The Functional Analysis Of E3 Ubiquitin Ligase RNF6 In The Development Of Hematological Malignancies

Part 1 Preliminary functional analysis of RNF6 in hematological malignanciesObjective:The function and mechanism of RNF6 has not been reported in hematological malignancies. In this study, the expression of RNF6 was measured in multiple myeloma(MM), leukemia cell lines and patients’ samples. Meanwhile, the function of RNF6 was analyzed when RNF6 was over-expressed or silenced by lentivirus in MM and leukemia cells, which may elucidate the function of RNF6 in hematological malignancies.Methods:(1) The m RNA levels of RNF6 in bone marrow from patients and heathy donors were detected by q-RT-PCR and RT-PCR;(2) The protein levels of RNF6 in MM(H929, KMS11, LP1, OCI-My5, OPM2 and RPMI-8226) and leukemia(AML2, HL60, NB4, THP1, K562, Jurkat and 697) cell lines were detected by immunoblotting;(3) The effects of over-expressed RNF6 on leukemia cell proliferation and CFC production were assessed by MTT and colony forming assays;(4) The effects of RNF6 silence on leukemia cell proliferation were assessed by sh RNA;(5) The sensitivity of RNF6-overexpressed cells to Bortezomib was verified by trypan blue staining.Results:(1) q-RT-PCR and RT-PCR revealed that the m RNA levels of RNF6 in bone marrow species from multiple myeloma and leukemia patients were higher than bone marrow species from healthy donors;(2) RNF6 proteins in MM and leukemia cell lines were higher than NBM;(3) Overexpression of RNF6 obviously promoted cell proliferation and colony forming by MTT and CFC assays;(4) Silence of RNF6 in leukemia cells markedly inhibited cell proliferation;(5) Over-expressed RNF6 could significantly decrease K562 sensitivity to Bortezomib.Conclusions:The expression of RNF6 in all of MM and leukemia patients’ samples and cell lines was abnormally high. At the same time, over-expression or silence of RNF6 markedly promoted or inhibited tumor cell proliferation. Taken together, RNF6 may be involved in the development of hematological malignancies. In addition, over-expression of RNF6 inhibited the sensitivity of K562 cells to Bortezomib, which suggested that RNF6 may be associated with drug resistance.Part 2 Modulation mechanism of RNF6 transcription by5-amino-8-hydroxyquinoline(5AHQ)Objective:5-amino-8-hydroxyquinoline(5AHQ) is a novel chemical with high anti-tumor activity against MM and leukemia. Our previous DNA Microarray study found that RNF6 m RNA level was obviously affected by 5AHQ. In this section, we will study how RNF6 is regulated by 5AHQ at the transcription level.Methods:(1) MM and leukemia cells were treated with 5AHQ for 24 hrs followed by the viability assessment by MTT assay;(2) MM(OCI-My5 and RPMI-8226) and leukemia(AML2 and K562) cells were treated with 5AHQ for 24 hours, and then cells were harvested and total proteins or total RNA were isolated. The protein and m RNA levels of RNF6 were detected by Immunoblotting and RT-PCR, respectively;(3) RNF6 promoter was predicted by UCSC website, and RNF6 promoter fragments were constructed by deletion mutation. And then the activity of the promoter mutants was analyzed by dual-luciferase reporter assay system;(4) The potential transcription factor binding sites in RNF6 core promoter were predicted by TFSearch software. RNF6 promoter activity was analyzed by site-directed mutation and deletion mutation;(5) CHIP was carried out to detect whether Pbx1 could bind to RNF6 promoter;(6) The promoter activity of RNF6 mediated by Pbx1/Prep1/MEIS1 was assessed by dual-luciferase reporter assay system;(7) Co-IP was performed to detect whether 5AHQ could affect the hetero-dimerization of Pbx1 and Prep1. At the same time, the activity of RNF6 core promoter treated by 5AHQ was measured by the luciferase assay.Results:(1) MTT assay showed that 5AHQ could significantly inhibit cell viability in MM and leukemia cells;(2) The m RNA and protein levels of RNF6 were down-regulated in MM and leukemia cells when cells were treated with 5AHQ;(3) Deletion mutation analysis indicated that-365/-99 was the core promoter region of RNF6 promoter, which exhibited the highest transcriptional activity;(4) Site-directed and deletion mutation analyses showed that the Pbx1 binding site was essential for RNF6 promoter activity;(5) CHIP assay indicated that Pbx1 could bind to RNF6 promoter;(6) The activity of RNF6 promoter was not up-regulated when Pbx1 only but up-regulated when Pbx1 and Prep1 or MEIS1 plasmids were co-transfected, which showed that Pbx1 could regulate RNF6 transcription when it formed heterodimers;(7) 5AHQ suppressed the hetero-dimerization of Pbx1 and Prep1. At the same time, the activity of RNF6 core promoter was down-regulated by 5AHQ.Conclusions:We further demonstrated that 5AHQ significantly inhibited cell viability in MM and leukemia cells, and RNF6 transcription could be regulated by 5AHQ through suppressing Pbx1/Prep1 hetero-dimerization. Meanwhile,-365/-99 was the core promoter of RNF6, and RNF6 transcription could be mediated by Pbx1.Part 3 Characterization of RNF6 ubiquitinationObjective:It is reported that RNF6 belongs to the RING finger protein family which can be auto-ubiquitinated. In this study, we investigated the ubiquitination of RNF6 and discussed the specific ubiquitination sites and deubiquitinases of RNF6 to further elucidate the mechanisms of RNF6 ubiquitination.Methods:(1) To identify which degradation pathway RNF6 involved in, cells were treated with lysosome inhibitors(NH4Cl and Chloroquine) and proteasome inhibitors(MG132 and Lactacystin);(2) The ubiquitination of RNF6 was detected by co-immunoprecipitation;(3) A series of RNF6 mutants with lysine(K) to arginine(R) were constructed by site-directed mutation, including single-lysine mutants, or three-lysine mutants as well as lysine-free mutant. And then the ubiquitination assay of these mutants was assessed by MG132 treatment;(4) CHX chase was performed to examine the half-life of WT, K0, K608 and K608 R protein;(5) The deubiquitination of RNF6 was detected by transfecting a series of deubiquitinase plasmids.Results:(1) The proteasome inhibitors MG132 and Lactacystin could accumulate RNF6 protein but not lysosome inhibitors NH4 Cl and Chloroquine, which indicated that RNF6 was degraded mainly by the proteasome pathway;(2) The polyubiquitin chains of RNF6 were clearly observed by Co-IP, and the polyubiquitination level of RNF6 was sharply increased when treated with proteasome inhibitors, which showed that RNF6 could be auto-polyubiquitinated;(3) The site-directed mutation analysis showed that the degradation of RNF6 by the proteasome could be blocked obviously when the lysine residues K608 of RNF6 were mutated, which indicated that K608 is the important ubiquitination site of RNF6. However, K0 with lysine-free could be still partly ubiquitinated, which suggested that some other amino acid residues could also mediate RNF6 ubiquitination probably, such as cysteine, serine and threonine;(4) CHX chase showed that K608 R could exhibit a longer half-life of RNF6;(5) Several DUBs including USP22 could up-regulate RNF6 protein.Conclusions:The RING finger protein RNF6 was mainly degraded via the ubiquitin-proteasome pathway. At the same time, RNF6 could be auto-ubiquitinated, and K608 was the important ubiquitination site of RNF6. And USP22 may be the deubiquitinase of RNF6.Part 4 RNF6 interacts with and stabilizes GRObjective:As an E3 ligase, RNF6 has been reported to induce atypical ubiquitination of AR and then promoted prostate cancer cell growth. In this study, we were intended to examine whether RNF6 could regulate GR in MM, which may further elucidate the function of RNF6 in MM development.Methods:(1) The protein expression profiles of GR and RNF6 were detected by immunoblotting in MM and leukemia cell lines, and the tendency of GR and RNF6 proteins was also examined when MM cells were treated with 5AHQ;(2) Transfection was carried out to study whether GR protein expression could be affected by RNF6;(3) The half-life of GR protein was analyzed by CHX chase in the presence or absence of RNF6;(4) Co-IP was subjected to examine whether RNF6 could interact with GR;(5) A series of RNF6 and GR deletion mutants were constructed by deletion mutation, and then Co-IP analysis was subjected to identify the specific interaction regions of GR and RNF6;(6) Co-IP was subjected to verify whether RNF6 could induce GR ubiquitination;(7) Immunoblotting was carried out to determine whether Dex-induced GR degradation was affected by RNF6 through Dex and MG132 treatments.Results:(1) The protein expression profiles of RNF6 and GR were similar and the tendency of RNF6 and GR proteins was also similar when MM cells were treated with 5AHQ, which indicated that RNF6 seemed to be correlated with GR;(2) RNF6 could up-regulate both exogenous and endogenous GR protein level, but not the m RNA level, which indicated that RNF6 regualted the post-modification of GR protein;(3) CHX chase showed that RNF6 could prolong the half-life of GR protein;(4) Co-IP assay demonstrated that RNF6 could interact with GR;(5) Deletion mutation analysis illustrated that 531-777 region of GR was essential for RNF6 binding, and 87-482 region of RNF6 could bind to GR;(6) Co-IP assay showed that RNF6 could induce atypical ubiquitination of GR;(7) Dex-induce GR degradation was suppressed by RNF6.Conclusions:RNF6 interacted with GR, and bound to the LBD domain of GR. In addition, RNF6, as the E3 ligase, could induce the atypical ubiquitination of GR and increased its stability. And RNF6 could suppress Dex-induced GR degradation.ConclusionsRNF6 is a RING domain-containing E3 ligase. Currently, there is no report on its biological function in blood cancers. In this study, we found that RNF6 is highly expressed in the bone marrow cells from MM and leukemia patients’ samples. RNA is also high in MM and leukemia cell lines. In addition, over-expression of RNF6 promotes leukemia cell proliferation while silence of RNF6 reduces leukemia viability. Notably, introduction of RNF6 attenuates K562 cell sensitivity to Bortezomib, suggesting RNF6 probably contributes to chemoresistance. Recently, it is reported that 5-amino-8-hydroxyquinoline(5AHQ) can inhibit the proliferation of leukemia and myeloma cells. We also found 5AHQ downregulates RNF6 transcription. Further studies demonstrate that-365/-99 is the core region of RNF6 promoter, wherein Pbx1 modulates RNF6 expression in combination with Prep1. Because RNF6 belongs to the RING finger protein family, we analyzed the auto-ubiquitination of RNF6. We found that RNF6 could be auto-ubiquitinated and it is degraded mainly via the ubiquitin-proteasome pathway. We next investigated the ubiquitination sites of RNF6 and we found that the lysine reside K608 was important for RNF6 ubiquitination. RNF6 can be still ubiquitinated when all lysines(K) are mutated, thus suggesting other amino acid residues(such as cysteine, serine and threonine) may also mediate RNF6 ubiquitination. Functionally, RNF6 could bind to GR and increased GR stability in MM cells. At the same time, RNF6 could induce the atypical polyubiquitination of GR, which thus probably regulate the GR signaling pathway. In conclusion, this study for the first time reported the functional analysis and mechanism of RNF6 in hematological malignancies, especially in MM and leukemia cells, which may provide the theoretical basis for developing the targeted drugs based on RNF6 in clinical.