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Original Article Mechanism of SKOV-3 Inducing Hmsc Homing

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Original Article Mechanism of SKOV-3 Inducing Hmsc Homing Int J Clin Exp Med 2017;10(4):6262-6272 www.ijcem.com /ISSN:1940-5901/IJCEM0020176 Original Article Mechanism of SKOV-3 inducing hMSC homing Dongmei Fan1*, Xiaojuan Xie2*, Pengwei Qi1, Xianan Yang1, Ximeng Jin1 Departments of 1Gynaecology and Obstetrics, 2Anesthesia, The First Affiliated Hospital of Henan University of Science and Technology, No. 24, Jinghua Road, Luoyang 471003, Henan, China. *Equal contributors. Received November 20, 2015; Accepted March 29, 2016; Epub April 15, 2017; Published April 30, 2017 Abstract: Chemokine SDF-1 and their receptor CXCR4 play a key role in tumor invasion and metastasis. Ubiquitylation and degradation of epithelial E-cadherin complex mediated by Cb1 proto-oncogene-like 1 (CBLL1, also known as HAKAI) may regulate cell proliferation. Ovarian cancer has the highest mortality among malignant tumors of female reproductive system. Human mesenchymal stem cells (hMSC) are mesenchymal stem cells derived from bone mar- row, and hMSC homing has been considered the most promising and effective way to treat cancers. A preliminary was conducted, based on the established lentiviral vectors LV3-CXCR4 siRNA and LV3-CBLL1 RNAi, and hMSC, human ovarian cancer cell SKOV-3, as well as human cervical cancer Hela cells, to investigate the mechanism of hMSC homing induced by SKOV-3. The study showed that SDF-1/CSCR-4 biological axis participated in hMSC hom- ing induced by SKOV-3. hMSC facilitated SKOV-3 cell migration to the damaged site, and repaired SKOV-3 lesions. Degradation of E-cadherin mediated by CBLL1 also played an important role in this process. Keywords: Human bone marrow mesenchymal stem cell homing, stromal cell derived factor-1, CXCR4, CBLL1, SKOV-3 Introduction receptor CSCR-4 participate in the metastatic process of some malignant tumors [6, 7]. Ovarian cancer has the highest mortality Ovarian cancer cells mainly diffuse towards among malignant tumors of the female repro- peritoneum. Since CXCR4 is expressed in ovar- ductive system, while the mechanism of metas- ian cancer cells, SDF-1/CXCD4 axis probably tasis remains unclear [1]. It is reported that participates in this process and leads to ovari- chemokines and their receptors play an im- an cancer progression [8, 9]. Human E3 ubiqui- portant role in tumor invasion and metastasis tin protein ligase HAKAI (aka CBLL1), coded by [2]. hMSCs are non-hematopoietic stem cells cbll1 gene, has a Ring-finger structure, which derived from bone marrow with abilities of participates in the ubiquitination of E-cadherin adhesion and self-renewal. hMSCs also have complex, endocytosis and degradation in epi- the following characteristics: 1) easy to be thelial cells, and regulates cell proliferation. The obtained and cultured, with high proliferation; two lysine residues on the cytoplasmic region 2) multipotency and able to differentiate into of E-cadherin are the ubiquitination site. The bone cells, cartilage cells, adipocytes, cardiac degradation of E-cadherin by proteasome is muscle cells, etc; 3) low immunogenicity and regulated by phosphorylation. The binding sites low rejection following transplantation [3]. on E-cadherin to Hakai contain several tyrosine hMSC has been considered the most promising residues, which can be phosphorylated by tyro- stem cells for the treatment of multiple diseas- sine kinase (such as Src and Met) to enhance es. hMSC homing refers to the process dur- the binding between the two proteins [10, 11]. ing which hMSC directionally migrates to and engrafts in the target tissues under the effects A preliminary study was conducted to investi- of multiple factors. Thus, hMSC homing is criti- gate the mechanism of SKOV-3 inducing hMSC cal for hMSC treatment and has gained wide- homing, based on the established lentiviral vec- spread attention [4, 5]. Chemokine stromal tors LV3-CXCR4 siRNA and LV3-CBLL1 RNAi cell-derived factor-1 (SDF-1) and its specific and using human bone marrow MSC, human Mechanism of SKOV-3 inducing hMSC homing Table 1. Grouping of experiment SDF-1 levels before and after CX- ELISA CR4 interference on hMSC migra- Different treatment Groups tion were detected using ELISA kit 1-a Normal Hela cell group (double sandwich method) (Lianke, 2-a Empty lentivirus transfected Hela cell group China). Purified stromal cell-derived factor (SDF-1) antibody was used as 3-a LV3-CXCR4 siRNA lentiviral transfected Hela cell group solid antibody to coat the micro- 4-a Normal SKOV-3 cell group plate. SDF-1 was added to monoclo- 5-a Empty lentivirus transfected SKOV-3 cell group nal antibody coated wells, bound 6-a LV3-CXCR4 siRNA Lentiviral transfected SKOV-3 cell group to HRP labeled SDF-1 antibody and formed antibody-antigen-enzy- Table 2. Scratch grouping me labeled antibody complex. After thorough Scratch Assay Groups Different Treatment washes, TMB substrates were added for color development. TMB turned into blue color 1-b Hela normal cell group catalyzed by HRP enzyme and changed to yel- 2-b hMSC: Hela 1:500 low under the effect of acid. The strength of 3-b hMSC: Hela 1:1000 color was positively correlated with SDF-1 lev- 4-b SKOV-3 normal cell group els in the samples. Absorbance (OD value) 5-b hMSC : SKOV-3 1:500 was detected under 450 nm wavelength using 6-b hMSC: SKOV-3 1:1000 spectrophotometer. SDF-1 concentration in sa- mples was calculated using standard curve. ovarian cancer cell SKOV-3 and human cervical The concentrations of standards were plotted cell Hela system, providing theoretical support as x-axis and OD values were plotted as y-axis. for application of hMSC in the treatment of The linear regression equation of standard ovarian cancer. curve was calculated. Sample OD values were plugged into equation to calculate sample Materials and methods concentrations, which was multiplied by dilu- tion factor to obtain the real SDF-1 concentra- Cells and lentiviral vectors tion in samples. Human bone marrow mesenchymal stem cells hMSC migration ability towards two types of (hMSC) were purchased from Guangzhou Saiye cells detected by scratch assay: Cell scratch Co. Human ovarian cancer cells SKOV-3. assay (repair) is a convenient way to detect Human cervical cancer cells Hela were pur- cell migration and repair ability [13]. In the chased from Chinese Academy of Sciences present study, on the plate cultured with in vitro (Shanghai). LV3-CXCR4 siRNA and LV3-CBLL1 SKOV-3 and Hela cells, a pipette tip was used RNAi lentiviral vectors were constructed by our to draw lines in the central area of cell culture institute. to remove the cells growing in the center. Cells were continued to be cultured until 24 h and Methods the culture dish was taken out to examine whether the cells in peripheral regions migrat- SDF-1 expression before and after CXCR4 ed to the central scratch area and determine interference on hMSC migration in two types the cell migration ability. Details of experimen- of cells detected by ELISA: Cultured SKOV-3 tal groups are listed in Table 2. and Hela cells were transfected with estab- lished LV-CXCR4 siRNA lentiviral vector [12] Steps of scratch assay are listed as follows: and details of experimental groups are listed in Table 1. After treatment, cells were collected Cells were cultured in RPMI-1640 containing and cell suspension was diluted with PBS (pH 10% FBS and seeded in 6-well plates at a cer- 7.4) to a concentration of 1,000,000/ml. After tain concentration. Different concentrations of several freeze-thaw cycles, cells were damaged hMSC were seeded in the upper chamber of and cellular contents were released. Cells were Transwell. Cells were cultured for approximately centrifuged for about 20 min (2000-3000 rpm) 24 h until monolayer cells reaching 70-80% and supernant was collected carefully. confluence. 6263 Int J Clin Exp Med 2017;10(4):6262-6272 Mechanism of SKOV-3 inducing hMSC homing Table 3. Transwell migration assay-grouping three days and cells were passaged every five Transwell Migration days. In this study, Transwell migration assay Variation Treatment Assay-Groups A was used to detect the effect of hMSC on 1-c Hela normal cell group migration ability of two types of cells. Detailed experiment steps are listed as follows: 2-c hMSC: Hela 1:500 group 3-c hMSC: Hela 1:1000 group All cell culture reagent and culture plates were 4-c SKOV-3 normal cell group placed at 37°C. 5-c hMSC: SKOV-31:500 group 6-c hMSC: SKOV-31:1000 group Cells were cultured till logarithm phase. Cells were digested, and washed with PBS and serum-free media successively. Cells were sus- Table 4. Immunohistochemistry grouping pended with serum free media, counted and 5 Groups Variation Treatment adjusted to a concentration of 2×10 /ml. 1-d Hela normal cell group 700 µl media containing 10% serum and differ- 2-d hMSC: Hela 1:500 group ent ratios of hMSC were added in the lower 3-d hMSC: Hela 1:1000 group chamber (experiment group shown in Table 3). 4-d SKOV-3 normal cell group 150 µl tumor cell suspension was added in the 5-d hMSC: SKOV-31:500 group upper chamber. Cells were cultured in the incu- 6-d hMSC: SKOV-31:1000 group bator for 24 h. The lower surface was soaked in 70% methanol Without changing media, a 2.5 ul sterile pipet solution, fixed for 40 min, stained with trypan tip was used to slightly scratch a line through blue and observed using a microscope. Five the well on the monolayer of cells (pipet tip ver- regions including the center and four corners tical to the bottom of well, not tilt, to make a were selected and mean was calculated to straight line in the same direction).
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