Scholarlink

<p>The scratch assay, also known as the wound healing assay, is a widely used in vitro method to study two-dimensional cell migration and is a particularly valuable tool for studying the migratory behaviour of adherent cancerous cells. In the present study, we have demonstrated the role of miR-186 in LN229, a human glioblastoma cell, using our newly developed COS assay method. LN229 lacks the expression of the MGMT (O6-methylguanine-DNA methyltransferase) gene due to methylation in its promoter region and is known as MGMT-negative cells. Lack of MGMT protein makes it suitable for studying the mechanism of glioma formation. Earlier studies have reported both tumour suppressor and Onco-miR roles of miR-186 in different types of cancer; moreover, both up- and downregulation of miR-186 are reported in cancerous cells. In a scratch assay, a major limitation is the identification of spots imaged during different time points. The method describes a standardized scratch assay for glioblastoma cell lines LN229. Scratches made through tips usually differ in width from 5 to 40%. Our new method generates scratch crossovers, which can be followed for monitoring cell migration at marked locations to overcome the width differences. This design improves conventional scratch assays by ensuring consistent imaging of the same location at multiple time points, thereby reducing variability and increasing the reliability of migration measurements. The role of miR-186 is most predominantly demonstrated in glioma, so we have also selected LN229 cells and exposed these cells to temozolomide (TMZ), an alkylating agent used as a chemotherapeutic agent in glioma. Exposure to TMZ substantially decreased the rate of wound closure and down-regulated the expression of miR-186. Similarly, transfection of miR-186 mimics in LN229 cells also substantially decreased wound closure. Downregulating the expression of miR-186 through transfection of the miR-186 inhibitor prevented the effect of temozolomide on wound closure in LN229 cells. In conclusion, using our newly developed COS assay method, we have demonstrated the tumour suppressor role of miR-186 in glioma cells and have identified upregulation in the expression of miR-186 after exposure to TMZ in LN229, which are MGMT-negative glioma cells.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

New Crossover Scratch Assay (COS Assay) Method for Quantifying Cell Migration at Marked Locations Identified Role of miR-186 and Temozolomide (TMZ) in Glioblastoma Cells

  • 期刊论文

  • 发表日期：

    2026年3月5日

查看全文
  • Prasenjit Srivastava,
  • Prabhat Kumar,
  • Pragati Raghuwanshi,
  • Nishant Singh,
  • Sadashiv,
  • Sanjay Yadav

摘要

The scratch assay, also known as the wound healing assay, is a widely used in vitro method to study two-dimensional cell migration and is a particularly valuable tool for studying the migratory behaviour of adherent cancerous cells. In the present study, we have demonstrated the role of miR-186 in LN229, a human glioblastoma cell, using our newly developed COS assay method. LN229 lacks the expression of the MGMT (O6-methylguanine-DNA methyltransferase) gene due to methylation in its promoter region and is known as MGMT-negative cells. Lack of MGMT protein makes it suitable for studying the mechanism of glioma formation. Earlier studies have reported both tumour suppressor and Onco-miR roles of miR-186 in different types of cancer; moreover, both up- and downregulation of miR-186 are reported in cancerous cells. In a scratch assay, a major limitation is the identification of spots imaged during different time points. The method describes a standardized scratch assay for glioblastoma cell lines LN229. Scratches made through tips usually differ in width from 5 to 40%. Our new method generates scratch crossovers, which can be followed for monitoring cell migration at marked locations to overcome the width differences. This design improves conventional scratch assays by ensuring consistent imaging of the same location at multiple time points, thereby reducing variability and increasing the reliability of migration measurements. The role of miR-186 is most predominantly demonstrated in glioma, so we have also selected LN229 cells and exposed these cells to temozolomide (TMZ), an alkylating agent used as a chemotherapeutic agent in glioma. Exposure to TMZ substantially decreased the rate of wound closure and down-regulated the expression of miR-186. Similarly, transfection of miR-186 mimics in LN229 cells also substantially decreased wound closure. Downregulating the expression of miR-186 through transfection of the miR-186 inhibitor prevented the effect of temozolomide on wound closure in LN229 cells. In conclusion, using our newly developed COS assay method, we have demonstrated the tumour suppressor role of miR-186 in glioma cells and have identified upregulation in the expression of miR-186 after exposure to TMZ in LN229, which are MGMT-negative glioma cells.