TOB1 Deficiency Enhances the Effect of Bone Marrow-Derived Mesenchymal Stem Cells on Tendon-Bone Healing in a Rat Rotator Cuff Repair Model

Background/Aims: This study investigated the effect of silencing TOB1 (Transducer of ERBB2, 1) expression in bone marrow-derived mesenchymal stem cells (MSCs) on MSC-facilitated tendon-bone healing in a rat supraspinatus repair model. Methods: Rat MSCs were transduced with a recombinant lentivirus encoding short hairpin RNA (shRNA) against TOB1. MSC cell proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The effect of MSCs with TOB1 deficiency on tendon-bone healing in a rat rotator cuff repair model was evaluated by biomechanical testing, histological analysis and collagen type I and II gene expression. An upstream regulator (miR-218) of TOB1 was determined in MSCs. Results: We found that knockdown of TOB1 significantly increased the proliferative activity of rat MSCs in vitro. When MSCs with TOB1 deficiency were injected into injured rat supraspinatus tendon-bone junctions, the effect on tendon-bone healing was enhanced compared to treatment with control MSCs with normal TOB1 expression, as evidenced by elevated levels of ultimate load to failure and stiffness, increased amount of fibrocartilage and augmented expression of collagen type I and type II genes. In addition, we found that the TOB1 3′ untranslated region is a direct target of miR-218. Similar to the effect of TOB1 deficiency, overexpression of miR-218 effectively promoted tendon-bone healing in rat. Conclusion: These results suggest that TOB1 may play a negative role in the effect of MSCs on tendon-bone healing, and imply that expression of TOB1 may be regulated by miR-218

Negative correlation of LIV-1 and E-cadherin expression in hepatocellular carcinoma cells.

LIV-1, a zinc transporter, is a mediator downstream of STAT3 both in zebrafish and mammalian cells, and is involved in epithelial-mesenchymal transition (EMT). Despite LIV-1 participates in cancer growth and metastasis, little is known about the association of LIV-1 with human liver cancer development. Therefore, the expression of LIV-1 mRNA was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) in 4 cultured cell lines (3 carcinoma and 1 normal liver cell lines), and the localization of LIV-1 protein was investigated by immunohistochemistry. Expression of LIV-1 protein was analyzed by Western blot both in 4 cultured cell lines and 120 liver tissues (100 carcinoma and 20 histologically normal tissues), and the relationship between its expression and clinicopathological finding was investigated in 100 hepatocellular carcinoma(HCC) tissues. Then stable siRNA expressing Hep-G2 cells were generated to assess the function of LIV-1 in liver cancer cells. We found that LIV-1 mRNA was more highly expressed in liver cancer cell lines compared to normal liver cell line. Western blot showed the expression of LIV-1 was higher in 61% liver carcinoma tissues than that in normal liver tissues. Down-regulated LIV-1 cells showed significant inhibition of proliferation in vitro and reduction of tumor growth in vivo. Furthermore, E-cadherin expression increased in LIV-1 siRNA expressing Hep-G2. These findings indicated that LIV-1 may induce the EMT in HCC cells

Correlation between clinicopathological findings and LIV-1 expression.

a<p>Analysed by Fisher’s exact test.</p>b<p>Analysed <b>χ</b>2 test.</p

LIV-1 mRNA and protein expression levels in three cancer cell lines (SMMC-7721, Hep-3B, Hep-G2) and one normal liver cells line (L02) were investigated by RT-PCR (A and B) and Western blot (C and D).

<p>The relative expression levels were expressed as the ratio of band intensity for LIV-1 relative to that for GAPDH. The expression levels of these cell lines were more intense than that in L02 cells. (E) Western blot showed the higher LIV-1 expression in HCC tissues compared to normal liver tissues. **<i>P</i><0.01.1: L02;2: SMMC-7721;3: Hep-3B;4: Hep-G2.</p

LIV-1 and E-cadherin expression in Hep-G2 cells after transfection with LIV-1 siRNA or scrambled siRNA.

<p>At 48 h post-transfection, the total RNA and protein were extracted and subjected to RT-PCR analysis (A and B) and Western blot (C and D). The relative mRNA and protein expression levels were expressed as the ratio of band intensity for LIV-1 relative to that for GAPDH. (E) Effect of silencing of LIV-1 on cell growth. At 0, 12, 24, 48 and 72 h post-transfection, the curve of cell growth was determined by MTT assay. Values were expressed as means ± SD, n = 6. *<i>P</i><0.05 compared to control group.1: untreated; 2: scrambled siRNA; 3: LIV-1 siRNA.</p

Upregulation of CSC-like properties in Hep3B cells with LX-2 CM.

<div><p>(A) Flow cytometry analysis the CSC surface marker CD133. The percentage of CD133 positive cells was increased in Hep3B cells upon LX-2 CM exposure. (B) Real-time PCR and (C) Western blot analysis of CSC transcriptional factors Bmi1 and Klf4 mRNA expressions in Hep3B cells. Both Bmi1 and Klf4 expressions were upregulated in Hep3B cells after LX-2 CM exposure.</p> <p>*p < 0.05, **p < 0.01, mean ± SEM.</p></div

Hep3B cells upon LX-2 CM exposure exhibit molecular changes which are consistent with EMT.

<div><p>(A) Real-time PCR analysis of E-cadherin, N-cadherin and Vimentin mRNA levels and (B) Protein expressions of E-cadherin, N-cadherin and Vimentin in Hep3B cells were analyzed by Western blotting, GAPDH as endogenous control. E-cadheirn expression was suppressed, while Vimentin and N-cadherin expressions were increased in Hep3B cells with LX-2 CM exposure. (C) Immunofluorescence analysis of β-catenin and Vimentin expressions. After LX-2 CM exposure, β-catenin was nuclear translocation and Vimentin was induced in Hep3B cells.</p> <p>*p < 0.05, ***p < 0.001, mean ± SEM.</p></div

MOESM1 of The protective or damaging effect of Tumor necrosis factor-α in acute liver injury is concentration-dependent

Additional file 1. Liver injury was aggravated in the CCl4-induced rats of TNF-α deficiency. (A) Gene sequencing diagram suggested TNF-α was successfully deleted by crispr/cas9 technique. Obviously, the five basic groups were deficient during the 205–210 gene segment of TNF-α−/− rat. (B) Serum ALT and (C) AST levels were examined by Roche Diagnostic kits in Hithachi Modular P Autoanalyser. The levels of ALT and AST were significantly elevated 24 h later after 1 ml/kg CCl4 was subcutaneously injected into Sprague–Dawley rats 24 h later (***p < 0.001). In the TNF-α−/− rats, serum of ALT and AST were elevated more than the TNF-α+/+ rats (*p < 0.01). (D) Hematoxylin-eosin–stained of liver paraffin sections analyzed that there was almost no hepatocellular steatosis in the rats without treatment of CCl4. And the steatosis was almost distributed all over the field of vision in the TNF-α−/− rats. It was more serious than the TNF-α+/+ rats