| Lung cancer is a common malignant tumor and the incidence of lung cancer shows ascendant trend around the world.With the pollution and destruction of environment,the society pays close attention for the high incidence and mortality of lung cancer.Despite progress has been made in the diagnosis and treatmeng recently,the prognosis of lung cancer is still poor.The overall5-year survival rate of lung cancer still has been around9%-14%.Relapse and metastasis is the major factor which affect the treatment effect and the main reason leading to patients death.Tumor metastasis is associated with many different kinds of mechanisms. Lung cancer metastasis is a complicated process driven by multiple genes and follows multiple steps. The regulation of metastasis-associated genes is the molecular basis for metastasis.. Therefor, characterizing the realationship between the new metastasis associated gene funtion and the metastasis of lung cancer become more important for rational designing of new drugs, developing clinical prognosis assay for lung cancer and learning more about the related molecular mechanism of progress of lung cancer.In recent years, many studies demonstrate that the transcription factor MTA1protein family have important influence on the tumor metastasis.MTA gene family, is finally found and named in the rat metastatic breast cancer by the differential screening cDNA fragment library.In the recent five years, the significance of MTA1in the development and occurrence in many kinds of cancers is generally well known.In addition to the lung cancer,the expression of MTA1generally increase around the human cancers. In the analysis of tumor tissues such as endometrial carcinoma uterus and pancreatic tumor,we can found that the over-expression of MTA1mRNA or MTA1protein has the positive correlation with tumor invasion and lymph node metastasis.But around the breast cancer、 liver cancer and oesophageal carcinoma,the expression of MTA1leads to poor prognosis. Our former research found that,MTA1jene is a very important metastasis associated gene. The over-expression of MTA1can promote the process of cell proliferation and cell division,and even the invasion and migration of cancer cell.So we conjecture that MTA1jene may be the essence of the characteristic behavior of lung cancer cell.On the other hand,microRNA are very important regulation small RNAs include siRNA(small interfering RNA)、miRNA(microRNA)、piRNA(piwi interacting RNA) and hsRNA(heterochromatin associated small RNA) which play important role in the tumor development.But until now,many tumor-related microRNA are still unknown.The micro RNA-related mechanism of tumor metastasis is not entirely clear.So we need do more studies on the specific microRNA and target genes in a certain environment for the development of specific drugs about the the diagnosis and treatment of cancer.We choose high-metastatic lung cancer cell line named95D in order to observe the infiltration and metastasis of the tumor with bioinformatics approaches and RNAi or by inhibit or over-express the expression of microRNA.And the realationship between this mechanism of invasion and migration and the related microRNA。In order to observe the infiltration and metastasis of the tumor, establish a high-metastatic lung cancer cell line expressing enhanced green fluorescent protein(EGFP).Therefore this project intended to use the in vitro cuture,from the same cell lines (PLA-801)with tha same qenetic background of hign and low metastatic human lung cancer stain95D and95C as the research object,bioinformatics method and the MTA1gene transfection or RNA interference,microRNA expression or inhibition.a target gene expression detection,cell function analysis technology,combined with lung cancer clinical tissue samples analysis to study the MTAlgene to promote the realationship between the mechanism of lung cancer cell invasion and metastasis by regulating the expression of micro RNA to clarify the molecular targets of MTA1promoying invasion and metastasis of lung cancer cells,and intended to show microRNA function by MTA1regulation.To inestigated the molecular mechanism of invasion and metastasis of lung cancer,looking for a potential the rapeutic target for beneficial exploration.Methods:1. pLVTHM/shMTAl and pLVTHM/vector lentivirus interference vector were constructed.Designed MTA1siRNA and non-specific interference fragment,using TCTCTTGAA stem-loop structure,in the5’end and3’ends, respectively, together with Mlu I and Cla I restriction sites,plus TTTTT termination sequence in the3’ends. Designed and synthesised the Oligo oligonucleotide sequence of the shRNA DNA. Designed and synthesised the upstream chain and downstream chain,then made them annealied to form the oligonucleotide.Then got sticky end linear pieces from the pLVTHM carrier by double-enzyme cleavage method using the endonuclease Clal and Mlul. Double digestion product electrophoresis1.5h by0.8%agarose gel electrophoresis. Obtained the linear fragment by plastic recycling method for the follow-up of ligation reactions.Purified the target fragment,make the pLVTHM linear pieces connected with MTA1double-stranded oligo. pLVTHM/shMTAl and pLVTHM/vector lentivirus interference vector were constructed.95D cell with MTA1silenced by stable RNA interference was established.Conversed the adapter-ligated fragments of pLVTHM-shMTA1and pLVTHM/vector and shaked overnight at37℃,pick a single colony in5ml LB medium with ampicillin penicillin resistance, identificatied the bacterium fluid by PCR. And put the positive ones to sequenced.2.After pLVTHM/shMTA1, pLVTHM/vector and pLVTHM lentivirus interference vector was constructed, we established95Dcells by stable RNA interference. Three stably cell clones were chosen for functional exploration.Extracted total RNA from the stable monoclonal cell. And reverse transcription into cDNA. Identified the expression of MTAljene and protein in the stable monoclonal cell by Real-time PCR and western blot.3. The influence of MTAlgene silencing on the growth characteristics of lung cancer cellsExponential phase cells (1×103cells/well) were planted in the96-well plate with100ul cell suspension per well. At1,2,3,4,5,6and7d after seeding, cells were treated with10ul CCK-8solution for1h at37℃. Spectrometric absorbance at wavelength of450nm was measured on a microplate reader.4. Wound migration assay Equal numbers of cells (2×105) were seeded into6-well culture plate, each group has three duplicate wells. The injury line was made with a sterile plastic pipette tip on cells cultured in plates at90%confluency. After being rinsed with phosphate-buffered saline (PBS), cells were cultured in complete medium and migration of cells into the wound was observed at different time points, and then photographs were taken under a40×objective.5. Clonegenic survival assayEqual numbers of cells (100) were seeded into6-well culture plate, each group has three duplicate wells. Make cells spread evenly.Then cells were incubated at37℃、5%CO2for growth. After two weeks of incubation, when colonies were visible, rinsed with phosphate-buffered saline (PBS). They were fixed with methanol,then dyed them with the application of sappanwood dyeing liquid15min.Count the number of clones formed under the microscope.The experiment was repeated three times. Then planting efficiency (PE) were calculated as follows: PE=colony number/inoculating cell numberx100%.6.Cell cycle assaysThe cells were harvested at90%confluency, washed in PBS, and then fixed with ice-cold70%ethanol at4℃overnight. When ready to stain with, cells were rinsed twice in PBS. Then cells were stained with PI at room temperature for30min. Cell cycle phase distribution was analyzed by flow cytometry.7.Transwell chamber assay for migrationThe study used cells at exponential phase, after being rinsed with phosphate-buffered saline (PBS).Cells was digested with trypsin to obtain single cell viability.Make the cell density to1-10×105.Put200μlcells suspension in internal chamber.Put complete medium in6-well culture plate. Then cells were incubated at37℃、5%CO212h for growth.Each group has three duplicate wells.Took up the internal chamber, wiped the cells which were not through the membrane.. They were fixed with methanol,then dyed them with the application of sappanwood dyeing liquid15min. Count the number of cells under the microscope.The experiment was repeated three times.8. Transwell chamber assay for invasionFirst,put symmetrical matrigel in the nternal chamber, all the steps were finished on the ice.Then Put200ul cells suspension in internal chamber.Put complete medium in6-well culture plate. The rest of the steps were the same as the previous experiment.9. Statistical analysisSPSS13.0software package was used to carry out statistical analysis. Results are presented as mean±SD. Comparison of mean was analyzed by One-Way ANOVA or Independent-Sample T Test. Factorial analysis was utilized for analyzing the results of CCK-8and wound migration assay. Student-Neuman-Keuls method was used for multiple comparing. A P value less than0.05was considered statistically significant.Results:1.We constructed the pLVTHM/shMTA1, pLVTHM/vector and pLVTHM lentivirus interference vector sucessfully, then we established95D cells by stable RNA interference. Three stably cell clones were chosen for functional exploration.2.After the MTA1jene was scilenced,the capacity of proliferation of he human lung cancer95D cell in vitro was no significant change.The95D/NC,95D/CTL-si and95D/MTA1-sil#cells were inoculated in96-well plates with continuous observation for7days. Growth curves were plotted according to the OD value at450nm. Factorial analysis showed that the ability of proliferation was no significantly decreased in the95D/MTA1-sil#cells(F=5.927, P=0.019).In vitro wound migration assay was carried out to detect the ability of cell migration in the presence or absence of MTA1gene expression. Factorial analysis showed that, width of injury line between these three groups was significantly different (F=139.429, P <0.01), indicating that knockdown of MTA1expression decreased the ability of in vitro migration. In vitro Clonegenic survival assay was carried out to detect the ability of cell proliferation, the result showed that the ability of proliferation was significantly decreased in the95D/MTAl-si1#cells (F=3.085,p>0.05).Flow cytometry analysis showed that, G2/M,S,gG0/G1peak were no significantly chaanged,.In vitro transwell chamber assay as carried out to detect the ability of cell migration and invasion.The result showed that abilities of migration and invasion were significantly decreased in the95D/MTA1-sil#cells((F=333.902, P<0.01;F=173.561, P<0.01).3.After the MTA1jene was scilenced, We used jene chip to detect the related microRNA in lung cancer cell95D.They are miR103、miR125b、miR194、 miR210、miR500、miR744。We also have identified the up regulation of miR125b and miR-194,on the contrary, We also have identified the down regulation of miR-210、miR-103and miR-500.Conclusion:1.After the MTA1jene was scilenced,the capacity of proliferation of he human lung cancer95D cell in vitro was no significant change.2. After the MTA1jene was scilenced,the cell cycles of the human lung cancer95D cell in vitro was no significant change.3. The capacity of migration and invasion was obviously decreased after the knockdown of MTA1expression. 4. After the MTA1jene was scilenced, We detected six related microRNAs in lung cancer cell95D used jene chip. They are miR103、miR125b、miR194、 miR210、miR500、miR744.5. We also have identified the up regulation of miR125b and miR194, the down regulation of miR210、miR500andmiR103used RT-PCR. |
PDF Full Text Request