[Home ] [Archive]   [ فارسی ]  
:: Main :: About :: Current Issue :: Archive :: Search :: Submit :: Contact ::
:: Volume 30, Issue 2 (6-2022) ::
Journal of Ilam University of Medical Sciences 2022, 30(2): 18-31 Back to browse issues page
Editing of the MALAT1 Gene in MDA-MB-361 Breast Cancer Cell Line using the Novel CRISPR Method
Soraya Ahmadi-Baloutaki1 , Abbas Doosti * 2, Mojtaba Jaafarinia3 , Hamedreza Goudarzi4
1- Dept of Genetic, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran
2- Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran , abbasdoosti@yahoo.com
3- Dept of Biology, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran
4- Dept of Genetic, Faculty of Basic Sciences, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran
Abstract:   (719 Views)
Introduction: Long non-coding RNAs play an important role in regulating gene expression, RNA processing, histone modification, and rearrangement of chromatin genes. These molecules can also be involved in many biological processes, such as organogenesis, cell differentiation, development, genome imprinting, quantitative compensation, and tumorigenesis. High expression of MALAT1 (a type of lncRNA) in many cancers, including breast cancer, indicates that a disorder of MALAT1 regulation is an important factor in the development of many types of cancer. Breast cancer is the most common cancer among women worldwide, and the invasion, as well as metastasis of this disease, are considered among the main causes of death. The present study aimed to knock out the MALAT1 gene in the MDA-MB-361 breast cancer cell line and evaluate its function and effects on the expression of genes associated with apoptosis.
Material & Methods: In this study, two types of sgRNA were designed by CHOPCHOP software for exon 1 of the MALAT1 gene. These sgRNAs were cloned separately into two CRISPR vectors to generate the recombinant vectors PX459-sgRNA1 and PX459-sgRNA2. Co-transfection of these two recombinant vectors into the MDA-MB-361 cancer cell line was performed using lipofectamine 2000. MALAT1 gene editing was investigated in the cells receiving recombinant vectors. The expression of genes related to apoptosis was analyzed by Real-Time PCR. Cell proliferation and apoptosis were assessed by MTT and flow cytometry methods, respectively.
(Ethic code: IR.IAU.M.REC.1399.010)
Findings: The MALAT1 gene was edited by the CRISPR method in MDA-MB-361 cells. The rate of cell proliferation in the cells of the treatment group, compared to the control groups, showed a significant decrease (P<0.05). Apoptosis levels were significantly increased in cancer cells the MALAT1 gene of which had been deleted. Moreover, the expression of BCL2 and survivin anti-apoptotic genes in treated (edited) cells was significantly reduced, compared to control cells (P<0.05). Increased expression of proapoptotic genes P53, BAK, BAX, and FAS was also observed in the edited cells (P<0.05).
Discussion & Conclusion: The results of this study confirm that the deletion of the MALAT1 gene has a significant effect on increasing apoptosis and reducing cell proliferation. A reduction in the expression of the MALAT1 gene can prevent the growth and proliferation of breast cancer cell lines. Therefore, it seems that the control of MALAT1 oncogene expression is useful and effective for controlling tumors.
Keywords: Apoptosis, Breast cancer cell line, CRISPR, MALAT1
Full-Text [PDF 1747 kb]   (212 Downloads)    
Type of Study: Research | Subject: biotechnolohgy
Received: 2021/06/21 | Accepted: 2021/12/18 | Published: 2022/06/5
1. Zahedi R, Molavivardanjani H, Baneshi MR, Haghdoost AA, Malekpourafshar R. Incidence trend of breast cancer in women of Eastern Mediterranean region countries from 1998 to 2019 a systematic review and meta-analysis. BMC Womens Health 2020; 20:53-62. doi: 10.1186/s12905-020-00903-z
2. Alziftawi NH, Shafie AA, Mohamedibrahim MI. Cost effectiveness analyses of breast cancer medications use in developing countries a systematic review. Expert Rev Pharmacoecon Outcomes Res 2020; 27:1-11. doi: 10.1080/14737167.2020.1794826.
3. Farhood B, Geraily G, Alizadeh A. Incidence and mortality of various cancers in Iran and compare to other countries a review article. Iran J Public Health 2018; 47:309-16.
4. Ghafourifard S, Tamizkar KH, Hussen BM, Taheri M. An update on the role of long non-coding RNAs in the pathogenesis of breast cancer. Pathol Res Pract 2021; 219. doi: 10.1016/j.prp.2021.153373.
5. Li He, Si qing Ma, Jin Huang, Xiao ping Chen, Hong hao Zhou. Roles of long noncoding RNAs in colorectal cancer metastasis. Oncotarget 2017; 8: 39859-76. doi: 10.18632/oncotarget.16339.
6. Zhou S, He Y, Yang S, Hu J, Zhang Q, et al. The regulatory roles of lncRNAs in the process of breast cancer invasion and metastasis. Biosci Rep 2018; 38. doi: 10.1042/BSR20180772.
7. Dey BK, Mueller AC, Dutta A. Long non coding RNAs as emerging regulators of differentiation, development, and disease. Transcription 2014; 5:e944014. doi: 10.4161/21541272.2014.944014.
8. Zhao M, Wang S, Li Q, Ji Q, Guo P, et al. MALAT1 a long non-coding RNA highly associated with human cancers. Oncol Lett 2018; 16:19-26. doi: 10.3892/ol.2018.8613.
9. Li X, Chen N, Zhou L, Wang C, Wen X, et al. Genome wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer. Am J Cancer Res 2019; 9:714-29.
10. Wang Y, Xue D, Li Y, Pan X, Zhang X, et al. The long noncoding RNA MALAT-1 is a novel biomarker in various cancers a meta analysis based on the GEO database and literature. J Cancer 2016; 7:991-1001. doi: 10.7150/jca.14663. eCollection 2016.
11. Dake C, Xu T, Chang HH, Song Q, Zhu Y, et al. The role of MALAT1 in cancer. J Cancer Sci Clin Ther 2019; 3: 5-27. doi: 10.26502/jcsct.5079016.
12. Peng R, Luo C, Guo Q, Cao J, Yang Q, et al. Association analyses of genetic variants in long non-coding RNA MALAT1 with breast cancer susceptibility and mRNA expression of MALAT1 in Chinese Han population. Gene 2018; 642: 241-8. doi: 10.1016/j.gene.2017.11.013.
13. Shen F, Zheng H, Zhou L, Li W, Xu X. Overexpression of MALAT1 contributes to cervical cancer progression by acting as a sponge of miR‐429. J Cell Physiol 2019; 234:11219-26. doi: 10.1002/jcp.27772.
14. Chen Y, Huang W, Sun W, Zheng B, Wang C, et al. LncRNA MALAT1 promotes cancer metastasis in osteosarcoma via activation of the PI3K-Akt signaling pathway. Cell Physiol Biochem 2018; 51:1313-26. doi: 10.1159/000495550.
15. Sun Z, Ou C, Liu J, Chen C, Zhou Q, et al. YAP1-induced MALAT1 promotes epithelial–mesenchymal transition and angiogenesis by sponging miR-126-5p in colorectal cancer. Oncogene 2019; 38:2627-44. doi: 10.1038/s41388-018-0628-y.
16. Ellis MJ, Ding L, Shen D, Luo J, Suman VJ, et al. Whole-genome analysis informs breast cancer response to aromatase inhibition. Nature 2012; 486:353-60. doi: 10.1038/nature11143.
17. Yoshimoto R, Mayeda A, Yoshida M, Nakagawa S. MALAT1 long non-coding RNA in cancer. Biochim Biophys Acta 2016; 1859:192-9. doi: 10.1016/j.bbagrm.2015.09.012.
18. Gayatri A, Diermeier S, Akerman M, Chang KC, Wilkinson LE, et al. Differentiation of mammary tumors and reduction in metastasis upon Malat1 lncRNA loss. Gens Dev 2016; 30:34-51. doi: 10.1101/gad.270959.115.
19. Mendell JT. Targeting a long noncoding RNA in breast cancer. N Engl J Med. 2016; 374:23:2287-9. doi: 10.1056/NEJMcibr1603785.
20. Louie E, Chen XF, Coomes A, Ji K, Tsirka S, et al. Neurotrophin-3 modulates breast cancer cells and the microenvironment to promote the growth of breast cancer brain metastasis. Oncogene 2013; 32: 4064-77. doi: 10.1038/onc.2012.417.
21. Vijai S, Gohil N, Garcia RR, Braddick D, Fofié CK. Recent advances in CRISPR‐Cas9 genome editing technology for biological and biomedical investigations. J Cell Biochem 2018; 119:81-94. doi: 10.1002/jcb.26165.
22. Carlos PQ, Aparicio-Prat E, Arnan C, Polidori T, Hermoso T, et al. Scalable design of paired CRISPR guide RNAs for genomic deletion. PLoS Comput Biol 2017; 13 doi: 10.1371/journal.pcbi.1005341.
23. Tianzuo Z, Rindtorff N, Betge J, Ebert MP, Boutros M. CRISPR/Cas9 for cancer research and therapy. Semin cancer biol. Academic Press 2019; 55: 106-119. doi: 10.1016/j.semcancer.2018.04.001.
24. Yuan R, Varanasi M, Mendes S, Yamagata HM, Wilson RD, et al. Poly (Beta-Amino Ester) Nanoparticles Enable Nonviral Delivery of CRISPR-Cas9 Plasmids for Gene Knockout and Gene Deletion. Mol Ther Nucleic Acids 2020; 20: 661-72. doi: 10.1021/acs.biomac.2c00137.
25. Qiong W, Meng WY, Jie Y, Zhao H. LncRNA MALAT1 induces colon cancer development by regulating miR‐129‐5p/HMGB1 axis. J Cell Physiol 2018; 233 6750-57. doi: 10.1002/jcp.26383.
26. Shi D, Zhang Y, Lu R, Zhang Y. The long non-coding RNA MALAT1 interacted with miR-218 modulates choriocarcinoma growth by targeting Fbxw8. Biomed Pharmacother 2018; 97: 543-50. doi: 10.1016/J.BIOPHA.2017.10.083.
27. Zhang R, Xia Y, Wang Z, Zheng J, Chen Y, et al. Serum long non coding RNA MALAT-1 protected by exosomes is up-regulated and promotes cell proliferation and migration in non-small cell lung cancer. Biochem Biophys Res Commun 2017; 490: 406–14. doi: 10.1016/j.bbrc.2017.06.055.
28. Zidan HE, Karam RA, El-Seifi OS, Elrahman TA . Circulating long non-coding RNA MALAT1 expression as molecular biomarker in Egyptian patients with breast cancer. Cancer genet 2018; 20: 32-7. doi: 10.1016/j.cancergen.2017.11.005.
29. Wenjian Y, Bai Y, Li Y, Guo L, Zeng P, et al. Upregulation of MALAT-1 and its association with survival rate and the effect on cell cycle and migration in patients with esophageal squamous cell carcinoma. Tumor Biol 2016; 37: 4305-12. doi: 10.1007/s13277-015-4223-3.
30. Quan-jun D, Xie LQ, Li H. Overexpressed MALAT1 promotes invasion and metastasis of gastric cancer cells via increasing EGFL7 expression. Life Sci 2016; 157: 38-44. doi: 10.1016/j.lfs.2016.05.041.
31. Ruilin L, Xue M, Zhang L, Lin ZQ. Long noncoding RNA MALAT1-regulated microRNA 506 modulates ovarian cancer growth by targeting iASPP. Onco Target Ther 2017; 10: 35-42. doi:10.2147/OTT.S112686.
32. Xie H, Liao X, Chen Z, Fang Y, He A, et al. LncRNA MALAT1 inhibits apoptosis and promotes invasion by antagonizing miR-125b in bladder cancer cells. J Cancer 2018; 8: 3803-11. doi: 10.7150/jca.21228.
33. Jijun L, Gao J, Tian W, Li Y, Zhang J. Long non-coding RNA MALAT1 drives gastric cancer progression by regulating HMGB2 modulating the miR-1297. Cancer Cell Int 2017; 17: 44-9. doi: 10.1186/s12935-017-0408-8.
34. Yi H, Lin J, Fang H, Fang J, Li C, et al. Targeting the MALAT1/PARP1/LIG3 complex induces DNA damage and apoptosis in multiple myeloma. Leukemia 2018; 32: 2250-62. doi: 10.1038/s41375-018-0104-2.
35. Chang J, Xu W, Du X, Hou J. MALAT1 silencing suppresses prostate cancer progression by upregulating miR-1 and downregulating KRAS. Onco Targets Ther 2018; 11: 3461–73. doi: 10.2147/OTT.S164131.
36. Khadijeh Z, Shademan M, Ghahramani Seno MM, Dehghani H. CRISPR/Cas9 knockout strategies to ablate CCAT1 lncRNA gene in cancer cells. Biol Proced Online 2018; 1: 1-12. doi: 10.1186/s12575-018-0086-5.
37. Said AH, Boshra MS, El Meteini MS, Shafei AE, Matboli M. lncRNA-RP11-156p1. 3, novel diagnostic and therapeutic targeting via CRISPR/Cas9 editing in hepatocellular carcinoma. Genomics 2020; 112: 3306-14. doi: 10.1016/j.ygeno.2020.06.020.
38. Tingting J, Wang F, Qiao B, Ren Y, Xing L, et al. Knockdown of lncRNA pandar by crispr-dCas9 decreases proliferation and increases apoptosis in oral squamous cell carcinoma. Front Mol Biosci 2021; 8: 68-75. doi: 10.3389/fmolb.2021.653787.
39. Shuai Z, Hua L, Liu YH, Sun XM, Jiang MM, et al. Inhibition of long non-coding RNA UCA1 by CRISPR/Cas9 attenuated malignant phenotypes of bladder cancer. Oncotarget 2017; 8: 9634-46. doi: 10.18632/oncotarget.14176.
40. Yi-Qiang Z, Pei JH, Shi SS, Guo XS, Cui GY, et al. CRISPR/Cas9-mediated knockout of the PDEF gene inhibits migration and invasion of human gastric cancer AGS cells. Biomed Pharmacother 2019; 111: 76-85. doi: 10.1016/j.biopha.2018.12.048.
Send email to the article author

Add your comments about this article
Your username or Email:


Ethics code: IR.IAU.M.REC.1399.010

XML   Persian Abstract   Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Ahmadi-Baloutaki S, Doosti A, Jaafarinia M, Goudarzi H. Editing of the MALAT1 Gene in MDA-MB-361 Breast Cancer Cell Line using the Novel CRISPR Method. sjimu 2022; 30 (2) :18-31
URL: http://sjimu.medilam.ac.ir/article-1-7147-en.html

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Volume 30, Issue 2 (6-2022) Back to browse issues page
مجله دانشگاه علوم پزشکی ایلام Journal of Ilam University of Medical Sciences
Persian site map - English site map - Created in 0.16 seconds with 31 queries by YEKTAWEB 4514