The Effects Of MiR-200c On Osteosarcoma Cells And Its Relationship With AKT2

BACKGROUND:Osteosarcoma is a bone tumor disease that often occurs in children and adolescents with a high degree of malignancy.It is prone to metastasis and easy to relapse in the early stage.The etiology of osteosarcoma is not yet clear,and there are no reliable,non-invasive biomarkers to detect the occurrence,development and prognosis of the disease.At present,the clinical treatment of osteosarcoma is mainly amputation combined with neoadjuvant chemotherapy,but these treatments have high treatment cost,long treatment period,inaccurate effect,low five-year survival rate and poor prognosis.Therefore,it is important to identify the cause and find effective biological markers.MicroRNAs(miRNAs)are a class of small non-coding RNAs that play a key role in the development,progression and metastasis of tumors.Due to their conservation,stability and easy separation in plasma,they have become ideal candidates for the development of plasma biomarkers.Therefore,miRNAs have become a hot research topic in recent years.The miR-200 family,which is a member of miRNAs,is currently thought to play a special role in human health and disease,with miR-200 c showing low levels of expression in a variety of tumors.According to research,miRNAs are often used after transcription to regulate genes.Studies have shown that miRNAs may regulate AKT2 gene expression.AKT2 belongs to a family of serine/threonine and plays an important role in the occurrence and development of diseases.A number of studies have shown that various miRNAs are involved in the presence of AKT2 in osteosarcoma.However,the relationship between miR-200 c and AKT2 in osteosarcoma has not been shown.This study aimed to preliminarily study the relationship between miR-200 c and AKT2 in osteosarcoma at the cellular level.It is hoped that by studying the relationship between the two,the mechanism of osteosarcoma development will be further understood,which will provide a new idea for the prevention,diagnosis and treatment of osteosarcoma.AIM:The aim of this study was to investigate the expression of miR-200 c in osteosarcoma cells and the effects of miR-200 c on proliferation,invasion and migration of osteosarcoma cells,and to further investigate the relationship between miR-200 c and AKT2 in osteosarcoma cells.METHODS:Four osteosarcoma cell lines(HOS,U-2OS,Saos-2,MG-63)and normal osteoblast NHOst were selected,and the expression level of miR-200 c in each cell line was detected by qRT-PCR.The osteosarcoma cell line was verified by qRT-PCR.The proliferation of osteosarcoma cell lines was detected by CCK8 method.The invasion and migration ability of osteosarcoma cell lines were determined by Matrigel and Transwell chambers.The TargetScan search program predicted the binding site of miR-200 c and AKT2,constructed different luciferase gene expression plasmids and detected luciferase activity.The expression of AKT2 gene in osteosarcoma cell lines was determined by RT-PCR.The relative content of AKT2 protein in osteosarcoma cell lines was determined by Western Blot.RESULTS:1.Compared with normal osteoblast NHOst,the expression of miR-200 c in the four osteosarcoma cell lines all decreased,which has statistically significant(P<0.05).Among them,miR-200 c expression was the lowest in MG-63,which has significant statistical significance(P<0.01).2.Compared with the MG-63/miR-NC group,the expression of miR-200 c gene was significantly increased in the MG-63/miR-200 c group(P<0.01).3.Compared with the MG-63/miR-NC group,the proliferative ability of osteosarcoma cells was obviously decreased in MG-63/miR-200 c group(P <0.01).4.Compared with the MG-63/miR-NC group,the invasion and migration ability of osteosarcoma cells was obviously decreased in MG-63/miR-200 c group(P<0.01).5.TargetScan predicted that miR-200 c can be combined with AKT2 3' URT region.6.Compared with the MG-63/miR-NC group,the luciferase activity of wild type(WT)was significantly decreased in the MG-63/miR-200 c group(P<0.01),while there was no significant difference in luciferase activity between the(MUT)groups(P>0.05).The wild type and mutant luciferase activities in the MG-63/miR-200 c group were statistically significant(P <0.05),compared with the MG-63/miR-NC group,the expression of AKT2 gene was significantly decreased in the MG-63/miR-200 c group(P<0.05).8.Compared with the MG-63/miR-NC group,the expression of AKT2 protein was significantly decreased in the MG-63/miR-200 c group(P<0.05).CONCLUSION:miR-200c is down-regulated in osteosarcoma cells,and it can inhibit the proliferation,invasion and migration of osteosarcoma cells by inhibiting the expression of AKT2.