| BackgroundOsteosarcoma(OS)is a highly aggressive malignant tumor that ranks first in skeletal malignancies.It is usually fatal for patients with osteosarcoma.Patients with osteosarcoma with metastasis or with osteosarcoma who have relapsed have a worse therapeutic effect.Osteosarcoma most frequently invades the metaphysis of the long bones of the limbs and is most likely to occur in the distal femur,proximal humerus,and proximal humerus.For patients over the age of 25,there are more types of bone sites,such as the clavicle,ribs,shoulder blades,and tibia.Although osteosarcoma occurs most frequently in patients 5 years of age and early adulthood,the incidence peaks again in the elderly(> 65).The occultity of osteosarcoma is often found in patients with accidental injuries,and can also be diagnosed by imaging.However,compared with the clinical manifestations of most neoplastic diseases,osteosarcoma has a stronger ability to invade and disperse.Osteosarcoma can spread to multiple bones and lymph nodes in the body.Osteosarcoma can be diagnosed by combining PET/CT and predicting the degree of necrosis at the site of invasion.When a distant metastasis occurs,the osteosarcoma can be divided into the lungs and the lungs at the metastatic site,and the most common site is the lungs.In addition to lung and extrapulmonary metastases with osteosarcoma,osteosarcoma can also invade adjacent bone by jumping metastasis.Jumping metastasis is also called synchronous regional bone metastasis.Its range of metastasis is local,but it may be a more serious metastatic complication for the body.Although local tumors can be controlled by conventional amputation surgery and postoperative chemotherapy,more than 80% of patients with osteosarcoma have metastasized at the time of diagnosis.When the osteosarcoma did not metastasize,the surgical treatment and complete postoperative treatment were performed.The 5-year survival rate was 50% in China,and the 4-year survival rate was 0% when lung and bone metastasis occurred during treatment.This is also the main cause of osteosarcoma-related death.In recent years,in international and domestic research on osteosarcoma,the role of various serum markers in the diagnosis of osteosarcoma and the progress of osteosarcoma in the body has been paid more and more attention.The role of alkaline phosphatase(ALP)and lactose dehydrogenase(LDH)in various biomarkers of serum is prominent,and the diagnosis of osteosarcoma is compared with various serum markers alkaline phosphatase(ALP).The highest value.Although the current study found that the occurrence and development of osteosarcoma is closely related to the patient’s race,gender,age,genetic changes and exposure,the exact pathophysiology and mechanism of osteosarcoma remains unclear.The molecular pathogenesis and genetics of osteosarcoma are enormous and extremely heterogeneous.The pathogenesis of osteosarcoma as a primary malignant tumor is extremely complicated,so to achieve a definitive diagnosis of osteosarcoma must be jointly studied through a comprehensive multidisciplinary approach.Phosphatase and tensin homolog(PTEN): is a tumor suppressor gene with diphosphatase activity,and loss of PTEN function leads to PI3K-AKT(phosphoinositide 3-kinase-RAC-α serine/threonine protein Activation of the kinase)pathway makes PTEN a potentially useful tumor marker for distinguishing between inert and aggressive diseases in patients with clinically localized tumors.In order to be able to use the state of PTEN as a biomarker for predicting tumor and tumor prognosis,an analytically validated assay has been developed which reliably and reproducibly detects PTEN loss in tumor tissue and blood tissue.The use of clinical grade analysis in tumor tissue has shown a strong correlation between the loss of PTEN and its protein and a strong correlation between PTEN loss and adverse pathological features and oncological outcomes.In advanced disease,assessing the PTEN status in a liquid biopsy predicts the response of the tumor to targeted therapy.Finally,studies have shown that PTEN may have other functions than the PI3K-AKT pathway,including the role of tumor growth by regulating immune responses and tumor microenvironment.In recent years,the function of PTEN as a metabolic regulator has attracted great attention.MicroRNAs(miRNAs)are small non-coding RNAs(about 22 nt in length)and are known to be important primary regulators of eukaryotic gene expression.Current research has demonstrated that miRNAs play a key role in the pathogenesis of many cancers,and that misregulation of miRNAs is a well-known feature of cancer.In recent years,miR-29 has become a key miRNA in the study of a variety of cancers,and a large number of studies have shown that it can regulate a variety of carcinogenic processes,including epigenetics,protein stability,metabolism,proliferation,apoptosis,metastasis,fiber,angiogenesis and immune regulation.Although miR-29 has been documented as a tumor suppressor in most studies,the controversy surrounding the reporting of miR-29 as an oncogene remains.ObjectivesIn this study,the role of miR-29 in the proliferation and metastasis of osteosarcoma cells was analyzed by comparing the expression levels of miR-29 in osteosarcoma and normal tissues.The experimental data were used to verify the role of PTEN in promoting the proliferation and metastasis of osteosarcoma cells by miR-29,and to analyze its relationship with clinical case parameters,providing new ideas for immunotherapy of osteosarcoma based on PTEN.MethodsThe collection of osteosarcoma and adjacent non-cancerous tissues from 60 patients with osteosarcoma who underwent osteosarcoma resection and pathological diagnosis from the Department of Adult Orthopaedics and Pediatric Orthopaedics of the First Affiliated Hospital of Zhengzhou University from February 2010 to August 2016.The patients with osteosarcoma and their families were approved,and the patient’s family signed the informed consent form for the operation before the patient underwent osteosarcoma resection.The goal was to ensure that all patients with osteosarcoma involved in the study were excluded from any other systemic tumor other than osteosarcoma.All patients with osteosarcoma did not receive radiotherapy or chemotherapy before surgery.The osteosarcoma tissues removed from the operation and the normal tissues not invaded by the adjacent tissues were collected.All fresh surgical specimens were rinsed with normal saline and immediately placed in liquid nitrogen at-80 °C.Stored in refrigerant for subsequent experiments.The purchased OS cell line(MG-63,U2 OS,143B,Saos-2)was cultured in a medium containing RPMI-1640 at a temperature of 37 ° C for subsequent experiments.The level of miR-29 in OS tissues and cells was detected by using RT-qPCR.Cell proliferation was measured by MTT assay and RT-qPCR.ResultsRT-qPCR assay was used to detect miR-29 levels in osteosarcoma tissues and cells and normal tissues.The results showed a significant increase in the mean level of miR-29 in osteosarcoma cancer tissues.The difference between the two groups was statistically significant(P<0.05).Compared with normal cells(hFOB1.19),the expression of miR-29 in osteosarcoma cells(MG-63,U2 OS,143B,Saos-2)was also The difference between the two groups was statistically significant(P < 0.05),which means that miR-29 was overexpressed in osteosarcoma and osteosarcoma cell lines.Cell proliferation was also measured by MTT assay and RT-qPCR.MTT results showed that the cell viability in the miR-29 mimetic group was enhanced compared to the control mock group.RT-qPCR analysis revealed increased expression of PCNA(a marker of cell proliferation)in the miR-29 mimetic group.Transwell-measured cell migration demonstrated that miR-29 mimics promoted the ability of osteosarcoma cells to migrate.The above experimental results indicate that miR-29 molecules can promote the proliferation of osteosarcoma cells.It suggests that miR-29 may be important for the progression of osteosarcoma.The effect of PTEN on osteosarcoma was examined by using Western blot analysis and RT-qPCR to assess PTEN protein and mRNA levels in MG-63 cells when restoring or inhibiting miR-29.PTEN expression decreased when miR-29 was restored,while PTEN expression increased when miR-29 was inhibited.It is suggested that miR-29 and PTEN expression are negatively correlated.The miR-29 mimetic and the PTEN vector were co-transfected into MG-63 cells to test the effect of PTEN in the regulation of osteosarcoma cells by miR-29.The results show that PTEN can reverse the promotion of miR-29.In conclusion,miR-29 can promote OS cell migration and proliferation and target PTEN.ConlulsionsThe expression level of miR-29 was increased in osteosarcoma tissues,and the expression of miR-29 and PTEN was negatively correlated in osteosarcoma.PTEN overexpression has been shown to inhibit proliferation and migration of osteosarcoma cells,and PTEN can also reverse the promotion of osteosarcoma progression by miR-29.miR-29 can negatively regulate the expression of PTEN in MG63 cells.It indicates that miR-29 may play a very important role in the occurrence,development,metastasis and survival of osteosarcoma,and it may provide a theoretical basis for the immunotherapy of osteosarcoma based on the expression of PTEN. |
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