The Role And Mechanism Research Of MiR-373 Expression In Esophageal Squamous Cell Carcinoma

Esophageal carcinoma (EC) is the eighth leading cancer worldwide and is characterized by its high mortality rate, with a less than 15% 5-year survival rate. Esophageal squamous cell carcinoma (ESCC) is the predominant pathological type of esophageal cancer in China, which has a high tendency for recurrence and metastasis. Surgical resection is the main treatment in the early stage. The patients of intermediate and advanced stage have no other choice than to receive radiotherapy and chemotherapy, which themselves are not very effective. However, due to a lack of apparent symptoms and early detecting methods, the patients are often diagnosed at a late stage. Therefore, it is of great importance to find an effective molecular diagnostic method to detect subclinical esophageal carcinoma. It is important for the early diagnosis and treatment of esophageal carcinoma to realize the mechanism of esophageal carcinoma development and explore the effective diagnostic and therapeutic targets.MicroRNAs (miRNAs) are a class of 19-22-nucleotide non-coding RNAs. miRNAs affect multiple pathways by binding to specific sites at 3’-UTR of target mRNAs. So far, miRNAs have been discovered in 206 organisms, ranging from microbes to animals. There are more than 2000 miRNAs published on the official website miRBase. More than 60% of human protein-coding genes are expected to be regulated by miRNAs. miRNAs play an important role in regulating cellular differentiation, proliferation, aggressiveness, angiogenesis and apoptosis. Abnormity of miRNAs are closely related to the occurrence of a variety of tumors. It has been shown that a variety of miRNAs participate in the process of esophageal cancer occurrence. miR-183, miR-142, miR-483 are regarded as oncogenes, while miR-375, miR-98, miR-214, miR-143 are down-regulated in ESCC, which serve as tumor suppressor genes.A large number of studies had shown that miR-373 played an important role in many types of malignant tumors. miR-373 was first discovered by Voorhoeve, who identified that miR-373 is a potential novel oncogene participating in the development of human testicular germ cell tumor by blocking the p53 pathway.As an oncogene, miR-373 regulates cell cycle by targeting PPP6C, and consequently, promotes HCC cell growth. In addition, circulating miR-373 might be a potential biomarker for detecting lymph node metastatsis in breast cancer. miR-373 also suppresses invasion and metastasis by targeting Rab22a gene in human ovarian cancer. It is reported that miR-373 is upregulated (tumor≥1.5-fold normal) in ESCC tumors by microarray analysis. However, the mechanism of miR-373 in ESCC is still unclear, especially its expression level in the plasma.Matrix metalloproteinases (MMPs) are a class of proteinase which could degrade extracellular matrix, break the histologic barrier around the tumor cells, and promote the malignant tumor cell invasion and metastasis. There is a balanced relationship between MMPs and tissue inhibitor of metalloproteinase (TIMPs) in the normal environment. Malignant tumors can disrupt the balance between MMPs and TIMPs, improve the ability of tumor invasion and metastasis. Recent studies have shown that there is a close relationship between tissue inhibitor of metalloproteinases-3 (TIMP3) and malignant tumors. As a tumor suppressor, low expression of TIMP3 can facilitate tumor growth, invasion, metastasis and suppress apoptosis. TIMP3 could be a promising biomarker of independent prognostic factor in bladder cancer and esophageal squamous cell carcinoma. Previous studies showed that TIMP3 was associated with invasion and metastasis in ESCC. And we predicted TIMP3 might be the target gene of miR-373 using bioinformatics software.In this study, we showed that miR-373 was upregulated in ESCC tissues and ESCC patients’plasma. After overexpressing or knocking down miR-373 by cell transfection, its effect on cell proliferation, apoptosis, migration and invasion was observed. We looked for direct target genes of miR-373, and investigated the role of miR-373 in the pathogenesis of ESCC, which provides a new theoretical basis of ESCC diagnosis and treatment.Part 1 miR-373 expression level in esophageal squamous cell carcinoma and its clinical significanceObjective:To investigate miR-373 expression in human ESCC tissue and plasma, analyze the relationship between miR-373 abnormal expression and clinical features, such as differentiation, T stage, lymph node metastasis, and TNM stage, and explore the correlation between miR-373 level in ESCC tissues and that in the plasma.Methods:The study included 63 patients who were first diagnosed with ESCC in Shandong Cancer Hospital and Taian Central Hospital from October 2012 to September 2014. ESCC tissues and adjacent normal tissues were collected from patients after surgical resection. Peripheral blood was collected from each patient before surgery. Plasma samples were also collected as controls from 39 healthy volunteers. Quantitative real time-PCR was performed to test miR-373 expression levels in 63 ESCC tissues and matched adjacent normal tissues. We also assessed miR-373 levels in the plasma from 63 ESCC patients and 39 healthy volunteers.Results:1. It showed that miR-373 expression level was considerably increased in 63 ESCC tissues compared with matched adjacent normal tissues(P<0.01). miR-373 expression level in tumor tissues was closely correlated with differentiation, tumor status, and lymph node metastasis (P<0.05)2. We assessed miR-373 level in the plasma from 63 ESCC patients and 39 healthy volunteers, which was much higher in ESCC patients than in healthy volunteers (P<0.01). There was also a good correlation between miR-373 level in plasma and T stage, as well as lymph node metastasis (P<0.05)3. There was a correlation between miR-373 level in ESCC tissues and that in the same individuals’plasma using Spearman correlation test (r=0.553; P<0.01). It was demonstrated that miR-373 expression in tissues was consistent with that in plasma.Conclusion:miR-373 was upregulated in ESCC tissues and ESCC patients’plasma. As an oncogene, miR-373 played an important role in ESCC.Part 2 Effects of miR-373 on the biological behaviors of ESCC cell linesObjective:To observe the effect of miR-373 on the biological behaviors of the manipulated cell lines, such as proliferation, apoptosis, migration andinvasion, and explore the relationship between miR-373 level and ESCC biological characteristics.Methods:miR-373 expression levels in four different esophageal squamous carcinoma cell lines were evaluated by quantitative real time-PCR. The highest expression level of miR-373 was observed in KYSE410 cells, and the lowest in ECA109 cells. As a result, KYSE410 and ECA109 were selected for further experiments. miR-373 mimics was used to enhance the level of miR-373 in ECA109 while miR-373 inhibitor was used to decrease the level of miR-373 in KYSE410. CCK-8 assay was used to evaluate the effect of miR-373 on ESCC cells’proliferation ability. Apoptosis assay was performed to measure cell apoptosis. Invasion and migration experiments were conducted using transwell chambers covered with or without matrigel.Results:1. miR-373 expression level decreased in the order of KYSE410> EC9706> TE-1> ECA109. KYSE410 cells, with the highest expression level, and ECA109 cells, with the lowest expression level, were selected for further experiments.2. miR-373 mimics increased proliferation ability in ECA109 cells compared with the negative control groups at 48h (P<0.05). And transfection after 72h, the difference is more clear (P<0.01). KYSE410 cells treated with miR-373 inhibitor showed a significant decrease in proliferation ability at 48h (P<0.05). Transfection after 72h, the difference is still statistical significance (P<0.05). These results indicate that miR-373 enhances proliferation in ESCC cell lines.3. The percentage of apoptotic cells in ECA109 cells, treated with miR-373 mimics, did not decrease significantly compared with the negative control groups (P>0.05) However, the rate of apoptotic cells in KYSE410 cells, treated with miR-373 inhibitor, was higher than that in the control cells (P<0.01), which indicates suppression of miR-373 level may lead to apoptosis.4. Migration in ECA109 treated with miR-373 mimics was enhanced significantly compared with the negative control group (P<0.01). So was the invasion ability (P<0.01). Migration and invasion in KYSE410 treated with miR-373 inhibitor declined significantly compared with the negative control groups (P<0.01). These observations demonstrate that miR-373 promotes migration and invasion in ESCC cell lines.Conclusion:Overexpression of miR-373 in ESCC cells enhances proliferation, migration, and invasion. On the other hand, suppression of miR-373 in ESCC cells decreases proliferation, migration, and invasion and also induces cell apoptosis.Part 3 Preliminary investigation of the mechanism of miR-373 in esophageal squamous cell carcinomaObjective:Although miRNAs don’t encode proteins, they bind to specific sites at 3’-UTR of target mRNAs in accordance to the principle of complementary base pairing, which leads to mRNA degradation or post-transcriptional suppression. We predicted the potential target genes of miR-373 using bioinformatics software. Having considered genes associated with invasion and metastasis in ESCC, we chose TIMP3 for further study and explore whether TIMP3 is the direct target of miR-373 mediating the effect on the biological behavior of ESCC cells.Methods:TIMP3 protein levels in ESCC cells were measured before and after transfection. Luciferase reporter assay was conducted to verify TIMP3 is the direct target of miR-373. Functional experiments were used to further prove that TIMP3 mediates the role of miR-373 in ESCC cell migration and invasion.Results:1. The protein level of TIMP3 significantly decreased in ECA109 cells after miR-373 mimics transfection. And miR-373 inhibitor increased TIMP3 expression in KYSE410 cells.2. Luciferase reporter assay was conducted by co-transfecting the reporter plasmids harboring TIMP3-3’UTR-WT and miR-373 mimics in human HEK293T cells, which caused 37% decrease compared with the negative controls. However, there were no significant differences among TIMP3-WT/NC, TIMP3-Mut/NC, and TIMP3-Mut/ miR-373.3. pcDNA3.1-TIMP3 without the 3’-UTR sequence and miR-373 mimics were co-transfected into ECA109 cells. It turned out that pcDNA3.1-TIMP3 could attenuate the migration and invasion ability induced by miR-373 mimics in ECA109 cells. Then, shRNA-TIMP3 and miR-373 inhibitor were co-transfected into KYSE410 cells, which showed that shRNA-TIMP3 could partly reverted the migration and invasion ability inhibited by miR-373 inhibitor in KYSE410.Conclusion:miR-373 promotes ESCC cell migration and invasion by targeting TIMP3.