| Objective: This thesis analyzes and explores the influence and possible mechanism of mi R34 a on the chemosensitivity of esophageal squamous cell carcinoma,and provides new ideas for enhancing the chemotherapy effect of esophageal squamous cell carcinoma by targeting key micro RNA.Methods:(1)CCK8 assay was used to detect the effect of mi R34 a on the chemosensitivity of esophageal squamous cell carcinoma(ESCC)cells.(2)The morphological changes of ESCC cells were observed after overexpression of mi R34 a and treatment with cisplatin by inverted microscope.(3)Plate cloning,q RT-PCR and Western blot technology were used to detect the effects of overexpression of mi R34 a combined with cisplatin on cell proliferation and apoptosis of esophageal squamous cell carcinoma cells and the expression of apoptosis-related proteins Bcl-2 and Bax in esophageal squamous cell carcinoma stem cell-like cells.(4)Western blot assay was used to detect the expression of FNDC3 B and PI3K/AKT signaling pathway related proteins in esophageal squamous cell cells after overexpression of mi R34 a and combined treatment with cisplatin;The expression of PI3K/AKT signaling pathway related proteins in esophageal squamous cell carcinoma stem cells-like cells treated with overexpression of mi R34 a and FNDC3 B combined with cisplatin were detected.(5)An animal model of human esophageal squamous cell carcinoma xenografted tumor in nude mice was established,and the effect of combined intervention of overexpression of mi R34 a and cisplatin on the growth of xenografted tumor and the morphology of tumor cells were observed by using hematoxylin-eosin staining technique.(6)The expression of proliferation-related protein Ki-67,apoptosisrelated protein Bcl-2 and Bax,and stemness-related protein BMI-1,CD44 and Nanog in transplanted tumors after overexpression of mi R34 a and cisplatin combined treatment were detected by immunohistochemistry.(7)Western blot analysis was detected the expression levels of FNDC3 B and PI3K/AKT signaling pathwayrelated proteins in transplanted tumors after mi R34 a was overexpressed and treated with cisplatin.(8)Statistical analysis was performed using SPSS 25.0 software.Results:(1)Under the action of different concentrations of cisplatin,the survival rate of esophageal squamous cell carcinoma cells decreased after overexpression of miR34a(P<0.05).(2)After overexpressing mi R34 a and treating with cisplatin,the density of esophageal squamous cell carcinoma cells was significantly reduced,and the cells gradually shrunk,became round and lost their normal shape.(3)Overexpression of mi R34 a combined with cisplatin decreased the colony formation ability of esophageal squamous cell carcinoma cells,up-regulated the expression of pro-apoptotic protein Bax in esophageal squamous cell carcinoma cells and esophageal squamous cell carcinoma stem cell-like cells,and down-regulated the expression of anti-apoptotic protein Bcl-2 expression(all P<0.05).(4)Four groups of esophageal squamous cell carcinoma xenograft models were successfully established in nude mice(Negative control lentivirus and sodium chloride group,overexpression of mi R34 a and sodium chloride group,negative control lentivirus and cisplatin group,and overexpression of mi R34 a and cisplatin group).Microscopy showed diffuse growth of tumor cells in each group and significant cellular dysplasia,with extensive necrosis observed in overexpression of mi R34 a combined with cisplatin group.(5)After overexpression of mi R34 a combined with cisplatin intervention,tumor growth was inhibited,and the expression levels of proliferation-related protein Ki-67,anti-apoptotic protein Bcl-2,stemness-related proteins BMI-1,CD44,and Nanog were downregulated,while the expression of pro-apoptotic protein Bax was upregulated in the transplant tumors(P<0.05 for all).(6)Overexpression of mi R34 a combined with cisplatin intervention decreased the expression levels of FNDC3 B and the phosphorylation level of key proteins in PI3K/AKT pathways in ESCC cells and transplant tumors.Furthermore,the phosphorylation level of key proteins in the PI3K/AKT pathway was higher in ESCC stemlike cells treated with overexpression of mi R34 a combined with FNDC3 B and cisplatin than in overexpression of mi R34 a combined with cisplatin group(P<0.05 for all).Conclusion:(1)Overexpression of mi R34 a increases the sensitivity of esophageal squamous cell carcinoma cells to cisplatin;(2)Overexpression of mi R34 a increases the sensitivity of esophageal squamous cell carcinoma xenografts to cisplatin in nude mice;(3)mi R34 a may inhibit the activation of PI3K/AKT pathway by negatively regulating FNDC3 B,thereby increasing the sensitivity of esophageal squamous cell carcinoma to cisplatin. |
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