| Objection: We focused on exploring the inhibition mechanism of miR-23 a in endometrial cancer.By detecting the expression of miR-23 a in endometrial tissues,para-carcinoma tissues and endometrial cancer cell lines,we searched for the downstream target of miR-23 a and its effect on SIX1,and analyzed the biological function of SIX1.Prove the regulation of miR-23 a on the SIX1 expression in endometrial cancer,provide a theoretical basis and basis for the clinical diagnosis and treatment of endometrial cancer.Method: Part 1: Study the expression levels of miR-23 a in endometrial tissues,para-carcinoma tissues,and endometrial cancer cell lines: 1)Firstly,we measured the expression levels of miR-23 a in 16 pairs of human tissues,all the specimens were obtained from the gynecology department of the Second Hospital of Tianjin Medical University.The patients who underwent surgical resection were collected fresh endometrial cancer tissues and para-carcinoma tissues without any intervention.The expression of miR-23 a in endometrial cancer tissues and para-carcinoma tissues was detected by qRT-PCR.2)Ishikawa cells and HEC1 B cell lines were cultured,and the expression of miR-23 a in each cell line was detected by qRT-PCR.The miR-23 a mimetic or miR-23 a ASO was transfected into Ishikawa cells or HEC1 B cells to validate the miR-23 a expression level.3)To test the role of miR-23 a in endometrial cancer cell lines,transfect miR-23 a mimetic or miRNA con into Ishikawa cells or HEC1 B cells,through cell function assays such as colony formation assay,scratch assay,transwell assay were performed to detect cell proliferation,migration and invasion in each group.Part2: SIX1 was proved to be a downstream target of miR-23 a and we analyzed the biological function of SIX1.1)First,we found SIX1 on the miR-23 a target list through software: microRNA.org,TargetScan,miRanda and other software.Then we conducted bioinformatics or luciferase reporter gene assay to prove the regulation of miR-23 a on the SIX1 expression.To test the possibility of the direct link between miR-23 a and SIX1,the SIX1 3’UTR with either a wild-type or a mutant miR-23 a target sequence downstream of the firefly luciferase gene was inserted.The pGL3-SIX1 UTR WT,pGL3-SIX1 UTR Mut and pGL3 constructs were transfected into HEC1 B cells,respectively,and the miR-23 a mimics or miRNA con was administered to detect luciferase activity.2)To verify the Correlation between miR-23 a and SIX1 expression in endometrial cancer tissues,we tested their expression in 30 endometrial cancer cases.We tested SIX1 expression by IHC,and we divided 30 cases into 2 groups: SIX1 low and high expression groups.In addition,we tested miR-23 a expression in the above two groups by qRT-PCR(SIX1 low and high expression groups: n = 15 each group).To analyze whether there is a correlation between the expression of miR-23 a and SIX1 in endometrial cancer tissues.3)To investigate the influence of SIX1 on the biological behaviors of endometrial cancer cells,SIX1 plasmid or siRNA was used to raise or reduce the expression of SIX1 in HEC1 B or Ishikawa cells,through cell function tests such as colony formation assay,scratch assay,transwell assay.The proliferation,migration and invasion of cells in each group were examined.4)SIX1 protein expression level was detected by treatment with different miR-23 a in a human endometrial cancer cell line by Western blotting.In HEC1 B cells,the expression level of SIX1 protein was detected by treatment with miR-23 a ASO or ASO con group;SIX1 protein expression level was detected by treatment with miR-23 a mimic or miRNA con in Ishikawa cells.Part3: Preliminary study of miR-23 a inhibits the development of endometrial cancer by targeting SIX1.1)Reversal of cell proliferation,migration and invasion by adding SIX1 siRNA or plasmid after simultaneous removal or overexpression of miR-23 a.The expression level of miR-23 a in different treatment groups was examined by qRT-PCR in HEC1 B or Ishikawa cells.The expression level of SIX1 protein in different treatment groups was examined by Western blotting.Cell function assays,such as colony formation assay,scratch assay,and transwell assay,were used to detect cell proliferation,migration,and invasion in different treatment groups.2)In order to verify the inhibitory effect of miR-23 a mimic and/or paclitaxel Taxol and determine whether miR-23 a regulates SIX1 expression in vivo,Ishikawa cells were injected subcutaneously in the right flank of nude mice.Tumor volume was measured in different treatment groups.SIX1 expression in mouse tumors was assessed by Western blot.Result: Part 1: 1)The miR-23 a expression level had an average of 42% reduction in endometrial cancer tissues compared with the paired para-carcinoma tissues.(P<0.05,respectively).2)The expression of miR-23 a was increased or decreased in miR-23 a mimic or miR-23 a ASO transfected into Ishikawa cells or HEC1 B cells(P < 0.05,respectively).3)Cell function experiments showed that overexpression of miR-23 a inhibited cell proliferation,migration,and invasion of Ishikawa cell line and HEC1 B cell line(P < 0.05,respectively): The results of colony formation assay showed that capacity for proliferation was significantly weakened in miR-23 a mimics transfection cells compared to control(P < 0.05,respectively);Besides,the result of scratch assay showed a significantly wider space in miR-23 a mimics transfection cells compared to the control(P < 0.05,respectively);Moreover,the result of transwell assay showed significantly fewer cells in miR-23 a mimics transfection cells compared to the control(P< 0.05,respectively).Part2: 1)We found SIX1 was on the list of miR-23 a targets suggested by microrna software(http://www.microrna.org/microrna/getGeneForm.do).To test the possibility of the direct link between miR-23 a and SIX1,the SIX1 3’UTR with either a wild-type or a mutant miR-23 a target sequence downstream of the firefly luciferase gene was inserted.The pGL3-SIX1 UTR WT,pGL3-SIX1 UTR Mut and pGL3 constructs were individually transfected into HEC1 B cells.miR-23 a significantly reduced the relative luciferase activity of the wild type SIX1 3’UTR(P <0.05),but miR-23 a could not reduce the relative luciferase activity of the mutant SIX1 3’UTR(P> 0.05).Our results indicate that miR-23 a may bind directly to the SIX1 3’UTR.2)There was a significant difference in the expression of miR-23 a between SIX1 low and high expression tissue samples(P < 0.05),indicating a negative correlation between miR-23 a and SIX1 expression in endometrial cancer tissues.3)Compared with the control group,the expression of SIX1 protein in HEC1 B or Ishikawa cells was increased or decreased in the SIX1 plasmid group or the siRNA group,respectively(P < 0.05).The results of colony formation assay showed that capacity for proliferation caused by the increased or reduced expression of SIX1 was significantly enhanced or weakened compared to the control in HEC1 B or Ishikawa cells,respectively(P<0.05).The results of scratch assay indicated the significantly smaller or bigger width caused by the raised or reduced SIX1 than that in the control of HEC1 B or Ishikawa cells,respectively(P<0.05).The results of transwell assay demonstrated the significantly more or fewer cells caused by the increased or decreased expression of SIX1 than that in the control in HEC1 B or Ishikawa cells,respectively(P<0.05).To sum up,the results indicated that over-expression or loss of SIX1 level promoted or inhibited cell proliferation,migration and invasion in vitro.4)Therefore,consistent with the previous results,through the different miR-23 a treatment in the two kinds of endometrial cancer cells,our results showed the SIX1 protein expression level was higher treated with miR-23 a ASO than that treated with ASO control in the HEC1 B cells(P<0.05).In addition,our results showed that the SIX1 protein expression level was lower treated with miR-23 a mimics than that treated with miRNA con in the Ishikawa cells.To sum up,miR-23 a could down-regulate the SIX1 expression in endometrial cancer cells.Part3: 1)We reversed the cell proliferation,migration and invasion abilities by adding SIX1 siRNA or plasmid following the loss or the over-expression of miR-23 a at the same time.The miR-23 a expression was reduced in the miR-23 a ASO group in HEC1 B cells(P<0.05),while the decreased miR-23 a expression was not statistically different between the SIX1 siRNA+miR-23 a ASO group and the miR-23 a ASO group(P>0.05).Similarly,the miR-23 a expression was increased in the miR-23 a mimics(P<0.05),while the increased miR-23 a expression was not statistically different between the SIX1 plasmid+miR-23 a mimics group and the miR-23 a mimics group(P>0.05).The results proved that the loss or the over-expression of miR-23 a was not increased or reduced by adding SIX1 siRNA or plasmid.Moreover,the SIX1 expression was increased or reduced following the loss or the over-expression of miR-23 a in the miR-23 a ASO or mimics group(P<0.05,respectively),and the SIX1 expression was reduced or increased by adding SIX1 siRNA or plasmid in HEC1 B or Ishikawa cells(P<0.05,respectively).The results demonstrated that miR-23 a directly targeted SIX1 in endometrial cancer cells.Besides,the results of clone formation,scracth assay and transwell assay showed that proliferation,migration and invasion abilities were strenthened or weakened,following the loss or the over-expression of miR-23 a in the miR-23 a ASO or miR-23 a mimics group in HEC1 B or Ishikawa cells(P<0.05,respectively),but the changed cells proliferation,migration and invasion abilities following the loss or the over-expression of miR-23 a could be rescued by the down-regulation or up-regulation of SIX1 in HEC1 B or Ishikawa cells(P<0.05,respectively).In a word,the results above indicated that miR-23 a could promote the development of endometrial cancer by targeting SIX1.2)The mean tumor volume in the Taxol group was significantly lower compared to the control group(P <0.05).The mean tumor volume was significantly lower for the miR-23 a mimics and miR-23 a mimics + Taxol group compared to the mimics control group(P <0.05,respectively).Besides,the mean tumor volume was significantly lower in the miR-23 a + Taxol group compared to the Taxol group(P <0.05).In addition,we evaluated SIX1 expression in mice tumors by western blot and showed that the expression of SIX1 was decreased due to miR-23 a mimics and/or Taxol(P <0.05).Moreover,compared to the Taxol group,the expression of SIX1 in miR-23 a mimics + Taxol group was decreased significantly(P<0.05).There was no significant difference in the body weight of mice treated with miR-23 a mimics and/or Taxol,none of the mice tested showed signs of other adverse effects as described in the Methods section,and no toxic effects were observed through blood counts or the observation of liver As well as renal function.These results demonstrated the antitumor effects and safety of miR-23 a mimics and/or Taxol.Taken together,these results above indicated that miR-23 a could inhibit chemo-resistance of endometrial cancer in vivo by targeting SIX1.Conclusion: In this study,we found that miR-23 a may be a suppressor gene in endometrial cancer cells,and abnormal changes in miR-23a-SIX1 interaction may be associated with the development of endometrial cancer.Our results indicate that SIX1 is regulated by miR-23 a.The up-regulated or down-regulated ability of cell proliferation,migration or invasion following the loss or over-expression of miR-23 a was restored by adding SIX1 siRNA or plasmid in vitro.miR-23 a might be a therapeutic target for endometrial cancer. |
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