Effect Of MiR-34a/FNDC3B On The Growth Of Transplanted Tumor In Nude Mice With Esophageal Squamous Cell Carcinoma And Its Possible Mechanism

Objective:Our research group’s previous experiments found that miR-34a was underexpressed in esophageal squamous cell carcinoma(ESCC),and inhibited the proliferation,invasion and migration of esophageal cancer cells by targeting FNDC3B.The purpose of this study is as follows:Objective:To investigate the effect of miRNA-34a and FNDC3B gene overexpressed recombinant lentiviral vector on the growth of transplanted tumor in ESCC nude mice and its possible mechanism.Methods:(1)Construction of Eca109 stable transfected cell lines:Human esophageal carcinoma Eca109cells were divided into four groups:Lentivirus negative control group(LV-control),miRNA-34a overexpression group(LV-miRNA-34a+LV-control),FNDC3B overexpression group(LVFNDC3B+LV-control),miR-34a and Mir-34a/FNDC3B co-overexpression group(LV-miRNA-34a+LV-FNDC3B).The overexpressed lentivirus vectors of miR-34a and FNDC3B were constructed respectively to transfect esophageal cancer Eca109 cells.(2)After 72h of lentivirus infection,the total protein of each group was extracted,and the m RNA expression level of miR-34a was detected by q RT-PCR.The protein expression level of FNDC3B was detected by Westernblot to confirm the transfection efficiency.(3)Construction of transplanted tumor model of nude mice:Twenty 4-week-old male nude mice were purchased and randomly divided into 4 groups with 5 mice in each group.Four groups of stably transfected Eca109 cells at the expansion stage were digested into single cells,and cell suspension was prepared,and 3×10~6cells per cell were injected into the right axillary subcutaneously of nude mice to establish the subcutaneously transplanted tumor model of ESCC nude mice.The body weight and xenograft tumor volume of nude mice were measured every 3 days since the injection of esophageal cancer cells,and the tumor growth curve was drawn.On the 21st day of tumor formation,nude mice were sacrificed.Part of transplanted tumor tissue was frozen in liquid nitrogen,and the other part was fixed with 10%formaldehyde solution and embedded with paraffin..(4)Hematoxylin-eosin staining(HE)was used to observe the microscopic morphology of the transplanted tumor.Immunohistochemistry(IHC)was used to detect the changes of Bmi-1,CD44 and Nanog proteins in transplanted tumors of ESCC nude mice.The changes of FNDC3B,PI3K/AKT pathway key molecules and MMP-2 and MMP-9 proteins in the transplanted tumor of ESCC nude mice were detected by Westernblot.(5)Bioinformatics technology was used to analyze the correlation between FNDC3B and MMP-2 and MMP-9 protein expression and the enrichment analysis of FNDC3B related pathway in esophageal cancer.Results:(1)After repeated optimization,the infection efficiency of Eca109 cells of esophageal cancer could reach 80%under the condition of MOI=10 and Hitrans G A supplemented with 8μg/m L of 1640basic medium,which was the best infection condition of virus.(2)q RT-PCR results showed that compared with negative control group,m RNA expression level of miRNA-34a in Eca109 cells in miRNA-34a overexpression group was significantly increased(P<0.05).Western blot results showed that compared with negative control group,the expression level of FNDC3B protein in Eca109 cells of recombinant lentivirus FNDC3B overexpression group was significantly increased(P<0.05).(3)The results of in vivo nude mouse tumor transplantation showed that the tumor formation rate of the four groups was 100%;The growth curve of transplanted mice showed that compared with the negative control group,the growth rate of transplanted mice significantly decreased after miRNA-34a overexpression,and significantly increased after FNDC3B overexpression.The high expression of miRNA34a reversed the promoting effect of FNDC3B on the growth of transplanted mice.(4)HE staining results showed that the transplanted tumor tissues in the four groups had the characteristics of cancer cells under the microscope,the shape of tumor cells was irregular,the atypia was obvious,and the mitotic images were common.(5)Immunohistochemical results showed that after miRNA-34a overexpression,dry related molecules Bmi-1,CD44 and Nanog were low expression in transplanted tumor tissues of nude mice,while Bmi-1,CD44 and Nanog were high expression in transplanted tumor tissues of FNDC3B overexpression group.High expression of miRNA-34a reversed the promoting effect of FNDC3B on the expression of dry related molecules in transplanted tumor tissues of nude mice.(6)Western blot results showed that highly expressed miRNA-34a inhibited the expression of FNDC3B in transplanted tumor of nude mice.And the expression of MMP-2 and II MMP-9 in transplanted tumor tissue of nude mice decreased after miRNA-34a overexpression.After overexpression of FNDC3B,the protein expressions of MMP-2 and MMP-9 in transplanted tumor tissues were significantly increased,and miRNA-34a was high enough to reverse the promoting effect of FNDC3B on the expression of invastion-related molecules in transplanted tumor tissues of nude mice.In addition,the results also showed that the phosphorylation level of key proteins in PI3K/AKT pathway decreased after miRNA-34a overexpression,while the phosphorylation level of key proteins in PI3K/AKT pathway increased significantly after FNDC3B overexpression.High expression of miRNA-34a reverses the promotion of FNDC3B to phosphorylation of key proteins of PI3K/AKT pathway in transplanted tumor tissue of nude mice.(7)Bioinformatics analysis showed that FNDC3B was positively correlated with the expression of MMP-2 and MMP-9 in esophageal cancer.The enrichment analysis of FNDC3B single gene pathway showed that PI3K-Akt signaling pathway was one of the major enrichment pathways.Conclusion:(1)miR-34a inhibited the growth of transplanted tumors in ESCC nude mice,and inhibited the expression levels of molecu Les related to dryness and invasion in transplanted tumors.(2)FNDC3B promoted the growth of transplanted tumor in ESCC nude mice,and promoted the expression of molecu Les related to dryness and invasion of transplanted tumor in ESCC nude mice.(3)miR-34a/FNDC3B may inhibit the growth and metastasis potential of transplanted ESCC nude mice through PI3K/AKT/GSK-3βsignaling pathway.