| Lung cancer is one of the world's highest morbidity and mortality of malignant tumor,is currently the biggest threat to human life and health.With the development of China's industrialization process and damage to the environment,the increasingly serious haze in recent years,in northern region,lung cancer has become the first threat in our country,statistical data shows that in 2012 lung cancer incidence and mortality were first to male,the second incidence and mortality to female,and lung cancer patients 5 years of survival rate is low,80% of the clinical diagnosis of lung cancer patients have lost the operation chance.Lung cancer easily metastasizes to brain and bone,bone metastasis is the main complications of lung cancer,main symptoms includs pain,pathologic fracture and metastatic spinal cord compression and high blood calcium and physical condition.Patients with lung cancer that has spread to the brain,bone,the prognosis is often poor,so in the initiation and development process lung cancer,screening the factors associated with lung cancer metastasis is closely related to the prognosis and quality of life to patients.The present study found that a variety of eukaryotes endogenous miRNA molecules,mainly involved in the mRNA interference phenomenon,as an important transcription regulatory factors in the virus,animal and plant species after the transcription of mRNA silence effect(post-transcriptional gene silencing,PTGS)plays a vital role.The present study has confirmed that miRNAs mainly through its seed sequence matches to target gene mRNA with complete or incomplete complementary pair,results in the mRNA degradation or the protein of target gene translation inhibition,and regulates the expression of one or more of the target gene,exerts its functions in the biological development and in the process of tumorigenesis and progression.Currently researches on the process of tumorigenesis and progression associated mi RNAs mainly focused on the human endogenous miRNA.More and more studies have showed that exogenous miRNA,in particular,mi RNA derived from the virus in the process of tumorigenesis and metastasis also play an important role.MiRNA derived from the virus generated in the process of virus infection which has the effect of RNA interference.In 2004,researchers have discovered the first virus encoded microRNA belonged to herpes virus family,in addition,more researches showed the virus encoded microRNAs targeted its mRNA to adjust different stages in the process of viral infection.Recent studies have shown that parts of the viral miRNA can also reshape the intracellular environment by interfering with human genes,make it more suitable for the need of infection and survive,such as it has been reported that Kaposi's sarcoma associated herpesvirus KSHV can use virus endogenous miRNA molecules regulate gene transcription.Our researches mostly focus on the functions of hsv2-miR-H9-5p and its target genes in lung cancer initiation and development process,the underlying molecular mechanism of interaction and biological function in the treatment of lung cancer.PART1 Screen and Verification of miRNAs that Associated with Lung Cancer Bone MetastasisObjective: Screening the differential expression miRNAs by comparing the lung cancer tissue to bone metastasis samples.Bioinformatics analysis combined with literature review for the purpose of the research.Enlargement the sample size of test,verification the differential expression of miRNA that we focused on by using fluorescent quantitative real time PCR.Methods: Using company Exiqon's miRNA microarray,with 10 cases of lung cancer bone metastasis specimens and 10 cases of primary lung cancer specimens compared to screen with the aim to obtain the differential expression miRNA.Bioinformatics analysis combined literature review,determined the next miRNA that is going to study.Using fluorescence quantitative PCR instrument Roche 480 II,QPCR validation the differnential expression,and further expanded the sample size to verify the microarray results.Results: We found several miRNA which expression were differential compared primary lung cancer with bone metastases.Conbined with literature review,we selected hsv2-miR-H9-5p as our research aim.QPCR validated the differential expression of hsv2-miR-H9-5p.Further expanded the samples validated the differential expression.Conclusion: In the process of bone metastasis of lung cancer,miRNAs that differentially expressed indicated they may play a role in lung cancer metastasis.Hsv2-miR-H9-5p expressed differentially,using real-time PCR to verify the microarray results and further expanded the sample size to validate differential expression.Microarray and quantitative PCR results showed hsv2-miR-H9-5p may play a function in the process of lung cancer bone metastasis,this miRNA molecule maybe serve the purpose of further research.PART2 Validation of Hsv2-miR-H9-5p Target Gene and its Function in Lung Cancer CellsObjective: Using the lung cancer cell lines LTEP-?-2 and SPC-?-1 confirmed the function of hsv2-miR-H9-5p in the process of lung cancer metastasis at cellular level.Explore the interaction of hsv2-miR-H9-5p and its target genes and the possible regulatory mechanism.At the same time research on the possible biological functions of target gene SOCS2.Methods: Transwell assay was performed to detect the function of hsv2-miR-H9-5p on cell migration,matrigel invasion experiments detected the influence of hsv2-miR-H9-5p on lung cancer cell invasion ability;CCK-8 assay analysed the influences of hsv2-miR-H9-5p overexpression on cell proliferation.Flow cytometry detected the effect of hsv2-miR-H9-5p on cell cycle distribution and apoptosis;bioinformatics analysis(miRDBase,RNAhybrid,targetscan,etc.)screened for the target genes of hsv2-miR-H9-5p,dual luciferase reporter assay to verify the results;research the effect of target gene SOCS2 on the biological behavior of lung cancer cell LTEP-?-2 and SPC-?-1.Results: Migration assay showed that hsv2-miR-H9-5p promoted cell migration;invasion assay showed that hsv2-mi R-H9-5p mimic promoted invasive ability of matrix gel in two groups of cells;CCK-8 assay showed that compared with negative control,hsv2-miR-H9-5p mimic promoted significantly cell proliferation in the two cell lines LTEP-?-2 and SPC-?-1;apoptosis was detected by flow cytometry which showed that apoptosis rate of hsv2-miR-H9-5p mimic group was less than that of negative control;cell cycle analysis showed no obvious difference;dual luciferase reporter assay showed that SOCS2 is the target gene of hsv2-miR-H9-5p,real-time quantitative PCR and western blot were used to further confirme the results at the mRNA and protein level of SOCS2;inhibited lung cancer cell proliferation,promoted cell apoptosis of lung cancer,weaked the migration and invasion of lung cancer cells because of SOCS2 overexpression.Conclusion: Our study showed that hsv2-miR-H9-5p can promote the malignant phenotype of lung cancer cells,SOCS2 gene mRNA 3'UTR region was a specific regulatory site of hsv2-miR-H9-5p derived from exogenous virus,the expression level of the encoding protein SOCS2 was regulated by hsv2-miR-H9-5p in tumors.There were more researches on the function of SOCS2 in different tumors and the functions were significantly different in different tumor types.In our presrnt study,we found that SOCS2 significantly inhibited the tumor malignant biological behavior in the two cell lines LTEP-?-2 and SPC-?-1.Our work showed that from the cellular level,virus through its encoding mi RNA at the transcription level regulated the endogenous gene expression which provides direct evidence,and further deepened the understand of the mi RNA derived from virus and its molecular mechanism in the progress of tumor progression. |
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