| Background and aim:Multiple Myeloma(MM),which is a malignant disease with abnormal hyperplasia of plasma cells in bone marrow.In recent years,with the development of social population aging and the update of experimental diagnosis technology,the incidence of MM in China has been rising,and the age of onset is becoming younger.There is no radical cure for MM.Although in the past years,the application of clinical first-line therapy in MM has significantly improved the prognosis of patients after using a large number of new drugs such as Bortezomib,Thalidomide,and Lenalidomide.But the recurrence of the disease,and drug resistance remians unable to overcome as to current clinical physicians.Therefore,it is of important practical significance and theoretical value to study the development of esophageal cancer from the molecular level to prevent the disease from the genetic level.miRNA is a non-coding single stranded,a small RNA,that is widely expressed in both prokaryotic and eukaryotic organisms,ranging from 20 nt to 25 nt.After the transcriptional,mi RNA regulations the expression of genes by a target m RNA 3 'UTR region combination causing the translation inhibition of target genes,oncogenes or tumor suppressor gene function,thus widely participating in tumor occurrence,development,invasion and metastasis.In recent years,the study found that the mi RNA involved in regulating tumor invasion and metastasis,regulating cell early development,cell differentiation,gene expression and regulation of metastatic tumor proliferation,and other key steps through multiple signaling pathways.More and more evidence shows mi RNA may serve as tumor potential biomarkers in circulating plasma or serum,it provides a study of direction for the diagnosis,the test of prognosis and the prediction of individual response and resistance to drug therapy,and the mechanism of tumor formation.mi R-125 b plays an important role in the occurrence and progression of tumors,but the role of mi R-125 b is not clear in myeloma.The function of mi R-125 b in myeloma and its molecular mechanism needsto be further explored.Is there any specific change in mi RNA expression in peripheral circulation of multiple myeloma patients? Is the abnormal expression of mi RNA associated with the diagnosis and the prognosis of the disease?What molecular mechanism does mi RNA regulating myeloma growth,invasion and metastasis? Is it possible to achieve the initial therapeutic effect of tumor by interfering with the regulation of mi RNA? In view of this,this topic is to study the expression of mi R-125 b in myeloma.The effects of mi R-125 b on the growth,invasion and apoptosis of myeloma cells were observed.Tere,We explored whether PHLPP2 and MKK7 were the target genes of mi R-125 b.The molecular mechanism of mi R-125 b in cell growth,invasion and apoptosis was discussed primary.Methods:The mi RNA expression of 6 cases of myeloma plasma samples and 6 healthy controls were detected by high-throughput sequencing,and the expression genes of differentially expressed genes were analyzed.Using Real-time PCR method to further verify,In addition,clinical indicators such as ISS staging,cytogenetics and FISH detection were analyzed.The expression level of mi R-125 b and its target genes in myeloma plasma tissues and myeloma cell lines was detected by Real-time PCR.The function of mi RNA-125 b in myeloma was detected by in vitro and in vivo experiments(CCK8 experiment,the clone formation experiment,flow cytometry,transwell migration and invasion experiment,immunohistochemical,immunofluorescence experiments,western blot and nude mice into tumor).The target gene of mi R-125 b was verified by lucife Rase reporter.Results:Part1: The establishment of plasma mi RNA expression profiles in multiple myelomaPlasma samples of myeloma were analyzed by high-throughput sequencing.In this study,a total of 29 differentially expressed mi RNAs genes were obtained.There were 13 differentially expressed genes that were up-regulated mi RNA genes.16 genes that were down-regulated.q RT-PCR was performed for 8 mi RNAs(mi R-125b-5p,mi R-483-3p,mi R-4326,mi R-6894-3p,mi R-4498,mi R-490-3p,mi R-7155-5p and mi R-937-3p)that were up-regulated in myeloma.Expression levels of 5mi RNAs(mi R-125 b,mi R-4326,mi R-4498,mi R-490-3p and mi R-7155-5p)consistent with chip results,confirmed the reliability of chip data.mi RNA-125 b has high accuracy in myeloma,the expression level of mi R-125 b was correlated with the stage of myeloma,and there was no correlation between age,gender,and karyotype.Part:2 The effect of mi R-125 b expression on the biological function of myeloma was Investigated.In this study,the expression of mi R-125 b cell lines(MM1.S cells,RPMI-8226 cells and U266 cells)and plasma cells were detected by real-time fluorescence quantitative PCR in myeloma.q RT-PCR showed that the expression level of mi R-125 b in human myeloma cell lines was significantly higher than that of human normal plasma cells,having a significant difference(p <0.05),demonstrating that mi R-125 b is an oncogenic mi RNA in myeloma.In order to verify whether mi R-125 b affects the biological behavior of myeloma cells.In this study,the synthesized mi R-125 b mimics and mi R-125 b inhibitor were transfected into myeloma cell lines(RPMI-8226 cells and U266 cells)to observe the proliferation,invasion and migration of cells.mi R-125 b inhibitor transfected into myeloma cell lines RPMI-8226 and U266 cell,then the cell proliferation,invasion and migration was significantly reduced,and mi R-125 b inhibitor promoted cell apoptosis.The above experimental results have shown that mi R-125 b promoted the proliferation,invasion and migration of myeloma cells.Part3:The biological function of mi R-125 b targeting PHLPP2 regulat ing Akt pathway in myelomaWe use bioinformatics methods(Target Scan,Star Base V2.0 and mi RDB)to predict the mi R-125 b integrating region of target the 3 ' PHLPP2 UTR to modulate its expression.In this part of the experiment,we have verified the PHLPP2(direct target effect of mi R-125b)by the fluorescence enzyme report.In order to further define PHLPP2 as the target gene of mi R-125 b,RT-PCR method was used to detect theeffect of mi R-125 b on PHLPP2 m RNA level,and the PHLPP2 m RNA level increased markedly after RPMI-8226 and U266 cells inhibit mi R-125 b.the expression of PHLPP2 m RNA was significantly decreased after mi R-125 b mimic effect.At the same time,the western blot experiment also showed that the level of PHLPP2 protein also increased significantly after the inhibition of mir-125 by RPMI-8226 and U266 cells.The PHLPP2 m RNA level decreased significantly after mi R-125 b mimic.Next,this study also detected a significant low expression of PHLPP2 in myeloma by Real-time PCR,and the statistical analysis revealed that mi R-125 b was negatively correlated with PHLPP2 expression in myeloma.All of results indicated that one of the direct target genes in mi R-125 b regulation was PHLPP2.Studies have shown that PHLPP2 can inhibit the Akt signaling pathway by Akt out of phosphorylation,and do we speculate whether the myeloma caused by mi R-125 b can further activate the Akt signaling pathway by targeting PHLPP2? To analyze this point,we observed the expression of Akt pathway-related protein by western blot.Results showed that mi R-125 b could promote the activity of Akt signaling pathway.mi RNA-125 b regulates the Akt pathway by inhibiting the expression of PHLPP2,facilitating the proliferation and metastasis of myeloma cells.Part4:Effect of mi R-125 b targeting MKK7 on the biological function of MAPK pathway in the control of myelomaIn order to further explore the mechanism of mi R-125 b in myeloma,we use bioinformatics methods(Targetscan,Starbase V2.0 and Mirdb)to predict another mi R-125 b target gene MKK7.We have verified MKK7(the direct targeting effect of mi R-125b)by using the Lucife Rase reporter experiment.In order to further define MKK7 as the target gene of mi R-125 b,RT-PCR method was used to detect the effect of mi R-125 b on MKK7 m RNA level,and western blot method was used to detect the expression of blot protein MKK7.The results indicated that one of the direct target genes in mi R-125 b regulation was MKK7.We speculate whether mi R-125 b can further regulate the development of myeloma caused by MAPK signaling pathway by targeting MKK7? In order to resolve this point,we used western blot to observe the expression of MAPK pathway JNK related proteins.The results showed thatmi R-125 b inhibited the expression of MKK7 and then regulated the MKK7/JNK pathway,which promoted the proliferation and metastasis of myeloma cells. |
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