Intestinal mRNA expression profiles associated with mucosal healing in ustekinumab-treated Crohn's disease patients: bioinformatics analysis and prospective cohort validation.

BACKGROUND: Variations exist in the response of patients with Crohn's disease (CD) to ustekinumab (UST) treatment, but the underlying cause remains unknown. Our objective was to investigate the involvement of immune cells and identify potential biomarkers that could predict the response to interleukin (IL) 12/23 inhibitors in patients with CD. METHODS: The GSE207022 dataset, which consisted of 54 non-responders and 9 responders to UST in a CD cohort, was analyzed. Differentially expressed genes (DEGs) were identified and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Least absolute shrinkage and selection operator (LASSO) regression was used to screen the most powerful hub genes. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the predictive performances of these genes. Single-sample Gene Set Enrichment Analysis (ssGSEA) was used to estimate the proportions of immune cell types. These significantly altered genes were subjected to cluster analysis into immune cell-related infiltration. To validate the reliability of the candidates, patients prescribed UST as a first-line biologic in a prospective cohort were included as an independent validation dataset. RESULTS: A total of 99 DEGs were identified in the integrated dataset. GO and KEGG analyses revealed significant enrichment of immune response pathways in patients with CD. Thirteen genes (SOCS3, CD55, KDM5D, IGFBP5, LCN2, SLC15A1, XPNPEP2, HLA-DQA2, HMGCS2, DDX3Y, ITGB2, CDKN2B and HLA-DQA1), which were primarily associated with the response versus nonresponse patients, were identified and included in the LASSO analysis. These genes accurately predicted treatment response, with an area under the curve (AUC) of 0.938. T helper cell type 1 (Th1) cell polarization was comparatively strong in nonresponse individuals. Positive connections were observed between Th1 cells and the LCN2 and KDM5D genes. Furthermore, we employed an independent validation dataset and early experimental verification to validate the LCN2 and KDM5D genes as effective predictive markers. CONCLUSIONS: Th1 cell polarization is an important cause of nonresponse to UST therapy in patients with CD. LCN2 and KDM5D can be used as predictive markers to effectively identify nonresponse patients. TRIAL REGISTRATION: Trial registration number: NCT05542459; Date of registration: 2022-09-14; URL: https://www. CLINICALTRIALS: gov .

KDM5D Histone Demethylase Identifies Platinum-Tolerant Head and Neck Cancer Cells Vulnerable to Mitotic Catastrophe.

Head and neck squamous cell carcinoma (HNSCC) is a major contributor to cancer incidence globally and is currently managed by surgical resection followed by adjuvant chemoradiotherapy. However, local recurrence is the major cause of mortality, indicating the emergence of drug-tolerant persister cells. A specific histone demethylase, namely lysine-specific demethylase 5D (KDM5D), is overexpressed in diverse types of cancers and involved in cancer cell cycle regulation. However, the role of KDM5D in the development of cisplatin-tolerant persister cells remains unexplored. Here, we demonstrated that KDM5D contributes to the development of persister cells. Aurora Kinase B (AURKB) disruption affected the vulnerability of persister cells in a mitotic catastrophe-dependent manner. Comprehensive in silico, in vitro, and in vivo experiments were performed. KDM5D expression was upregulated in HNSCC tumor cells, cancer stem cells, and cisplatin-resistant cells with biologically distinct signaling alterations. In an HNSCC cohort, high KDM5D expression was associated with a poor response to platinum treatment and early disease recurrence. KDM5D knockdown reduced the tolerance of persister cells to platinum agents and caused marked cell cycle deregulation, including the loss of DNA damage prevention, and abnormal mitosis-enhanced cell cycle arrest. By modulating mRNA levels of AURKB, KDM5D promoted the generation of platinum-tolerant persister cells in vitro, leading to the identification of the KDM5D/AURKB axis, which regulates cancer stemness and drug tolerance of HNSCC. Treatment with an AURKB inhibitor, namely barasertib, resulted in a lethal consequence of mitotic catastrophe in HNSCC persister cells. The cotreatment of cisplatin and barasertib suppressed tumor growth in the tumor mouse model. Thus, KDM5D might be involved in the development of persister cells, and AURKB disruption can overcome tolerance to platinum treatment in HNSCC.

Increased Mars2 expression upon microRNA-4661-5p-mediated KDM5D downregulation is correlated with malignant degree of gastric cancer cells.

Gastric cancer (GC) is the fifth most common malignancy and the third leading cause of cancer-related mortality worldwide. Methionyl-tRNA synthetase 2 (Mars2) has been suggested as a biomarker indicating poor prognosis of cancers. This study focuses on the function of Mars2 in GC and the responsible molecules. Mars2 was highly expressed in GC patients according to a transcriptome analysis and the data from the public database, and its high expression was confirmed in the acquired GC cell lines. Downregulation of Mars2 significantly weakened the proliferation, resistance to death, migration and invasion of GC cells. The H3K4me3 modification level was increased in the promoter region of Mars2, which was attributed to reduced abundance of lysine demethylase 5D (KDM5D) in the Mars2 promoter. MicroRNA (miR)-4661-5p was identified as an upstream regulator of KDM5D. Downregulation of miR-4661-5p led to an increase in the expression of KDM5D while a decline in the expression of Mars2, which reduced the malignant behaviors of GC cells; however, the malignant behaviors of GC cells was restored after further inhibition of KDM5D. To conclude, this study suggested that increased Mars2 expression upon miR-4661-5p-mediated KDM5D downregulation is correlated with malignant degree of GC cells.

KDM5D inhibit epithelial-mesenchymal transition of gastric cancer through demethylation in the promoter of Cul4A in male.

Gastric cancer is one of the top causes of cancer-related death around the world, and poor prognosis of gastric cancer is due to the lack of early detection and effective treatment especially in male. Here, we first revealed the role of histone lysine-specific demethylase 5D (KDM5D) in gastric cancer in male. KDM5D was associated with the metastasis of gastric cancer because of its critical role in the epithelial-mesenchymal transition of gastric cancer cells. Downregulation of KDM5D in gastric cancer cells significantly increase the number of migrated or invaded cells due to the increasing expressions of mesenchymal markers. Downregulation of KDM5D also promotes tumor formation of gastric cancer cell in vivo. For mechanism, downregulation of KDM5D could inhibit the demethylation in the promoter of CUL4A, which lead to the increasing expression of ZEB1 and decreasing expressions of p21 and p53. Collectively, KDM5D performed its role in metastasis of gastric cancer through demethylation in the promoter of CUL4A, and it suggested us a novel target in gastric cancer treatment in male.

Resistance to docetaxel in prostate cancer is associated with androgen receptor activation and loss of KDM5D expression.

The androgen receptor (AR) plays an essential role in prostate cancer, and suppression of its signaling with androgen deprivation therapy (ADT) has been the mainstay of treatment for metastatic hormone-sensitive prostate cancer for more than 70 y. Chemotherapy has been reserved for metastatic castration-resistant prostate cancer (mCRPC). The Eastern Cooperative Oncology Group-led trial E3805: ChemoHormonal Therapy Versus Androgen Ablation Randomized Trial for Extensive Disease in Prostate Cancer (CHAARTED) showed that the addition of docetaxel to ADT prolonged overall survival compared with ADT alone in patients with metastatic hormone-sensitive prostate cancer. This finding suggests that there is an interaction between AR signaling activity and docetaxel sensitivity. Here we demonstrate that the prostate cancer cell lines LNCaP and LAPC4 display markedly different sensitivity to docetaxel with AR activation, and RNA-seq analysis of these cell lines identified KDM5D (lysine-specific demethylase 5D) encoded on the Y chromosome as a potential mediator of this sensitivity. Knocking down KDM5D expression in LNCaP leads to docetaxel resistance in the presence of dihydrotestosterone. KDM5D physically interacts with AR in the nucleus, and regulates its transcriptional activity by demethylating H3K4me3 active transcriptional marks. Attenuating KDM5D expression dysregulates AR signaling, resulting in docetaxel insensitivity. KDM5D deletion was also observed in the LNCaP-derived CRPC cell line 104R2, which displayed docetaxel insensitivity with AR activation, unlike parental LNCaP. Dataset analysis from the Oncomine database revealed significantly decreased KDM5D expression in CRPC and poorer prognosis with low KDM5D expression. Taking these data together, this work indicates that KDM5D modulates the AR axis and that this is associated with altered docetaxel sensitivity.

Two Splice Variants of Y Chromosome-Located Lysine-Specific Demethylase 5D Have Distinct Function in Prostate Cancer Cell Line (DU-145).

One of the major objectives of the Human Y Chromosome Proteome Project is to characterize sets of proteins encoded from the human Y chromosome. Lysine (K)-specific demethylase 5D (KDM5D) is located on the AZFb region of the Y chromosome and encodes a JmjC-domain-containing protein. KDM5D, the least well-documented member of the KDM5 family, is capable of demethylating di- and trimethyl H3K4. In this study, we detected two novel splice variants of KDM5D with lengths of 2650bp and 2400bp that correspond to the 100 and 80 kDa proteins in the human prostate cancer cell line, DU-145. The knockdown of two variants using the short interfering RNA (siRNA) approach increased the growth rate of prostate cancer cells and reduced cell apoptosis. To explore the proteome pattern of the cells after KDM5D downregulation, we applied a shotgun label-free quantitative proteomics approach. Of 820 proteins present in all four replicates of two treatments, the abundance of 209 proteins changed significantly in response to KDM5D suppression. Of these, there were 102 proteins observed to be less abundant and 107 more abundant in KDM5D knockdown cells compared with control cells. The results revealed that KDM5D knockdown altered the abundance of proteins involved in RNA processing, protein synthesis, apoptosis, the cell cycle, and growth and proliferation. In conjunction, these results provided new insights into the function of KDM5D and its splice variants. The proteomics data are available at PRIDE with ProteomeXchange identifier PXD000416.

The epigenetic downregulation of LncGHRLOS mediated by RNA m6A methylase ZCCHC4 promotes colorectal cancer tumorigenesis.

BACKGROUND: m6A modification is currently recognized as a major driver of RNA function that maintains cancer cell homeostasis. Long non-coding (Lnc) RNAs control cell proliferation and play an important role in the occurrence and progression of colorectal cancer (CRC). ZCCHC4 is a newly discovered m6A methyltransferase whose role and mechanism in tumors have not yet been elucidated. METHODS: The EpiQuik m6A RNA methylation kit was used to detect the level of total RNA m6A in six types of digestive tract tumors. The Kaplan-Meier method and receiver operating characteristic curve were used to evaluate the prognostic and diagnostic value of the newly discovered m6A methyltransferase, ZCCHC4, in CRC. The effects on CRC growth in vitro and in vivo were studied using gain- and loss-of-function experiments. The epigenetic mechanisms underlying ZCCHC4 upregulation in CRC were studied using RIP, MeRIP-seq, RNA pull-down, and animal experiments. RESULTS: We reported that the ZCCHC4-LncRNAGHRLOS-KDM5D axis regulates the growth of CRC in vitro and in vivo. We found that ZCCHC4 was upregulated in primary CRC samples and could predict adverse clinical outcomes in patients with CRC. Mechanistically, ZCCHC4 downregulated LncRNAGHRLOS to promote CRC tumorigenesis. As a downstream molecule of LncRNAGHRLOS, KDM5D directly controls CRC cell proliferation, migration, and invasion. CONCLUSION: This study suggests that the ZCCHC4 axis contributes to the tumorigenesis and progression of CRC and that ZCCHC4 may be a potential biomarker for this malignancy.

Copy number loss of KDM5D may be a predictive biomarker for ATR inhibitor treatment in male patients with pulmonary squamous cell carcinoma.

A limited number of patients with lung squamous cell carcinoma (SCC) benefit clinically from molecular targeted drugs because of a lack of targetable driver alterations. We aimed to understand the prevalence and clinical significance of lysine-specific demethylase 5D (KDM5D) copy number loss in SCC and explore its potential as a predictive biomarker for ataxia-telangiectasia and Rad3-related (ATR) inhibitor treatment. We evaluated KDM5D copy number loss in 173 surgically resected SCCs from male patients using fluorescence in situ hybridization. KDM5D copy number loss was detected in 75 of the 173 patients (43%). Genome-wide expression profiles of the transcription start sites (TSSs) were obtained from 17 SCCs, for which the cap analysis of gene expression assay was performed, revealing that upregulated genes in tumors with the KDM5D copy number loss are associated with 'cell cycle', whereas downregulated genes in tumors with KDM5D copy number loss were associated with 'immune response'. Clinicopathologically, SCCs with KDM5D copy number loss were associated with late pathological stage (p = 0.0085) and high stromal content (p = 0.0254). Multiplexed fluorescent immunohistochemistry showed that the number of tumor-infiltrating CD8+ /T-bet+ T cells was lower in SCCs with KDM5D copy number loss than in wild-type tumors. In conclusion, approximately 40% of the male patients with SCC exhibited KDM5D copy number loss. Tumors in patients who show this distinct phenotype can be 'cold tumors', which are characterized by the paucity of tumor T-cell infiltration and usually do not respond to immunotherapy. Thus, they may be candidates for trials with ATR inhibitors.

X and Y Differences in Melanoma Survival Between the Sexes.

Marked differences in survival from melanoma are noted between men and women that cannot be accounted for by behavioral differences. We and others have provided evidence that this difference may be due to increased expression of immune-related genes from the second X chromosome because of failure of X inactivation. In the present review, we have examined evidence for the contrary view that survival differences are due to weaker immune responses in males. One reason for this may be the loss of Y chromosomes (LOY), particularly in older males. The genes involved may have direct roles in immune responses or be noncoding RNAs that regulate both sex and autosomal genes involved in immune responses or tumor growth. Loss of the KDM6C and KDM5D demethylases appeared to common genes involved. The second factor appears to be the activation of androgen receptors (AR) on melanoma cells that increase their invasiveness and growth. Induction of T-cell exhaustion by AR that limits immune responses against melanoma appeared a common finding. The development of treatments to overcome effects related to gene loss on Y poses challenges, but several avenues related to AR signaling appear worthy of further study in the treatment of metastatic disease.

Sex-biased gene expression during neural differentiation of human embryonic stem cells.

Sex differences in the developing human brain are primarily attributed to hormonal influence. Recently however, genetic differences and their impact on the developing nervous system have attracted increased attention. To understand genetically driven sexual dimorphisms in neurodevelopment, we investigated genome-wide gene expression in an in vitro differentiation model of male and female human embryonic stem cell lines (hESC), independent of the effects of human sex hormones. Four male and four female-derived hESC lines were differentiated into a population of mixed neurons over 37 days. Differential gene expression and gene set enrichment analyses were conducted on bulk RNA sequencing data. While similar differentiation tendencies in all cell lines demonstrated the robustness and reproducibility of our differentiation protocol, we found sex-biased gene expression already in undifferentiated ESCs at day 0, but most profoundly after 37 days of differentiation. Male and female cell lines exhibited sex-biased expression of genes involved in neurodevelopment, suggesting that sex influences the differentiation trajectory. Interestingly, the highest contribution to sex differences was found to arise from the male transcriptome, involving both Y chromosome and autosomal genes. We propose 13 sex-biased candidate genes (10 upregulated in male cell lines and 3 in female lines) that are likely to affect neuronal development. Additionally, we confirmed gene dosage compensation of X/Y homologs escaping X chromosome inactivation through their Y homologs and identified a significant overexpression of the Y-linked demethylase UTY and KDM5D in male hESC during neuron development, confirming previous results in neural stem cells. Our results suggest that genetic sex differences affect neuronal differentiation trajectories, which could ultimately contribute to sex biases during human brain development.

Determination of sex in cDNA samples from mice.

The need to take sex into account in biomedical research is now recognized and mandated by funding institutions. In laboratory rodents, such as mice, sexing is usually performed anatomically or by genotyping using multiplex or simplex PCR techniques on genomic DNA. Here we present a simple RT-PCR-based method targeting Kdm5c and Kdm5d to determine genetic sex in mouse cDNA samples, allowing for retrospective sex determination.

Human Sex Matters: Y-Linked Lysine Demethylase 5D Drives Accelerated Male Craniofacial Osteogenic Differentiation.

Female sex is increasingly associated with a loss of bone mass during aging and an increased risk of developing nonunion fractures. Hormonal factors and cell-intrinsic mechanisms are suggested to drive these sexual dimorphisms, although underlying molecular mechanisms are still a matter of debate. Here, we observed a decreased capacity of calvarial bone recovery in female rats and a profound sexually dimorphic osteogenic differentiation in human adult neural crest-derived stem cells (NCSCs). Next to an elevated expression of pro-osteogenic regulators, global transcriptomics revealed Lysine Demethylase 5D (KDM5D) to be highly upregulated in differentiating male NCSCs. Loss of function by siRNA or pharmacological inhibition of KDM5D significantly reduced the osteogenic differentiation capacity of male NCSCs. In summary, we demonstrated craniofacial osteogenic differentiation to be sexually dimorphic with the expression of KDM5D as a prerequisite for accelerated male osteogenic differentiation, emphasizing the analysis of sex-specific differences as a crucial parameter for treating bone defects.

KDM5D inhibits the transcriptional activation of FKBP4 by suppressing the expression of E2F1 in colorectal cancer in males.

Colorectal cancer (CRC) remains the most frequently diagnosed malignancy and also a major contributor to cancer-related death throughout the world. Here, we first revealed the role of histone lysine-specific demethylase 5D (KDM5D) in CRC in males. KDM5D expression in tumor and adjacent tissues of male CRC patients was investigated using immunohistochemistry and RT-qPCR, and the correlation between its expression and patients' prognosis was analyzed. Downregulation of KDM5D in CRC patients was associated with poor prognoses. Overexpression of KDM5D significantly inhibited the growth and metastasis of CRC in vitro and in vivo. The downstream mechanism of KDM5D in CRC was investigated using bioinformatics analysis, and the regulatory relationship was confirmed by ChIP-qPCR and luciferase reporter assays. KDM5D suppressed E2F1 expression by mediating H3K4me3 demethylation. E2F1, highly expressed in CRC, promoted the expression of FKBP4 at the transcriptional level by binding to the FKBP4 promoter. Finally, rescue experiments revealed that overexpression of FKBP4 significantly reversed the inhibitory effect of KDM5D on CRC growth and metastasis. Collectively, KDM5D exerted an anti-tumor and anti-metastatic in CRC through demethylation in E2F1 and suppression of FKBP4 transcription, which might represent a novel target in CRC treatment in male.

KDM5D predicts response to docetaxel chemotherapy in metastatic castration resistant prostate cancer patients.

BACKGROUND: The administration of docetaxel chemotherapy is one therapeutic option to delay disease progression and increase overall survival in metastatic castration resistant prostate cancer (mCRPC). However, about 15% of patients are primary resistant to chemotherapy and hence would benefit from an alternative mCRPC treatment. Despite intensive research, there are no robust clinical validated biomarkers to predict mCRPC therapy response. Thus, the aim of the study was to determine KDM5D expression in archival radical prostatectomy specimens of patients medicated with docetaxel at time of mCRPC development in order to correlate KMD5D expression with treatment response. METHODS: We used in situ hybridization (ISH) (RNA scope 2.5 HD) to determine KDM5D expression in tissue samples of 28 prostate cancer patients. KDM5D status was correlated to chemotherapy response (PSA and radiographic response). RESULTS: Data revealed that KDM5D is significantly overexpressed in tumor cells (P<0.0001) but also in benign cells (P<0.02) of those patients who responded to chemotherapy compared to non-responders. CONCLUSIONS: To summarize, KDM5D is a promising novel biomarker predicting response to docetaxel chemotherapy already at the time of localized disease and thus potentially avoiding metastatic biopsies in the mCRPC stage of disease.

Increased Expression of Y-Encoded Demethylases During Differentiation of Human Male Neural Stem Cells.

Human neural stem cells (hNSCs) have long been used as an in vitro model to study neurogenesis and as candidates for nervous system therapy. Many parameters have been considered when evaluating the success of transplantation, but sex of donor and recipients is often not discussed. We investigated two commercial NSC lines, the female hNSC-H9 and male hNSC-H14, and we observed faster growth rates in the male cells. At 4 days of differentiation, male cells presented a significant increase in expression of DCX, an immature neuronal marker, while female cells showed a significant increase in RMST, a long noncoding RNA, which is indispensable during neurogenesis. In addition, expression of neural markers MAP2, PSD95, SYP, DCX, and TUJ1 at day 14 of differentiation suggested a similar differentiation potential in both lines. The most significant differences at day 14 of differentiation were the expression levels of RELN, with almost 100-fold difference between the sexes, and MASH1, with more than 1,000-fold increase in male cells. To evaluate whether some of the observed differences may be sex related, we measured the expression of gametologous genes located on the X- and Y-chromosome. Most noticeable was the increase of Y-encoded demethylases KDM6C (UTY) and KDM5D during differentiation of male cells. Our results indicate that attention should be paid to sex when planning neurogenesis and transplantation experiments.

SeXY chromosomes and the immune system: reflections after a comparative study.

BACKGROUND: Sex bias in immune function has been contributed in part to a preponderance of immune system-related genes (ISRG) on the X-chromosome. We verified whether ISRG are more abundant on the X chromosome as compared to autosomal chromosomes and reflected on the impact of our findings. METHODS: Consulting freely accessible databases, we performed a comparative study consisting of three complementary strategies. First, among coding X/Y-linked genes, the abundance of ISRG was compared to the abundance of genes dedicated to other systems. Genes were assigned considering three criteria: disease, tissue expression, and function (DEF approach). In addition, we carried out two genome-wide approaches to compare the contribution of sex and autosomal chromosomes to immune genes defined by an elevated expression in lymphatic tissues (LTEEG approach) or annotation to an immune system process, GO:0002376 (GO approach). RESULTS: The X chromosome had less immune genes than the median of the autosomal chromosomes. Among X-linked genes, ISRG ranked fourth after the reproductive and nervous systems and genes dedicated to development, proliferation and apoptosis. On the Y chromosome, ISRG ranked second, and at the pseudoautosomal region (PAR) first. According to studies on the expression of X-linked genes in a variety of (mostly non-lymphatic) tissues, almost two-thirds of ISRG are expressed without sex bias, and the remaining ISRG presented female and male bias with similar frequency. Various epigenetic controllers, X-linked MSL3 and Y-linked KDM5D and UTY, were preferentially expressed in leukocytes and deserve further attention for a possible role in sex biased expression or its neutralisation. CONCLUSIONS: The X chromosome is not enriched for ISRG, though particular X-linked genes may be responsible for sex differences in certain immune responses. So far, there is insufficient information on sex-biased expression of X/Y-linked ISRG in leukocytes to draw general conclusions on the impact of X/Y-linked ISRG in immune function. More research on the regulation of the expression X-linked genes is required with attention to 1) female and male mechanisms that may either augment or diminish sex biased expression and 2) tissue-specific expression studies.

KDM5D-mediated H3K4 demethylation is required for sexually dimorphic gene expression in mouse embryonic fibroblasts.

Males and females share the same genetic code, but gene expression profile often displays differences between two sexes. Mouse embryonic fibroblasts (MEFs) have been used to experiment as a useful tool to test gene function. They have also been characterized by gender-based differences in expressed genes such as Y-linked Sry or X-linked Hprt. However, there is no report on sex differences in global gene expression. Here, using the next-generation RNA sequencing, we compared the comprehensive transcriptome of MEFs derived from two sexes. In comparison with the female group, the male group up-regulated 27 differentially expressed genes (DEGs), in which a male-specific histone demethylase KDM5D gene is included, and 7 DEGs were down-regulated. Based on the results by searching the ENCODE analysis, it was shown that the expression of 15 genes identified is potentially regulated by the methylation of H3K4me1 or H3K4me3. Interestingly, we demonstrated that both of H3K4 methylation are induced by knocking down KDM5D, which causes changes in patterns of eight DEGs found in male MEFs. Collectively, these data not only suggest an importance of KDM5D-mediated demethylation of H3K4 involved in the sexually dimorphic gene expression in male MEFs, but also may provide information regarding sex-dependent changes in gene expression when MEFs are used for experiments.

Investigation of Histone Lysine-Specific Demethylase 5D KDM5D) Isoform Expression in Prostate Cancer Cell Lines: a System Approach.

BACKGROUND: It is now well-demonstrated that histone demethylases play an important role in developmental controls, cell-fate decisions, and a variety of diseases such as cancer. Lysine-specific demethylase 5D (KDM5D) is a male-specific histone demethylase that specifically demethylates di- and tri-methyl H3K4 at the start site of active gene. In this light, the aim of this study was to investigate isoform/transcript-specific expression profiles of KDM5D in three prostate cancer cell lines, Du-145, LNCaP, and PC3. METHODS: Real-time PCR analysis was performed to determine the expression levels of different KDM5D transcripts in the prostate cell lines. A gene regulatory network was established to analyze the gene expression profile. RESULTS: Significantly different expression levels of both isoforms were found among the three cell lines. Interestingly, isoform I was expressed in three cell lines while isoform III did only in DU-145. The expression levels of both isoforms were higher in DU-145 when compared to other cell lines (P<0.0001). The observed expression profile was determined by using regulatory network analyses. CONCLUSION: The present study, for the first time, not only showed the expression profiles of KDM5D isoforms in prostate cancer cell lines but also evaluated the effects of the gene regulatory network on the expression profile of this gene.