USP35 Activated By MiR Let-7a Inhibits Cancer Cell Proliferation

MicroRNAs (miRNAs) are 20-25 nucleotides in length, non-coding and endogenous RNA molecules, since Ambros et al first found heterochronic gene lin-4 in nematode in 1993, the mechanisms of miRNAs function cause a great deal of attention. MicroRNAs expression and functions are linked to important biological processes in development, cell differentiation, regulation of cell cycle and apoptosis and involved in a variety of pathological processes including cardiovascular diseases, neurological diseases, diabetes, cancer, etc. It is predicted miRNA has been able to regulate approximately 30% of human genes expression. MiRNAs have primarily been demonstrated to mediate gene expression at the posttranscriptional level either by inducing mRNA degradation or repressing translation via association with sequence-specific microRNA recognition target sites in target mRNA untranslated regions (UTRs) and coding regions. Much evidence demonstrates that miRNAs negatively regulate partially complementary target mRNAs at the transcriptional level by sequences at the mRNA promoter regions. However, an emerging experimental studies reveal that microRNAs can additionally function to activate gene expression at posttranscription or translation level by direct and indirect mechanisms. Overall, results indicate that miRNAs can be regulate the expression of downstream target genes through different mechanisms to affect important biological process.MiR let-7 was first identified as a heterochronic switch gene in Caenorhabditis elegans. It is abundantly detectable in later stages of development. MiR let-7 encodes a 21-nucleotide RNA that control expression of heterochronic genes lin-14, lin-28, lin-41, lin-42 and daf-12 through complementary to elements in the 3’untranslated regions. As a member of miR let-7 family, miR let-7a has been widely studied and has a critical role for tumor growth and progression. Previous work has shown reduced expression levels of miR let-7a in some cancer types, such as lung、breast、prostate and colon cancer compared to normal, the decreased miR let-7a expression is linked to increased tumorigenicity and poor prognosis. High expression of miR let-7a in cancer cells can significantly inhibit cell proliferation、invasion、tumorigenesis and metastasis. It has been reported that miR let-7a exerts its anti-tumor activity by mediating the expression of downstream target genes, such as K-RAS、HMGA2、EZH2、 c-myc, etc.In recent years, miR let-7a has made great progress at antitumor effect, the precise mechanisms of miR let-7a in the pathogenesis of human cancers still remain unclear. Identification regulatory substrates and function route of miR let-7a is essetial to elucidate anti-tumor mechanisms、related tumor development and tumor treatment strategy.Using cDNA microarray detection in the human prostate cancer cell line LNCaP after transfection with miR let-7a mimics or miR let-7a inhibitor, analyzed the change of gene expression profile induced by miR let-7a mimics or inhibitor. Several differentially expressed genes regulated by miR let-7a were screened, of which USP35 was changed significantly in LNCaP cells with miR let-7a mimics or miR let-7a inhibitor. As a newly identified member of deubiquitylases, Ubiquitin specific protease 35 (USP35) can specific recognize target ptoteins and catalyze the removal of ubiquitin from the ubiquitinated substrates. So far little is known about the information and study of USP35, previous work have demonstrated:1) overexpression of USP35 inhibit PARK2-mediated mitophagy.2) genome-wide array comparative genomic hybridization studies have confirmed that genome-wide copy number of USP35 changed in breast cancer.3) USP35 significantly increased in radiofrequency radiation. There is no information about the regulatory mechanisms of USP35 gene expression, its function in tumorgenesis as well as clinical significance.This study was first detected the relationship between miR let-7a and USP35 in some commonly-used cell lines and clinical samples, results confirmed that USP35 is a new target gene of miR let-7a and miR let-7a up-regulates the expression of USP35. Functional experiments showed miR let-7a exerts its tumor proliferation inhibition roles through regulating the expression of USP35. Subsequently, detecting the role of USP35 in tumor proliferation, results showed that overexpression of USP35 inhibited cell proliferation significantly compared with their respective controls, in contrast, knockdown of USP35 showed a significant increase of cell proliferation, xenograft models of nude mouse showed USP35 can regulate tumorigenic capacity of cancer cells in vivo, and impair the tumorigenesis in nude mice.In addition, we screened target substrates of USP35, using immunoprecipitation and mass spectrometry analysis, we got a crucial protein-TNFAIP3 interacting protein 2 (ABIN-2) and identified that USP35could stabilize ABIN-2 by deubiqutination and inhibit TNFa-induced NF-kB activation by stabilizing ABIN-2 protein,In summary, these findings focus on miR let-7a-mediated expression of USP35 and potentially molecular mechanisms of USP35 as a tumor repressor, and illustrate biological function of USP35 in tumor proliferation. Our current findings identify a regulatory network of miR let-7a-USP35-ABIN-2 pathway, which may serve as potential therapeutic targets to help us control the human cancer progression.Part Ⅰ. MiR Iet-7a positively regulates the expression of USP35[Object]To study the relationship between miR let-7a and USP35 in various cancer cell lines and different clinical tumor samples, further identify results of gene chip, and confirm miR let-7a up-regulates the expression of USP35.[Methods]1. Transfect miR let-7a mimics or inhibitor into prostate cancer cell line LNCaP and used qRT-PCR and Western blot to detect the expression of USP35.2. Determine expression of miR let-7a and USP35in commonly-used human cancer cell lines and their corresponding non-malignant cell lines using qRT-PCR and Western blot, respectively.3. Determine USP35 protein expression after overexpression of let-7a mimics in H1299 cells (expressing lower miR let-7a) or let-7a inhibitor in HeLa cells (expressing higher miR let-7a) using Western blot.4. Examine the expression of miR let-7a and USP35 in human cancer tissues and the corresponding noncancerous normal tissues using qRT-PCR and Western blot, analyzing the relationship between miR let-7a and USP35.5. MiR let-7a mimics and control mimics were transfected into LNCaP cells, after 24h, silence plasmid of USP35 and the control plasmid sh-CTL were co-transfected into LNCaP cells.Examine the cell growth in cells by MTT and colony formation assay.[Result]1. The detection of USP35 mRNA and protein expression level in LNCaP cells by qRT-PCR and Western blot showed that let-7a mimics increased, whereas let-7a inhibitor decreased both mRNA and protein level of USP35.2. qRT-PCR results showed that expression level of miR let-7a was much lower in the most cancer cell lines than that in non-malignant epithelial cells, consistent with the expression trend of miR let-7a, Western blot results showed that expression level of USP35 was much lower in the most cancer cell lines than that in non-malignant epithelial cells.3. The detection of USP35 protein expression level by Western blot showed USP35 expression was much higher after overexpression of let-7a mimics in H1299 cells and USP35 expression was much lower after overexpression of let-7a inhibitor in HeLa cells.4. qRT-PCR results showed that expression level of miR let-7a was much lower in the cancer tissues than that in the normal tissues. qRT-PCR and Western blot showed the mRNA and protein level of USP35 was also significantly decreased in the cancer tissues compared with the adjacent normal tissues. Statistical analysis showed that USP35 expression was positively correlated with miR let-7a level in breast cancer tissues.5. MTT and Colony formation analysis showed that MiR let-7a mimics in LNCaP cells inhibited cell proliferation and reduced colonies numbers compared with control cells.silence plasmid of USP35 reduced the effect of miR let-7a mimics in LNCaP cells.[Conclusion]1. MiR let-7a positively regulates the expression of USP35.2. MiR let-7a exerts its ability of inhibiting tumor proliferation through regulating the expression of USP35.Part Ⅱ. Research on the role of USP35 inhibits tumor proliferation[Object]In this part, we tested whether USP35 have the ability of inhibiting tumor proliferation of cancer cells by experiments in vitro and nude mice model experiment.[Methods]1. Construct USP35 over-expressing plasmid pcDNA3.1-USP35. pcDNA3.1-USP35 and the control plasmid were transient transfected into H1299 and LNCaP cells, after 48h, the changes of USP35 expression were verified by Western blot.2. USP35 silence plasmid (sh-USP35-1, sh-USP35-2, sh-USP35-3, sh-USP35-4) and the control plasmid sh-CTL were transient transfected into HeLa and LNCaP cells, after 48h, the changes of USP35 expression were verified by Western blot, screening the best silencing plasmid.3. Over-expressing plasmid of USP35 and the control plasmid were transient transfected into H1299 and LNCaP cells, silence plasmid of USP35 and the control plasmid sh-CTL were transient transfected into HeLa and LNCaP cells.Examine the cell growth in cancer cells with overexpression and knockdown of USP35 by MTT.4. Over-expressing plasmid of USP35 and the control plasmid were transient transfected into H1299 and LNCaP cells, silence plasmid of USP35 and the control plasmid sh-CTL were transient transfected into HeLa and LNCaP cells.Examine the ability of colony formation in cancer cells with overexpression and knockdown of USP35 by colony formation assay.5. To study whether the up-regulated USP35 can regulate tumorigenic capacity of cancer cells in vivo, we established xenograft models of nude mouse with the application of the constructed over-expressing and control H1299 cells and observed the tumorigenic capacity and weight.[Result]1. The USP35 overexpression plasmid pcDNA3.1-USP35 were successfully established. Western blot results showed that overexpression and knockdown plastid of USP35 change significantly.2. MTT results showed that overexpression of USP35 inhibited cell proliferation significantly compared with their respective controls in both H1299 and LNCaP cells. In contrast, HeLa and LNCaP cell lines with USP35 knockdown showed a significant increase of cell proliferation3. Colony formation analysis showed that ectopic expression of USP35 in H1299 and LNCaP cells led to significant reduced colonies numbers compared with their control cells. In contrast, silenced expression of USP35 caused an increase of the numbers of colonies in HeLa and LNCaP cells.4. Xenograft models of nude mouse exhibited overexpression of USP35 can impair tumorigenic capacity of cancer cells in vivo.[Conclusion]USP35 can inhibit cell proliferation process in cancer cells, and may play a vital role as a tumor suppressor gene in tumor development process.Part III. Research on molecular mechanisms of USP35 inhibits NF-κB activation by deubiquitinating ABIN-2 protein.[Object]As a novel member of DUB family, USP35 can catalyze the removal of ubiquitin from the ubiquitinated substrates.but its specific target substrates is still poorly understood. The major object of this part was to clarify its target substrates and possible molecular mechanisms.[Methods]1. LNCaP and H1299 cells were transfected with HA-tagged ubiquitin (HA-Ub) alone or co-transfected with pcDNA3.1-USP35. Verify The whole proteins ubiquitination of whole proteins by immunoprecipitation.2. H1299 cells were transiently transfected with USP35 overexpression plasmid and empty vector respectively. the whole proteins were detected by immunoprecipitation and mass spectrometry analysis, then screened potential USP35-interacting proteins.3. Construct USP35-interacting protein ABIN-2 over-expression plasmid Flag-ABIN-2. Over-expression USP35 alone or in combination with Flag-ABIN-2 were transient transfected into HEK293T and H1299 cells, after 48h, Lysates were verified by coimmunoprecipitation.4. Lysates of LNCaP and HeLa cells were detected by coimmunoprecipitation to examine endogenous interaction of USP35 and ABIN-2.5. HA-tagged ubiquitin and Flag-ABIN-2 were cotransfected into H1299 cells in the presence of USP35 overexpression plasmid or control vector, investigate the effect of USP35 on ABIN-2 ubiquitination by coimmunoprecipitation.6. Over-expressing plasmid of USP35 and the control plasmid were transient transfected into H1299 cells, silence plasmid of USP35 and the control plasmid sh-CTL were transient transfected into LNCaP cells, cells were treated with 50 ng/mL CHX for 0,2,4,8,12 and 24h.Examine the effect of USP35 on the half-life of ABIN-2 in cancer cells by Western blot.7. Over-expressing plasmid of USP35 and the control plasmid were transient transfected into H1299 cells, silence plasmid of USP35 and the control plasmid sh-CTL were transient transfected into LNCaP cells. After 42h, the cells were treated 6h with or without proteasome inhibitor MG132. Examine ABIN-2 protein expression by Western blot.8. Examine the expression of USP35 and ABIN-2 in above-mentioned human cancer tissues and the corresponding noncancerous normal tissues using Western blot, analyzing the relationship between USP35 and ABIN-2.9. NF-κB-dependent luciferase reporter gene were transient transfected into H1299 and LNCaP cells with over-expressing plasmid of USP35 or the control plasmid, after 42 h, cells were treated with or without TNFα (2ng/mL).6 h later, investigate the effect of USP35 on the regulation of TNFα-induced NF-κB activation using NF-κB-dependent luciferase reporter gene assay.10. NF-κB-dependent luciferase reporter gene were transfected into HeLa and LNCaP cells with USP35 shRNA alone or with USP35 shRNA and ABIN-2 expression plasmids. Cells were treated 6h with TNFα (2ng/mL), investigate TNFα-induced NF-κB activation using NF-κB-dependent luciferase reporter gene assay.[Result]1. The immunoprecipitation results showed that overexpression of USP35 markedly reduced the ubiquitination of whole proteins in LNCaP and H1299 cells.2. Detection of immunoprecipitation and mass spectrometry analysis proved that ABIN-2 is a potential USP35-interacting protein.3. Verify by coimmunoprecipitation that there is a physical interaction between USP35 and ABIN-2 proteins.4. Verify by coimmunoprecipitation that endogenous USP35 could bind to endogenous ABIN-2.5. The coimmunoprecipitation results showed overexpression of USP35 dramatically decreased the ABIN-2 ubiquitination.6. Detection of cycloheximide (CHX) chase assay showed that overexpression of USP35 significantly extended the half-life of ABIN-2, while silenced expression of USP35 shortened the half-life of ABIN-2.7. Proteasome inhibition experiment results showed proteasome inhibitor MG132 could block ABIN-2 expression level that USP35 overexpression increased ABIN-2 expression while USP35 knockdown decreased its expression, USP35 could protect ABIN-2 from proteasome-dependent degradation.8. Western blot showed protein level of ABIN-2 was also significantly decreased in the cancer tissues compared with the adjacent normal tissues. Statistical analysis showed that USP35 protein expression was positively correlated with ABIN-2 protein expression in lung and breast cancer tissues.9. Detection of NF-κB-dependent luciferase reporter gene assay proved that over-expressing plasmid of USP35 in LNCaP and H1299 cells could inhibit TNFa-induced NF-κB activation.10. Detection of NF-κB-dependent luciferase reporter gene assay proved that silenced USP35 expression in LNCaP and HeLa cells could extremely enhance TNFa-induced NF-κB activation. The enhancing NF-κB activation was alleviated after cotransfection with ABIN-2.[Conclusion]1. USP35 could up-regulate the expression of ABIN-2 by promoting ABIN-2 deubiquitination and decreasing its proteasomal degradation.2. USP35 could inhibit TNFa-induced NF-κB activation by stabilizing ABIN-2 protein.