Abstract
ObjectiveLAMA3 is a widely studied methylated gene in multiple tumors, but the relationship between chemotherapy resistance in ovarian cancer is unclear. In this study, LAMA3 methylation was predicted by bioinformatics, and the ability of LAMA3 methylation to predict the chemotherapy resistance and prognosis of ovarian cancer was confirmed in experiments.MethodsMultiple databases have performed the bioinformatics analysis of methylation and transcription factor binding site (TFBS) on the promoter region of LAMA3 gene. Pyrosequencing detected the methylation of LAMA3. QRT-PCR and immunohistochemistry detected the expression of LAMA3. Real Time Cell Analyzer (RTCA) detects changes in cell proliferation, migration and invasion ability. Flow cytometry was used to detect apoptosis.ResultsCPG islands of 176 bp, 134 bp, 125 bp and 531 bp were predicted in the promoter region of LAMA3 gene. The 4 prediction results are basically overlapped. 7 transcription factor binding sites were predicted, and the one with the highest score was on the predicted CpG island located in the proximal promoter region. LAMA3 hypermethylation and low expression are both associated with chemotherapy resistance and poor prognosis in ovarian cancer. LAMA3 methylation was negatively correlated with expression. After upregulation of LAMA3, the proliferation ability of chemoresistant ovarian cancer cell decreased, while the ability of apoptosis, invasion and migration increased.ConclusionLAMA3 hypermethylation is associated with chemotherapy resistance and poor prognosis. As a typical CpG island gene, LAMA3(cg20937934) and LAMA3(cg13270625) hypermethylation is negatively correlated with low expression. LAMA3 promotes the invasion, migration and apoptosis of SKOV3DDP. In the future, the mechanism of LAMA3 methylation in ovarian cancer will need to be further studied.
Highlights
Ovarian cancer is one of the three most common malignant tumors of the female reproductive system, and the mortality rate is the highest in gynecological malignancies [1]
CNNPromoter results showed that there are 7 transcription factor binding sites in the upstream promoter region of the Laminin subunit Alpha 3 (LAMA3) gene, and the one with the highest score is the predicted CpG island located in the proximal promoter region (The top four scores are: position:4700, SCORE:1.123; position:3000,SCORE:0.71; position:2300, SCORE:0.686; position:1600,SCORE:0.635; position:900, SCORE:0.669;)
LAMA3 hypermethylation is associated with chemotherapy resistance and poor prognosis In order to investigate the accuracy of bioinformatics prediction of CpG island methylation in the promoter region of LAMA3 gene, this study selected two locis in the LAMA3 TSS200 region for methylation detection
Summary
Ovarian cancer is one of the three most common malignant tumors of the female reproductive system, and the mortality rate is the highest in gynecological malignancies [1]. Chemotherapy resistance greatly reduces the survival rate of ovarian cancer patients, which is an important reason for the high mortality [5]. Chemotherapy resistance in ovarian cancer is related to many factors such as genetics, epigenetics, somatic gene mutations, immune system and tumor microenvironment abnormalities [7, 8]. Abnormal DNA methylation alters gene expression and affects gene function by interfering with the binding of specific transcription factors to the recognition positions in gene promoters [10, 11]. Chemotherapy drugs kill tumor cells by causing DNA damage, inhibiting proliferation, and promoting cell apoptosis. Abnormal methylation levels of the promoters of these related pathway genes can affect the sensitivity of tumor cells to chemotherapy drugs
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