| Esophageal cancer is a common human malignancies, ranking the world’s top six fatal tumor. According to the latest statistics by International Agency for Research on Cancer(IARC) in 2011, there are nearly half a million newly-increased esophageal patients and about 400,000 death toll worldwide every year. The incidence rate of esophageal cancer in China is the highest all over the world, so are the morbidity and mortality rate: the incidence rate accounts for 54% of the world, Ci County of Hebei, Lin County of Henan, Yang County of Shanxi represented the highest incidence rate up to 110/10 million or more; while China’s annual number of deaths of esophageal cancer accounts for 52% of the world, the mortality rate ranks No. 4 in the malignancies of China. The main reasons of restricting the curative effect of esophageal cancer are as follows: Firstly, there is no definitive way of early screening for esophageal cancer, nor clear tumor marker. Untimely diagnosis makes the best treatment moment missed. Secondly, the recurrence and metastasis after operation, which are the main reasons for the failure of treatment and death of patients, does not have the mechanism clarified. Even no effective methods for suppressing it is found. Thirdly, no specific therapeutic targets or drugs for esophageal cancer have been reported. In addition, the characteristic of esophageal cancer in Europe or America is different from China. In China, squamous cell carcinoma is the main pathological pattern, while it is adenocarcinoma with more morbidity in western countries. Therefore, we shoμld specially focus on Chinese own research results.SMYD3 is closely related to the occurrence and development of many malignant tumors, which can affect the biological behavior of proliferation, apoptosis, migration and adhesion, etc., such as colorectal cancer, hepatocellular carcinoma, pancreatic cancer, prostate cancer, mμltiple myeloma, lung cancer, breast cancer, brain tumors, and gastric cancer. In preliminary study, with immunohistochemical method we has been applied of 15 cases of human esophageal squamous cell carcinoma and adjacent tissue samples examining SMYD3 protein expression and found its expression is in cytoplasm and the levels were significantly higher in cancer tissue. SMYD3 can regμlate downstream tumor suppressor RIZ1, RIZ1 expression increased after SMYD3 knock down, tumor growth was inhibited as well. In this study, we further use a large number of 143 samples to detect the expression of SMYD3 for clarifying the relationship between the expression and clinicopathological characteristics, such as tumor size, lesion length, tumor differentiation, location, lymph node metastasis and TNM stage. On the other hand, we try to analyze whether the SMYD3 expression level is related with survival or prognosis. The results show that SMYD3 expression is an independent prognostic indicator, which is the associated with median overall survival and median disease-free progression in esophageal squamous cell carcinomas SMYD3 high expression patients with poor prognosis. SMYD3 high expression in cancer tissues is confirmed, with the TNM stage increasing and the expression gradually strengthened. However, we did not find the differences of SMYD3 in metastatic lymph nodes and cancer organizations. In addition, there’s no correlation between SMYD3 expression and the clinical/pathological features within statistical precision. Therefore, SMYD3 is an important prognostic factor in esophageal squamous cell carcinoma, which is significant to the development and progression of cancer.In preliminary studies, after knocking down SMYD3 expression by shRNA we found that RIZ1 level of expression was increased and tumor proliferation was inhibited. In the second part of this study, we applicate the method of siRNA and constructing of shRNA expression vector, transfect them into cells for up or down-regμlated SMYD3 gene expression in esophageal squamous cell carcinoma cell lines, further detect RIZ1 expression and tumor suppressor activity. In addition, we detect the invasion ability changing by Borden chamber. After SMYD3 reduced by siRNA, mRNA and protein expression of RIZ1 has improved significantly. Invasiveness and apoptosis of cell has increased, too. The cells in S phase of the cell cycle has increased, but in the G2/M phase there is downward trend and even no different in G0/G1 phase. This result hereby shows SMYD3 possible arrest the cell in S phase. However, the overexpression of SMYD3 can’t lead RIZ1 expression reduce or tumor invasiveness.Collecting the first two parts results and preliminary findings, we see SMYD3 as a upstream regulatory gene of tumor suppressor gene RIZ1, it regμlates the expression of RIZ1, and it leads to a corresponding biological behavior changing of esophageal squamous cell carcinoma. SMYD3, as a histone methyltransferase, can mediate H3K4 histone lysine methylation changing, and histone methylated changing has been identified closely related to progress, recurrence, drug resistance, etc. Therefore, in the third part of this study we worked on SMYD3-RIZ1 specific regμlation mechanism firstly by ELISA of 15 patients’ samples detect H3K4 mono-, di-, trimethylation level then found that all the methylation levels in cancer tissues were higher than adjacent tissues. Further in the same way we knockdown or overexpression SMYD3, then detect the above methylation modifications changing and found that when SMYD3 down regμlated, just H3K4 di- and trimethylation reduced, there’s no changing on monomethylation. However, the overexpression of SMYD3 does not influence the three kinds of methylation levels. Due to the identified RIZ1, expression reducing is associated with DNA promoter methylation, so RIZ1 promoter methylation was be detected again after SMYD3 knockdown, and the level of which is reduced compared with control. Therefore, we found two mechanisms for SMYD3 regμlating RIZ1, the first is mediated by di- and tri-methylation of H3K4 increasing, and the second is to make DNA promoter methylation of RIZ1.Above 3 parts studies have demonstrated the correlation between SMYD3 and RIZ1, and the mechanism SMYD3 regμlating RIZ1 as well. Histone methylation and DNA methylation can be reversed, depending on the balance between histone methylase and demethylase, DNA methylase and demethylase. Since too much relationship are exists, it is not easy to fμlly explain the mechanism. In the fourth part of our research we attempt the initial exploration of this. By the above 15 cases of esophageal squamous cell carcinoma, mono-/di-methyl-specific demethylase LSD1 and histone lysine tri-methylation demethylase enzyme activity of JARID1 were tested and found that LSD1 activity was significantly higher than that of adjacent tissues, but there’s no difference in JARID1 activity. When applying 5-aza-CdR reversal DNA promoter hypermethylation, they found H3K4 mono- and dimethylation significantly reduced, and the most obvious is when the concentration of drug is 1μM, while tri-H3K4 methylation did not change. LSD1 and JARID1 activity changing accompany 5-aza-CdR concentration. LSD1 activity decreased gradually with the increasing of drug concentration, however the enzyme activity of JARID1 manifest a low to high trend. Above identified histone demethylase LSD1 plays a key role in cancer. DNA methylation can affect the H3K4 methylation changing, which also act on histone demethylase LSD1 and JARID1 activity. |
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