MiR-200a Expression In Osteosarcoma And Its Clinical Significance

BackgroundOsteosarcoma is a primary malignant bone tumor that occurs in children and adolescents.Its incidence is at the top of primary bone tumors.Most osteosarcomas occur in the long tubular bones of the extremities,especially in the the proximal metaphysis of distal femur and tibia.Due to the rapid growth of tumor tissue,strong invasion ability,early metastasis and other characteristics,the surrounding normal bone tissue is often eroded by the tumor,which has a huge impact on the patients’ life.With the advancement of surgical technology,the concept of neoadjuvant chemotherapy and the improvement of postoperative radiotherapy and chemotherapy,traditional amputation has been replaced by neoadjuvant chemotherapy combined with limb salvage surgery.The five-year survival rate of patients with osteosarcoma has increased significantly to 60~70%,but because of the highly invasive and early metastasis of osteosarcoma and resistance to chemotherapy drugs,together with the current absence of tumor markers with high sensitivity and specificity,whose result is when most of osteosarcoma patients come to visit have entered the late stage of cancer,so the patient’s tumor recurrence rate and mortality still did not significantly improve.Therefore,there is an urgent need to explore the potential molecular mechanisms involved in the development of osteosarcoma in order to early diagnosisand early treatment of osteosarcoma to improve the prognosis and quality of life of osteosarcoma patients.Dendritic cells(DCs)are well known as a class of specific antigen-presenting cells that are widely distributed in humans and are commonly distributed in blood,tissues,and lymphoid organs.They are considered to be the center of the immune system.The function is to bridge the specific immune system and the non-specific immune system,which can generate a strong immune response.In the past decade,DC-based cancer immunotherapy has been carried out around these functions of DC,aiming to develop immunotherapy strategies for cancer through vaccination.To this end,we must better understand DC immune biology and the latest advances in applied science,regulation of the innate immune system and adaptive immune system and tumor microenvironment,in order to improve its huge anti-tumor immunotherapy potential.MicroRNAs are non-coding RNA molecules with a regulatory function of about19 ~ 25 nucleotides in length.mi RNAs regulate the expression of target genes by inducing messenger RNA(mRNA)degradation by complementary pairing with the 3’untranslated region(UTR)base of the target mRNAs or by inhibiting mRNA translation by incomplete base pairing with the seed sequence.Abnormal expression of miRNAs has been observed in human cancers and has been shown to be involved in a variety of key cellular processes,including cell differentiation,proliferation,and metabolism.In addition,miRNA expression profiles in plasma and serum samples have been used to accurately classify human cancers,suggesting that miRNAs may be used as markers for cancer diagnosis and prognosis.DCs are composed of subpopulations with different development and functions,and it has been found that the development of different DC families is regulated by the network of different cytokines and transcription factors.In addition,the response of DC is strictly regulated to maintain the homeostasis of the immune system.In the immune system,miRNAs function at checkpoints during hematopoietic development and cell subpopulation differentiation,which modulate effector cell function and involve maintaining homeostasis.DCs are regulated by miRNAs.In the past decade,miRNAs have made great progress in regulating the development and function ofDCs.Based on the current status of osteosarcoma treatment,dendritic cell-based immunotherapy can be developed as a very promising treatment.ObjectivesThis study compared the expression levels of miRNA-200 a in osteosarcoma cell lines and normal osteoblasts,osteosarcoma tissues and paracancerous tissues,peripheral blood of osteosarcoma patients and healthy volunteers,and analyzed the relationship between the miRNA-200 a expression level of cancer tissues of osteosarcoma patients and clinic pathological parameters to provides a theoretical basis for the immunotherapy of osteosarcoma based on dendritic cells.MethodsWe selected 40 patients with osteosarcoma who had undergone osteosarcoma resection and pathological diagnosis from October 2015 to October 2017 in the Orthopedics and Pediatric Surgery department of the First Affiliated Hospital of Zhengzhou University.We did the research with the consent of osteosarcoma patients and their families,all patients need to exclude other system tumors other than osteosarcoma.We collected 5 ml of peripheral venous blood of the patients on the day of surgery,and collecting cancerous tissues,para-cancer tissues,and tissues that have not been invaded by cancer during surgery,including 20 males and 20 females,aged between 10 and 48 years.The fresh surgical specimens were resected and washed with normal saline and quickly stored in a low temperature refrigerator at-80°C.In addition,clinical data such as tumor size,lymph node metastasis,and tumor infiltration were collected.At the same time,5 ml of blood samples which collected from outpatient examinations were stored in a low-temperature refrigerator at-80°C until use.Human U2 OS osteosarcoma cell line and human hFOB1.19 osteoblast cell line were cultured.The expression level of miRNA-200 a in specimens and cells was detected by qRT-PCR,and their relationship with clinical data was analyzed.Results1.Detection of miRNA-200 a expression in osteosarcoma patients with cancer tissue and osteosarcoma cell linesqRT-PCR assay was used to detect the expression level of miRNA-200 a in tumor tissue and adjacent tissues of osteosarcoma,human osteosarcoma cell line U2 OS and human osteoblast cell line hFOB1.19.The results showed that the expression of miRNA-200 a in cancer tissue was low.In the adjacent tissues,the difference between the two groups was statistically significant(P=0.0462).The expression of miRNA-200 a in osteosarcoma cells was lower than that in human osteoblasts.The difference between the two groups was statistically significant(P=0.0021).This means that miRNA-200 a is lowly expressed in osteosarcoma tissue and osteosarcoma cell lines.In addition,the differences in expression levels of mi RNA-200 a in osteosarcoma tissues between different sexes,different ages,different tumor sizes,different tumor growth sites,different pathological types,metastasis,and different survival conditions were compared.The results showed that the death group and metastasis group The expression level of mi RNA-200 a was significantly higher than that of surviving and non-metastatic groups(P<0.05),suggesting that miRNA-200 a may play an important role in the occurrence,development,metastasis,and survival of osteosarcomas.2.Detection of miRNA-200 a Expression in Peripheral Blood of Patients with OsteosarcomaThe qRT-PCR assay was used to compare the expression levels of miRNA-200 a in the serum of 40 patients with osteosarcoma and 40 healthy people who examined at the outpatient clinic.The results showed that the expression of miRNA-200 a in the serum of patients with osteosarcoma was higher than that in the healthy people in the outpatient examination.There was a statistically significant difference between the two groups(P<0.05).ConclusionsmiRNA-200 a is down-regulated in the tumor tissue of osteosarcoma patients,and its expression in the serum is up-regulated,indicating that it may play an important role in the occurrence,development,metastasis,and survival of osteosarcoma.It can provide a theoretical basis for the immunotherapy of osteosarcoma based on dendritic cells.