Mechanisms Study Of E2F1 Involving In The Biolocical Function Of Esophageal Squamous Cell Carcinoma By Regulating MicroRNAs

Background and ObjectiveEsophageal cancer in humans occurs worldwide and ranks sixth as a cause of cancer mortality. Two main histological types have been identified, esophageal squamous cell carcinoma(ESCC) and esophageal adenocarcinoma(EAC). ESCC is the main histological subtype of esophageal cancer in China. The prognosis and outcome of esophageal cancer is very poor, and the overall 5-year survival rate is less than 20%. So it is very important to investigate the mechanism of ESCC and find novel target for early diagnosis and therapy.DNA damage response(DDR) is an important anti-tumor barrier, maintaining the integrity of genome by counteract the genotoxic stress in vivo and in vitro. DDR initiates a series of DNA damage checkpoint pathway, including sensors, transducers and effectors. As one of the most important effectors, E2F1 transcription factor can regulate both G1/S cell cycle arrest and apoptosis in DDR, thus became more important. Althouth p53 also has these two roles in DDR, p53 gene is mutant in most human tumors. Therefore, E2F1 is particularly important.E2F1 is one of the E2 Fs transcription factors family, which is the dowmsteam effectors of tumor suppressor retinoblastoma protein(Rb). E2 Fs determine the timely expression of numerous genes essential for entry and through S phage of cell cycle. It is reported now that E2F1 can also regulate DNA synthesis and replication, mitotic checkpoints, DNA damage repair, self-renewal, develepment and differentiation. As the first discovered and studied most extensively member of E2 Fs family, E2F1 can also regulate apoptosis and antophage. In human tumors, the inhibition of E2F1 by Rb is often disrupted through several different mechanisms. These changes lead to abnormal release of E2 Fs, thereby inducing transcriptional activation of E2 Fs target genes, ultimately leading to cell abnormalities. It is reported E2F1 is deregulated in most human tumors including ESCC. E2F1 expression is shown to be associated with the lower survival of patients and the worse histological grade. Besides the expression research, study on the E2F1 function in is rare esophageal squamous cell carcinoma. Therefore, study the functions and specific mechanisms of E2F1 deeply in esophageal squamous cell carcinoma is of great significance.micro RNAs(mi RNAs)are about 21-23 nt noncoding RNAs that regulate the gene expression at post-transcriptional level. mi RNAs regulate almost of all biological processes, and their expression is often detected in human malignancies. mi RNAs participate in tumors by target oncogenes or tumor suppressors such as p53, Myc, E2F1, Ras, and BCL-ABL etc.. Conversely, mi RNAs expression can also be transcriptional regulated by a variety of oncogenes or tumor suppressor genes including E2F1. E2F1 upregulated mi RNAs, can in turn play as the negative feedback regulatory factor of E2F1 pathway through targeting or inhibiting the expression of E2F1 to maintain cell homeostasis. Moreover, mi RNAs can either target DDR related genes such as ATM, ATR, Chk1 and Ch K2 etc., or transcriptional regulated by these genes including E2F1. Howerer, there have no studies reported that E2F1 regulates mi RNAs invoving in DDR in ESCC.It has reported that mi R-203 and mi R-26 b act as tumor suppressors in human malignancies, and they regulated cell cycle arrest or apoptosis by regulating different target genes. But there are so far no studies about mi R-203 and mi R-26 b involves in DDR. Meanwile, there are fewer reports about the transcriptional regulation of both of the mi RNAs. By bioinformatic analysis, we found that there are E2F1 binding sites in the promoter of the mi R-203 gene and the host gene of mi R-26 b. So we predicated that mi R-203 and mi R-26 b may be regulated by E2F1. This paper aims to investigate the molecular mechanisms of mi R-203 and mi R-26 b expression regulated by E2F1 in ESCC, and the function of these factors influence ESCC cell growth and DDR.The main results of this paper are as bellows1. The induction of mi R-203 by cisplatin is dependent on E2F1 in ESCC1.1 Western blot and q RT-PCR assay were used to detect the expression of γ-H2 X and E2F1, and the time and dose dependence of mi R-203 expression by cisplatin treatment in ESCC. We found that DDR was comfirmed according to the expression of DDR markerγ-H2 X. The expression of mi R-203 was detected simultaneously with the upregulation of E2F1 protein. Moreover, mi R-203 was induction by cisplatin for time and dose denpedency. It is indicated that mi R-203 was induced by cisplatin in DDR in ESCC, and the effect may be associated with E2F1.1.2 Western blot and q RT-PCR assay were used to detect the expression of E2F1 and mi R-203 after overexpression or RNA interference of E2F1 expression in ESCC. The results showed that the expression of mi R-203 was increased after E2F1 overexpression in EC109 and Kyse450 cells. When E2F1 expression was RNA interference by E2F1 in EC109 and Kyse450 cells,the expression of γ-H2 X was decreased with E2F1,implied that the DDR was associated with E2F1. It’s more important that the induction of mi R-203 by cisplatin was blocked by E2F1 interference. These results confirm that the induction of mi R-203 by cisplatin is mediated by E2F1.1.3 Luciferase reporter and Ch IP assay were carried out to study the transcriptional regulation and related mechanisms of mi R-203 by E2F1. Through bioinformatic analysis, we found three putative E2F1 binding sites upstream the transcriptional start sites of mi R-203, located at-501/-486(BS1),-439/-449(BS2) and-202/-194(BS3), respectively. Luciferase reporter assay showed that E2F1 overexpression increased the luciferase activity of the reporter vector containing the three E2F1 binding sites. The deletion mutation of BS2 sites resulted in complete loss of the E2F1 mediated luciferase activity. Ch IP assay showed that E2F1 antibody was specifically recruited to the amplicon containing the BS2 sites, and cisplatin treatment led to enhancement of the binding activity. These results prove that E2F1 binds to the BS2 site of mi R-203 promoter region directly, and upregulates the expression of mi R-203 at transcriptional level.2. The influence of mi R-203 to the biological function of esophageal squamous cells2.1 q RT-PCR and CCK8 assay were used to detect the expression of mi R-203 in ESCC cell lines and the affect of mi R-203 to ESCC cell proliferation. Through detection of the basal expression levels of mi R-203 in four ESCC cell lines, we found that mi R-203 expression was lowest in EC109 cells, and highest in EC9706 cells. Transfection of the mi R-203 mimics inhibited the proliferation of EC109 cells.2.2 Flow cytometry and Annexin V-FITC analysis were conducted to detect roles of mi R-203 on cell cycle and apoptosis in ESCC. We found that compared with NC,overexpression of mi R-203 in EC109 cells resulted in the percentage of cells in G1 phase accumulation significantly, and S phase cells reduced significantly. In contrast, when blocking the expression of mi R-203 in EC9706 cells, the G1 phase cells decreased significantly, while the G1/M phase and S phase cells increased significantly. In addition, we found that mi R-203 had no effect on apoptosis of ESCC cells. It indicates that mi R-203 may function as regulators of cell cycle rather than apoptosis to regulate the proliferation of ESCC cells.2.3 Western blot assay was used to detect the expression levels of CDK6, p Rb and E2F1 through mi R-203 mimics overexpression in EC109 or Kyse450 cells. The results showed that the expression of CDK6, p Rb and E2F1 were inhibited by mi R-203 in ESCC cells. It indicates that mi R-203 negatively regulates the expression of p Rb by inhibiting CDK6, and ultimately reduces the expression of E2 F. Therefore, there may be a feedback loop between mi R-203 and E2F1 connecting by CDK6.3. The induction of mi R-26 b by cisplatin is dependent on E2F1 in ESCC3.1 QRT-PCR assay was conducted to detect the expression level of mi R-26 b treated with differente concentration of cisplatin or at differente time point. The results indicated that the expression of mi R-26 b was increased after treated with cisplatin in EC109 and Kyse450 cells. Moreover, the induction of mi R-26 b by cisplatin was for time and dose denpedency. Combined with the results in chapter Ⅱ that E2F1 and γ-H2 X epression were also induced by cisplatin in EC109 and Kyse450 cells, it indicated that mi R-26 b was induced by cisplatin in DDR in ESCC, and the effect may be associated with E2F1.3.2 QRT-PCR assay was used to detect the expression of mi R-26 b after E2F1 overexpression or RNA interference in ESCC cells. We found that the expression of mi R-26 b was increased afer E2F1 overexpression, and the induction of mi R-26 b was blocked after E2F1 RNAi. Results were indicated the induction of mi R-26 b by cisplation is mediated by E2F1.3.3 Luciferase reporter and Ch IP assay were carried out to study the transcriptional regulation and related mechanisms of mi R-26 b by E2F1. We found that E2F1 directly binds to the promoter region of the host gene CTDSP1 of mi R-26 b and activates transcriptional activity of mi R-26 b.4. The effect of mi R-26 b on biological function of ESCC cells4.1 QRT-PCR assay was used to measure the expression levels of mi R-26 b in cancer tissues and adjacent non-tumor tissues of 33 ESCC patients. We found that the expression level of mi R-26 b was lower in cancer tissues than in adjacent non-tumor tissues of 33 ESCC patients.4.2 CCK8 and flow cytometry assays were conduct to detect the affect of mi R-26 b on ESCC cells proliferation and cell cycle. We found that mi R-26 b can inhibit the proliferation of ESCC cells significantly. Compared with NC, overexpression of mi R-26 b in EC109 cells resulted in G1 phase cells accumulation significantly. In contrast, when blocking the expression of mi R-26 b in EC9706 cells, the G1 phase cells decreased significantly. It indicates that mi R-26 b can induce the G1/S phase cell cyle arrest in ESCC cells.4.3 Flow cytometry assays were conduct to detect the affect of cisplatin on cell cycle treated for indicate time in ESCC cells. We found that the percentage of G1 phase cells accumulation significantly after cisplatin treatment for indicate time in Kyse450 cells, while the percentage of S phase cells reduced significantly, but G2 phase cells almost no change. Blocked the expression of mi R-26 b in Kyse450 cells combined with cisplatin treatment, the enhancement of G1 phase cells did not happen. The results indicated that cisplatin can induce G1/S phase cell cycle arrest, but the induction eddect was depenedent on mi R-26 b expression.4.4 Western blot assay was performed to measure the expression levels of Rb, ATM and E2F1. The results showed the expression of Rb, ATM and E2F1 were inhibited by mi R-26 b in ESCC cells. It indicates that ATM and Rb may be the target genes of mi R-26 b. Moreover, mi R-26 b may reduce E2F1 expression by targeting Rb or ATM, so there may be a feedback loop among them.In summary, the results showed that E2F1 can transcriptional regulation mi R-203 and mi R-26 b in ESCC cell lines. In contrast, mi R-203 and mi R-26 b also can reduce E2F1 exprssion through different target genes. It indicated that mi R-203 and mi R-26 b were new members of E2F1 regulation network. E2F1 induced G1/S cell cycle arrest by regulating mi R-203 and mi R-26 b, which affected the proliferation of ESCC cells. Moreover, they involved in cisplatin induced DDR in ESCC cells, and may act as cheakpoint in early DDR. This paper revealed that E2F1-induced mi RNAs involved in the regulation of the biological function of ESCC, and help us to understand the mechanisms of the development of ESCC, providing new evidence for the treatment of ESCC.