| Tongue cancer is the most common malignant tumor in oral cancer.Due to the frequent movement of the tongue,and it is rich in lymphatic vessels,nerves and blood vessels,tongue cancer is more likely to cause local invasion and distant metastasis than other head and neck tumors.Decreased cell-to-cell adhesion is a key step in tumor invasion and metastasis.Cadherin is an adhesion molecule between epithelial cells that plays an important role in establishing and maintaining cell-to-cell junctions.A large number of reports have reported that cadherin is closely related to a variety of tumor invasion and metastasis.Therefore,the aim of this study was to transcriptome sequencing and analysis of the tongue cancer high metastatic cell line constructed by the research group,and select differentially expressed cadherins to study.ObjectivesIn this study,we transcribed and sequenced the self-constructed tongue cancer high-metastasis cell line LN2(second-generation tongue cancer cell high-transfer strain,which was derived from the lymphatic metastasis of the CAL27 cell line of the nude mouse tongue cancer model,transfer rate 25%),tongue cancer high metastatic cell line LN4(fourth-generation tongue cancer cell high-transfer strain,which was derived from the lymphatic metastasis of the CAL27 cell line of the nude mouse tongue cancer model,transfer rate 63%)and CAL27 cell lines,and analyzed differentially expressed cadherin superfamily genes.Based on this,we hypothesized the large differentially expressed cadherin family genes may play an important role in tongue cancer cell lines.Therefore,we explored the role of differentially expressed cadherin genes in tongue cancer cell lines in order to provide a theoretical basis for early molecular diagnosis and targeted therapy of tongue cancer.Materials and MethodsPart ?: the tongue cancer cell line CAL27 and the self-constructed tongue cancer high metastatic cell line LN2 and LN4 were sequenced and analyzed,and the expression of CDH11 gene was significantly decreased in LN2 and LN4 high metastatic cell lines.To further verify the transcriptome sequencing results,Real time-PCR was used to detect mRNA expression in CAL27 and LN4 cell lines.Part ?: CAL27 and TCA8113 tongue cancer cell lines were infected with lentivirus LV-CDH11 to construct a stable overexpressing CDH11 cell line.The overexpression of mRNA and protein in stable overexpressing cell lines was verified by Real time-PCR and Western blotting.The site where CDH11 was expressed in the overexpressing cell line was observed by a cellular immunofluorescence assay.Part ?: The effects of overexpression of CDH11 gene on the function of tongue cancer cell lines CAL27 and TCA8113 were verified by CCK8 proliferation assay,plate cloning assay,transwell chamber assay,tumor stem cell granule assay,adhesion assay,and through human oral epithelial cell migration assay.ResultsPart ?: transcriptome sequencing of tongue cancer high metastatic cell line established in the previous study group showed that CDH11 expression in tongue cancer high metastatic cell line LN4 was lower than that of tongue cancer high metastatic cell line LN2;tongue cancer high metastatic cell line LN2 expression Lower than CAL27 cell line.The Real time-PCR results of CAL27 cell line and tongue cancer high metastatic cell line LN4 showed that CDH11 was lower than CAL27 cell line in tongue cancer high metastatic cell line LN4.Part ?: After infection of tongue cancer cell lines with lentivirus,real time-PCR and Western blotting results showed that the mRNA and protein expression levels of CDH11 in overexpression group of CAL27 and TCA8113 cell lines were higher than those in vector group.Cellular immunofluorescence experiments confirmed that CDH11 overexpression was present on the membrane of CAL27 and TCA8113 cells in the overexpression group relative to the vector group.Part ?: Compared with the vector group,the CCK8 proliferation experiment,the plate cloning experiment,the transwell chamber experiment,and the tumor stem cell granule assay results of the overexpression group were not statistically significant;while in the adhesion assay the adhesion ability of the overexpression group is stronger than the vector group;in the through human oral epithelial cell migration experiment,the ability of the overexpression group to pass through the transwell chamber is weaker than that of the vector group.ConclusionThe expression of CDH11 was down-regulated in the high-metastasis of tongue cancer cells LN2 and LN4.It was confirmed that overexpression of CDH11 has no effect on the proliferation,migration and invasion of CAL27 and TCA8113 tongue cancer cell lines,but it can enhance the adhesion of CAL27 and TCA8113 cell lines and decreased the ability of TCA8113 cells to penetrate human oral epithelial cells for migration. |
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