Real-time Quantification Of MiRNAs By RNA-tailing And Primer-extension RT-PCR In Gastric Carcinoma

ObjectiveGastric cancer,the second most common cancer in the world,causes nearly one million deaths worldwide per year.In china,gastric cancer ranked the top common cancer with 4000,000 new patients and 260,000 death per year.Unfortunately,gastric cancer is usually diagnosed at an advanced staged and the prognosis is poorthe overall relative 5—rear survival rate is currently less than 20%.Many etiological factors can increase the risks of gastric cancer,including H.pylori infection,food,environmental and genetic factors.H.pylori is the main risk factor in about 80%or more of gastric cancers.The role of H.pylori on gastroduodenal diseases has been proposed,but the detailedmolecular pathway remains unclear.Recently years,several oncogenes and tumer supressors have been found in gastric cancer,such as p53,APC,C-met,FGF2,CDH1 and heparinase.However,the molecular events leading to gastric malignancy and the genetic components that are altered at the inception and the course of the neoplasm are largely unknown.At present, the relationship between microRNA and the development of the tumor and the potential diagnostic value of cancer research has become the new hot spots.MicroRNAs are a family of 21-25 nucleotide small RNAs that,at least for those few that have characterized targets,negatively regulate gene expression at the post-transcription level.Recently,many studies revealed miRNAs play key roles in cell proliferation,apoptosis,development,differentiation and metabolism.Indeed,miRNA abrrent expression has been previously found in various types of hunman cancers,and miRNA signatures were associated with specific clinicobiological features.In addition, miRNAs are proved to function as oncogenes and tumor suppressor genes through post-transcriptional regulation on their target genes.Therefore,it is necessary to indentify deregulation miRNAs as new biological makers that can be used to screen high-risk pations in order to allow better gastric cancer detection,earlier intervention and increase the likelihood of successful treatment.In this study,we investigate the miR-18b,miR-21,miR-148b and miR-365 expression levels in gastric cancer,and try to unravel the biological pathways of miRNAs on gastric tumorigenesis.However,as an important growth regulator of cell cycle regulation and molecular,miRNA has become a new generation of potential tumor marker.The purpose of this study through SYBR Green Real-time quantitative RT-PCR detection of miR-18b,miR-21,miR-148b and miR-365 express levels in gastric cancer. we have established a simple method to detect the miRNAs expresion levels.Total RNA was polyadenylated by poly(A)polymerase,and then cDNA was synthesized by a specific reverse transcriptase primer and reverse transcriptase using the poly(A)-tailed totoal RNA as templates.And then the results of SYBR Green real-time PCR were analysis by the Relatively quantitative 2^{-ΔΔCt}method.Materials And MethodsAll hunman tissue samples were obtained from surgical specimens of 12 patients with gastic carcinoma from 2007 to 2008 at Department of Surgerical Oncolgy,The First Hospital of China Medical University,china.All tissues,including gastric carcinoma and corresponding adjacent normal tissue,were divided into two parts and preserved in liquid nitrogen for 30 min after removing from the body,then stored at -80℃.Informed cosent was takenen from all subjects.Small-sized RNAs(<200bp)were extracted and polyadenylated by poy(A) polymerase.One-base anchored olig-dT primers with 40nt nt extension at their 5'-ends were used to reverse transcribe the poly(A)-tailed miRNA,then were amplified by SRBR Green real-time PCR using miRNA specific primers.And then the results were analysised By 2^{-ΔΔCt}method.Results 一,The optimized of 2^{-ΔΔCt} analysis methodsThe results of SYBR Green real-time PCR were analysis by the Relatively quantitative 2^{-ΔΔCt} method.This analysis methods requires that the difference of the standard curve slope between the target gene(miR-18b,miR-21,miR-148b and miR-365)and housekeeping gene(U6 SnRNA)should be less than 0.01.miR-18b, miR-21,miR-148b,miR-365 and U6 snRNA were quantified at 260nm wavelength UV and were diluted 10-fold gradient:105,104,103,102,101,respectively.The results showed that the difference between the slope 0.062,0.003,0.004,0.093 respectively, were less than 0.01,consistent with the analysis conditions.二,Expression of miRNAs in gastric carcinomaIn order to confirm the level of miRNAs in human gastric cancer,we examined using SYBR Green real-time RT-PCR,the expression of miR-18b,miR-21,miR-148b,miR-365 and U6 snRNA in 12 matched paired of gastric tumoral and non-tumoral tissuses from patients.The results showed that miR-18b,miR-21 and miR-365 was up-regulated in 6,7,8of 12 Samples with gastric cancer respectively.And miR-148b was down-regulated in 7of 12 Samples with gastric cancer.Conclusions1.RNA-tailing and primer-extension RT-PCR is a convenient and reliable method to detect miRNAs which may play an important role in the development of gastric carcinoma.2.The expression levels of miR-148b in gastric cancer tissues was lower compared to normal tissues which forecast miR-148b may play a key role as tumor suppressor gene.The expression levels of miR-18b,miR-21,miR-365 in gastric cancer tissues was higher compared to normal tissues which forecast miR-18b,miR-21,miR-365 may play a key roles as tumor oncogenes.