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Manuscript ID : ASCIJ-2307-1516 (R1) Visit : 555 Page: 147 - 160

10.22034/ascij.2023.1992183.1516

Article Type: Original Research

Functional evaluation of EZH2 Gene Silencing Effect on β-catenin Marker Expression in YM-1 Squamous Cell Carcinoma Cell Line of Esophageal Cancer

Subject Areas : Journal of Animal Biology

Fatemeh Nourmohammadi 1 , Mohammad Mahdi Forghani Fard 2 * , Vajihe Zarrinpour 3 , Mohammad Reza Abbaszadegan 4

1 - Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran
2 - Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran
3 - Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran
4 - Medical Genetics Research Center, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

Received: 2023-07-23 Accepted : 2023-07-31 Published : 2024-02-20

Keywords: β-catenin, EZH2, shRNA, Esophageal squamous cell carcinoma,

Abstract :

Esophageal cancer is the sixth cause of cancer death in the world and the eighth most common cancer in the world. One of the most characteristic features of this cancer is the resistance to chemo-radiotherapy and the high percentage of recurrence in patients. Beta-catenin marker is one of the main factors of metastasis and invasion in patients. EZH2 gene is a regulatory factor of gene expression and increases the proliferation of tumor cells and maintains the pluripotency of stem cells. EZH2 is aberrantly expressed in a variety of malignant cancers. β-Catenin is a structural component of adherens junctions along with cadherin and the main signaling agent of the Wnt pathway in the nucleus. Aberrant expression of beta-catenin often leads to cancer and metastasis. In this study, the EZH2 gene silencing vector was cloned in Escherichia coli bacteria and then extracted, and the EZH2 gene was silenced in YM-1 esophageal cancer cells. Then RNA extraction and cDNA synthesis were evaluated after confirming EZH2 gene silencing, beta-catenin expression using Real-time PCR technique. Cell migration test was used to check the invasion rate of cells after genetic manipulation. The results showed a decrease in beta-catenin expression in cell lines induced by EZH2 silencing. EZH2 silencing significantly reduced the growth and migration rate of esophageal cancer cells. The results indicate the role of EZH2 gene in regulating beta-catenin gene expression in esophageal squamous cell cancer. Considering that tumor spread is one of the most important factors in the malignancy of ESCC cancer, and on the other hand, these features of esophageal cancer cause premature death of patients due to metastasis and invasion, finding an effective marker in controlling cancer stem cells can be promising for the treatment of ESCC patients.

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Full-Text:


زیست­شناسی جانوري، سال شانزدهم، شماره دوم، زمستان 1402، صفحات 160-147، فاطمه نورمحمدی و همکاران

 

مقاله پژوهشی

بررسی عملکردی اثر فاکتور رونویسی EZH2 بر بیان مارکر β-catenin در رده سلولی کارسینومای سلول‌های سنگفرشی مری YM-1

فاطمه نورمحمدی1، محمدمهدی فرقانی فرد1*، وجیهه زرین پور1، محمدرضا عباس­زادگان2

1- گروه زیست­شناسی، واحد دامغان، دانشگاه آزاد اسلامی، دامغان، ایران

2- مرکز تحقیقات ژنتیک پزشکی، دانشکده علوم پزشکی مشهد، مشهد، ایران

*مسئول مکاتبات:  forghanifard@gmail.com

تاریخ دریافت:01/03/1402                      تاریخ پذیرش: 09/05/1402

DOI: 10.22034/ascij.2023.1992183.1516

چکیده

سرطان مری به عنوان ششمین عامل مرگ و میر در اثر سرطان در جهان و هشتمین سرطان شایع در جهان می‎باشد. از شاخص- ­ترین ویژگی­های این سرطان مقاومت به شیمی-رادیو تراپی و درصد بالای عود مجدد در بیماران می‌باشد. مارکر بتا-کاتنین یکی از اصلی­ترین عوامل متاستاز و تهاجم در بیماران می‌باشند. ژن EZH2 یک فاکتور تنطیمی بیان ژن بوده و تکثیر سلول‌های تومور را افزایش می‌دهد و پرتوانی سلول‌های بنیادی را حفظ می‌کند. EZH2 به طور غیرطبیعی در انواع سرطان‌های بدخیم بیان می‌شود. بتا-کاتنین یک جزء ساختاری از اتصالات چسبنده به همراه کادهرین و عامل اصلی سیگنال­دهی مسیر Wnt در هسته است. بیان نابجای بتا-کاتنین (β-catenin) اغلب منجر به سرطان و متاستاز می‌شود. در این مطالعه وکتور خاموش‌سازی ژن EZH2 در باکتری اشرشیا کولی کلون و سپس استخراج شده و در سلول‌های رده سرطانی مری YM-1 ژن EZH2 خاموش شده است. سپس استخراج RNA و سنتز cDNA پس از تایید خاموش‌سازی ژن EZH2 بیان بتا-کاتنین با استفاده از تکنیک Real-time PCR مورد ارزیابی قرار گرفت. تست سلولی مهاجرت سلولی برای بررسی سرعت تهاجم سلول‌ها بعد از دستکاری ژنتیکی استفاده شد. نتایج، کاهش بیان بتا-کاتنین را در رده­های سلولی تحت القای خاموش‌سازی EZH2 نشان داد. خاموش‌سازی EZH2 بطور معنی­داری میزان رشد و مهجرت سلول‌های سرطانی مری را کاهش داد. نتایج حاکی از نقش ژن EZH2 در تنظیم بیان ژن بتا-کاتنین در سرطان سلول‌های سنگفرشی مری می‌باشد. با توجه به این که انتشار تومور یکی از مهمترین عوامل بدخیمی سرطانESCC می‌باشد و از طرفی این ویژگی­های سرطان مری به علت متاستاز و تهاجم موجب مرگ زودرس بیماران می­گردد، یافتن مارکر موثر در کنترل سلول‌های بنیادین سرطانی می‌تواند نویدبخش درمان بیماران ESCC باشد.

کلمات کلیدی: سرطان سلول‌های سنگفرشی مری، بتا-کاتنین، EZH2، shRNA.

Functional Evaluation of EZH2 Gene Silencing Effect on Β-Catenin Marker Expression in YM-1 Squamous Cell Carcinoma Cell Line of Esophageal Cancer

 

Fatemeh Noormohammadi1, Mohammad Mahdi Farzani Fard1*, Vajiheh Zarrinpour1, Mohammad Reza Abbaszadegan2

 

1- Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran

2- Medical Genetics Research Center, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

*Corresponding Author Email: forghanifard@gmail.com

Abstract

Esophageal cancer is the sixth cause of cancer death in the world and the eighth most common cancer in the world. One of the most characteristic features of this cancer is the resistance to chemo-radiotherapy and the high percentage of recurrence in patients. Beta-catenin marker is one of the main factors of metastasis and invasion in patients. EZH2 gene is a regulatory factor of gene expression and increases the proliferation of tumor cells and maintains the pluripotency of stem cells. EZH2 is aberrantly expressed in a variety of malignant cancers. β-Catenin is a structural component of adherens junctions along with cadherin and the main signaling agent of the Wnt pathway in the nucleus. Aberrant expression of beta-catenin often leads to cancer and metastasis. In this study, the EZH2 gene silencing vector was cloned in Escherichia coli bacteria and then extracted, and the EZH2 gene was silenced in YM-1 esophageal cancer cells. Then RNA extraction and cDNA synthesis were evaluated after confirming EZH2 gene silencing, beta-catenin expression using Real-time PCR technique. Cell migration test was used to check the invasion rate of cells after genetic manipulation. The results showed a decrease in beta-catenin expression in cell lines induced by EZH2 silencing. EZH2 silencing significantly reduced the growth and migration rate of esophageal cancer cells. The results indicate the role of EZH2 gene in regulating beta-catenin gene expression in esophageal squamous cell cancer. Considering that tumor spread is one of the most important factors in the malignancy of ESCC cancer, and on the other hand, these features of esophageal cancer cause premature death of patients due to metastasis and invasion, finding an effective marker in controlling cancer stem cells can be promising for the treatment of ESCC patients.

 

Keywords: Esophageal squamous cell carcinoma, EZH2, shRNA, β-catenin.

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