Alteration Of Long Non-coding RNA Expression In Mouse Lung Tumorogenesis Induced By Urethane

Background and objectLong non-coding RNA(lncRNA) are defined as transcripts longer than200ntwithout coding capacities, but play an important role in the regulation of geneexpression. Multiple lines of evidence indicate that lncRNA have a variety of regulationways by interacting with its partner DNA, RNA, and/or protein. Although thepathogenic mechanism of lncRNA remain unclear, a large number of clinicalobservations and experimental evidence indicate that lncRNA can regulate geneexpression at transcription and post-transcription levels and widely take part in speciesevolution, embryonic development, stem cell maintenance, cell proliferation anddifferentiation and tumorigenesis. Compared with variety of malignant tumors, theincidence and mortality of lung cancer was in the first place, it serious threat the healthof people. At present, the sputum cytology, imaging and other traditional diagnostictechniques in the diagnosis of lung cancer lack of specificity with other lung diseases,such as pneumonia, tuberculosis and so on. Therefore, to find a high specificity andsensitivity of diagnostic methods is an important way to improve the survival rate ofpatients with lung cancer. With the development of the molecular biology of cancerresearch, the diagnosis of lung cancer at the molecular level is possible. The existing studies have shown that lncRNA play an important role in tumorigenesis anddevelopment，the dysregulation of lncRNA has been regarded as a primary feature ofsome human cancers. Such as MALAT-1were overexpressed in non-small cell lungcancer, PCGEM1overexpression in prostate cancer cells. However, the expression andfunctional significance of lncRNA in lung cancer progression remains unclear.Therefore, study the molecular mechanism of lung cancer and detection specificmolecular biomarkers for lung cancer, which will have important significance for theearly diagnosis of lung cancer. Our primary studies, base on the urethane-inducedmouse lung cancer model to study the altered expression of lncRNA in the developmentprocess of lung cancer. And then, select the specific expression lncRNA in thedevelopment process of lung cancer as potential molecular biomarkers of lung cancer,and provide some experimental evidence for lncRNA involved in chemicalcarcinogenesis.Methods1. Establish the mouse lung cancer model The treatment group received fourweekly intraperitoneal urethane((1000mg/kg in200μl of phosphate-buffered saline(PBS)) injections, the control group received four weekly intraperitoneal PBS. Micewere killed at three months and six months after urethane initiation respectively.2. Pathological analysis The biopsy of normal lung tissue and lung tumor tissuewere observed under the microscope after HE staining.3. Expression profiles of lncRNA Using the mouse non-coding RNA microarraysto analyze the dynamic expression of a large subset of non-coding RNAs (ncRNAs)in the urethane-induced mouse.4. Analysis the different experss of lncRNA in expression profiles Using theScreening criteria of5-fold up-or down-regulated in their expression level andprobability values (P <0.01) to select different express lncRNA.5. qRT-PCR to verify the microarray results Using the qRT-PCR to verify theexpression level of lncRNA whether consistent with the microarray results.6. qRT-PCR detection lncRNA expression levels Using the qRT-PCR to detection the expression levels of lncRNA in the urethane-induced mouse.7. Statistial analyses All statistical analysis was done with SPSS17.0software.Values are expressed as mean±S.D. All experiments were done at least three times.Differences between groups were analyzed by double-sided Student’s t-test orMann-Whitney tests for the compare of two groups, according to data feature. Thedifferences were considered to be significant when P<0.05.Results1. Establish the mouse lung cancer model The control group of mice did not findlung tumor. The incidences of lung cancer in the mice after urethane-treated threemonths were85%, the average tumor number of each mouse was2.81±2.04, andthe average tumor size was0.68±0.20. The incidences of lung cancer in the miceafter urethane-treated six months were100%, the average tumor number in eachmouse was6.00±2.07, and the average size of the tumor was1.76±1.34.2. Pathological analysis All tumors exhibited a homogeneous appearance at thestereomicroscope, and pathological analysis of the tumors showed that all wereclassifiable as adenomas.3. Analysis the different experss of lncRNA in expression profiles Using theScreening criteria of5-fold up-or down-regulated in their expression level andprobability values (P <0.01) to select different express lncRNA, we identified42lncRNA that statistically, differently expressed in lung tumor of urethane-inducedgroup compare to its non-tumor lung tissue and normal lung tissue of control group.These included14lncRNA that were overexpressed and28lncRNA that wereunderexpressed in lung tumor of urethane-induced group.4. qRT-PCR to verify the microarray results The result show that theexpression level of lncRNA was consistent with the microarray results.5. qRT-PCR detection lncRNA expression levels The express level of AF498294is both upregulated in the lung tissue of mice after urethane-treated three monthsand six months, the express level of AK017233and AK017306are downregulated.Compare to the lung tissue of the control group, the express level of AF498294isalso upregulated. Conclusion1. Mouse lung tumorogenesis induced by urethane, we use the mouse lung cancermodel as the basis for futher research.2. The expression of lncRNA significant change in urethane-induced mouse lungmodel, suggest that lncRNA involved in the occurrence and development of themouse lung.3. The lncRNA of AF498294, AK017233and AK017306were different expression inthe two didderent stage of lung cancer, they may be potential molecular markersfor the diagnosis of lung cancer.