| Background:Chronic myeloid leukemia(CML)is a malignant disease originated from bone marrow pluripotent hematopoietic stem cells and characterized by abnormal proliferation of granulocytes in peripheral blood and bone marrow.Its main characteristic is the formation of Ph chromosome,that is,the ABL gene on chromosome 9 and the BCR gene on chromosome 22 form the BCR-ABL fusion gene due to chromosome translocation.The gene encoding the BCR/ABL fusion protein has tyrosine kinase activity,and sustained to abnormal phosphorylation of downstream target protein tyrosine residues,form a composite signal cascade,make to multiple cells dysfunction,including abnormal adhesion,apoptosis ability weakened,cell proliferation and migration induced,cell differentiation ability of inhibition.CRKL(vcrk sarcoma virus CT10 oncogene homologue(avian)-like),a member of CRK adapter protein family,is ubiquitously expressed and conserved across eukaryotic organisms.It is composed by one N-terminal Src homology2(SH2)domain,one N-terminal SH3(SH3N)and one C-terminal SH3(SH3C)domains.Mi R-124-3p is a highly conserved mi RNA,which binding to the 3?-untranslated region(3?-UTR)of targeted m RNAs to regulate gene expression either directly degrading m RNA or suppressing posttranscriptional protein translation.In recent years,it has been exported that the decreased expression level of mi R-124-3p is related to cell differentiation,apoptosis,proliferation,metastasis and metabolism.Long non-coding RNA has been a hot topic in recent years,playing a role in transcriptional regulation,DNA damage repair,chromatin recombination and other biological functions.Competing endogenous RNAs(ce RNA)are a new pathway to regulate tumor progression and metastasis,and also the main mechanism by which Lnc RNAs play their biological functions.Lnc RNA binds mi RNAs reactive elements(MREs),binds mi RNAs through "sponge" competition,prevents them from binding to m RNA,regulates gene expression,and forms Lnc RNA-mi RNA-m RNA dialogue regulation mechanism.Our previous study found that CRKL reduced the proliferation,migration and invasion of K562 cells and promoted cell differentiation.In order to study the molecular mechanism of CRKL,we screened the variational Lnc RNAs after CRKL knockout by microarray.Among them,the change of Lnc RNA119766 was very obvious.Lnc RNA119766 is a long non-coding RNA with a length of 2173 bp,located on human chromosome 7.Lnc RNA119766 may play an important role in the biological function of CRKL.It was reported that mi R-124-3p was down-expression in CML.Bioinformatics prediction analysis found that mi R-124-3p had potential negative targeted regulation on CRKL and Lnc RNA119766.Lnc RNA119766 may be used as ce RNA to regulate the expression of CRKL through the adsorption of mi R-124-3p and then regulate the occurrence and development of CML.The interaction and mechanism of CRKL/mi R-124-3p/Lnc RNA119766 axis in CML have not been reported.Objective:1.To investigate the expression of CRKL,mi R-124-3p,Lnc RNA119766 in clinical samples of CML patients;2.Verified the ce RNA network in CRKL,mi R-124-3p,Lnc RNA1197663.To definite the influence of CRKL,mi R-124-3p on proliferation,migration and invasion of K562 cell;4.To explore the molecular mechanism of Lnc RNA119766-mi R-124-3p-CRKL axis regulate the CML cell biological behavior.Methods:1.The different expression of lnc RNAs between sh RNA-CRKL-K562 and sh RNA-NC-K562 cell was detected by microarray.2.q RT-PCR was used to detect the expression of CRKL,mi R-124-3p,Lnc RNA119766 in CML and normal people,analyzed the correlation with between them;3.q RT-PCR were used to detected the affects mi R-124-3p on CRKL m RNA,Lnc RNA119766 level and Western blot(WB)detected the effects of mi R-124-3p on CRKL protein expression level;4.To construct wild-type psi CHECK-2-CRKL-3?-UTR-WT,psi CHECK2-Lnc RNA119766-WT and mutant psi CHECK-2-CRKL-3?-UTR-MUT,psi CHECK2-Lnc RNA119766-MUT,dual luciferase reporter experiment were used to checked the affects of mi R-124-3p on CRKL-3?-UTR,Lnc RNA119766 in vitro;5.RNA immunoprecipitation assay(RNA IP,RIP)was performed to detect the enrichment levels of mi R-124-3p and Lnc RNA119766 by AGO2 antibody,and to verify the targeted negative regulation of mi R-124-3p on Lnc RNA119766.6.We transiently transfected PCDH-EF1-MCS-T2A-Puro and PCDH-EF1-MCS-T2A-Puro-CRKL into K562 cells,then to detected the expression level of CRKL m RNA and protein by q RT-PCR and WB.In addition,we detected the effects of CRKL overexpression on mi R-124-3p and Lnc RNA119766 by q RT-PCR;7.We transiently transfected the PCDH-EF1-MCS-T2A-Puro and PCDH-EF1-MCS-T2A-Puro-Lnc RNA119766 into K562 cell,q RT-PCR checked the expression level of Lnc RNA119766 and the influence of Lnc RNA119766 overexpression on mi R-124-3p and CRKL m RNA,meanwhile,the expression level change of CRKL protein was detected by Western blot;8.q RT-PCR was used to detect the effect of CRKL down-regulation on the expression levels of mi R-124-3p and Lnc RNA119766.9.According to the full-length sequence of Lnc RNA119766,three small si RNA interference sequences targeting Lnc RNA119766 were designed and synthesized.Meanwhile,unrelated sequences were designed as negative control.We transiently transfected si Lnc RNA119766 and negative control si NC into K562 cells.The expression level of Lnc RNA119766 and the effect of down-regulation of Lnc RNA119766 on the expression level of mi R-124-3p and CRKL m RNA were detected by q RT-PCR.The effect of the down-regulation of Lnc RNA119766 on CRKL protein expression was detected by WB;10.CCK-8 measured the influence of CRKL and mi R-124-3p on K562 cell proliferation ability;11.Transwell method was used to detect the effects of CRKL and mi R-124-3p on the migration and invasion ability of K562 cells;12.The Rescue experiment verified whether the up-regulation of CRKL reversed the inhibitory effect of mi R-124-3p on the migration and invasion of K562 cells.13.WB measured the influence of CRKL,mi R-124-3p,Lnc RNA119766 on PI3K/AKT pathway in K562 cells;14.Co-immunoprecipitation(Co-IP)were used to measure the interaction of CRKL and PI3K;15.PI3 K specific inhibitor LY294002 blocked PI3K/AKT pathway,then we checked the influence of CRKL,mi R-124-3p,Lnc RNA119766 on PI3K/AKT pathway and the effect on K562 cells migration abilities.Results:1.We found that 111 lnc RNAs were down-regulated(Fold change<0.5),and 288 lnc RNAs were up-regulated >1.5)through microarray after CRKL knockout.2.Compared with normal people,the expression levels of CRKL and Lnc RNA119766 in CML patients were increased,while the expression levels of mi R-124-3p was decreased.CRKL was positively correlated with Lnc RNA119766 and negatively correlated with mi R-124-3p,and Lnc RNA119766 was negatively correlated with mi R-124-3p.3.q RT-PCR and Western blot results showed that the up-regulation of mi R-124-3p could promote the expression level of CRKL m RNA,protein and Lnc RNA119766,while the down-regulation of mi R-124-3p could inhibit the expression level of CRKL m RNA,protein and Lnc RNA119766.4.Dual luciferase reporter demonstrated that compared with NC mimic,the cotransfection of mi R-124-3p mimic with psi CHECK-2-CRKL-3-UTR-WT/ WT 1/WT2 reduced the ratio of Photinus pyralis luciferase activity to Renilla reniformis luciferase activity.When the two binding sites were mutated,the inhibition of mi R-124-3p on the relative luciferase activity was basically eliminated.This indicated that mi R-124-3p regulated CRKL by targeting and binding CRKL-3?-UTR at two binding sites,and CRKL was the direct target gene of mi R-124-3p.At the same time,the co-transfection of mi R-124-3p with psi CHECK2-Lnc RNA119766-WT ? psi CHECK2-Lnc RNA119766-WT3 reduced the ratio of Photinus pyralis luciferase activity to Renilla reniformis luciferase activity.After the mutation of this binding site,the inhibition of mi R-124-3p on the relative luciferase activity was basically eliminated,and the co-transfection of mi R-124-3p with WT1,WT2,MUT1 and MUT1 basically had no effect on luciferase activity.The above results indicated that Lnc RNA119766 was the direct target gene of mi R-124-3p,and mi R-124-3p mainly directly targeted the third binding site of Lnc RNA119766 to inhibit the expression of Lnc RNA119766;5.RIP results showed that compared with Ig G antibody group,the expressions of mi R-124-3p and Lnc RNA119766 in the AGO 2 antibody group were significantly increased,and mi R-124-3p could target and regulate Lnc RNA119766.6.CRKL-overexpressed cell lines were obtained by transiently transfected PCDH-EF1-MCS-T2A-Puro and PCDH-EF1-MCS-T2A-Puro-CRKL,and the upregulation of CRKL promoted the expression of Lnc RNA119766 and inhibited the expression of mi R-124-3p,the down-regulation of CRKL inhibited the expression of Lnc RNA119766 and promoted the expression of mi R-124-3p.7.Lnc RNA119766 overexpressed cell lines were obtained by transiently transfected PCDH-EF1-MCS-T2A-Puro and PCDH-EF1-MCS-T2A-PuroLnc RNA119766,and the up-regulation of Lnc RNA119766 induced the level of CRKL and reduced the level of mi R-124-3p,the down-regulation of Lnc RNA119766 inhibited the expression of CRKL and promoted the expression of mi R-124-3p.8.CCK-8 results showed that CRKL up-regulation promoted the proliferation of K562 cells.Mi R-124-3p overexpression inhibited the proliferation ability of K562 cells,and mi R-124-3p down-regulated promoted the proliferation ability of K562 cells.9.Transwell results showed that up-regulation of CRKL promoted the migration and invasion of K562 cells.Mi R-124-3p overexpression decreased the migration and invasion ability of K562 cells,and mi R-124-3p down-regulated promoted the migration and invasion ability of K562 cells.10.Rescue results showed that compared with the mi R-124-3p mimic group,the expression level of CRKL in the mi R-124-3p +PCDH-CRKL group was increased and the cell migration ability was enhanced.Compared with the PCDH-CRKL group,the CRKL expression level of mi R-124-3p + PCDH-CRKL group was decreased and the cell migration ability was weakened.Thus it indicated that exogenous PCDH-CRKL does not contain 3'-UTR,and mi R-124-3p has no effect on exogenous CRKL,and only inhibited the expression of endogenous CRKL.Therefore,the overexpression of CRKL can reverse the inhibition of mi R-124-3p on the migration ability of K562 cells.It is further demonstrated that mi R-124-3p negatively regulates CRKL expression by targeting CRKL-3'-UTR and plays its biological function.11.The immunoprecipitation result showed that CRKL directly binds to PI3 K.12.Mi R-124-3p,CRKL and Lnc RNA119766 affect the PI3K/AKT signaling pathway.The down-regulation of CRKL and Lnc RNA119766 and the up-regulation of mi R-124-3p inhibited the expression of PI3 K and p-AKT.The up-regulation of CRKL,Lnc RNA119766 and the downexpression of mi R-124-3p induced the expression of PI3 K and p-AKT.13.After blocking PI3K/AKT pathway by PI3 K inhibitor LY294002,the promoting effects of mi R-124-3p,CRKL and Lnc RNA119766 on the PI3K/AKT pathway were inhibited.Moreover,the induce of up-regulation of CRKL and Lnc RNA119766,and the down-regulation of mi R-124-3p on the migration of K562 cells were inhibited.This indicates that Lnc RNA119766-mi R-124-3p-CRKL axis regulates the migration of K562 cells through the PI3K/AKT pathway.Conclusion:1.The expression levels of CRKL and Lnc RNA119766 in CML patients increased,while the expression levels of mi R-124-3p decreased.CRKL was positively correlated with Lnc RNA119766 and negatively correlated with mi R-124-3p,and Lnc RNA119766 was negatively correlated with mi R-124-3p.At the clinical sample level,the three were in accordance with the regulatory relationship of ce RNA.2.Lnc RNA119766 acted as a ce RNA to sponge mi R-124-3p increasing CRKL expression in K562 cells.3.CRKL and mi R-124-3p affected the malignant behavior of K562 cells.4.Lnc RNA19766-mi R-124-3p-CRKL axis affected the malignant behavior of K562 cells by regulating the PI3K/AKT signaling pathway. |
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