Based on the analysis via TCGA and clinical samples data, the expression of SIRT6 showed negatively correlated with the expression of mir-125b. After knocking-out the expression of SIRT6 through CRISPR/Cas9, HCC cells showed the decreased cell viability and invasiveness, which had the similar function upon the overexpression of the miR-125b. The function induced by overexpression of miR-125b can be rescued by the restoration of SIRT6. Further experiments demonstrated that the HCC cells showed the significant cellular senescence and apoptosis upon overexpression of miR-125b or knockout SIRT6, which is in accordance with the compromised cell malignancy. Thus, we conclude that, by targeting SIRT6, miR-125b can function as a tumor suppressor to induce the cellular senescence and apoptosis in hepatocellular carcinogenesis and could provide a novel insight for HCC treatment.
Keywords: CRISPR/Cas9; SIRT6; hepatocellular carcinoma; microRNA; senescence.
Conflict of interest statement
None.
Figures
Figure 1
MiR-125b is aberrantly expressed in HCCs and is associated with sirtuin family. A. The miR-125b expression level in clinical samples was analyzed by qPCR. B. miR-125b is differentially expressed in HCC patients and HTSeq-Counts data acquired from TCGA database, which is shown with a logarithmic conversion (log2counts). Unpaired _t_-test. C. miR-125b gene is related with the overall survival rate of HCC patients. Red line: high expression, blue line: low expression. Data is acquired from TCGA database and analyzed via LinkedOmics bioinformatics [23]. D. Schematic illustrations showing exact position and numbers of miR-125b binding sites of certain sirtuin family members. E. The predicted binding site of miR-125b in the 3’UTR of SIRT2, SIRT3, SIRT5 SIRT6 and SIRT7 mRNA is analyzed via TargetScan bioinformatics [24]. F. Western blots shows the protein level of SIRT1-SIRT7 in cells transfected with miR-125b mimic or control mimic in HepG2 cell lines. G. The expression of SIRT1-SIRT7 in HCC patients. The results, based on TCGA database, were acquired from GEPIA bioinformatics [25].
Figure 2
SIRT6 is the target of miR-125b and is up-regulated in HCCs. A. Dual-luciferase reporter assays. Sequences encoding wild-type and mutated fragments, mutated sequence, shown in the right panel, of the SIRT6 3’-UTR were inserted into a luciferase reporter plasmid. MiR-125b mimic was co-transfected with reporter plasmid into 293T cells. Relative renilla luciferase expression was normalized to firefly luciferase. B. Western blots shows the SIRT6 protein level in cells transfected with miR-125b mimic or control mimic. C. SIRT6 protein in tissue samples were measured by western blotting (adjacent non-tumor tissue (left lane) versus tumor tissue (right lane)). β-actin was used as loading control. D. Immunohistochemistry (IHC) shows the SIRT6 expression in adjacent non-tumor tissues (left) and tumor tissues (right). E. The level of SIRT6 mRNA in tissue samples was detected by qRT-PCR. Data were analyzed using 2-ΔCT method in tissue samples and normalized to the expression of β-actin. F. SIRT6 expression in HCC patients and HTSeq-Counts data acquired from TCGA database, which is shown with a logarithmic conversion (log2counts). Unpaired _t_-test. G. SIRT6 gene is related with the overall survival rate in HCC patients. Red line: high expression, blue line: low expression. Data is acquired from TCGA database and analyzed via Linked Omics bioinformatics [23].
Figure 3
The SIRT6 and miR-125b knockout induced by CRISPR/Cas9 affects the cell proliferation. A. Schematic illustration on miR-125b knockout design. The orange character represents the mature miR-125b sequence and blue character represents the sgRNA sequence. B. Genome sequencing on pre-miR-125b-1 and pre-miR-125b-2. The expression of miR-125b is successfully eliminated by CRISPR/CAS9 via two sgRNAs targeting both the genomic region of pre-miR-125b-1 and pre-miR-125b-2 separately. C. Genome sequencing on exon 4 of SIRT6. D. The SIRT6 expression in SIRT6 knockout HepG2 cell lines induced by CRISPR/Cas9 was determined via western blot assay. E. The cell proliferation of miR-125b and SIRT6 knockout cell lines was investigated by colony formation assay. Upper lane: HepG2 cell lines. Lower lane: Sk-hep1 cell lines.
Figure 4
MiR-125b-SIRT6 axis affects the cell migration of HCC cells. A. The effect of miR-125b on the migration of HepG2 and Sk-hep-1 cell lines. A representative photograph from three independent experiments is shown one. The plot shows the results of wound healing assay as mean ± SD of open wound area. Statistical analysis was performed using _t_-test. B. The effect of SIRT6 on the migration of HepG2 and Sk-hep-1 cell lines. C. The cell migration ability of 125b overexpression HepG2 and Sk-hep-1 cell lines upon SIRT6 transfection.
Figure 5
MiR-125b-SIRT6 axis affects the cell invasion on HCC cells. A. The cell invasion ability of miR-125b on HepG2 and Sk-hep-1 cell lines via transwell assay. The invasive cells stained by crystal violet were shown in a representative photograph. The plot data showing the invasive cell counts were presented as the mean ± SD, n=3. Statistical analysis was performed using _t_-test. B. The cell invasion ability of SIRT6 on HepG2 and Sk-hep-1 cell lines. C. The cell invasion ability of 125b overexpression HepG2 and Sk-hep-1 cell lines upon SIRT6 transfection.
Figure 6
MiR-125b induces HCC senescence and apoptosis via inhibiting SIRT6. A. Senescence-associated β-galactosidase staining was used to exhibit senescence-like phenotypes in SIRT6 knockout HepG2 and Sk-hep-1 cell lines. Cells with positive staining were shown in a representative photograph. Positive staining cell counts were presented as the mean ± SD, n=3. Statistical analysis was performed using _t_-test. B. SA-β-gal staining assay showing the cell senescence rate in miR-125b overexpression cells and miR-125b overexpression HepG2 and Sk-hep-1 cells upon SIRT6 transfection. C. Western blots shows the SIRT6, P16, Bax and Caspase3 protein level in cells of each group as treated above. β-actin was used as loading control.
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