Experimental Study On The Effect Of MiR-21 On The Regulation Of Osteosarcoma And Its Cancer Stem Cells

Osteosarcoma is the largest primary malignant bone tumors harming to the teen ages.Previously,surgery(amputation)is the main therapy to osreosarcoma.Because patients will not only have to bear with physical and mental trauma caused by mutilated body,but also their survival rate of 5 years is less than 20%,so is a disaster for patients and their families.Even now,using a variety of limb salvage operations combined with auxiliary chemotherap before and aftery operations,can greatly improve the survival rate of five years,and retain limb function of patients at a large degree,so as to improve the quality of patients' life,but there are still nearly 1/4 to 1/3(40%-50%in literature)patients whose survival period is not more than 5 years.How to improve the efficiency of chemotherapy and reduce the recurrence and metastasis of osteosarcoma is the focus of clinical research in the treatment of osteosarcoma.Recently,many studies have focused on the field of cancer stem cells.Cancer stem cells have unlimited proliferation,self-renewal and multilineage differentiation ability,and cancer stem cells are in a diastasis period,and insensitive to chemotherapy.After chemotherapy,cancer stem cells increasing significantly,is the main reason that results the chemo-resistance and recurrence of tumor.The recurrence and metastasis of osteosarcoma is the cause of the existence of osteosarcoma stem cells after the comprehensive treatment of limb salvage operations combined with preoperative and postoperative auxiliary chemotherap.At present,the development of diagnosis and treatment of osteosarcoma is in a bottleneck,how to effectively kill cancer stem cells,reduce the proportion of cancer stem cells in solid sarcoma,is an effective way to improve the treatment effect.CD133 is a transmembrane glycoprotein that has been found in hematopoietic stem cells and normal stem cells,and was later found in most cancer stem cells membrane,as a broad spectrum markers is applied to the resolution and separation for cancer stem cells.At present,CD 133 is the conventional recognition of the key identification of cancer stem cells in osteosarcoma,so we also take CD 133 gene expression as a characteristics marker of cancer stem cell in our experiment.MicroRNAs(miRNAs)is a family of non encoding small molecules RNAs(the average length of approximately 22 nucleotides),which play an important role in the regulation after gene transcription level,usually caused by translation inhibition,target degradation and gene silencing,so they have important significance on the process of life.Many of them,as the proto oncogenes or tumor suppressor genes,play an important role in the occurrence,development and metastasis of miRNA.MiR-21,as a cancer causing miRNA,plays an important role in the proliferation,invasion and metastasis of different malignant tumors.Studies have confirmed that miR-21 is highly expressed in osteosarcoma cells,and miR-21 promotes the growth and metastasis of osteosarcoma.At the same time,the study confirmed that miR-21 in a variety of malignant tumor stem cells have abnormal expression,and through the interaction of protein or gene to regulate the malignant tumor stem cell function;in addition,studies have shown that miR-21 inhibitors(antisense oligonucleotides)can improve the sensitivity of some tumor cells to radiotherapy drugs.VEGF is a heparin binding growth factor specific for vascular endothelial cells,which is the main factor to promote angiogenesis in the human body.During tumor growth,under the action of a variety of tumor promoting factors,the expression of VEGF and its receptor in tumor tissue shoots up,and there is a large amount of irregular angiogenesis in tumor tissue,which provides sufficient nutrients to the tumor growth,and access to the invasion and metastasis for the tumor,and is involved in various process of tumor growth,invasion and metastasis.Studies have confirmed that VEGF plays an important role in the development and metastasis of osteosarcoma.In other malignant tumors,miR-21 can promote the growth and metastasis of tumor by enhancing the expression of VEGF.Therefore,in our research about the role of miR-21 in the formation,development,metastasis and cancer stem cell of osteosarcoma,we make use of VEGF as a control to analysis the role of miR-21,and analyse the correlation of the regulation of miR-21 and VEGF to cancer stem cells of osteosarcoma.Objective:Through studying the changes of VEGF gene and CD133 gene expressions in two different osteosarcoma cells(1543 and 1423 cells)and the changes of growth and invasion speed of osteosarcoma cell after blocking miR-21 and inhibiting VEGF,the research wants to confirme that miR-21 has a regulatory effect on osteosarcoma and its cancer stem cells.And through analyzing regulation on osteosarcoma and its cancer stem cells comparing between miR-21 and VEGF,we want to investigate the mechanism and the law of regulation of miR-21 on osteosarcoma and its cancer stem cells,in order to find out the clinical treatment that can effectively inhibit the occurrence and development of osteosarcoma stem cells and reduce the proportion of cancer stem cells in osteosarcoma.Method:1 cells and cell culture:The human osteosarcoma CRL-1543 and CRL-1423 cell lines were selected as experimental cells and cultured according to the cell culture medium recommended by ATCC and the culture conditions and procedures.2 Realtime-PCR experiment2.1 The cultivation of two cell linesAll cells were divided into three experimental groups as Oh(initial)control group,8h blocking group,24h blocking group.And each group includes 2 holes of 1543 cells and 2 holes of 1423 cells.All cells were cultured according to the above-mentioned cell culture method.2.2 Treatment of two cell linesAfter three groups of cells were incubated in CO2 incubator for 24 hours,the initial control group was harvested after the cells were completely adhered.Then,miR-21 antisense oligonucleotide strand(has-mir-21-5p)and VEGF receptor inhibitor were added to the two wells of the other two groups,respectively,and then the culture plate was put back into the incubator.Cells were collected at 8 and 24 hours after treatment,respectively,as the 8-hour inhibition group and 24-hour inhibition group.RNA was extracted from each cell sample of each experimental group,then cDNA was prepared by reverse transcription.Real time-PCR was performed to detect the expression of VEGF and CD 133 genes in each sample.3 Cell immunohistochemical assay3.1 The cultivation of two cell linesTwo 8-hole chamber sliders,were labeled as CD 133 and VEGF.Two rows of cells was cultured in each chamber slider,1543 cell in the upper row and 1423 cell in the lower row,and each hole containing 10000 cells with 0.5ml appropriate culture medium.The cells were placed in the incubator for 24 hours until they were completely adherent.3.2 Treatment of two cell linesThen,the following treatment was performed respectively to four wells of cells of each row.The cells of the first hole were treated as negative control,the second hole as positive control,the third hole as VEGF(-)group with VEGF receptor inhibitor being added in,the fourth hole as miR-21(-)group being transfected by miR-21 blocking agent(has-mir-21-5p).After being incubated in the incubator for 24h,the treated cells were performed immunohistochemical staining was to detect the expression of VEGF and CD 133 in each sample.4 Cell scratch test4.1 The cultivation of two cell linesThree experimental groups were divided into blank control group,miR-21 blocking group,and miR-21 blocking +VEGF inhibition group.Each group consisted of 2-hole of 1543 cells and 2-hole of 1423 cells,and the cell culture method was carried out according to the above-mentioned cell culture method.4.2 Treatment of two cell linesAll cells were incubated for 24 hours in the incubator,until the cells were completely adhered.The blank control group wasn't added any blocking agent,and the second group was added the miR-21 blocking agent,and the third group was added the miR-21 blocking agent and VEGF receptor inhibitor.The twelve-hole cell culture plate was incubated in the incubator again,until the cells almost grew full of the bottom of the hole.Then all the cells were carried out the scratch test to compare the difference of the proliferation ability of each group cells.Result:1.PCR real-time experimental results:1.1 The expression of VEGF gene and CD 133 gene in each cell line:After VEGF being inhibited,the expression of VEGF in 1543 cell and 1423 cell was decreasing(P<0.05),whereas the expression of CD 133 was significantly increased in 1543 and 1423 cells(P<0.05).After miR-21 being blocked,the expression of VEGF gene and CD133 gene in 1543 cells decreased(P<0.05),while the expression of VEGF gene and CD 133 gene in 1423 cells was significantly increased(P<0.05).1.2.The variation with time of VEGF gene's and CD 133 gene's expression in each cell line:(1)After VEGF being inhibited,both VEGF gene's and CD 133 gene's expression in 1543 cells were first increased,but with the progress of time,the expression decreased gradually,and the expression of VEGF gene had been lower than that of the initial control group after treatment of 24h;after miR-21 being blocked,the expression on VEGF gene and CD133 gene in 1543 cells was decreasing,and with the progress of time it had no significant change.(2)After being treated,1423 cell expressed CD 133 gene and VEGF gene significantly differently at the two time points(8h and 24h).After VEGF being inhibited for 8 hours,the expression of CD 133 gene reached 4.13 times of that of the initial control group,while at the inhibition of 24 hours,the expression of CD 133 gene was reduced dramatically to 79%of that of the initial control group.2.Results of cell immunohistochemical assay:2.1 1543 cell is VEGFlow/CD133high cell,and 1423 cell is VEGFhigh/CD133low?cell.2.2 In 1543 cell,after inhibition of VEGF,the expression of VEGF gene was decreasing,while the expression of CD 133 gene was increased;and after blocking the miR-21,the expression of both VEGF gene and CD133 gene were down-regulate.2.3 In 1423 cell,after inhibition of VEGF,the expression of VEGF gene was decreasing,while the expression of CD133 gene was increased;and after blocking the miR-21,the expression of both VEGF gene and CD 133 gene were obviously up-regulate.3.Results of cell scratch test:3.1 The proliferation rate of 1543 cell was significantly faster than that of 1423 cells.3.2 The two experimental groups(inhibition of miR-21 group and simultaneous inhibition of both miR-21 and VEGF)of 1543 cells were significantly slower than the control group(P<0.05),but there was no significant difference in the growth rate between the two experimental groups.3.3 The cell growth rate of 1423 cells in the miR-21 inhibitor group was slower than that of the control group(P<0.05).Simultaneously,the growth rate of the cells with two inhibitors was the slowest,and the difference was statistically significant(P<0.05).Conclusion:1.Blocking miR-21 has a more significant regulatory effect to osteosarcoma cells with VEGF gene low expression,and the inhibition of VEGF has a more significant regulatory effect to osteosarcoma cells with VEGF gene high expression.2 The expression of CD 133 gene was up-regulated in two osteosarcoma cell lines after being inhibited VEGF,and VEGF probably inhibit the expression of CD 133 gene in osteosarcoma cells.3 miR-21 plays a role in osteosarcoma with VEGF low expression that can promote the occurrence and proliferation of cancer stem cells,thereby it increases the proportion of cancer stem cells in osteosarcoma.In this part of the osteosarcoma cells,blocking miR-21 can significantly reduce the expression of CD 133 gene which is the marker of osteosarcoma stem cells,therefore it may have the effect of reducing tumor stem cells.4.For 1423 cells with high expression of VEGF,the inhibition of VEGF can regulate the expression of CD 133 gene in two-way.And it shows as suppression of long time after enhancement for a short period of time.Significance:Our study found that miR-21 antisense oligonucleotides being transfected into osteosarcoma cells with VEGF low expression can downregulate the expression of VEGF and CD 133 in osteosarcoma cells,thus inhibit tumor growth,invasion and metastasis,and reduce the proportion of cancer stem cells,so it can greatly improve the efficiency of chemotherapy for osteosarcoma.VEGF receptor inhibitors are likely to be an effective treatment to osteosarcoma with VEGF high expression,which can inhibit the growth,invasion and metastasis of tumor cells,and reduce the population and proportion of cancer stem cells in osteosarcoma,thereby enhance the sensitivity to chemotherapy of osteosarcoma.To sum up,our study provides valuable basic research information for the treatment of osteosarcoma with different molecular characteristics,which is helpful for guiding clinical medication and treatment.Based on our research,we can use the corresponding therapeutic agents for osteosarcoma with different molecular features(high expression of VEGF or low expression of VEGF),and help to achieve accurate medication and treatment.