| Background: Esophageal cancer is one of common human malignancies and is the sixth most common cause of cancer mortality and the eighth most common type of cancer worldwide. Despite advances in the diagnosis and treatment of this type of cancer, the survival rate at 5 years is poor. Almost 90% of esophageal cancer is esophageal squamous cell carcinoma(ESCC) and adenocarcinoma. In our country, ESCC is more common than other types. Although a lot of researches have been done on ESCC, the molecular mechanisms underlying ESCC have not yet been fully unraveled. Micro RNAs are a class of single-stranded, non-coding RNAs. They are 17-25 ribonucleotides in length. mi RNAs suppress m RNA expression at the post-transcriptional level by binding to the 3’-untranslated region(UTR) of target m RNAs. Aberrant expression of specific mi RNAs has been investigated in many human cancers and mi RNAs function as a novel class of oncogenes and tumor suppressor genes. Some researches show thatmi RNAs could play important roles in tumorigenesis and progression of ESCC. For example, mi R-21 has been shown to be significantly upregulated in ESCC tissues and to regulate cell proliferation and invasion through the suppression of phosphatase and tensinhomolog(PTEN). However, mi R-203 and mi R-205 have been reported to be downregulated in ESCC and regulate tumor proliferation by Np63 and ZEB2, respectively. Researches on the functions of mi RNAs in ESCC will help to find new detectable diagnostic and therapeutic marker for ESCC.Aims:(1) To detect the mi RNA expression profiles between ESCC tissues and corresponding normal esophageal tissues.(2) To investigate the expression level of mi R-1 and to explore possible mechanism of mi R-1 in ESCC by vivo and vitro researches.Methods:(1) The mi RNA expression profiles were detected and differentially expressed mi RNAs were identified in ESCC tissues and corresponding normal esophageal tissues using mi RNA microarray.(2) The accuracy of the mi RNA microarray results was verified by quantitative real-time RT-PCR, due to the absolute fold change, mi R-1 was selected for further analysis.(3) RT-q PCR was performed to confirm the expression levels of mi R-1 in 64 pairs of primary ESCC samples. And the correlation of mi R-1 downregulation with clinicopathological characteristics was investigated.(4) RT-q PCR was performed to confirm the expression levels of mi R-1 in ESCC cells.(5) ESCC cells were transfected with mi R-1 mimics or inhibitors respectively, the effects of mi R-1 on biological behavior of ESCC cells were detected by MTT assay, colony formation assay, the scratch test and transwell assays.(6) Nude mouse transplantation tumor experiment was conducted to detect the oncogenicity of ESCC cells treated by mi R-1 agomir, mi R-1 agomir control and saline.(7) The target genes regulated by mi R-1 were predicted by bioinformatics web sites, PREX1 was selected for further analysis.(8) Luciferase reporter assay was performed to confirm the effect of mi R-1 on PREX1.(9) RT-q PCR was performed to confirm the expression levels of PREX1 m RNA in ESCC cells.(10) ESCC cells were transfected with mi R-1 mimics, PREX1 m RNA level was detected by RT-q PCR and protein level was detected by Western blot.(11) Immunohistochemical staining was performed to detect the expression of PREX1 in ESCC tissues, and the correlation of PREX1 with mi R-1 wasinvestigated.Results:(1) A paired t-test was used to assess the differentially expressed mi RNAs between the cancer and normal esophageal tissue samples and differences at P<0.05 were considered statistically significant. With the 2-fold cut-off point, 27 downregulated and 16 upregulated mi RNAs were detected in the ESCC tumor tissue samples compared with their normal counterparts. However, with the 4-fold cut-off point, 15 downregulated and 9 upregulated mi RNAs were detected in the ESCC tumor tissue samples compared with their normal counterparts.(2) Hierarchical cluster analysis of these 43 mi RNAs was performed. The analysis revealed that the cancer tissue samples were grouped separately from the normal esophageal tissue samples and this created 2 major cluster branches. To confirm the results from microarray analysis, 9 mi RNAs were selected for validation by RT-q PCR. All the mi RNAs examined showed a pattern of up- or downregulation similar to that obtained by microarray analysis. Due to the absolute fold change, mi R-1 was selected for further analysis.(3) mi R-1 was significantly downregulated in the primary ESCC samples compared with the corresponding normal esophageal tissue samples. Among all the samples, 90.6%(58/64) of the ESCC tissue samples showed a 2-fold lower expression of mi R-1. And the downregulation of mi R-1 significantly correlated with tumor invasion and an advanced clinical stage.(4) mi R-1 was significantly downregulated in 5 ESCC cells. mi R-1 over-expression by transfection inhibited the cell growth rate, colony forming ability, scratch repair rate and invasion ability, then down-regulation of mi R-1 exhibited the opposite effect. In nude mouse transplantation tumor experiment, mi R-1 overexpression inhibited oncogenicity of ESCC cells.(5) Target genes of mi R-1 were predicted using the 3 databases respectively. Among these, 181 genes were common to all 3 databases. The predicted target genes of mi R-1 were then classified by GO enrichment analysis. The results showed that the significantly enriched target genes of mi R-1 were associated with transcription regulator activity, sequence-specific DNA binding, regulation of transcription and vasculature development.(6) Luciferase reporter assay showed that mi R-1 over-expression remarkably repressed the expression of luciferase from a vector containing the wild-type(PREX1 3’UTR) mi R-1 binding site, but had no effect on mutant and negative controlvector.(7) The RT-q PCR results revealed that there was a negative correlation between mi R-1 and PREX1 expression in ESCC cells. RT-q PCR and western blotting analysis revealed that mi R-1 over-expression caused degradation of PREX1 m RNA and its protein levels.(8) Immunohistochemical staining showed that PREX1 was significantly upregulated in the primary ESCC samples compared with the corresponding normal esophageal tissue samples with cytoplasm localization. The statistical analysis showed there was a negative correlation between mi R-1 and PREX1 expression in ESCC tissues.Conclusions:(1) mi R-1 was markedly downregulated in ESCC and inhibited ESCC cell proliferation, invasion and migration.(2) PREX1 was a target gene of mi R-1 and its expression was negatively regulated by mi R-1. All of this is useful to investigate new potential therapeutic target for treatment of ESCC. |
PDF Full Text Request