Study On The Inhibitory Effect Of Dual-Targeting MiR-21 And CXCR4 On Malignant Glioma

Brain tumors are the third leading cause of cancer-related death in adults.Among various types of brain tumors,malignant gliomas(MG)is a highly permeable tumor in the central nervous system,whose cells can transfer from the primary lesions to the surrounding normal brain tissue and distant parts.The high invasiveness of glioma is the key to the poor clinical prognosis.Therefore,elucidating the molecular and biological mechanisms of gliomas may contribute to develop new treatments for this difficult disease.MicroRNA(miRNA)is single-stranded,short,non-coding RNA that is involved in many biological processes,including tumorigenesis,cell proliferation,apoptosis,invasion and metastasis.Studies have shown that miR-21 overexpression is determined in malignant glioma and interference ofits expression can significantly inhibit cell viability.Previous studies have shown that increased miR-21 expression can lead in the increased viability and activity of glioma cells.However,the molecular mechanism by which miR-21 functions in glioblastoma is unclear.CXC family chemokine receptor(CXCR4)is a chemokine receptor,which has been shown to be involved in lymph node metastasis in multiple cancers.CXCR4 expression is closely related to tumor invasion and recurrence,the study reported.In addition to affecting metastasis,CXCR4 can also regulate the growth and apoptosis of tumor cells and other biological functions,including malignant glioma.However,further studies are needed to elucidate the molecular mechanism by which CXCR4 regulates the development of malignant gliomas.The PI3K/AKT and RaF/MEK/ERK pathways are two of the most common abnormal pathways in cancers,and play a key role.Co-activation of these two pathways was found in many different tumor types,including melanoma,prostate cancer,and colorectal cancer.Studies have shown that there are other interaction sites between PI3K/AKT and Raf/MEK/ERK pathways.As a result,the two signaling pathways are interconnected,and further understanding how they work could provide more effective new therapies for cancer treatment.This aim for the study of effect of dual targeting miR-21 and CXCR4 on biological behaviour and mechanism in malignant glioma,first qRT-PCR was used to detect the miR-21 and CXCR4 expression in malignant glioma tissues and cells;At cellular level,lentiviral vectors(interferencing miR-21 and/or CXCR4)were constructed and MTT,flow cytometry,scratch,Transwell,and Western blot assays were performed to measure the effect of dual targeting miR-21 and CXCR4 on cell proliferation,apoptosis,migration,invasion and the PI3K/AKT and Raf/MEK/ERK signaling pathway in malignant glioma cells;In vivo experiments,malignant glioma xenograft models were established to explore the effect of dual-targeting miR-21 and CXCR4 on the growth of malignant glioma xenograft and the molecular mechanism.HE staining assays were used to detect the glioblastoma tissue pathological morphology.This study is mainly divided into three parts:Part I:Expression of miR-21 and CXCR4 in malignant glioma;Part II:Effect of double-targeting miR-21 and CXCR4 on the biological characteristics of glioblastoma cells;Part III:Inhibitory effect of double-targeting miR-21 and CXCR4 on tumorigenesis of malignant glioma cells in nude mice.Main Content:The first part:Expression of miR-21 and CXCR4 in malignant gliomaMethods1.Expression of miR-21 and CXCR4 mRNA in malignant glioma tissues and cells was measured by qRT-PCR.2.Expression of CXCR4 protein in malignant glioma tissues and cells detected by western blot.ResultsThe results from qRT-PCR and Western blot showed that compared with normal tissue adjacent to tumors,miR-21 and CXCR4 expression was significantly increased in malignant glioma tissues.Compared with HAc cells,miR-21 and CXCR4 expression was significantly increased in malignant glioma cells U251 and U87.The second part:Effect of double-targeting miR-21 and CXCR4 on the biological characteristics of glioblastoma cellsMethods1.Lentiviral vectors targeting interferring miR-21 and/or CXCR4 expression were established.Recombinant lentiviruses were packaged and infected U251 and U87 cells after titration to obtain the stable cell lines.The expression of miR-21 and CXCR4 in stable cell lines was detected by qRT-PCR and Western blot.2.The effect of double-interfering miR-21 and CXCR4 on malignant glioma cell proliferation,apoptosis,migration and invasion was determined by MTT,flow cytometry,scratch and Transwell assays.3.Expression of the PI3K/AKT pathway related-proteins p-AKT and AKT,and the Raf/MEK/ERK signaling pathway related-proteins ERK1/2 and p-ERK1/2 were detected by Western blot in malignant glioma cells.Results1.The results from qRT-PCR and Western blot analyses showed that the expression of miR-21 in anti-miR-21 U251 and U87 cells was significantly decreased compared with the control group.Compared with the control group,CXCR4 mRNA and protein expression in anti-miR-21 U251 and U87 cells was significantly reduced.miR-21 and CXCR4 expression was significantly reduced in anti-miR-21+sh-CXCR4 U251 and U87 cells compared with the control group.The results indicated that miR-21 interference,CXCR4 interference and miR-21 and CXCR4 dual interference stable cell lines were successfully constructed.2.In U251 and U87 cells,compared with knockdown of miR-21 or CXCR4 expression,the effect of double-interfering miR-21 and CXCR4 on suppressing proliferation,migration and invasion,promoting cell apoptosis was more apparent in U251,U87 cells.3.In anti-miR-21 and sh-CXCR4 U251 and U87 cells,compared with control group,p-AKT protein expression was significantly reduced.Compared with anti-miR-21 and sh-CXCR4 U251 and U87 cells,in anti-miR-21+sh-CXCR4 U251 and U87 cells,p-AKT protein expression was significantly reduced.AKT protein expression had no obvious difference.Compared with the control group,the expression of p-ERK1/2 proteins in the anti-miR-21 group and sh-CXCR4 group was significantly reduced,while there was no significant difference in the expression of ERK1/2 proteins.However,compared with the anti-miR-21 and sh-CXCR4 U251 and U87 cells,p-ERK expression in the anti-miR-21+sh-CXCR4 group was significantly reduced.It was shown that compared with interference of miR-21 or CXCR4,double-interfering miR-21 and CXCR4 could inhibit the activity of the PI3K/AKT and Raf/MEK/ERK signaling pathways more effectively.The third part:Inhibitory effect of double-targeting miR-21 and CXCR4 on tumorigenesis of malignant glioma cells in nude miceMethods1.U251 cells were used to construct the xenograft model of glioblastoma in nude mice,and the volume of xenografts was detected regularly.After 30 days,nude mice were sacrificed and the weight of xenograft was detected.2.qRT PCR was used to detect the expression of miR-21 and CXCR4 in anti-miR-21,sh-CXCR4 and anti-miR-21+sh-CXCR4 malignant glioma xenografts,3.The pathological morphology of malignant glioma xenograft was observated by HE staining.4.Western blot was performed to mearure the effect of miR-21 interference,CXCR4 interference and double-targeting miR-21 and CXCR4 on the PI3K/AKT and Raf/MEK/ERK signaling pathway in malignant glioma xenografts.Results1.Compared with the control group,anti-miR-21,sh-CXCR4 and anti-miR-21+sh-CXCR4 treatments significantly inhibited the growth of glioblastoma transplanted tumor,while anti-miR-21+sh-CXCR4 treatments significantly inhibited the growth of glioblastoma transplanted tumor compared with the control group,anti-miR-21 group and sh-CXCR4 group.2.Compared with the control group,miR-21 expression was significantly reduced in the anti-miR-21 group and anti-miR-21+sh-CXCR4 group,while miR-21 expression was not significantly different in the sh-CXCR4 group.Compared with sh-CXCR4 group,miR-21 expression was also significantly decreased in anti-miR-21+sh-CXCR4 group.There was no significant difference in CXCR4 expression between the anti-miR-21 group and the anti-miR-21+sh-CXCR4 group.Compared with the anti-miR-21 group,the expression of CXCR4 in the anti-miR-21+sh-CXCR4 group was also significantly reduced.3.HE staining showed that cancer cell density was reduced,cell shape became round,the number of nucleoli was decreased and nuclear was weakly stained in anti-miR-21,sh-CXCR4 and anti-miR-21+sh-CXCR4 malignant glioma transplanted tumor tissues,compared with control group.Moreover,the anti-miR-21+sh-CXCR4 treatment has the most powerful effect on cells.4.Compared with the control group,p-AKT protein expression was significantly decreased in anti-miR-21,sh-CXCR4 and anti-miR-21+sh-CXCR4 malignant glioma transplanted tumor tissues and the leves of p-AKT was lowest in anti-miR-21+sh-CXCR4 group,but there was no significant difference in the expression of AKT protein.Compared with the control group,the expression levels of p-ERK1/2 protein in the anti-miR-21 group and sh-CXCR4 group were significantly reduced,while the expression levels of p-ERK1/2 protein in the anti-miR-21+sh-CXCR4 group were significantly reduced compared with anti-miR-21 group and sh-CXCR4 group.There was no significant difference between the ERK1/2 proteins.These data suggested that inteference of miR-21 or CXCR4 could inhibited the activities of the PI3K/AKT and Raf/MEK/ERK pathways and double-interfering miR-21 and CXCR4 has the strongest effect.Conclusion1.The expressions of miR-21 and CXCR4 in glioblastoma tissues and cells U251 and U87 were significantly higher than those in normal tissues and HAc cells.These results suggested that miR-21 and CXCR4 played an important role in the occurrence and development of glioblastoma.2.Both in vivo and in vitro experiments confirmed that compared with the interference of miR-21 or CXCR4,double-interfering miR-21 and CXCR4 could more effectively inhibit the proliferation,migration,invasion and growth of transplanted tumor and promote cells apoptosis,which may be related to the PI3K/AKT and Raf/MEK/ERK signaling pathways.Therefore,double-targeted miR-21 and CXCR4 may be ideal therapeutic targets for malignant gliomas.