Identification Of Differentially Expressed Serum MiRNAs And The Clinical Application In Osteosarcoma Patients

Background:Osteosarcoma is one of the most common tumours in adolescent. Due to the high degree malignancy of osteosarcoma, the majority patients has occurred micrometastases before the clinical diagnosis. Currently, the combination of surgical resection and chemotherapy is the most effective treatment and prolongs the survival of patients with resectable osteosarcoma. At present, the Diagnostic methods of OS in clinical includes the clinical symptoms, the imaging detection and the biopsy technique. Unfortunately, The specificity of these traditional methods is not high, most early-stage OS are asymptomatic and difficult to detect. Given the specific tumor markers and prognostic factors of osteosarcoma is so little that studies of the early diagnosis and prognosis in osteosarcoma has an important clinical significance. More and more studies show that, miRNAs are closely related with cancer, they can not only act as an important tumor marker in early diagnosis of tumor, but also as an important prognostic factor in the treatment of cancer. Accumulating experimental evidences indicate that miRNAs also play an important role in OS, the expression levels of miRNA in patients tissue occurred a significant change compared with the normal tissue, which suggest that miRNAs could be used as a biomarker for cancer detection. The studies of circulating miRNAs in OS is limited, Thus, we hypothesized that there may be specific miRNA expression profiling in the serum of osteosarcoma patients, and the expression of these miRNA in the OS may be varied during the course of treatment. On one hand, We hope that this expression profiles can be used as a non-invasive diagnostic markers, on the other hand, by studying the role of those abnormal changes miRNA both in tissues and serum from osteosarcoma patient, which can provide guidance for the clinical treatment of osteosarcoma.Objective:The purpose of this project is to detect the specific expression of miRNA profiling which may be present in the serum of patients with osteosarcoma, At the same time we examined the sensitivity of these miRNA for chemotherapy and surgery in the process of osteosarcoma treatment.Methods:An initial screen of miRNA expression was performed in four groups of serum samples by the technology of low-density chip, the four groups of serum samples were taken from OS patients (5ml from patients without treatment,5ml from patients with chemotherapy, 5ml from patients with surgery and 5ml from age-and gender-matched controls). A TaqMan probe-based stem-loop quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay was employed to confirm the expression of selected miRNAs in serum samples from 30 patients without any treatment and 40 controls, We also select five miRNA to detect their sensitivity for chemotherapy and surgery in the treatment of osteosarcoma process.Results:The microarray results showed that 316,304,293 and 233 miRNAs were detected in patients without treatment, patients with chemotherapy, patients with surgery and controls, respectively. Serum miRNA expression profile in OS patients were significant different from controls and 32 serum miRNAs (11 miRNAs was significantly upregulated and 21 miRNAs was significantly decreased) were markedly regulated in OS patients without treatment compared to controls. The qRT-PCR result identified a profile of three serum miRNAs:miR-199a, miR-32 and miR-328 to be significantly changed (with the fold change ranged from 2-4) in the serum of OS group without treatment compared to controls, which suggesting that the combination of these three kinds of miRNA the OS may be used as a potential early diagnostic markers. In this issue, we are also particularly concerned the sensitivity of miRNA to chemotherapy and surgery from OS serum, The qRT-PCR result suggest that miR-199a and miR-32 are resistance to chemotherapy but sensitive to the surgery.Conclusions:Human serum contains large amount miRNAs and the expression profile of miRNAs in OS patients were significant different from controls, We determined a profile of three serum miRNAs were regulated in OS patients, The three miRNA group may be used as a novel blood-based biomarker for the diagnosis of OS. We are also particularly concerned that miR-199a and miR-32 are resistance to chemotherapy but sensitive to the surgery, which may have a relationship with the prognosis of OS patients. miR-199a have significant change in both OS serum and tissues, and can affect biological functions of MG63, which may play an important role in the development and treatment of osteosarcoma.