| Part 1 Expression of miR-320 and PBX3 in gliomaObjectiveGliomas are the most common brain tumors.They are characterized by morphological and genetic complexity,and they infiltrate diffusely into normal brain parenchyma.This feature renders all current therapeutic strategies ineffective.Although,radiotherapy or chemotherapy are the main treatment measures of glioma,this tumor progress rapidly and has a certain resistance to chemoradiotherapy.Despite advances in treatment modalities,but poor prognosis of glioma remains unchanged.It is an urgent clinical challenge to identify sensitive and specific early biomarkers for treatment measures and prognosis,but the key problem is to clarify the mechanism of glioma development.MicroRNA(miRNA)are a class of short,endogenous,non-coding RNA molecules that bind with the 3'-untranslated regions(3'-UTRs)of target mRNA,regulating the expression of gene.Researches have shown the importance of miRNA in the regulation of gene expression during apoptosis and cell proliferation.miRNA have also been found to be critical roles in tumor biology,thus they have been established as new and essential oncogenes and tumor suppressor genes.Aberrant expression of these miRNA was implicated in tumor development.Previous investigations have indicated that miR-320 is involved in the development of many tumors.For example,miR-320 has been proved to play a critical role in suppressing tumor angiogenesis through the silencing of Neuropilin 1 expression,thus miR-320 might be a potential anti-angiogenic or anti-cancer therapeutic.In addition,miR-320 has been found to inhibit cell proliferation and induces apoptosis in breast,prostate,hepatocellular carcinoma,colorectal adenoma and submucosal invasive carcinoma tissues.However,until now,the molecular mechanisms underlying the involvement of miR-320 in glioma remain poorly unknow.Pre-B-cell leukemia homeobox(PBX),a family of transcription factors,has been reported to play an important role in tumor growth.It usually binds to specific DNA sequences by interacting with other homologous proteins such as HOX,resulting in transcription activation or suppression of the target genes.Increased expression of PBX is closely correlated with tumor growth and progression in malignancies including ovarian cancer,melanoma and prostate cancer.Interference of interaction between PBX/HOX can induce apoptosis,leading to tumor growth inhibition of ovarian cancer,kidney cancer,non-small cell lung cancer,and breast cancer.Therefore,the PBX family is likely to be closely associated with malignant behavior of the tumor cells and could be a target molecule for cancer treatment.PBX3,a member of the human PBX family,has been continuously reported to be associated with tumor growth and progression.It was reported that expression level of PBX3 is significantly increased in malignant prostate cancer tissues.In 2013,PBX3 was found to be an important cofactor of HOXA9 in leukemogenesis.Direct targeting of HOXA/PBX3 impairs leukemia growth and sensitizes cells to standard chemotherapy.Recently,PBX3 was also reported to be upregulated in gastric cancer and to regulate cell proliferation.Together,the data suggest that PBX3,as an oncogene in some leukemias and solid cancers,is an important factor for regulating malignant biological characters of cancer cells.MAPK pathway plays a key role in many biological processes,including cell proliferation,tumor invasiveness,stem-like phenotypes,as well as resistance to chemotherapy.Previous research has indicated that PBX3 is able to promote migration and invasion of cancer cells via activation of MAPK signaling pathway.Although,researches have confirmed that PBX3 is closely correlated with various cancers,the few direct evidences confirm that PBX3 is related with glioma.In order to study the effect of miR-320 and PBX3 in the development and progressiong of glioma,and to analyze the role of miR-320 in the regulation of PBX3 in glioma,this article will start from the following two aspects:(1)The expression of miR-320 in glioma tissues:miR-320 expression was determined using qRT-PCR in glioma tissues and adjacent tissues.Expression of miR-320 was significantly reduced in glioma tissues in comparison with that of adjacent tissues.(2)The expression of PBX3 in glioma tissues:PBX3 expression was determined using qRT-PCR in glioma tissues and adjacent tissues.Expression of PBX3 was significantly increased in glioma tissues in comparison with that of adjacent tissues.Western blot analysis also showed that PBX3 expression was significantly increased in glioma tissues compared with adj acent tissues.Methods1 miR-320 expression was determined using qRT-PCR in glioma tissues and adjacent tissues.2 PBX3 expression was determined using qRT-PCR in glioma tissues and adjacent tissues.Western blot was used to determine the expression of PBX3 in glioma tissues and adjacent tissues3 All experiments were performed at least three times,and all samples were analyzed in triplicates.Statistical diference between each group was assessed by Student's t test and ANOVA analysis using SPSS 12.0 software.P<0.05 was considered to be statistically significant.Results1 The expression of miR-320 was significantly reduced in glioma tissues in comparison with that of adjacent tissues.2 The expression of PBX3 was significantly increased in glioma tissues in comparison with that of adjacent tissues.Western blot analysis also showed that PBX3 expression was significantly increased in glioma tissues compared with adjacent tissues.Conclusions1 The expression of miR-320 was significantly reduced in glioma tissues in comparison with that of adjacent tissues.The results show that miR-320 may be a tumor suppressor gene in glioma.The results suggest that miR-320 may inhibit the development of glioma.2 The expression of PBX3 was significantly increased in glioma tissues in comparison with that of adjacent tissues.The results show that PBX3 may have the effect of oncogene in glioma.The results suggest that PBX3 may promote the development of glioma.3 The expression of miR-320 and PBX3 was negative correlation in glioma tissues.This suggest that miR-320 may regulate the expression of PBX3 in gliomas.Part 2 miR-320 regulates the expression of PBX3 in gliomaObjectivemiR-320 is a non-coding RNA molecule with 18-25 nucleotides.Its function is to regulate the expression of gene at post-transcriptional level.miRNA abnormal regulation occurs in a variety of tumors.The regulation is achieved by combining miRNA with the 3 'end translation region(3' UTR)of mRNA,resulting in the degradation of mRNA.There are two complementarities between miRNA and target molecules.Complete complementation leads to RISC degradation,but incomplete complementation inhibits mRNA transcription,and then leads to instability and degradation of target molecules.Since transcription can be regulated even in incomplete pairing,a single miRNA can regulate multiple mRNAs,while a single mRNA can be regulated by multiple miRNA.As a member of the miRNA family,miR-320 is associated with the occurrence and development of many tumors and has the function of tumor suppressor gene in some tumors.Some studies have found that the expression of miR-320 was reduced in colorectal cancer,gastric cancer,breast cancer,bladder cancer,and human retinoblastoma.miR-320 has the biological functions of regulating tumor cell proliferation,invasion and metastasis of tumor cells,tumor cell apoptosis and chemotherapy resistance.PBX3 is a member of the PBX transcription factor family.Previous studies have confirmed that PBX3 plays a crucial role in the development of tumor.The expression of PBX3 is up-regulated in gastric cancer,colorectal cancer,prostate cancer and leukemia,indicating that PBX3 is carcinogenic.PBX3 is regulated by other miRNA in different tumors,such as miR-33a-3p,miR-495 and miR-497.Recent studies have found that PBX3 is regulated by miR-320 in multiple myeloma.The objective of this study is to predict the target molecule of miR-320 by on-line tools.The regulatory effect of miR-320 on target PBX3 was determined by the transfection of miR-320 mimics.Finally,the luciferase report was used to verify the combination of miR-320 with PBX3 3'UTR.Methods1 Cell Culture:U87 and U251 glioma cell lines were purchased from Chinese Academy of Sciences Cell Bank.Cells were cultured in Dulbecco's Modified Eagle's Medium(DMEM;Sigma Aldrich,St.Louis,MO,USA)supplemented with 10%fetal bovine serum.Cultures were maintained at 37 ? in a humidified atmosphere with 5%CO2.2 Cell Transfection:miR-320 mimics and NC(negative rna control)plasmids were transformed into U87 and U251 glioma cells.3 The expression of miR-320 and PBX3 after transfection of miR-320 mimics plasmid was detected by real-time fluorescence quantitative PCR.4 The expression of PBX3 at protein level after transfection of miR-320 mimics plasmid by western blot.5 Luciferase assays(1)miR-320 target molecules were predicted by targetscan online tools.(2)The 3'-UTR of PBX3 was amplifed and cloned downstream of frefy luciferasecoding region in the pMirReport vector.Mutations were introduced into the potential miR-320 binding sites using the QuikChange site-directed mutagenesis kit.Firefy luciferase reporters,Renilla luciferase pRL-TK vector and miR-320 mimics were co-transfected into the U87 and U251 cells.Cells were collected 36 h after transfection and assayed for luciferase activity using the Dual-Luciferase Reporter Assay System 6 Statistical analysis:All experiments were performed at least three times,and all samples were analyzed in triplicates.Statistical diference between each group was assessed by Student's t test and ANOVA analysis using SPSS 12.0 software.P<0.05 was considered to have statistically significant.Results1 By utilizing a targetscan online tool,according to the principle of sequence complementary pairing,we find that there is a sequence fragment of PBX3 gene 3'-UTR that may be complementary to miR-320.2 The expression of miR-320 in U87 and U251 glioma cells after transfection of mir-320 mimics plasmid and NC plasmid was detected by real-time fluorescent quantitative PCR.The expression level of miR-320 in U87 and U251 glioma cells after transfection of miR-320 mimics was significantly higher than that of NC group.At thesame time,the expression of PBX3 mRNA in U87 and U251 glioma cells transfected with miR-320 mimics was significantly lower than that of NC group.Therefore,miR-320 can inhibit the expression of PBX3.3 The expression of PBX3 at protein level in U87 and U251 glioma cell lines transfected with miR-320 mimics and NC plasmid was detected by western blot.The expression level of PBX3 protein in U87 and U251 glioma cell lines transfected with miR-320 mimics was significantly lower than that of NC group.Consistent with the level of PBX3 at the mRNA level.it is further confirmed that miR-320 inhibits the expression of PBX3.4 Luciferase reporters,Renilla luciferase pRL-TK vector and miR-320 mimics were co-transfected into the U87 and U251 cells.Luciferase reporter assay showed that over-expression of miR-320 led to a marked decrease of Renilla luciferase activity,which was specifcally abolished by the mutation of the corresponding anti-seed sequence in 3' UTR of PBX3.These results suggested that miR-320 directly modulate PBX3 expression by direct binding.Conclusions1 By utilizing a targetscan online tool,according to the principle of sequence complementary pairing,we find that there is a sequence fragment of PBX3 gene 3'-UTR that may be complementary to miR-320.2 miR-320 can regulate the expression of PBX3 in glioma,.3 miR-320 can regulate the expression of PBX3 by directly binding with PBX3 3'-UTR.Part 3 Effects of miR-320 on proliferation,apoptosis of glioma cellsObjectiveGliomas are the most common tumors in the nervous system.They originate from glial cells and trend to invade peripheral brain tissues.Most of the tumors are characterized by invasive growth,the operation is not easy to be completely cut,and the effect of treatment is poor.It is known that the ultimate cause of glioma development are unlimited proliferation and apoptosis inhibition,which provides important inspiration for the further study of the molecular mechanism of glioma pathogenesis.miRNA is an endogenous small fragment of non-coding RNA that plays a role in post-transcriptional regulation of gene expression.More and more studies have shown that miRNA is involved in the core signal transduction of glioma and can be used as a potential biomarker and therapeutic target.miR-320,a member of the miRNA family,is down-regulated in a variety of tumors,Ssuch as breast cancer,mesothelioma,liver cancer(hepatobiliary duct cancer)and hepatocellular carcinoma,non-small cell lung cancer and small cell lung cancer,colon and colorectal cancer,prostate cancer,Oral and cervical cancer.miR-320 is involved in the occurrence and development of cancer and has potential clinical application value.The MAPK signal pathway is an important signal transduction pathway in cells,which can transfer extracellular signals into cells and nuclei,and participate in the biological processes of cell proliferation,differentiation,transformation,and apoptosis.Phosphorylated MAPK kinase can activate MAPK or ERK in a single time,and activated ERK can translocate into the nucleus to activate some transcription factors,induce cell cycle progression and transcription of anti-apoptotic gene.The abnormal signal pathway leads to cell transformation and resistance to apoptosis.Therefore,this pathway is an attractive target for gliomas.Studies have shown that this signaling pathway is closely related to the occurrence and development of tumor.Previous studies have shown that PBX3 can promote the growth of colorectal cancer cells by activating the MAPK signaling pathway.Therefore,we speculate that PBX3 may also promote glioma by activating MAPK pathway.To investigate the effect of miR-320 on the proliferation and apoptosis of glioma cells and to provide the theoretical and experimental basis for the clinical application of miR-320,This article will carry on the research from the following three aspects:(1)The effect of miR-320 on proliferation of glioma cells:MTT and clone formation experiment showed a significant reduction in proliferation after transfection of miRNA-320 mimics in glioma cells.Flow cytometry was used to detect the distributionof cell cycle.In U87 and U251 cells.The G0/G1 phase fraction in miRNA-320 mimicsgroup was significantly increased.(2)miRNA-320 induces apoptosis in glioma cell lines:Flow cytometry showed that the apoptosis rate of glioma cells in miR-320 mimics group was significantly higher than that in NC group.At the same time,the expression of apoptotic protein cleaved caspase 3 in miR-320 mimics group was significantly higher than that in NC group.(3)MiR-320 overexpression or the low expression of PBX3 inhibit MAPK pathway activation in glioma cells:Western blot showed that the phosphorylation levels of RAF-1,p38,ERK1/2,ERK5 and JNKin miRNA-320 mimics group were significantly lower than NC group,and the phosphorylation levels of RAF-1,p38 and ERK1/2 in shPBX3 group were significantly lower than those in NC group.Methods1 Cell Culture:U87 and U251 glioma cells were cultured in Dulbecco's Modified Eagle's Medium,and supplemented with 10%fetal bovine serum.Cultures were maintained at 37 ? in a humidified atmosphere with 5%CO2.2 Cell proliferation assays:The proliferation of U87 and U251 glioma cells transfected with miR-320 mimics and NC plasmid was detected by MTT method.Clone forming experiment was used to detect clone forming ability in miR-320 mimics group and NC group.3 Cell cycle analysis:The cell cycle distribution of U87 and U251 glioma cells transfected with miR-320 mimics and NC plasmid was detected by flow cytometry.4 Cell apoptosis assays:Flow cytometry was used to detect the apoptosis of U87 and U251 glioma cells after the transfection of miR-320 mimics and NC.5 Western blot assay:western blot detected the expression of apoptotic protein caspase 3 and cleaved caspase 3 in miR-320 mimics group and NC group;western blot detected the expression of p-Raf-1?Raf-1?p-p3 8?p38?p-ERK1/2?ERK1/2?p-ERK5?ERK5?p-JNK?JNK in miR-320 mimics group and NC group;western blot detected the expression of p-Raf-1?Raf-1?p-p38?p38?p-ERK1/2?ERK1/2 in shPBX3 group and NC group.6 Statistical analysis:All experiments were repeated at least three times,and all samples were analyzed in triplicates.Statistical diference between each group was assessed by Student's t test and ANOVA analysis using SPSS 12.0 software.P<0.05 was think having statistically significant.Results1 MTT assay showed a significant reduction in proliferation after transfection of miRNA-320 mimics in glioma cells.Clone formation test also showed that the clone formation number of miR-320 mimics group was significantly lower than that of NC group.To further study whether the proliferation of glioma cells is related to the change of cell cycle,the cell cycle distribution was analyzed by flow cytometry.The number of glioma cells transfected with miR-320 mimics in G0/G1 phase increased significantly.2 The apoptosis rate of U87 and U251 glioma cells in miR-320 mimics group was significantly higher than that in NC group,and the number of apoptotic cells was significantly increased.In addition,transfection of miR-320 mimics increased the expression of cleaved caspase 3.3 The Raf-1,p38,ERK1/2,ERK5 and JNK phosphorylation level of U87 and U251 glioma cells in miR-320 mimics group was significantly lower than that in NC group by western blot assay.To address whether miR-320 functions through targeting PBX3,The expression of PBX3 was inhibited by shPBX3,the low expression of PBX3 signifcantly reduced the phosphorylation level of Raf-1,p38 and ERK1/2.Taken together,the results indicate that miR-320 may suppress glioma cell growth through targeting PBX3 and regulating MAPK pathway.Conclusions1 miR-320 inhibits glioma cell proliferation by regulating cell cycle distribution.2 miR-320 induced apoptosis in glioma cells.3 miR-320 may suppress glioma cell growth through targeting PBX3 and regulating MAPK pathway. |
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