Detecting The Expression Of MicroRNAs In CRC By Fluorescence Quantitative PCR

ObjectiveColorectal cancer(CRC) is the third commonest form of cancer occurring worldwide,and it affects men and women almost equally.In the western developed countries the incidence has already rises to the second commonest.Colorectal cancer is the third most and second leading cause of cancer death in he United Stated.In China,with the development of economy and the change of eating habit,the incidence of colorectal cancer increases every year.Because of lower malignant biological behavior,the 5-year survival of patients with colorectal cancer after resection is 40-60%,which is much higher than other malignant tumors.But it still is the one of most leading death cancer in our country.So,it is very essential to further raise the diagnostic level for colorectal cancer.Early diagnosis,early and standard operation treat is the key to beat colorectal cancer.MicroRNAs are small 19 to 22 nucleotide sequences of RNA that participate in the regulation of cell differentiation,cell cycle progression,and apoptosis.MicroRNAs act much like small interfering RNA,annealing with RISC,to cleave messenger RNA, and microRNAs exert translational inhibition that is incompletely understood.They are important factors in tumorigenesis and have been the subject of research in many types of cancers,including colorectal cancer.Recent studies support an important role of microRNAs in the initiation and progression of human malignancies.Some microRNAs may function as oncogenes or tumor suppressors.Changes in the expression of many microRNAs have been observed in colorectal tumor tissues and cell lines.Some of them are significantly down-regulated,for example miR-143,miR-145,let-7,miR-34a,etc.and others such as miR-31,miR-21,etc.are up-regulated.Scientists have demonstrated that transfection of miR-143 and miR-145 precursors into human colorectal cancer cell lines led to significant growth inhibition in a dose-dependent manner.Moreover,some chemotherapeutic drugs can affect the expression level of colorectal cancer related miRNAs.It was observed that the expression level of miR-34 in colorectal cancer cell lines increased after adriamycin treatment.Researchers suspect miRNAs may be implicated in the process that chemotherapetic drug works.Ascertaining the precise mechanism of miRNAs will make a supplement to the etiological and pathological theories of colorectal cancer and suggest a promising future therapeutic strategy.After reading domestic and foreign literature,we selected miR-365,miR-18b, miR-21and miR-148b as research targets.Florescence quantitative RT-PCR was used to detect the expression level of microRNAs in colorectal tumor tissues as well as compared normal mucosa.Then we analysis the relationship between the clinical pathological features of colorectal cancer and the expression level of microRNAs.That all we did aimed at finding a new kind of biomarker to help early diagnosis and anti-metastasis treatment of colorectal cancer.Materials and MethodsThirty patients with colorectal cancer were selected in the No.1 hospital of China medical university from December 2007 to May 2008.The group consisted of 12 men and 18 women.After informed consent was obtained,29 couples of tumor tissues and compared non-tumor tissues were selected.All samples were immediately stored at liquid nitrogen 30minutes and then transferred to -80℃fridge.Total RNA isolation was performed with the mirVana miRNA isolation kit(Ambion) according to the manufacturer's instruction.RNA concentration and purity were controlled by UV spectrophotometry.(A260/A280 between 1.8-2.0).And then all RNA was polyadenylated by poy(A) polymerase with ploy(A) kit(Ambion) according to the instruction of manufacture. cDNA was synthesized from the total tailed RNA by gene-specific primer,which was one-base anchored olig-dT primers with 40nt nt extension at their 5'-ends. Real-time PCR was performed using Roto-gene 3000 instrument.The 20ul reaction mixture includes 10ul SYBR ExTaq Premix,1.6ul RT product,0.8ul Primer,7.6ul ddH20.Reaction were incubated in a 36-well optical plate at 95℃for 5 seconds. followed by 45 cycles of 95℃for 5,58℃for 20 seconds and 72℃for 30 seconds.The threshold cycle data were determined using default threshold setting.Data analysis was performed by the relatively quantitative 2-△△Ct method according to the instruction of Roto-gene 3000 real-time amplification.Results1.the RNA tailed poly(A) primer intension RT-PCRmicroRNA is long about 22nt,which may be shorter than a primer,so we must do our best to design a suitable couple of primer before PCR amplification.After reading the foreign and domestic literatures,we selected RNA tailed primer intension RT-PCR to detect the microRNAs.We successfully detected the 4 microRNAs in colorectal cancer tissues as well as compared non-tumor tissues.Sequencing the product of RT-PCR,the sequence of target were obtained.This method was effective and cheap and should be recommended.2.relationship between the expression of miR-18b,miR-21,miR-148b,miR-365 and clinicopathologic featuresStatistical differences between the expression of miR-18b and infiltration depth was observed,and there were no differences between the other 3 kinds of microRNAs and clinicopathologic features.We found that the up-regulation of miR-18b had a same tend as the infiltration depth increases.Maybe the high expression of miR-18b was a sign presenting more malignant in colorectal cancer.Conclusions1.RNA-tailing and primer-extension RT-PCR method is a reliable,inexpensive method to detect miRNAs and should be recommended in the future. 2.There was statistical difference between the expression level of miR-18b and the infiltration depth in colorectal cancer.High expression of miR-18b may indicate a more malignant biological behavior.