| Esophageal cancer is a common malignant tumor. Its current incidence is 19.34/100,000 population in China, ranking sixth in new cancer cases, and accounting for 7.27% of all cancer. Its mortality rate is 8.96%, which is the fourth most cause of cancer deaths nationwide. According to the histological features, esophageal cancer can be divided into five types, of which the most common is esophageal squamous cell carcinoma(ESCC), accounting for about 90 percent, followed by esophageal adenocarcinoma, accounting for about 7 percent, other types are rare. More than 80% of patients with ESCC are in the middle or late stages at diagnosis because of no obvious symptoms and effective detection methods in early esophageal cancers. Its 5-year survival rate is less than 20%. Therefore, the early diagnosis and treatment and looking for effective molecular markers for diagnosis of patients is of great significance.Small RNAs(micro RNAs, mi RNAs, mi Rs) are noncoding single-stranded RNAs of 19 to 24 nucleotides in length. These small RNAs can cause gene silencing effect in the post-transcriptional level and participate in the physiological function such as regulating cell proliferation, differentiation and apoptosis, by combining with 3’UTR region of target gene m RNA to suppress its translation or made its degradation, Thus it is closely related to the occurrence and development of tumor. Mi R-1 is encoded by the mi R-1-133 cluster which has two copies(at 18q11 and 20q13) in the human genome producing identical mature mi R sequences for mi R-1 and mi R-133. It was originally identified as a muscle-specific micro RNA enriched in cardiac and skeletal muscles that played important roles during cardiogenesis and myogenesis. More recently, many functional studies have demonstrated that mi R-1 is a tumor suppressor in various cancers. Studies have shown that mi R-1 has aberrant expression, its expression is low in many tumors such as colon cancer, bladder cancer and kidney cancer, and it acts as a tumor suppressor. However, little information is available about its effects and mechanisms on ESCC.A number of studies confirm that LIM and SH3 protein 1(LASP1) and Transgelin-2(TAGLN2) play very important role in malignant tumor proliferation, invasion and development, and prove that they are the target genes of mi R-1 in many tumors. However, studies of the two genes in esophageal squamous carcinoma and their relationship with mi R-1 rarely reported.This study used quantitative real-time reverse transcription-polymerase chain reaction(q RT-PCR) to detect the expression level of mi R-1 in ESCC and its adjacent normal esophageal squamous tissues(NESTs), and analyzed the relationship between mi R-1 expression level and ESCC clinicopathological features. Eca109 cells were treated with mi R-1 mimics, inhibitors or their controls respectively. The bio-function(proliferation, cell apoptosis and cell cycle, metastasis and invasion) of mi R-1 in Eca109 cells was also assayed by MTT proliferation assay, flow cytometry(FCM) assay and Transwell assay. The predicted target genes LASP1 and TAGLN2 were determined by Luciferase report assay. Also we detected the expression of LASP1 and TAGLN2 in ESCC specimens and analyzed its relationship with mi R-1 expression level. And in the last, we discuss the possibility of plasma mi R-1 as the tumor marker. The main research contents and results were shown as follows:PartⅠThe expression of mi R-1, LASP1 and TAGLN2 and their relationship with clinicopathological features in ESCC.Objective: To investigate the expression of mi R-1、LASP1 and TAGLN2 in human esophageal squamous cell carcinoma(ESCC) tissues and discuss its relationship with clinicalpathological features in ESCC.Methods: q RT-PCR was adopted to detect the expression of mi R-1 at the transcription level in 55 ESCC specimens and its adjacent para-cancerous tissues. Retrospective analysis was used to analyze the relationship between mi R-1 expression and clinicopathological features. Clinicopathological features including age, gender, tumor location, TNM stage, histological grade, lymph node metastasis and tumor embolus. And q RT-PCR and immunohistochemistry were adopted to detect the expression of LASP1 and TAGLN2 in ESCC specimens which were associated with tumor invasion and metastasis. Also its relationship with clinicopathological features were analysed. In addition, the correlationship between mi R-1 and LASP1 and TAGLN2 were analyzed.Results: 1 The expression of mi R-1 in ESCCs and the adjacent NESTs. We used q RT-PCR to detect the expression of mi R-1 in ESCCs and the adjacent NESTs, and found that mi R-1 expression was significantly reduced in the ESCC tissues as compared with that in NESTs(P<0.05). 2 Correlation between mi R-1 expression and clinicopathological characteristics of ESCC. In 55 ESCC specimens, mi R-1 expression had no correlation with patient age, gender, tumor location and tumor size(P>0.05), but was significantly associated with regional lymph node metastasis, histologic classification and vessel invasion(P=0.002, P =0.000, P =0.022). 3 The expression of LASP1 and TAGLN2 in ESCCs and the adjacent NESTs. We used q RT-PCR and immunohistochemistry to detect the expression of LASP1 and TAGLN2 in 55 pairs of ESCCs and its adjacent NESTs, which were associated with tumor invasion and metastasis. We found that their expression were significantly increased in the ESCC tissues as compared with that in NESTs(P<0.05). 4 Correlation between the expression of LASP1 and TAGLN2 and clinicopathological characteristics of ESCC. In 55 of ESCC specimens, LASP1 and TAGLN2 expression had no correlation with patient age, gender, tumor location and tumor size(P>0.05),but was significantly associated with regional lymph node metastasis, histologic classification and vessel invasion(P=0.000,P=0.000,P=0.008 and P=0.000,P=0.001,P=0.008). 5 The expression levels of LASP1 and TAGLN2 were both inversely correlated with mi R-1 expression in ESCC tissues. The Spearman rank correlation test analysis found that the percentage of high expression of mi R-1 is 66.7%(16 cases/24 cases) in ESCC specimens which have low expression of LASP1(24 cases). However, the percentage of low expression of mi R-1 is 87.1%(27 cases/31 cases) in ESCC specimens which have high expression of LASP1(31 cases). Mi R-1 is negatively correlated with LASP1 expression(r=-0.45,P<0.05). The percentage of high expression of mi R-1 is 50.0%(12 cases/24 cases) in ESCC specimens which have low expression of TAGLN2(24 cases). However, the percentage of low expression of mi R-1 is 74.2%(23 cases/31 cases) in ESCC specimens which have high expression of TAGLN2(31 cases). Mi R-1 is negatively correlated with TAGLN2 expression(r=-0.382,P<0.05).Conclusion: 1 Mi R-1 expression was significantly reduced in the ESCC tissues as compared with that in NESTs. However, LASP1 and TAGLN2 are significantly increased in the ESCC tissues as compared with that in NESTs. 2 The expression of mi R-1, LASP1 and TAGLN2 are correlated to TNM stages, lymph node metastasis, histologic classification and vessel invasion. 3 The expression level of mi R-1 is inversely correlated with the expression of LASP1 and TAGLN2 in ESCC tissues, which suggests that mi R-1 may play important role in migration and invasion of ESCC.Part The effectⅡ s of mi R-1 on the biological behaviour of ESCC cells in vitro.Objective: To investigate the effects of mi R-1 on the proliferation, apoptosis, cell cycle, migration and invasion of ESCC cells in vitro.Methods: Eca109 cells were treated with mi R-1 mimics, inhibitors or their controls, respectively. The bio-function of mi R-1 in ESCC was also assayed by MTT proliferation assay, FCM and invasion assay.Results: 1 The results of mi R-1 on proliferation of ESCC cell Eca109. The results of MTT test showed that, the proliferation rate of cells in mimics-mi R-1 group were significantly decreased than that in mimics-mi R-1 control group, and the proliferation rate of cells in inhibitor-mi R-1 group were significantly increased than that in inhibitor-mi R-1 control group(P<0.05), which suggests that mi R-1 had inhibitory effect on esophageal squamous cancer cell proliferation. 2 The effects of mi R-1 on cell apoptosis and cell cycles of ESCC cell Eca109. The results of FCM showed that there were no significantly changes in Eca109 cells between mimics-mi R-1 group and mimics-mi R-1 control group or inhibitor-mi R-1 group and inhibitor-mi R-1 control group after transfection with different transfectants for 48h(P<0.05). 3 The effects of mi R-1 on migration and invasion of ESCC cell Eca109. The results of transwell assay showed that, the number of Eca109 cells that migrated through the microporous membrance or invaded through the Matrigel in mimics-mi R-1 group was less that that in mimics-mi R-1 control group(P<0.05), and the number of cells in inhibitor-mi R-1 group was more than that in inhibitor-mi R-1 control group(P<0.05), which suggested that mi R-1 may effect the Eca109 cells on cell migration and invasion.Conclusions: mi R-1 may inhibit the proliferation, migration and invasion capability of Eca109 cells, but has no effect on cell apoptosis and cell cycle.Part Ⅲ The study of regulatory relationship of mi R-1 on LASP1 and TAGLN2 expression in ESCC.Objective: To investigate the regulatory relationship of mi R-1 on LASP1 and TAGLN2 expression in ESCC.Methods: The candidate targets of mi R-1 were predicted by bioinformation website and software. And their possible target sequences were analysed combined with mi R-1 functions in ESCC cells for further screening. The predicted target genes LASP1 and TAGNL2 were determined by luciferase report. We detected the effects of mi R-1 on LASP1 and TAGNL2 in Eca109 cells by Western-blot.Results: 1 The potential target genes of mi R-1 were predicted by bioinformatics softwares and luciferase report assay were used to valid the target effect of mi R-1 on LASP1 and TAGLN2. Bioinformatics softwares predicted that mi R-1 combined with 3’UTR of LASP1 and TAGLN2, the two genes are possible target genes of mi RA-1. And we used luciferase report assay to detect the combined activity between mi R-1 and LASP1/TAGLN2. The result showed that compared to control, transfection with mi R-1 mimics could exhibited a striking reduction of luciferase activity(42% and 31%, P<0.05), which suggested that LASP1 and TAGLN2 were target genes of mi R-1, mi R-1 had a negative control on LASP1 and TAGLN2 through binding their 3’UTR. 2 The effects of mi R-1 on the expression of LASP1 and TAGLN2 in ESCC cells Eca109. Western-blot assay was used to detect the changes of LASP1 and TAGLN2 after transfection for 48 h. The results showed that, the protein expression of LASP1 and TAGLN2 in mimics-mi R-1 group was lower than that in mimics-mi R-1 control group, and the protein expression in inhibitor-mi R-1 group was higher that in inhibitor-mi R-1 control group(P<0.05).Conclusions: 1 mi R-1 may surpress the expression of LASP1 and TAGLN2 by combining with their 3’UTR, which indicates that LASP1 and TAGLN2 are target genes of mi R-1. 2 mi R-1 plays anti-metastatic function in ESCC patients may probably through targeting LASP1 and TAGLN2.Part Ⅳ The diagnostic value of plasma mi R-1 for ESCC.Objective: To investigate the expression level of mi R-1 in plasma and its diagnostic value for ESCC.Methods: Quantitative real-time reverse transcription-polymerase chain reaction(q RT-PCR) was used to detect the expression of plasma mi R-1 in two different groups including twenty-three primary ESCC patients and eleven healthy people as the control. Statistical analysis was applied to evaluate the relationship between mi R-1 and the clinicopathological data of ESCC patients. The mi R-1 expression level were also detected in ten ESCC patients preoperatively and 7-10 days postoperatively. Receiver operating characteristic(ROC) curve was used to estimate its diagnostic value.Results: 1 The expression of mi R-1 in plasma of ESCC patients and healthy control people. The results of q RT-PCR showed that the expression of mi R-1 was significantly decreased in the patients group compared with the control group(P < 0.01). 2 The expression level of mi RA-1 in plasma before and after surgery. The expression of mi R-1 in plasma 7-10 days after surgery was significantly increased than that before surgery for ESCC patients. 3 The relationship between mi R-1 expression in plasma and clinicopathological features of ESCC. The plasma expression level of mi R-1 was irrelevant with age, gender, tumor location and tumor size(P>0.05), but was significantly associated with lymph node metastasis status(P=0.005), and inversely correlated with histologic classification although there was no statistical difference(P=0.083). 4 The diagnostic value of mi R-1 expression in plasma. Receiver operating characteristic(ROC) curve was used to estimate its diagnostic value. For discriminating ESCC from healthy people using mi R-1 as the biomarker for early diagnosis, the specificity was 87%, the sensitivity was 81.8%, and the area under ROC curve was 0.881 [95%confidence interval, 95%CI: 0.743-1](P<0.01).Conclusions: 1 The expression of mi R-1 in plasma is significantly downregulated in ESCC patients, and may be upregulated after surgery. 2 Plasma mi R-1 is expected to become a new molecular marker for ESCC. |
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