The Tumor-suppressive Mechanisms Of CHD5in Lung Cancer

The1p36.3is frequently deleted in a range of human cancers Chromodomain helicase DNA binding protein5(CHD5) is a potent tumor suppressor that serves as a master regulator of a tumor-suppressive network locate on1p36.3.Examination of the role played by CHD5in a wide range of human cancers is warranted. There are many studies on tumor suppressor mechanism of CHD5gene in a range of cancer. Currently, very few studies have investigated CHD5in lung cancer.Thus; it is very significant to investigate the regulatory mechanism underlying aberrant CHD5expression in lung cancers.The CHD5as a new tumor suppressor gene located in the1p36block, was known as a part of the1p36chromosome. When the CHD5off work, the normal mechanism of cancer prevention will be cut off.It is illustrate that CHD5as a network control switch was deeply associated with a variety of human cancer. The CHD5in our body paly the role like a circuit breaker, regulating the tumor suppressor ability of our cells, when it is disconnected, the tumor occurs. It is very important to explore the role and tumor suppressor mechanism of CHD5in different human cancers. Base on molecular research and clinical practice to determine whether this gene has the function of chromatin remodeling that impact on the overall level of gene is very important. These studies will have a significant impact on understanding of tumorigenesis mechanisms to pave the way for the birth of the better diagnosis and treatment. CHD5gene specificity in expression but various in different cells, More than90%of CHD5that related to ESTs from a cDNA library of brain and nerve tissue. Because of previous study reveal that the expression of CHD5is very low but neural system so the analysis was focus on them. However, with the development of detection technology, more and more papers show that:CHD5was also have a relative lower expression other than tissue from neural system.In the present study, we first detected the CHD5mRNA level of seven cases lung cancer cell lines (H1299, A549, H1650, H358, HCC827, of H460, H661)using QPCR. Additionally, these samples were detect the protein expression level by Western-blot technique. The genome-wide hypomethylation and the abnormal methylation of CpG island were a common phenomenon in human tumors. In which, the abnormal methylation of CpG island methylation that makes some tumor suppressor gene inactivation is the key to cancer. Therefore, we detect CHD5gene promoter methylation levels in cell line that exposure and without exposure to5-aza-2-deoxycytidine. Furthermore,we random detect CHD5gene promoter methylation levels in three cell line(H1299, A549, H460) by using specific methylation sequence to explore the relationship between CpG island methylation and lung cancer.Next, we take pENTR-223.1-CHD5as a template for PCR amplification, and gain the coding frame of full-length CHD5from it. CHD5fragment and pEGFP-C1vector were double digested and then purified digestion products, connect the digestion products, we transfected constructed plasmid into human lung cancer cell lines (A549and of H1299), Following, we obtain cell lines with stably expressing EGFP-CHD5protein and EGFP protein by using G418screening. The cell proliferation curve and colony formation rate in cell lines were analyzed to explore the relationship between CHD5expression level and cell proliferation or colony formation.Finally, the A549-EGFP-CHD5and A549-EGFP cells that obtained in G418screening were injected into two groups of immune-deficient mice. Based on the recording of subcutaneous nodule growth/timing curve, we try to explore the role of CHD5in the developing of cancer.All cell lines examined exhibited significantly lower levels of CHD5gene expression relative to normal lung tissues. The lung cancer cell lines (A549, HI299, and H460) were examined to determine the methylation status of the CHD5promoter. There were strong methylation sites in both the cell lines and lung cancer tissue samples examined. To further confirm whether CHD5expression was silenced by methylation, we analyzed the methylation status of the CHD5promoter region in A549, H460, and H1299cells upon5-aza-dC treatment. A striking regional demethylation of the CHD5promoter in A549, H1299, and H460cells resulted, with an average reduction of40.85%of methylated CpGs.We examined the proliferation of these cell lines and found a major difference in the rate of cell proliferation between A549-EGFP-CHD5and A549-EGFP cells.Similarly, the proliferation rate of H1299-EGFP-CHD5cells was lower than that of H1299-EGFP cells. Next, A549-EGFP-CHD5, A549-EGFP, H1299-EGFP-CHD5, and H1299-EGFP cells were plated for colony formation. We found significant reductions that averaged47.83±4.6%for A549-EGFP-CHD5compared to A549-EGFP, and56.39±5.3%for H1299-EGFP-CHD5compared to H1299-EGFP).Compared with normal tissues, CHD5mRNA expression in lung tissue or lung cancer cell lines were significantly decreased, This result was also further proved by Western blot technology on protein level. It indicated that the decline of CHD5expression level was closely related to the development of lung cancer. Furthermore, lung cancer cell line that was exposure to5-Aza-2’-deoxycytidine-treated have a higher expression levels of CHD5and a lower CpG island hypermethylation. It suggest that the hypermethylation of the CHD5promoter region is maybe an important mechanism for the decline expression of CHD5.CHD5expression level of A549and H1299lung cancer cell proliferation was significantly inhibited in the basic experiment of the CHD5tumor suppressor gene function research, this effective was also present in cell colony formation after improve CHD5expression. This illustrates CHD5is more likely to be a tumor suppressor gene in lung cancer. It is indicated that CHD5is a very valuable tumor suppressor gene in lung cancer. However, we can not rule out the synergistic effect of these genes in other intracellular environment or company with CHD5. Meanwhile, hypermethylation of the other carcinogenic may be another explanation for leading to tumor.The high incidence of CHD5promoter hypermethylation in lung cancer can be explored not only as a lung cancer diagnosis but also prognosis prediction. To this end, it is important to characterize the CHD5promoter methylation in association with clinical characteristics, such as age, gender, smoking,tumor grade,and differentiation. Of course, more samples should be used to confirm this result in the following studies.