Study On Expression And Biological Mechanism Of DLC-1、ROCK1 In Non-small Cell Lung Cancer

Lung cancer is a most common malignant tumor with high prevalence and mortal ity all over the world, of which non small cell lung cancer (NSCLC) accounts for over 80%. Lots of study have been focused on the pathogenesis, early diagnosis and treat ment against NSCLC. However, the molecular mechanisms underlying its pathophysi ology remain obscure and need to be further investigated to develop novel therapeutic startegies in the future.DLC-1 gene, a new tumor-suppressor gene, is located on chromosome 8 of 21.3-22 areas. DLC-1 was found in the study of liver cancer. Then, the expression and pathogenesis of DLC-1 in colorectal cancer, breast cancer, pancreatic cancer and stomach cancer and other malignant tumors were developed gradually. The expression of DLC-1 present a low level or even loss in malignant tumor. To restore its expression level can inhibit tumor cell proliferation, migration, apoptosis, invasion, and apoptosis. Therefore, It can be a new molecular and provides a new train of thought for everyone and provide a new thought for everyone in the field of cancer research. It can provide a newer and more effective clinical treatment of tumor by mastering the DLC-1 gene biology function and molecular mechanism of action, restoring and enhancing the function of the protein. However, there are few reports of the gene about the relationship between its expression and survival and its specific mechanism of inhibitory effect in lung cancer. And studies have found that protein and DLC-1 no correlation between ROCK1 protein expression in cancer tissues, RhoA/ROCK signaling pathway may be the main mechanism of DLC-1 mechanism. Based on the above reasons, our study intends to study the differential expression of DLC-1 between carcinoma tissue and para-carcinoma tissue in lung cancer, and to explore the relevance with the clinical index, and to reveal the relationship between the DLC-1 and lung cancer development. We also construct eukaryotic expression plasmid pcDNA3.1-DLC-1. On this basis, we used the liposome to transfect the lung cancer cell line with plasmid pcDNA3.1-DLC-1. We observed the inhibitory effect of DLC-1 on cell growth, invasion of lung cancer. The studies have found that DLC-1 gene can control the tumor growth by regulating the RhoA/ROCK signaling pathway. So we also study the relationshiop with ROCK gene and the RhoA/ROCK signaling pathway. We explore its possible mechanism in order to provide new clinical thought and theory basis for the diagnosis and treatment of lung cancer. This study will be divided into three parts.Part One The expression and clinical significance of DLC-1,ROCK1 in NSCLC ObjectiveWe explore the expression of deleted in liver cancer 1 (DLC-1) and Rho-kinase 1(ROCK1) in non-small cell lung cancer to analysis the relationship between clinical pathological features and the expression of DLC-1 and ROCK1, therefor to explore the correlation between DLC-1 and ROCK1.Methods1. An analysis of 68 case non-small cell lung cancer patients from January 2011 to June 2012 in binzhou medical school affiliated hospital was carried out retrospectively. All cases were confirmed by pathological examination of surgical operation.68 carcinoma tissues and 68 adjacent tissues were collected.2. The expression of DLC-1,ROCK 1 in carcinoma and adjacent tissues was detected by Envision immunohistochemical technique.3. We analyzed the relationship between clinical pathological features and the expression of DLC-1 and ROCK1, the correlation between DLC-1 and ROCK1.4. All patients were followed up by the end of March 2015.5. Survival period was calculated from the date of surgery to the end of March 2015. We compared different surial differences between groups according to the results of follow-up.Results1. Immunohistochemical results showed that DLC-1 showed a low expression or a missing expression in non-small cell lung cancer. Positive DLC-1 expression rate of carcinoma tissues was 38.24%, positive DLC-1 expression rate of adjacent tissues was 75% with statistically significant difference(X2=14.256,P<0.01).Positive ROCK1 expression rate of carcinoma tissues was 57.35%, there was no expression of ROCK1 in adjacent tissues with statistically significant difference(X2=45.378,P<0.01).2. The expression of DLC-1,ROCK 1 was correlated with tumor differentiation, lymph node metastasis, TNM staging, rather than with sex,smoking history and pathological type. Through the correlation analysis, the expression of ROCK 1 in DLC-1 positive group was 34.62%(9/26), and the expression rate of ROCK1 in DLC-1 negative group was 71.43%(30/42). There was negative correlation between DLC-1 and ROCK1(r=-2.359, P=0.04).3. The 3 year survival rate in DLC-1 protein high expression group(88%) was obviously higher than that of low expression group (57.2%)with statistically significant difference(P=0.031).ConclusionLower or misssing expression of DLC-1 and higher expression of ROCK1 protein may play an important role in the occurence and development of NSCLC. Detecting the expression of DLC-1 and ROCK1 protein maybe useful for evaluating the biological behavior and prognosis of NSCLC.Part 2 The expression and correlation of DLC-1,ROCK1 in non-small cell lung cancer tissue and cell lines。ObjectiveTo explore the mRNA and protein level expression of deleted in liver cancer 1(DLC-1) and Rho-kinase 1(ROCK1) and the correlation between DLC-1 and ROCK1 in non-small cell lung cancer.Methods1. We collected 23 cases fresh non-small cell lung cancer tissue and 23 cases tissue adjacent to carcinoma. Lung cancer cell line H1299, A549 and Glua were prepared.2. Reverse transcription polymerase chain reaction (Real time RT-PCR) was used to detect mRNA expression of ROCK 1, DLC-1 in fresh lung cancer tissues.3. Using RT-PCR and Western Blot to detect mRNA and protein expression of ROCK1, DLC-1 in lung cancer cell lineResults1. The real time RT-PCR result showed that the mRNA expression of DLC-1 in lung cancer tissues is significantly lower than that in tissue adjacent to carcinoma,the mRNA expression of ROCK1 in lung cancer tissues is significantly higher than that in tissue adjacent to carcinoma.There was negative correlation between DLC-1 and ROCKl with statistically significant difference.2. The DLC-1 mRNA and protein expression of H1299 cell line was positive, that of A549 and Calu6 were positive.ConclusionDLC-1 showed a low expression or a missing expression in lung cancer tissues and lung cancer cell line.ROCK1 showed a high expression or a missing expression in lung cancer tissues and lung cancer cell line. RhoA/ROCK signaling pathway may be the main mechanism of DLC-1.Part 3 Construction of pcDNA3.1-DLC-1 recombinant vector and biological effects of transfection cellsObjectiveTo explore expression of deleted in liver cancer 1(DLC-1) and Rho-kinase I(ROCK1) in non-small cell lung cancer and the correlation between DLC-1 and ROCK1.Methods1. We extracted total RNA of the lung cancer cell line H1299 by reverse transcriptase cDNA synthesis; Application of RT-PCR gene amplification DLC-1 complete open reading frame, insert the eukaryotic expression vector pcDNA3.1.2. We used endotoxin in extraction method to extract the good expression vector, transfected transiently A549, Calu6 cells with liposome. After 48hours extracted RNA, transcripted reversely cDNA, identified the quality of RNA and cDNA, designed the prime of ROCK1 and confirmed the ROCK1 gene expression changes. We used Transfection of A549, Calu6 with empty plasmid pcDNA3.1 as controls.3. Endotoxin in extraction method to extract has built good expression vector, liposome transient transfection A549 cells, using transwell Chambers experiment to assess their impact on the ability of tumor cell invasion.4. We diluted the A549, Calu6 cells which was transfected well to 200 cells/10 ml, inoculated into 6 orifice. And we dyed the cells with crystal violet stain after the cells completed to clone growth, then we observed the cell growth and infection. Transfection of A549, Calu6 with empty plasmid pcDNA3.1 as controls.5. We stained the A549 cells which was transfected well with DAPI and detected the cell cycle change with FACS. Transfection of A549 with empty plasmidpcDNA3.1 as controls.Results1. The pcDN A3.1-DLC-1 recombinant vector was constucted successfully.2. The over-expression DLC-1 in A549,Calu6 could downregulate the expression of ROCK1. After transfection pcDNA3.1-DLC-1, the DLC-1 expression at the mRNA level had a higher effect, the results have significant difference, (P<0.05). After expressing DLC-1, ROCK1 mRNA level was significantly suppressed, after expressing DLC-1 in A549, Calu6, The expressing of ROCK 1 mRNA level drops in the control group 33%,47% respectively, the results have significant difference, (P< 0.05).3. After over expression of DLC-1 in lung cancer cell line A549,Calu6, the invasion in vitro experiments in cell membrane of the experimental group and the control group statistics were 26.8±1.3、67.8±5.2, the results have significant difference, (P<0.05). Clone formation experiment suggests A549, Calu6 cell clone formation ability decreased significantly, the results have significant difference, (P<0.05); DLC-1 also could block the G1/S phase transformation in lung cancer cells.ConclusionDLC-1 gene has a negative regulation role on lung cancer cells. It was related with cell proliferation and cell cycle division. DLC-1 may play an important role in the occurrence and development of NSCLC through ROCK1 signaling pathway.