The Study Of HMGN5 Gene Expression In Lung Cancer Tissue And The Mechanism On Tumorigenesis

The incidence and mortality rates of lung cancer are extremely high in the world. The highest cancer mortality rate is one of the top ten malignant tumors, lung cancer's 5-year overall survival rate is 13% to 15%. The incidence of lung cancer is still rising incidence of lung cancer, but the treatment effect of lung cancer is still not ideal in recent years, therefore the research of lung cancer is focused on the new chemotherapy drugs, molecular targeted drugs, the application of drug therapy and probing into the pathogenesis of lung cancer in order to find potential biological marker for the personalized treatment of lung cancer. Multiple genes and multiple steps are involved in the occurrence and the developed process of lung cancer. So the process by studying the abnormal expression of cancer genes, finding a diagnostic and therapeutic gene has a great clinical significance.As a sequence-specific post-transcriptional gene silencing, RNAi is a means which is induced 21-23 double-stranded RNA composed of sense and antisense RNA corresponding mRNA, so that mRNA of the specific genes in cells are degradated, leading to gene silencing. RNAi is a reverse genetics technology, which plays an important role in the study of functional genes in the post-genomic times. Aiming at genetic research in the "knockout" technology, RNAi technology for "ticking down" the target gene or target protein, has been widely used in signal transduction,gene function and research in cancer gene therapy,so dose in mammalian cells. In view of the RNAi technology is of great efficiency and simplicity in inhibition of target gene expression. RNAi becomes an effective means of gene function research. RNAi can selectively inhibit the expression of genes related to human disease in the study of gene function, so it has a broad prospect in the study of gene function and the treatment of human diseases. Researchers in-country and abroad have successfully used RNAi technology to selectively inhibit or down-regulate the expression of lung cancer related genes which play important roles in the development of lung cancer, such as oncogenes,anti-oncogenes and genes related to apoptosis, in order to achieve gene therapy purposes.HMGN5 is a member of the HMGN family,it is highly homologous with the NSBP1. King has discovered HMGN5 in 2001. Human gene HMGN5 is 8600bp long and it has 5 introns and 6 exons, and its complete cDNA sequence is 1865bp long. HMGN5 encodes a kind of human nuclear protein-nucleosome-binding protein that includes 282 amino acids, and its molecular weight is 31000 bp in theory. HMGN5 expression length is different between human and mice because man's HGMN5 that consists of 6 exons which is shorter.Although the molecular mechanism HMGN5 is not clear, but the literature reports that HMGN5 has potential relevance in disease and normal cell function. For example, over-expression experiments and gene chip analysis showed that HMGN5 can serve as a factor inducing differentiation of mouse embryonic stem cells. HMGN5 expresses highly in human squamous cancer,prostate cancer,adenocarcinoma of aging mice,highly metastatic breast cancer MDA-MB-435HM cells, so that the experimental results show that the HMGN5 is associated with the tumor. It is endogenous disorder that HMGN5 protein expresses increasedly in tumor tissue. In addition, HMGN5 in X chromosome, containing a disproportionately large number of genes may be related to mental function, as well as behavior and cognitive function in late evolutionary stage.By optimizing the reaction of chromatin, HMGN5 constantly changes the cell phenotype of environment. Highly acidic terminal and abnormal long terminal influence the interaction between connection chromosomes and histone, so that HMGN5 can more effectively expand the highly ordered structure of chromosomes. Direct competition between connection histone and HMGN5 provides a new mechanism model for regulationing dynamic structure of chromosome. Clarifing how HMGN5 influences chromosome structure and the specific modes of this competition mechanism is needed to be studied in depth. HMGN5 plays an important role in maintaining the integrity of chromatin fibers within cells, but the abnormal expression of HMGN5 is harmful to the cells. HMGN5 and the tumor have a potential relationship. It has been reported that HMGN5's expression level in bladder cancer,prostate cancer and other tumor tissues were significantly higher than normal tissue, but studies in lung cancer has not yet been clearly reported. So further research is how HMGN5 gene affects lung cancer occurrence and development, and we can seek a breakthrough in clinical treatment of lung cancer by that way. Objective:To investigate HMGN5 gene expression level in lung cancer tissue, and the connection with the clinical and pathological features; to build siRNA of HMGN5 in order to recombine lentiviral vectors; The influence on RNA interference silence HMGN5 gene in lung cancer cells proliferation,cell cycle and for ability of lung tumor. RNAi silencing HMGN5 genes in lung cacer cells A549 and H1299, and inhibition of HMGN5 gene working on tumor formation in nude mice, provide a theoretical basis of gene targeting therapy.Methods:To detect HMGN5 mRNA expression in lung cancer and besides the cancer tissues by Real-time PCR method, and analyse the relativity between the expression of HMGN5 and clinicopathological features of lung cancer; Design targeted HMGN5 gene siRNA interfereNce sequence; Build synthetic vectors with designed sequences; Choose positive and correct-sequencing plasmid to package virus; Let packaged virus infect human lung cancer cells A549,H1299, and verify silencing efficiency of human lung cancer cells A549,H1299 by Real-time PCR and Western blot methods; To inhibit HMGN5 gene expression in lung cancer A549 and H1299 cells by RNAi technology, at the same time, HMGN5 siRNA recombine lentiviruses to infect human lung cancer cells, through that we can draw a conclusion what HMGN5 changes of the proliferation ability of human lung cancer cell, after detecting the silencing expression of HMGN5 by using MTT; Detect the silencing HMGN5 expression and how human lung cancer cell DNA synthesis rate changes using Brdu; After application of colony formation method validating HMGN5 silence, the change of human lung cancer cell colony formation ability was detected; Using flow cytometry test cell cycles change in order to explore HMGN5's biological functions in lung cancer cells; Tumor model with human lung cancer A549 cells subcutaneous was built in nude mice, by observing the size and weight of the tumor in order to observe the influence on lung cancer in vivo formation ability by HMGN5 gene silencing.Results:Real-time PCR results showed that expression levels in the lung cancer tissue (4.60±0.47) was significantly higher than in adjacent tissue (1.02±0.55) (P<0.05); HMGN5 siRNA built lentiviral vector was successfully constructed. Real-time PCR and Western blot results showed that the experimental designed siRNA target sequence express silently in human lung cancer cells A549 and H1299 cells, and the silence efficiency of HMGN5 is 80%; Recombined HMGN5 siRNA lentiviral vectors have effected on human lung cancer cells A549 and H1299 cells, mRNA and protein of HMGN5 gene expression levels were significantly decreased; MTT,Brud and cell clone experimental results showed that the ability of cell proliferation, DNA replication and colony formation of transfected HMGN5-siRNA A549 and H1299 cell was significantly lower than the control group; Flow cytometry results showed that A549 and H1299 which transfected HMGN5-siRNA were arrested in G0/G1 phase, so that cell division was slow down; successfully established subcutaneous A549 nude mouse model in vivo experiment results showed that mice injected with HMGN5-siRNA's transfered tumor weight was significantly lower than the control group. Tumor formation ability of lung cancer cells in vivo was significantly inhibited.Conclusions:HMGN5 was expressed highly in lung cancer tissue; HMGN5 siRNA interference plasmids were successfully built, and it can inhibit mRNA and protein expression of HMGN5 gene; HMGN5 siRNA lentiviral vectors can inhibit the ability of cancer cell proliferation and colony formation, leading to cell arresed in G0/G1 phase in order to slow down cell division. So it can draw a conclusion that lung cancer HMGN5 genes may have relationship with development of lung cancer. HMGN5 can be used as a new target of cancer gene therapy, which provides new ideas and theoretical research base for treatment of lung cancer.