SETD7, H3K4me2, ZBTB20, and CDKN2D level in HCC was determined by TMA IHC.

<p>The two rows on the left indicate HE staining for HCC tumor tissues and paired ANLTs, whereas the two rows on the right indicate IHC staining for SETD7, H3K4me2, ZBTB20, and CDKN2D protein in HCC tumor tissues and paired ANLTs (<i>n</i> = 225). (Original magnified 100×; Inserted figures magnified 400×).</p

Increased Expression of <i>SETD7</i> Promotes Cell Proliferation by Regulating Cell Cycle and Indicates Poor Prognosis in Hepatocellular Carcinoma

<div><p>Purpose</p><p>To investigate the role of SET domain containing 7 (SETD7) in hepatocellular carcinoma (HCC) and determine whether SETD7 can be used as a predictor of overall survival in HCC patients.</p><p>Methods</p><p>mRNAs and proteins of <i>SETD7</i> and related genes in HCC tumor samples and paired adjacent non-tumorous liver tissues (ANLTs) (n = 20) or culture cells were determined by quantitative real-time PCR and Western blot. Cell proliferation and apoptosis with SETD7 knockdown SMMC-7721 cells or SETD7 overexpressed HepG2 cells were analyzed by CCK8 assay or flow cytometry. Gene expression alterations in SETD7 knockdown of SMMC-7721 cells were determined by digital gene expression (DGE) profiling. Defined data on patients (n = 225) with HCC were retrieved for the further study. Tissue microarrays (TMAs) were performed using paraffin tissues with tumor and ANLTs. SETD7 and related proteins were determined by TMAs immunohistochemistry. Statistical analyses were conducted to associate SETD7 expression with tumor features and patient outcomes, as well as related proteins expression.</p><p>Results</p><p>SETD7 expression was significantly higher in HCC tumor tissues than in ANLTs. SETD7 overexpression in vitro can promote HepG2 cell proliferation, whereas SETD7 knockdown can inhibit SMMC-7721 cell proliferation by regulating the cell cycle. SETD7 expression was significantly correlated with five genes expression. Increased SETD7 is associated with metastasis, recurrence, large tumor size, and poor tumor differentiation, and indicates poor prognosis in HCC patients.</p><p>Conclusions</p><p>SETD7 plays a critical role in HCC, and its immunohistochemistry signature provides potential clinical significance for personalized prediction of HCC prognosis.</p></div

Expression of <i>SETD7</i> in HCC tumor tissues, paired ANLTs and cell lines.

<p>(A) qRT-PCR analysis was performed to analyze <i>SETD7</i> expression in 20 pairs of HCC tumor tissues and ANLTs, the data shown are the mean of –ΔCT, and the expression of <i>SETD7</i> in HCC is significantly higher than that in ANLTs (P<0.05); (B) Western blot assay was performed to detect <i>SETD7</i> expression in 20 pairs of HCC tumor tissues and ANLTs; (C) Gray value of Western blot result (P<0.05); (D) Western blot assay was performed to analyze <i>SETD7</i> expression in normal hepatocyte cell line (HL-7702) and liver cancer cell lines (HepG2, SMMC-7721, QGY-7703, HCC-0010, and Bel-7404); (E). Typical IHC staining of SETD7 in HCC tumor tissues and ANLTs (with figures on the left magnified 100× and figures on the right magnified 400×); (F) Box plots indicate the IHC scores of SETD7 in HCC tumor tissues and ANLTs [mean, 3.204 (SD, 0.092) vs 1.427 (SD, 0.083), P<0.01].</p

<i>SETD7</i> expression changes liver cancer cell proliferation by regulating the cell cycle.

<p>(A) Western blot analysis was performed to detect the interference efficiency of siRNA after transfected with si-<i>SETD7</i>-1, si-<i>SETD7</i>-2, si-<i>SETD7</i>-3, or scramble RNA (NC) in SMMC-7721. (B) Western blot analysis was performed to detect <i>SETD7</i> expression in HepG2 transfected with pGV141-<i>SETD7</i> (OV-<i>SETD7</i>) or pGV141 plasmid (Control). (C, D) CCK8 array was used to assess proliferation in <i>SETD7</i> knockdown in SMMC-7721 cells or <i>SETD7</i> overexpression in HepG2 cells. *, P<0.05; **, P<0.01 by student’s t test. (E, F) Cell cycle was assessed in <i>SETD7</i> knockdown in SMMC-7721 cells or <i>SETD7</i> overexpression in HepG2 cells by flow cytometry assay.</p

Correlation between the SETD7 and clinicopatologic variables in HCC.

<p>Correlation between the SETD7 and clinicopatologic variables in HCC.</p

Kaplan–Meier survival analysis of the correlation between <i>SETD7</i> expression in patients with HCC and OS.

<p>Patients with high <i>SETD7</i> expression (solid line) exhibited decreased OS.</p

Correlation between the SETD7 and H3K4me2, ZBTB20, CDKN2D expression in HCC.

<p>Correlation between the SETD7 and H3K4me2, ZBTB20, CDKN2D expression in HCC.</p

Estrogen receptor competitive binding assay.

<p>ESCs were incubated in the presence of E₂ or puerarin solutions for 1 h. After removal of the medium, [³H]-E₂ binding capacity was measured. (A) Total binding of [³H]-E₂ to ERs in ESCs; the concentrations of [³H]-E₂ used were 0, 0.1950, 0.3906, 0.7813, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200 and 400 nmol/L. (B) Non-specific binding of [³H]-E₂. (C) Specific binding of [³H]-E₂, calculated by subtracting the non-specific binding values from the total binding values. (D) Measurement of [³H]-E₂ binding to ERs in the presence of varying concentrations of E₂ (0, 0.0061, 0.0183, 0.0549, 0.1646, 0.4938, 1.4815, 4.4444, 13.3333 and 40 µmol/L) or puerarin (0, 0.0061, 0.0183, 0.0549, 0.1646, 0.4938, 1.4815, 4.4444, 13.3333 and 40 µmol/L). The figures represent data from three experiments; each experiment was performed in triplicate, and data are represented as the mean ± SD.</p

Puerarin Suppresses Proliferation of Endometriotic Stromal Cells Partly via the MAPK Signaling Pathway Induced by 17ß-estradiol-BSA

<div><h3>Background</h3><p>Puerarin is a major isoflavonoid compound extracted from <em>Radix puerariae</em>. It has a weak estrogenic action by binding to estrogen receptors (ERs). In our early clinical practice to treat endometriosis, a better therapeutic effect was achieved if the formula of traditional Chinese medicine included <em>Radix puerariae</em>. The genomic and non-genomic effects of puerarin were studied in our Lab. This study aims to investigate the ability of puerarin to bind competitively to ERs in human endometriotic stromal cells (ESCs), determine whether and how puerarin may influence phosphorylation of the non-genomic signaling pathway induced by 17ß-estradiol conjugated to BSA (E₂-BSA).</p> <h3>Methodology</h3><p>ESCs were successfully established. Binding of puerarin to ERs was assessed by a radioactive competitive binding assay in ESCs. Activation of the signaling pathway was screened by human phospho-kinase array, and was further confirmed by western blot. Cell proliferation was analyzed according to the protocol of CCK-8. The mRNA and protein levels of cyclin D1, Cox-2 and Cyp19 were determined by real-time PCR and western blotting. Inhibitor of MEK1/2 or ER antagonist was used to confirm the involved signal pathway.</p> <h3>Principal Findings</h3><p>Our data demonstrated that the total binding ability of puerarin to ERs on viable cells is around 1/3 that of 17ß-estradiol (E₂). E₂-BSA was able to trigger a rapid, non-genomic, membrane-mediated activation of ERK1/2 in ESCs and this phenomenon was associated with an increased proliferation of ESCs. Treating ESCs with puerarin abrogated the phosphorylation of ERK and significantly decreased cell proliferation, as well as related gene expression levels enhanced by E₂-BSA.</p> <h3>Conclusions/Significance</h3><p>Puerarin suppresses proliferation of ESCs induced by E₂-BSA partly via impeding a rapid, non-genomic, membrane-initiated ERK pathway, and down-regulation of Cyclin D1, Cox-2 and Cyp19 are involved in the process. Our data further show that puerarin may be a new candidate to treat endometriosis.</p> </div

Phospho-proteomic profiling of E₂-BSA and puerarin effects on ERK1/2 phosphorylation in ESCs and confirmed by western blot analysis.

<p>Cells were treated as indicated. A. AKT, Chk2 and ERK1/2 were activated by E₂-BSA and suppressed by puerarin. B. Western blot analysis of E₂-BSA on phosphorylation in ESCs. Cells were treated with vehicle (DMSO) or E₂-BSA (10⁻⁸ mol/L) for 15 min. AKT, Chk2 and ERK1/2 were activated by E₂-BSA. Expression was normalized to total ERK1/2.</p