In this study prostate cancer cell line model was used selecting three type of them (Pc3, DU145 &amp; LNCaP) , gene transfecting was done by using the liposome into the cell culture cells then detecting the healing ability by the Wound Healing or (scratching) test that shows clear effect of ELK1 gene in this cells comparing with the control cell transfected with the knock down gene, using for each type of cells 6 repeats ,for each type there was two groups 1st for control ELK1 and the 2nd was knock down or (sh) ELK1, all works was done in Johns Hopkins University / School of Medicine / Pathology Department ( MD, USA

Al-Saadi, Ali Hmood

Aljarah, Ali Kadhim

Auda, Nazar Aziz

English

International Institute for Science, Technology and Education (IISTE): E-Journals

Journal of Natural Sciences Research                                                                                                                                                www.iiste.org ISSN 2224-3186 (Paper)   ISSN 2225-0921 (Online) Vol.4, No.22, 2014  94 The Elk1 Gene Effect on Prostate Cancer Cell Line Wound Healing Ability  Ali Kadhim Aljarah 1*, Ali Hmood Al-Saadi2, Nazar Aziz Auda3. 1 Babylon University / collage of science for Women, PO box 435, Al-Hillah city, Babylon , Iraq 2 Babylon University / collage of science 3 Baghdad University / collage of science *Email of corresponding author alialjarrah78@gmail.com Abstract In this study prostate cancer cell line model was used selecting three type of them (Pc3, DU145 & LNCaP) , gene transfecting was done by using the liposome into the cell culture cells then detecting the healing ability by the Wound Healing or (scratching) test that shows clear effect of ELK1 gene in this cells comparing with the control cell transfected with the knock down gene, using for each type of cells 6 repeats ,for each type there was two groups 1st for control ELK1 and the 2nd was knock down or (sh) ELK1, all works was done in Johns Hopkins University / School of Medicine / Pathology Department ( MD, USA)  Introduction The scratch assay was performed to compare the influence control ELK1 plasmid against sh ELk1 ( knock down ),that was transfected to the three prostate cancer cell type that we used in our study  DU145, Pc3 & LNCaP cells  were seeded in a high density at 12-well plates, and grown till they reach more than 80% influence then transfected with the control ELK1 plasmid against sh ELk1 ( knock down), Then the monolayer cells were physically wounded by scratching the surface with a pipette tip (200 µl) as uniformly and straight as possible. The images of cells invading the scratch were captured at indicated time points (24 hr.) using Inverted Microscope model CK40 Culture Microscope. The pictures were evaluated by measuring the difference in the area of the wounds with a ImageJ software and migration rate expressed as percentage of scratch closure was calculated as follows: % of scratch closure= a-b/a, where (a) is a distance between edges of the wound, and (b) is the distance which remained cell-free during cell migration to close the wound. The experiments were repeated in triplicate wells at least two times.(5) In other word using 3 type of   prostate cancer cell line PC3 , Du145 & LNCaP , then compareing between the control plasmid of ELK1 and the knock down sh ELK1  for each type we toke three picture for each well (12 well plate divide into 6 for control & 6 for sh) that mean 36 picture for each type of cell 3 repeats for each well then we transfer to excel taking the mean for each and by using program software called Image J for analyzing each one take the mean for each 6 of sh & the other 6 of control for comparing. (1,2) cells are seeded into a multi-well assay plate and allowed to attach, spread, and form a confluent monolayer. A pin tool or needle is used to scratch and remove cells from a discrete area of the confluent monolayer to form a cell-free zone into which cells at the edges of the wound can migrate. (3,4) Materials and methods The prostate cancer cell line culture stocks from  (ATCC Company / USA) All transfection was done by using lipofectamin2000 of Invitrogen / USA Growing medium for prostate cancer cell line was RPMI from Dako /USA Elk-1 shRNA Plasmid (h), Elk-1 (h)-PR from SantaCruz Biotechnology  / USA Wound healing Assay Protocol Prior to the assay: 1) Cells should be cultured to confluence or near (>90%) confluence in either 6 well dishes or 35 mM dishes. 2) Depending on the conditions, cell should be rinsed with PBS and starved in low serum media (1.5 ml; 0.5% - 0.1% serum in DMEM) overnight. On the day of the assay: 3) Prepare 10ml of base media with any additives. Sterile filter the mixtures and place in sterile 15 ml falcon tubes. Store at 4oC. Warm up before using. 4) Draw a line with a marker on the bottom of the dish. 5) Using a sterile 200 μl pipet tip, scratch three separate wounds through the cells moving perpendicular to the line drawn in the step above. See the figure for arraignment of the scratches. 6) Rinse the cells (very gently as sheets of the cells may lift off if you are not careful) with PBS and replace with 1.5 ml of media containing any additives (agonist, inhibitors, activators, ect…). 7) Take a picture using phase contrast and 10X. Do this just above and just below each line. This will help orient your measurements. Make certain the line just appears in each picture. Name each picture 6hr2LA– This indicates the 6th hour, dish 2, Left scratch (vs right or center) above the line (vs B for below). 8) Take another picture at 6, 12 and 24 hours. After each measurement, replace the old media with the media/additives that you prepared in step 3. Journal of Natural Sciences Research                                                                                                                                                www.iiste.org ISSN 2224-3186 (Paper)   ISSN 2225-0921 (Online) Vol.4, No.22, 2014  95 All data in the table 1 & figure (1-3)and images( 1-6)shows  that the transfection work successfully and there is an effect of ELK1 on the wound healing or cell activity and motility through wound healing. It appears that the ELK1 plasmid increased the wound healing process in vitro. (2) References 1- Chia-Herng Y., Yung-Wei C. Jai-Nien T., Bor-Show T. Jiuan-Jen S., Wen-Hung H. Jer-Yuh L. and Jin-Ming H. 2012, Expression of Protein Kinase C α and the MZF-1 and Elk-1 Transcription Factors in Human Breast Cancer Cells, Chinese Journal of Physiology 55, 2012. 2- Yeong-Chin J., Yung-Wei C., Yieng-How C., Jin-Ming H., Pei-Yu C., Jiuan-Jen S., Wen-Hung H., Jer-Yuh L., and Li-Sung H., 2012, Expression of Protein Kinase Cα and the MZF-1 and Elk-1 Transcription Factors in Human Bladder Transitional Cell Carcinoma Cells. Chinese Journal of Physiology 55(2) 2012. 3-  Friedl, P.; Hegerfeldt, Y.; Tusch, M. 2004, Collective cell migration in morphogenesis and cancer. Int. J. Dev. Biol. 48, 441-449. 4- Yarrow, J.C.; Perlman, Z.C.; Westwood, N.J.; Mitchison, T.J. 2004, A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods. BMC Biotechnol., 4, 21. 5- Keren I. Hulkower , Renee L. Herber. 2011, Cell Migration and Invasion Assays as Tools for Drug Discovery, Pharmaceutics. 3, 107-124.   Image 1    PC3 control ELK1 Wound Healing Assay Journal of Natural Sciences Research                                                                                                                                                www.iiste.org ISSN 2224-3186 (Paper)   ISSN 2225-0921 (Online) Vol.4, No.22, 2014  96  Image 2    PC3 sh ELK1 Wound healing assay.   Image 3    DU145 control ELK1 Wound Healing Assay Journal of Natural Sciences Research                                                                                                                                                www.iiste.org ISSN 2224-3186 (Paper)   ISSN 2225-0921 (Online) Vol.4, No.22, 2014  97  Image 4    DU145 sh ELK1 Wound healing Assay.   Image 5   LNCaP control ELK1 Wound healing Assay. Journal of Natural Sciences Research                                                                                                                                                www.iiste.org ISSN 2224-3186 (Paper)   ISSN 2225-0921 (Online) Vol.4, No.22, 2014  98  Image 6    LNCaP sh ELK1 Wound healing Assay. DU145 cntDU145 shPC3 cntPC3 shLNCp cntLNCp sh56008164833102601037050375295300791346979753290594876391664023849446005230216366940154192964767944555897444704786487616277524257318474765964587050645166826125196182458457462664813130664759245833436604850256631654023514885306663875344428495913863288948758332375000661688433842593916221210112205057280154762429647677744810295570636173024557649904399056683072947596556725612314617755963574703838573071148934554887504241645657815774845784580653247114554667124354774853957774503297516385247441925214381252908456107994763075.16761882814653721.55745749.92640548.917mean10.84891210110.75202168410.459565584ratio179698.3165697.9831566635.96233399.4268695678.41784706.9424std0.03202720.0295319760.0915660.0377163590.1210770.136571719stdr4.23548E-113.7916E-078.82401E-10p value Table 1 Wound Healing Assay result data for DU145, PC3 & LNCaP prostate cancer cell lines. Journal of Natural Sciences Research                                                                                                                                                www.iiste.org ISSN 2224-3186 (Paper)   ISSN 2225-0921 (Online) Vol.4, No.22, 2014  99  Figure 1 Comparison between the two plasmid of ELK1 ( sh & control) on PC3 prostate cancer cells for wound healing assay  Figure 2 Comparison between the two plasmid of ELK1 (sh & control) on DU145 prostate cancer cells for wound healing assay  Journal of Natural Sciences Research                                                                                                                                                www.iiste.org ISSN 2224-3186 (Paper)   ISSN 2225-0921 (Online) Vol.4, No.22, 2014  100  Figure 3 Comparison between the two plasmid of ELK1 ( sh & control) on LNCaP prostate cancer cells for wound healing assay   The IISTE is a pioneer in the Open-Access hosting service and academic event management.  The aim of the firm is Accelerating Global Knowledge Sharing.  More information about the firm can be found on the homepage:  http://www.iiste.org  CALL FOR JOURNAL PAPERS There are more than 30 peer-reviewed academic journals hosted under the hosting platform.   Prospective authors of journals can find the submission instruction on the following page: http://www.iiste.org/journals/  All the journals articles are available online to the readers all over the world without financial, legal, or technical barriers other than those inseparable from gaining access to the internet itself.  Paper version of the journals is also available upon request of readers and authors.   MORE RESOURCES Book publication information: http://www.iiste.org/book/  IISTE Knowledge Sharing Partners EBSCO, Index Copernicus, Ulrich's Periodicals Directory, JournalTOCS, PKP Open Archives Harvester, Bielefeld Academic Search Engine, Elektronische Zeitschriftenbibliothek EZB, Open J-Gate, OCLC WorldCat, Universe Digtial Library , NewJour, Google Scholar   

The Elk1 gene effect on prostate cancer cell line wound healing ability

https://www.iiste.org/Journals/index.php/JNSR/article/download/17632/17634

The Elk1 gene effect on prostate cancer cell line wound healing ability

Abstract

Similar works

Full text

Available Versions

International Institute for Science, Technology and Education (IISTE): E-Journals