| MicroRNAs(miRNAs)are 18-22 nucleotides of single-stranded RNA small molecules which bind to the 3 'UTR or 5'UTR region of the target gene mRNA through complementary base-pairing principle.They play a role in inhibiting mRNA function by interfering with mRNA translation,or directly degrading mRNA.It has been widely reported that miRNAs are involved in the regulation of tumor occurance and progression.Salivary adenoid cystic carcinoma(SACC)is derived from the salivary gland epithelial cells,and a relatively common salivary gland malignant tumor.Salivary adenoid cystic carcinoma has the following main biological characteristics:the relatively slow growth,high tendency of invasion,easy to spread along the nerve,local recurrence and distant metastasis along the blood to the lung and other organs,unsatisfactory chemotherapy and radiotherapy effect and long-term poor prognosis and so on.Recent studies have revealed thay miR-101-3p could regulate different types of tumors.For example,the anti-tumor effect of miR-101-3p has been reported in a variety of tumors,but the specific mechanisms are not fully elucidated.Studies have shown that miR-101-3p is down-regulated in tumors such as gastric cancer,breast cancer,oral squamous cell carcinoma and liver cancer.MiR-101-3p can play a role in promoting apoptosis and metastasis of tumor cells.In our study,miR-101-3p expression was down-regulated in both SACC tissues and SACC cell lines.After overexpression of miR-101-3p,the invasion,proliferation,cloning and formation of SACC cells were significantly inhibited,and the apoptosis of SACC cells was also increased.We found that miR-101-3p targeted proto-oncogene Pim-1(Moloney murine leukemia virus 1).Inhibition of SACC-83 and SACC-LM cells proliferation and invasion ability by miR-101-3p over-expression could be rescued by enhanced Pim-1.Pim-1 not only down-regulated the protein levels of survivin,cyclin D1 and?-catenin,but also enhanced the sensitivity of SACC-83 and SACC-LM cells to cisplatin.In conclusion,the results revealed the mechanism of miR-101-3p/Pim-1 in the occurrence and development of SACC,and may provide new ideas for the treatment of SACC.This study mainly includes the following three experiments.Experiment 1 Study on miR-101-3p inhibition of salivary adenoid cystic carcinoma growth and metastasis in vivo and in vitroObjective:To investigate the expression of miR-101-3p in human salivary adenoid cystic carcinoma and salivary adenoid cystic carcinoma cell lines.Upregulate or downregulate the expression of miR-101-3p by in vitro and in vivo experiments.To observe the effects of 101-3p on SACC cells function.Methods:MiR-101-3p expression in 30 human salivary adenoid cystic carcinoma specimens and 10 human normal parotid gland specimens were detected by real-time PCR.MiR-101-3p expression in the human salivary adenoid cystic carcinoma cell line SACC-83 and the more malignant cell sublines SACC-LM were also detected by real-time PCR.Constructed miR-101-3p overexpression or silencing SACC cell lines by lentivirus,miR-101-3p mimics and miR-101-3p inhibitor.Cell invasion,apoptosis,cloning formation,cell proliferation and nude mouse transplantation tumor experiments were observed.Results:The expression of miR-101-3p in SACC tissues was significantly lower than that in normal tissues,and the expression of miR-101-3p in SACC-LM cells was significantly lower than that in SACC-83 cells.The invasion ability of SACC-83 and SACC-83 cells was significantly suppressed after increased miR-101-3p expression.SACC-LM and SACC-83 cells showed stronger invasion ability when inhibited miR-101-3p compared with the control group.At the same time,the miR-101-3p overexpression group had increased SACC-LM and SACC-83 cells apoptosis.After increased miR-101-3p expression,the cell colony formation of SACC-LM and SACC-83 were significantly suppressed.CCK-8 cell proliferation assay showed that miR-101-3p overexpression significantly inhibited cell proliferation of SACC-LM and SACC-83,and the inhibition of miR-101-3p expression significantly enhanced the cell proliferation ability.In nude mice xenografts assays,tumor growth,tumor volume and tumor mass of the miR-101-3p overexpression group were significantly lower than those in the control group.Immunohistochemical staining showed that Ki67 expression in miR-101-3p overexpressing SACC tumors was significantly lower than that in the control group.The protein levels of Pim-1,survivin,cyclin D1 and?-catenin in miR-101-3p up-regulated group were significantly down-regulated than those in the control group.Conclusion:MiR-101-3p was down-regulated in human salivary adenoid cystic carcinoma tissues and adenoid cystic carcinoma cell lines.MiR-101-3p inhibited SACC cell invasion,proliferation and cell cloning formation,and increased SACC cell apoptosis.MiR-101-3p inhibited the growth of SACC transplanted tumors in nude mice.The expression of Ki67,Pim-1,survivin,cyclin D1 and ?-catenin were inhibited in xenograft in nude when upregulated miR-101-3p.Experiment 2 Study on the mechanism of miR-101-3p inhibited the proliferation and invasion of SACC cells by targeting Pim-1Objective:To explore the target gene of miR-101-3p and to study the effect of miR-101-3p on SACC cells by inhibiting the target gene.Methods:The target gene of miR-101-3p was predicted by TargetScan and miRanda,and the proto-oncogene Pim-1 was selected as the study object by comparison and comprehensive analysis.The expression of miR-101-3p and Pim-1 in human salivary adenoid cystic carcinoma and normal parotid gland tissues were detected by real-time PCR.Compare the expression of miR-101-3p and Pim-1 in human salivary adenoid cystic carcinoma to study the corelation between the two.We used luciferase reporter gene to verify whether Pim-1 was the target gene for miR-101-3p.Western blot was used to detect the expression of Pim-1,survivin,cyclin D1 and ?-catenin in SACC cell lines.The effects of Pim-1 and miR-101-3p on the proliferation and invasion of SACC cells were observed by CCK-8 cell proliferation assay and cell invasion assay.Results:The expression of Pim-1 in human salivary adenoid cystic carcinoma was significantly higher than that in normal parotid gland tissues.Compared with miR-101-3p expression in human salivary adenoid cystic carcinoma,we found that the expression of Pim-1 and miR-101-3p were negatively correlated.In order to further explore the relationship between miR-101-3p and Pim-1,we constructed a wild-type and mutant Pim-1 luciferase reporter gene plasmid,and it was proved that Pim-1 was the target gene of miR-101-3p.The levels of survivin,cyclin D1,and?-catenin in SACC-83 and SACC-LM cells transfected with miR-101-3p mimics and miR-101-3p inhibitor were detected by Western blot.The results were similar to those in in vivo experiments.The expression of survivin,cyclin D1 and ?-catenin was down-regulated by miR-101-3p mimics,and up-regulated by miR-101-3p inhibitors.The expression of Pim-1 in SACC-83 and SACC-LM cells was knocked down with Pim-1 siRNA,and the proliferation of SACC-83 and SACC-LM cells was significantly inhibited.After reducing the expression of miR-101-3p,the proliferation of SACC-83 and SACC-LM cells inhibited by Pim-1 siRNA did not significantly increased.SACC-83 and SACC-LM cells transfected with the Pim-1 plasmid increased cell proliferation ability became stronger.And this enhanced proliferation capacity was not suppressed by overexpression of miR-101-3p.The cell invasion assay showed that up-regulation of Pim-1 expression could promote cell invasion,and rescue cell invasion suppression induced by miR-101-3p over-expression.Conclusion:Pim-1 was the target gene of miR-101-3p.MiR-101-3p inhibited the expression of survivin,cyclin D1 and ?-catenin in SACC-83 and SACC-LM cells.MiR-101-3p inhibits proliferation and invasion of ACC cells by directly down-regulating Pim-1.Experiment 3 MiR-101-3p increased the sensitivity of SACC cells to cisplatinObjective:To study the effect of miR-101-3p on drug sensitivity of SACC cellsMethods:SACC-83 and SACC-LM cells were treated with 10 ?M cisplatin for 24 hours.The expression of miR-101-3p was detected by real-time PCR.The expression of Pim-1 was detected by Western blot.The effect of miR-101-3p on apoptosis and proliferation inhibition of SACC-83 and SACC-LM cells induced by cisplatin was examined by flow cytometry and CCK-8.Results:The expression levels of miR-101-3p and Pim-1 in SACC-83 and SACC-LM cells were detected by real-time PCR and Western blot.The expression levels of miR-101-3p and Pim-1 were detected 24 hours after treatment with SACC-83 and SACC-LM cells with 10 ?M cisplatin.The expression of miR-101-3p was down-regulated and the expression of Pim-1 was up-regulated in SACC-83 and SACC-LM cells treated with cisplatin.SACC-83 and SACC-LM cells were transfected with miR-101-3p mimics and treated with 10?M cisplatin for 24 hours and detected by flow cytometry compared with untransfected cells.The apoptosis of SACC-83 and SACC-LM cells with high expression of miR-101-3p was significantly increased,and the cell proliferation ability was significantly inhibited.These results indicate that miR-101-3p increased the drug sensitivity of SACC cells to cisplatin.Conclusion:MiR-101-3p increased the sensitivity of SACC cells to cisplatin. |
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