| Objective and backgrond:Lung cancer is one of the fastest-growing morbidity and mortality, and the greates tthreaten human health, malignant tumors.According to a new study of the national cancer registry Center in 2014,, male lung cancer mortality is 49.27% ranked first place in all malignant tumors;and female lung cancer mortality rates is 21.66%, ranked second in all malignant tumors(breast cancer 25.89%)in the country.Lung cancer incidence and mortality in cities are 20.48% and 27.05% ranked first place throughout the country;meanwhile in the countryside lung cancer incidence and mortality in the country is 18.5% and 22.42% ranked the first place too.With the advancement of technology and researchers’ in-depth study, although we have more and more awareness about lung cancer, but in the prevention and treatment of lung cancer is still the major problem we are currently facing.While many lung cancer patients with clinical symptoms of medical treatment, most already in advanced,studying on the pathogenesis of lung cancer, finding the early diagnosis of lung cancer in molecular biology, and exploreing therapeutic target for lung cancer is not only producing great value in the clinical, but also providing a more positive meaning for society more importantly.Lung cancer from happening to development is a process of gradual accumulation, generally due to the disorders regulation of cell growth and changes in growth patterns, causing precancerous cells and genetic changes.Lung cancer is an accumulation of genetic damage, is a multi-step, multi-gene involved in the process, including the activation of oncogenes, inactivation of tumor suppressor genes, and the disorder of epigenetic regulation, regulation of transcription level, post-transcriptional level, resulting in cell tissues and organs lose their nature and function,which produce a lot of damage to the human body.Lnc RNA is in the form of RNA in regulating gene expression levels on multiple levels, in General, more and more lnc RNA which are widely found to participate in genome regulation, generation, development and metastasis of tumors has played an important role.Although lnc RNA h19 expression have a close relationship with other tumors, including liver cancer, colorectal cancer, ovarian cancer, prostate cancer, breast cancer, and but the literature in non-small cell lung cancer is still rare.Therefore, this study was designed to study the correlation between the abnormal expression of long non-coding RNA h19 in non-small cell lung cancer,and its impact on the biological behavior of cancer cells.Methods:⑴ Collecting resected cancer specimens and paraneoplastic normal lung tissue samples of non-small cell lung cancer from 64 patients who have not received chemotherapy and whose postoperative pathologic diagnosis is non-small cell lung cancer.the tissues which are cleaned by Ice water and salt and quick-frozen 24 hours are saved in-80 ℃freezer.⑵Checking the expression of h19 in the 64 cases of non-small cell lung cancer by q RT-PCR:Firstly, using Trizol extract total RNA in cancer tissue and normal tissue,Then using agarose gel electrophoresis to check RNA quality, ensure the integrity of RNA. Design reverse transcription primers and the upstream and downstream primers of lnc RNA h19, and reverse transcription is based on reverse transcription Kit manual steps. The last,checking the expression of lnc RNA h19 by q RT-PCR.⑶According to each group’s expression to analysis their relevance to the risk factors of lung cancer.⑷Select A549 lung cancer cell lines and normal human bronchial epithelial cell line, to cell culture.⑸Using Trizol extract total RNA,Design reverse transcription primers and the upstream and downstream primers of lnc RNA h19 and β-actin,and reverse transcription.Cecking the expression of h19 in i lung cancer cell lines and bronchial epithelial cells by QRT-PCR.and comparing h19 expression in lung cancer cell lines with normal expression in bronchial epithelial cells.⑹Constructing overexpress plasmid vectors: p CDNA3.1-lnc RNA h19:firstly,According to the above method to design primers for amplification,and checking it with agarose gel electrophoresis.Ch ecking it again with agarose gel electrophoresis and double digestion when constructing overexpress plasmid vectors p CDNA3.1-lnc RNA h19 successfully.The last using the Gene sequencing to ensure the target gene truthfully.⑺G418 selects stable expression cell line A549:First selecting the concentration of antibiotic G418,Then selecting stable expression cell line A549-p CDNA3.1-lnc RNAh19.The last,using QRT-PCR checks the expression of target gene to confirm whether the cell lines established successfully.⑻Checking the migration capabilities of cell line A549-p CDNA3.1-lnc RNAh19 by scarification, and transfected with empty vector in the negative control group and only joined the transfection reagent in blank control group for comparison.⑼Checking the proliferative ability of cell line A549-p CDNA3.1-lnc RNAh19 by MTT, and transfected with empty vector in the negative control group and only joined the transfection reagent in blank control group for comparison.⑽Checking the invasive ability of cell line A549-p CDNA3.1-lnc RNAh19 by transwell, and transfected with empty vector in the negative control group and only joined the transfection reagent in blank control group for comparison.Results: 1 Lnc RNA h19 expression in non-small cell lung cancer significantly higher than the adjacent tissues(P<0.05).2 Expression of lnc RNA H19 in non small cell lung cancer is related to lymph node metastasis(P<0.05)but not related to age, sex, smoking history and tumor types.3.Overexpression of lnc RNA h19 in non-small lung cancer can increase cell ability of migration, invasion, proliferation.Conclusion: 1.Lnc RNA h19 which express significantly higher than that of normal lung tissue in non-small cell lung cancer significantly higher than that of normal lung tissue, has a great relationship with the occurrence of tumors, and cancer-promoting genes function.2.Expression of lnc RNA H19 in non small cell lung cancer i, which is related to lymph node metastasis(P<0.05)but not related to age, sex, smoking history and tumor types,increases lymph node metastasis in the cancers.3.Overexpression of lnc RNA h19 in non-small lung cancer,which can increase cell ability of migration, invasion, proliferation,may be a new target for treatment in non-small cell lung cancer. |
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