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Original Article Scinderin Is a Novel Transcriptional Target of BRMS1 Involved in Regulation of Hepatocellular Carcinoma Cell Apoptosis

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Original Article Scinderin Is a Novel Transcriptional Target of BRMS1 Involved in Regulation of Hepatocellular Carcinoma Cell Apoptosis Am J Cancer Res 2018;8(6):1008-1018 www.ajcr.us /ISSN:2156-6976/ajcr0077479 Original Article Scinderin is a novel transcriptional target of BRMS1 involved in regulation of hepatocellular carcinoma cell apoptosis Xiaojing Qiao1, Yiren Zhou1, Wenjuan Xie1, Yi Wang1, Yicheng Zhang1, Tian Tian1,3, Jianming Dou1, Xi Yang1, Suqin Shen1, Jianwei Hu2, Shouyi Qiao1, Yanhua Wu1 1School of Life Sciences, Fudan University, Shanghai 200433, P. R. China; 2Endoscopy Center and Department of General Surgery, Zhongshan Hospital of Fudan University, Shanghai 200032, P. R. China; 3Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, EH89XD, Scotland Received April 6, 2018; Accepted May 18, 2018; Epub June 1, 2018; Published June 15, 2018 Abstract: Tumor metastasis suppressor factor BRMS1 can regulate the metastasis of breast cancer and other tumors. Here we report scinderin (SCIN) as a novel transcriptional target of BRMS1. SCIN protein belongs to the cy- toskeletal gelsolin protein superfamily and its involvement in tumorigenesis remains largely illusive. An inverse cor- relation between the expression levels of BRMS1 and SCIN was observed in hepatocellular carcinoma (HCC) cells and tissues. On the molecular level, BRMS1 binds to SCIN promoter and exerts a suppressive role in regulating SCIN transcription. FACS analysis and caspase 9 immunoblot reveal that knockdown of SCIN expression can sensitize HCC cells to chemotherapeutic drugs, leading to suppression of tumor growth in vivo. Consistently, overexpression of SCIN protects cells from apoptotic death, contributing to increased xenografted HCC cell growth. In summary, our study reveals SCIN as a functional apoptosis regulator as well as a novel target of BRMS1 during HCC tumorigen- esis. Inhibition of SCIN might bring a potential cancer therapy approach. Keywords: BRMS1, scinderin, cell apoptosis, hepatocellular carcinoma, transcriptional regulation Introduction Herein, we further identified Scinderin SCIN( , also known as adseverin) as a novel transcrip- Human breast cancer metastasis suppressor 1 tional target of BRMS1. SCIN is located in (BRMS1) was first discovered and cloned in 7p21.3 and encodes a Ca2+-dependent actin 2000 by Seraj et al. [1]. Later on, it has been filament severing and capping protein. SCIN demonstrated that BRMS1 plays a suppres- protein was discovered in chromaffin cells in sive role in regulating tumor metastasis in 1990 but its biological functions remain largely breast cancer, melanoma, non-small cell lung illusive [13]. As a member of the gelsolin super- cancer, hepatocellular carcinoma, among oth- family, SCIN has the core structure of gelsolin- ers [2-5]. In consistent with these findings, the like domains and is involved in vesicle trans- expression level of BRMS1 has been found to port, exocytosis via regulating the polymerization be significantly down-regulated in multiple met- and disassembly of F-actin network [14-16]. In astatic tumor tissues [6-9]. It has been shown addition, SCIN also exhibits activities in regulat- that BRMS1 mainly acts as a component ing other cellular processes such as cell differ- of Sin3a•HDAC complex, which regulates entiation, osteoclastogenesis, among others chromatin status and further affects gene [17-19]. The involvement of SCIN in tumor devel- transcription efficiency [10, 11]. We have previ- opment was first investigated in the megakaryo- ously reported two well-defined tumor-metasta- blastic leukemia cells [20]. Miura et al. later sis related genes, osteopontin (OPN) and found that SCIN expression is significantly death-associated protein kinase 1 (DAPK1), increased in the cisplatin-resistant urothelial which were under transcriptional control of cancer patients by comparison with cisplatin- BRMS1 in hepatocellular carcinoma (HCC) sensitive control group [21]. Knockdown of cell lines [9, 12]. SCIN led to an increase of mitochondria-medi- SCIN suppresses HCC cell apoptosis ated cell apoptosis in vitro. Recently, the 95°C for 5 min hot start and 40 cycles of 95°C expression of SCIN has been shown to be up- for 5 sec, 58°C for 10 sec and 72°C for 20 sec. regulated in human prostate cancer, lung carci- Samples with no cDNA templates were used as noma and gastric cancer, suggesting an onco- negative control to rule out contamination in genic role of SCIN in regulating tumor growth each run. Melting curves were analyzed to con- [22-24]. However, the molecular mechanism firm the specificity of the PCR product. Primers accounting for abnormal SCIN expression in for BRMS1: forward, 5’-ACTGAGTCAGCTGCG- tumor remains unknown. GTTGCGG-3’; reverse, 5’-AAGACCTGGAGCTGC- CTC TGGCGTGC-3’. Primers for SCIN: forward, In our study, several lines of evidence demon- 5’-TCTGCGTTCCTGACTGTTC-3’; reverse, 5’-GA- strated that SCIN expression is transcriptional- CCTCCTTTCTTTGATGTTCC-3’. 18S rRNA was ly suppressed by BRMS1 in HCC cells. The used as the internal control. Primers for 18S association relationship between BRMS1 and rRNA: forward, 5’-GTAACCCGTTGAACCCCATT-3’; SCIN expression in HCC tissues and cells was reverse, 5’-CCATCCAATCGGTAGTAGCG-3’. studied through western blot analysis and the transcriptional mechanism was illuminated th- Cell culture and transfection rough dual luciferase assay and ChIP experi- ment. To further investigate the function of HCC cell lines SK-Hep1, Huh7, QGY-7701, Hep- SCIN, lentivirus carrying shRNA against SCIN 3B, SMMC-7721 and human embryonic kidney or recombinant plasmid overexpressing SCIN cell line 293T (HEK293T) were cultured with were utilized. It is found that knockdown of Dulbecco’s modified Eagle’s medium (DMEM), endogenous SCIN sensitized HCC cells to apop- supplemented with 10% fetal bovine serum tosis, whereas expression of SCIN protected (Gibco, USA) in 5% CO2 humidified atmosphere cells from apoptotic cell death. Moreover, SCIN at 37°C. Cells of 70% confluency were trans- promoted HCC cell growth in xenografted tumor fected with indicated plasmids or siRNA us- mice. Taken together, our findings character- ing Lipofectin 2000 (Invitrogen, USA) accord- ized SCIN as another functional downstream ing to the manufacturer’s instructions. Small effector of BRMS1, providing another molecu- interfering RNA against BRMS1 as previously lar pathway accounting for BRMS1’s tumor sup- described was used [9] and the target sequ- pressive role in HCC cells. ence of si-SCIN was: 5’-CGAGATGAGCTGACA- ACAT-3’. In experiments evaluating cells’ sensi- Materials and methods tivity to apoptosis stimulation, etoposide (Sig- ma, USA) and cisplatin (Abcam, USA) were Tumor specimens directly added to complete medium with indi- cated dosages. Fresh surgical specimens of HCC, including tumor tissues and the neighboring pathologi- Western blot analysis cally nontumorous liver tissues, were obtain- ed from liver cancer patients at Zhongshan Protein samples were separated by different Hospital, Shanghai, China. Written informed concentrations of SDS-PAGE and then trans- consent was obtained from all these patients. ferred to PVDF membranes. Membranes were Tissue samples were immediately frozen in incubated in 5% fat-free milk to avoid non-spe- liquid nitrogen after surgery and later stored cific binding. After blocking, membranes were at -80°C. incubated with specific primary antibodies against different proteins at 4°C overnight. Quantitative real-time PCR (qRT-PCR) Membranes were then washed in TBST for four times and incubated with HRP-conjugated sec- RNA extracted from tissues or cultured cells ondary antibody for 1 h at room temperature. using TRIzol reagent (Life Technologies, USA) Later on, membranes were washed in TBST for was used for reverse transcription using Pri- four times before being visualized by enhanced meScirptTM RT reagent and gDNA Eraser kit chemiluminescence (Pierce, USA) on a molecu- (Takara, Japan). Quantitative PCR analysis was lar imager ChemiDoc XRS+ system (Bio-Rad). performed with the CFX Connection detec- Antibodies used include anti-BRMS1 (Abcam), tion system (Bio-Rad, USA) using SYBR Green anti-SCIN (Abcam), anti-caspase 9 (cell signal- Supermix kit (Takara). Cycle parameters were ing technology, USA), anti-β-actin (Sigma), anti- 1009 Am J Cancer Res 2018;8(6):1008-1018 SCIN suppresses HCC cell apoptosis Myc (Sigma), anti-Flag (Sigma), peroxidase- Biosciences, CA, USA). The apoptotic cell popu- conjugated goat anti-mouse IgG and goat anti- lation corresponds to cells in sub-G1 phase. rabbit IgG (Jackson, USA). Each result was representative of three inde- pendent experiments with triplicate samples Chromatin immunoprecipitation (ChIP) for each condition. The ChIP assay was performed according to Cell proliferation assay our previous work [12]. Briefly, HEK293T cells were seeded at 1×107/dish in 100 mm dishes Cells were plated in 96-well plates at a density and then transfected with Myc-tagged BRMS1 of 1500 cells/well. In the serum deprivation plasmid or empty vector. At 36 h after transfec- assay, medium was replaced by DMEM after tion, cells were treated with 4% formaldehyde 24 hours’ culture with complete medium. Cell (Amresco, USA) to make them crosslinked and proliferation was detected using CCK8 assay. fixed. Cell nuclei were first released by lysis Generally, culture medium was replaced by 0.5 buffer in pH 8.1 and then sonicated. Genomic mg/mL CCK8 (Dojindo, Japan) diluted in DM- DNA fragment was immuno-precipitated by EM. Cells were then kept in 37°C for 4 hours anti-Myc antibody at 4°C overnight after treat- and absorbance at a wavelength of 490 nm ing with protein
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