Screening Of Gene Mutations Associated With Bone Metastases In Non-small Cell Lung Cancer Patients And The Influence Of GDP-mannose-4,6-dehydratase To Proliferation And Apoptosis In Non-small Cell Lung Cancer Cells

【Background】Lung cancer,as known as primary bronchial lung cancer,is one of the pulmonary malignancies with highest morbidity and mortality.It is indicated by National Cancer Center that 733 thousand new lung cancer cases and 610 thousand lung cancer deaths would occur in China in 2015.The morbidity and mortality rate in male ranks first of all kinds of cancers in China,and the morbidity rate in female ranks second while the mortality rate ranks first of all the cancers.In world-wide,The morbidity and mortality rate of lung cancer in male ranks first of all kinds of cancers and the morbidity rate in female ranks third while the mortality rate ranks second.According to pathological type,lung cancer can be dividedto small cell lung cancer,non-small cell lung cancer which contains adenocarcinoma,squamous cell carcinoma,and large cell carcinoma.As there are no obvious symptoms in early stage of lung cancer,the majority of patients have been in local advanced or even late stage when the tumor is been diagnosed,losing the chance of radical surgery.Bone is one of the blood transfer sites of lung cancer and half of lung cancer patients with skeletal metastases will encouter bone-related events.In the event of bone-related events,the patient’s survival time can be reduced by half.Non-small cell lung cancer accounts for 75%-80%of the total number of lung cancer with a poor general prognosis,whose 5-year survival rate is 15%-16%.The standard treatment of lung cancer includes surgery,radiotherapy,and chemotherapy which is into the bottleneck period.With the development of molecular targeted drugs and immunological checkpoint inhibitors,lung cancer treatment strategy has undergone tremendous changes,ushering in a new dawn.A number of drive genes involved in the occurrence and development of lung cancer which is a complex process.At present,the main target genes for non-small cell lung cancer include epidermal growth factor receptor sensitive mutations,anaplasticlymphoma kinase and c-ros oncogene 1 fusion,human epidermal growth factor receptor 2,mutations,BRAF V600 E mutation,high level MET amplification or MET exon 14 skipping mutation,RET rearrangement and so on.In fact,there are still many unknown driver genes.Looking for a new driver gene or therapeutic target is the current hot spot in lung cancer research.The cancer genome atlas is a collection of a large number of human cancer sequencing results,especially lung cancer,designed to systematically analyze the human cancer gene mutation map for the diagnosis and treatment of cancer andnew strategy.483 cancer-related genes have been screened by analyzing the TCGA database and we want to detect them in non-small cell lung cancer with bone metastases by using the next generation sequencing.From the adenocarcinoma RNA sequencing results of lung cancer in TCGA database,we find that GDP-mannose-4,6-dehydratase is one of the significant difference genes.GMDS is one of the key enzymes in the process of fucosylation,involving in the synthesis of GDP-fucose with GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase.Abnormal fucosylation has been founded in lung,colorectal,breast,ovarian,liver and pancreatic cancers and is closely related to the occurrence and development of cancers.Gene GMDS is located on chromosome 6,encoding 372 amino acids.Defective activity of GMDS leads to leukocyte adhesion deficiency type II syndrome.There are GMDS mutations in primary and metastatic colorectal carcinomas,whereas GMDS mutations are significantly higher in the metastatic foci than in the primary foci,which may be associated with progression of the disease.The deletion of GMDS exon in colorectal tumor cell line HCT116 leads to a functional defect that allows it to escape the immune surveillance of natural killer cells.Other key enzymes in fucosylation are also closely related to cancers.Metastatic colorectal tumor cell line sw620 expresses higher levels of FX than original tumor cell line sw480,enhancing tumor-endothelial adhesion.Lung adenocarcinoma cell line CL1-5 highly expresses FUT8,increasing proliferation,invasion and metastasis of cancer.The expression level of GMDS in non-small cell lung cancer and its role in the development and progression of lung cancer have not been reported.Therefore,we want to detect the expression level of GMDS in non-small cell lung cancer patients by immunohistochemistry,and to study the role of GMDS in the development of non-small cell lung cancer and its possible mechanism to explore whether GMDS can be used as a target in non-small cell lung cancer.【Objective】Todetect 483 cancer-related genes in non-small cell lung cancer patients with bone metastases in order to find out new lung cancer-related gene mutation.To detect the expression level of GMDS in non-small cell lung cancer patients to find whether there is a GMDS mutation in non-small cell lung cancer cell lines.To study the influence of GMDS in the proliferation and apoptosis of non-small cell lung cancer by silencing GMDS with a lentivirus-mediated RNA inference.【Methods】The 483 cancer-related genes was detected in 8 non-small cell lung cancer patients with bone metastases by next-generation sequencing platform Hiseq2000_PE75.Expression of GMDS was detected by immunochemistry in 10 human lung cancer tissues and the tissues around edge of the cancer.The mutation of GMDS was been detected by PCR and the expression of GMDS was been observed by real time PCR.The expression of GMDS was been silenced by lentivirus-mediated RNA inference,then the viability of cells was detected by MTT assay and the number of cell apoptosis was detected by flow cytometry.【Results】Part1:3620 gene mutations were identified anda number of gene mutations were detected in lung cancer-related signaling pathways.In the meanwhile,consistent mutations were found in 16 genes,and the mutation of hepatocyte nuclear factor 1 alpha（1394₁395ins8）was found in 6 patients（75%）,the mutation of APC（A818G）was found in 5 patients（62.5%）and the mutation of CD22（G1703A）was found in 4 patients（50%）.The results of Sanger sequencing of HNF1α and CD22 were consistent with the results of NGS sequencing.Part2: The expression of GMDS was3.597±1.908 in the lung cancer tissues,0.453±1.119 in adjacent tissues by immunochemistry.The expression level of GMDS in the lung cancer tissues was significantly higher than that in adjacent tissues（P<0.01）.The expression of GMDS of A549 cell in GMDS-si RNA group was 0.28±0.017,compared to 1.03±0.097 in the control group,which was significantly different（P<0.01）.The expression of GMDS of H1299 cell in GMDS-si RNA group was 0.19±0.022,compared to 1.00±0.004 in the control group,which was also significantly different（P<0.01）.The OD of H1299 cell in GMDS-Si RNA group after 96,120 hour culture was 0.464±0.019 and 0.453±0.038 while it was 0.815±0.051 and 0.969±0.012 in the control group,which is significant difference（P<0.01）.The OD of A549 cell in GMDS-Si RNA group after 96,120 hour culture was 0.744±0.005 and 0.858±0.041 while it was 1.105±0.026 and 1.441±0.042 in control group,which is significantly different（P<0.01）.The percentage of H1299 cell ofGMDS-Si RNA group in phase G1,S,G2 is 46.15±1.20%,50.80±0.82%,3.05±0.41%,compared to 27.96±0.93%,59.40±0.75%,12.64±1.21% in the control group respectively,which is significantly different（P<0.01）.The percentage of A549 cell of GMDS-Si RNA group in phase G1,G2 is 58.2±1.32%,10.62±0.80,compared to54.83±0.41%,14.42±0.42% in the control group respectively,which is significantly different.The percentage of apoptotic cell of H1299 cell in GMDS-Si RNA group is14.90±0.39%,higher than 3.83±0.23% in the control group（P<0.01）.The percentage of apoptotic cell of A549 cell in GMDS-Si RNA group is 23.01±0.45%,higher than3.59±0.13% in the control group（P<0.01）.【Conclusion】NGS results show that gene mutations in multiple lung neoplasms associated signaling pathways are involved in the development of non-small cell lung cancer.Besides,the consistent mutations such as HNF1α（1394₁395ins8）,CD22（G1703A）and APC（A818G）would be the potential genes accounting for bone metastasis in lung cancer.Moreover,the expression of GMDS in the lung cancer tissues is significantly high.Silencing GMDS expression of NSCLC cell lines,significantly inhibits cell proliferation,and promotes apoptosis.GMDS may be an important indicator involved in proliferation and apoptosis of non-small cell lung cancer and it may be a potential target for treatment.