| Part Ⅰ Study on miRNA-125a inhibiting proliferation of Laryngeal cancer cellObjective Our previous study found that overexpressed miRNA-125a can inhibit the proliferation of laryngeal carcinoma Hep2 cell line.In this part of the study,a series of in vitro cell function tests were conducted to investigate the effects of miRNA-125a on the proliferation of laryngeal cancer Hep2 and FaDu cell lines,and to find possible target genes.Method The laryngeal cancer Hep2 and FaDu cell lines were transfected with miRNA-125a mimic and inhibitor respectively.Colony formation assay and soft agar colony formation assay were used to observe the effect of miRNA-125a overexpression or inhibition on proliferation of laryngeal cancer Hep2 and FaDu cell lines,the effects of miRNA-125a overexpression or inhibition on laryngeal cancer Hep2 and FaDu cell lines were analyzed by Brdu and flow cytometry.Western blot,qRT-PCR,GFP reporter gene and Dual luciferase activity assay were used comprehensively to study the regulation of miR-125a on possible target genes in laryngeal cancer cells.Finally,changes of cell proliferation ability after knocking out the target gene ErbB3 were observed at the cellular level by MTT assay,colony formation assay and soft agar colony formation assay.Result Both colony formation assay and soft agar colony formation assay indicated that miRNA-125a expression significantly inhibited the proliferation of laryngeal cancer Hep2 and FaDu cell lines;Brdu assay showed that miR-125a can reduce the proportion of S phase cells in laryngeal cancer Hep2 and FaDu cell lines.miR-125a may inhibit the laryngeal carcinoma cells proliferation by blocking the cell cycle;flow cytometry results suggested that miR-125a can arrest the laryngeal cancer Hep2 and FaDu cell lines in G0/G1 phase,and decrease in S phase and G2/M phase cells;By western blot,qRT-PCR,GFP reporter gene and dual luciferase activity assay,we found that miR-125a has a direct negative regulation on ErbB3,and miR-125a can significantly inhibit ErbB3 expression thereby inhibiting laryngeal cancer cells proliferation.Finally,we used MTT assay,clone formation assay,and soft agar colony formation assay to verify the transfection of miR-125a-inhibitor at the cellular level.After knocking out the target gene ErbB3,the proliferation of laryngeal carcinoma cells was significantly inhibited compared with the non-knockout group.Conclusion The down-regulation of miR-125a expression in laryngeal cancer tissue suggests that it may play a role as tumor suppressor gene in laryngeal cancer;miR-125a may inhibit the proliferation of laryngeal cancer by acting on the target gene ErbB3 downstream;miR-125a may inhibit the proliferation of laryngeal cancer cells by affecting the cell cycle.Part Ⅱ Screening differential expression profiles of miRNAs associated with laryngeal lymph node metastasis by using gene chip technologyObjective Using miRNA chip to screen differential miRNAs associated with lymph node metastasis of laryngeal squamous cell carcinoma,provides a new direction for the study of laryngeal metastasisMethod Five cases of laryngeal squamous cell carcinoma were each collected from lymph node metastasis group and non-metastasis group.miRNA microarray gene chip analyses were performed on 10 specimens,to obtain miRNA differential expression profile in laryngeal carcinoma tissues of lymph node metastasis group and non-metastasis group.Using the Chip Significance Analysis of Microarrays software(SAM),to screen the miRNAs with significant differential expression in laryngeal squamous cell carcinoma tissue with or without lymph node metastasis.Result A total of 22 miRNA expressions were significantly up-regulated and 387 miRNA expression was significantly down-regulated in tumor tissues of lymph node metastasis group compared with non-metastasis group.Conclusion There are significant differential expression of miRNA in laryngeal squamous cell carcinoma with or without lymph node metastasis.These differential expressions of miRNA may play an important role in the metastasis and progression of laryngeal cancer and lay a preliminary foundation for relative studies of laryngeal cancer metastasis.Part Ⅲ Real-time quantitative PCR(qRT-PCR)was used to verify the results of gene chip analysisObjective Real-time quantitative PCR(qRT-PCR)was used to validate differential miRNAs associated with lymph node metastasis of laryngeal squamous cell carcinoma screened by gene chip.Method A total of 40 cases of laryngeal squamous cell carcinoma were randomly selected from the specimen bank of Guangdong Provincial People’s Hospital.20 cases with lymph node metastasis and 20 cases without lymph node metastasis.The qRT-PCR experiments were performed with U6 as the internal reference to detect Expression levels of miR-125a-5p.miR-144-3p,miR-193a-3p,miR-24-3p and miR-203 in 40 cases of laryngeal squamous cell carcinoma.Result qRT-PCR results validated that the expression levels of miR-125a-5p,miR-144-3p,miR-193a-3p,miR-24-3p,and miR-203 were significantly down-regulated in laryngeal squamous cell carcinoma,and the expression differences in laryngeal cancer tissue with or without lymph node metastasis has its statistical significance(P<0.05).Conclusion miR-125a-5p,miR-144-3p,miR-193a-3p,miR-24-3p and miR-203 were significantly different between the lymph node metastasis group and the non-metastasis group of laryngeal squamous cell carcinoma,which may be associated with lymph node metastasis of laryngeal squamous cell carcinoma,laying a preliminary molecular biological basis for further study of lymph node metastasis of laryngeal squamous cell carcinoma... |
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