The Preliminary Studies Of Biological Characteristics And Mechanisms Of Histone Deacetylase 5 On Non-Small Cell Lung Cancer

Background:Lung cancer has a highly mortality, accounting for 80% of non-small cell lung cancer. Most patients when firstly diagnosed have been already reached advanced lung cancer. Relapse and metastasis are still the formost causes. Major radiation and chemotherapy for increasing survival rate of lung cancer patients have reached their bottle neck. It's necessary in clinical medicine to probe into the mechanism of tumorigenesis and cancer development in a molecular level, to seek novel safe and effective therapy and improve outcome of lung cancer patients.In recently years, acetylization of histone as a pivotalfactor for gene regulation has received much attention. Acetlization of histone is catalyzed by Histone acetyltransferases (HATs) and Histone deacetylases (HDACs), which is a pare of antagonist enzymes. They transfer and dissociate lyophobic acetyl to the lysine residual on the N terminal, increase and decrease electrostatic attraction and sterical hinder, thus weaken and strengthen the interaction between histone and DNA, leading to the activation or suppression gene transcription. Currently,18 HDACs have been discovered, and they participate in tumorgenesis and cancer development for different degrees in different tumors.Human HDAC5 gene belongs to typeⅡHDACs, and has been found with low expression level in many tumors.But there hasn't been any report about whether the low expression level of HDAC5 contribute to the tumorigenesis and cancer development of non-small cell lung cancer cells yet.In this study, we have researched different expression of mRNA and protein of HDAC5 in non-small cell lung cancer and para-cancerous tissues, in an attempt to discover the relationship between HDAC5 and non-small cell lung cancer. Besed on this result, wild type HDAC5 (HDAC5wt) gene was transfected into the lung adenocarcinoma NCI-H1299 cell line to monitor the effection of proliferation, apoptosis and migration, and we will preliminarily investigate the effcetion of biological characteristics and mechanisms of HDAC5 on NSCLC.Objectives:1. To determine the expression of HDAC5 in non-small cell lung cancer and para-cancerou tissues, in an attempt to disclose the relationship between HDAC5 and non-small cell lung cancer. 2. To determine the influence of HDAC5 overexpression on the proliferation, apoptosis, migration in lung adenocarcinoma NCI-H1299 cell, and defined that HDAC5 is contributed to the tumorigenesis and cancer development in NSCLC.3. To probe the mechanism of HDAC5 overexpression on NCI-H1299 cell migration.Methods:1.28 cases of non-small cell lung cancer and para-cancerous tissues with clear pathologic type have been collected. Real-time RT PCR and Western blotting had been used to detect mRNA and protein expression of HDAC5 in cancer and para-cancerous tissues.2. FuGene HD non-liposome transfection reagent was adopted to transfer HDAC5 wt into NCI-H1299 cell, G418 was used for screening stable transfected cell lines. Real-time RT-PCR and Western blotting were used for identification of successful transfection. MTT assay was used to determine the proliferation after HDAC5 overexpression. DAPI staining was used to detected cell apoptosis; flow cytometry technology was used for inspecting influence of HDAC5 overexpression on NCI-H1299 cell cycling and apoptosis. Scratch wound healing assay and transwell migration assay are used to inspect influence of HDAC5 overexpression on NCI-H1299 migration.3. Real time RT-PCR was used to detected expression level of MMP-2,MMP-9 mRNA in NCI-H1299 cell before and after transfection; and we used Phalloidine-Rhodamine staining method to examine the expression of F-actin pseudopodia in these three groups.Results:1. Different expression of mRNA and protein in human non-small cell lung cancer and para-cancerous tissues.Collectting 28 NSCLC tissue samples from cardiothoracic surgery department of Changhai hospital in Second military medicine university, during April to November 2009. All the samples have been diagnosised as NSCLC. There are 20 adenocarcinomas and 8 squamous lung carcinomas; including 15 men and 13 women; between 41 years old and 70 years old, and the average age is 57.8. Result of real-time RT PCR shows that expression of HDAC5 was significantly lower in 71.4%(20/28) cancer tissues than in para-cancerous tissues(p<0.05,lung cancers vs.para-cancerous tissues). Result of Western blotting shows that HDAC5 protein expressed lower in cancer tissues than para-cancerous tissues, and in accordance with the result of real-time PCR.2. Effect of overexpression of HDAC5wt on the proliferation, apoptosis and migration of lung adenocarcinoma NCI-H1299 cell.Overexpression of HDAC5wt gene in stable lung adenocarcinoma NCI-H1299 cell significantly increased the HDAC5 mRNA and protein level. MTT results suggested that during a 5-day growth, there was no difference in OD490 value within 24h; on day 2 to day 5, the OD490 value of experimental group was significantly lower than normal control group and negative group. The result of DAPI staining showed that karyopyknosis and distortion were significant in experimental group than in normal control group and negative group; we used the flow cytometry technology to study the effection of HDAC5 on cell cycling and cell apoptosis, there was no difference among these three groups in cell cycling and early period of apoptosis rate; but in late period of apoptosis, the apotosis rate is significantly higher in experimental group (14.13±2.9) than in normal control group (7.3±3.55) and the negative control group (8.16±0.37) (p<0.05). Scratch wound healing assay shows significantly shorter migration distance in the experimental group than in the other two control groups; Transwell migration assay revealed the number of trans-membrane migration cells was significantly lower in the experimental group (158.3±19.42) than in normal control group (247±12.12) and negative control group (236±14.93)(p<0.05).3. Research on the mechanism of HDAC5 on adenocarcinoma H1299Real time RT-PCR shows that expression of MMP9 is significantly lower in the experimental group (0.32±0.07) than in normal control group (0.98±0.17) and negative control group (1.29±0.33) (p<0.05), but there was no difference in the expression of MMP2 within the three groups; Phalloidine-Rhodamine staining shows that F-actin pseudopodia was expressed lower in the experiment group than in the other two control groups.Summary:1. HDAC5 gene and protein expression in NSCLC cancer tissues is significantly lower than that in para-cancerous tissues, and is assumed to be related with the tumorigenesis and cancer development of NSCLC.2. HDAC5 gene can prohibit the proliferation, migration ability of NCI-H1299 cell, and promote its apoptosis, effecting it's biological function. 3. The synergic action between MMP-9 and F-actin is very important during the suppression of cell migration with HDAC5.Conclusion:The expression of HDAC5 is lower in NSCLC cancer tissues than in para-cancerous tissues. HDAC5 overexpression can prohibit the proliferation, migration ability of lung adenocarcinoma NCI-H1299 cell, and promote its apoptosis, thus influence tumorigenesis and cancer development of NSCLC.