Downregulation of CAMK2N1 due to DNA Hypermethylation Mediated by DNMT1 Promotes the Progression of Prostate Cancer

Background Calcium/calmodulin-dependent protein kinase II inhibitor I (CAMK2N1) is one of the tumor 22 suppressor genes in prostate cancer (PCa) and is significantly downregulated in PCa tissues compared 23 to benign and normal prostate tissues. Reduced expression of CAMK2N1 is positively correlated with 24 PCa progression. However, the mechanism of CAMK2N1 silencing in PCa is still unclear. The 25 promoter region of CAMK2N1 contained abundant CG loci, providing a great possibility for DNA 26 methylation. Consequently, we postulated that epigenetic modification resulted in the abnormal 27 expression of CAMK2N1 in PCa. Firstly, as the related signaling pathways in CAMK2N1 upregulated or downregulated PCa cells. Finally, 35 functional assays including wound healing, invasion and migration assay, and xenograft model in nude 36 mice were used to investigate the effect of DNMT1/CAMK2N1 interaction on the progression of PCa. CAMK2N1 was highly methylated in PCa cells and tissues compared to normal prostate epithelial 39 cells, normal prostate tissues and benign prostatic hyperplasia BPH tissues. The hypermethylation of 40 CAMK2N1 was associated with the clinicopathological characteristics in PCa patients. The reduced 41 expression of CAMK2N1 can be restored by 5-Aza-CdR treatment via demethylation. Moreover, we 42 confirmed that DNMT1 formed a positive feedback loop with CAMK2N1 in PCa cells. CAMK2N1 43 expression was downregulated by DNMT1-mediated DNA methylation, which reversely induced 44 DNMT1 expression through the AKT or ERK signaling pathway. The results of in vitro and in vivo 45 experiments demonstrated that CAMK2N1 inhibited PCa cell invasion, migration and proliferation and 46 these effects were reversed by DNMT1. 48 DNMT1-mediated hypermethylation of CAMK2N1 not only downregulates gene expression but also 49 promotes the progression of PCa, which could be served as a potential predictive biomarker. Science and Technology; MSP: methylation-specific PCR; PCa: prostate cancer; PSA: prostate- specific antigen; qRT-PCR: quantitative reverse transcription polymerase chain reaction; SD: standard 413 deviation; si-CA: si-CAMK2N1; TCGA: The Cancer Genome Atlas; WB: western blot.


Background 21
Calcium/calmodulin-dependent protein kinase II inhibitor I (CAMK2N1) is one of the tumor 22 suppressor genes in prostate cancer (PCa) and is significantly downregulated in PCa tissues compared 23 to benign and normal prostate tissues. Reduced expression of CAMK2N1 is positively correlated with 24 PCa progression. However, the mechanism of CAMK2N1 silencing in PCa is still unclear. The 25 promoter region of CAMK2N1 contained abundant CG loci, providing a great possibility for DNA 26 methylation. Consequently, we postulated that epigenetic modification resulted in the abnormal 27 expression of CAMK2N1 in PCa. 28

Methods 29
Firstly, we determined the DNA methylation level of CAMK2N1 in prostate cell lines and clinical 30 specimens by bisulfite sequencing (BS), pyrosequencing and The Cancer Genome Atlas in silico 31 analysis. Subsequently, we explored the expression of CAMK2N1 and its DNA methylation level by 32 qRT-PCR, western blot, immunofluorescence, BS and methylation-specific PCR in PCa cells after 5-33 Aza-CdR treatment or DNMT1 gene modification. Moreover, we analyzed DNMT1 expression as well 34 as the related signaling pathways in CAMK2N1 upregulated or downregulated PCa cells. Finally, 35 functional assays including wound healing, invasion and migration assay, and xenograft model in nude 36 mice were used to investigate the effect of DNMT1/CAMK2N1 interaction on the progression of PCa. 37 Results 38 CAMK2N1 was highly methylated in PCa cells and tissues compared to normal prostate epithelial 39 cells, normal prostate tissues and benign prostatic hyperplasia BPH tissues. The hypermethylation of 40 CAMK2N1 was associated with the clinicopathological characteristics in PCa patients. The reduced 41 expression of CAMK2N1 can be restored by 5-Aza-CdR treatment via demethylation. Moreover, we 42 confirmed that DNMT1 formed a positive feedback loop with CAMK2N1 in PCa cells. CAMK2N1 43 expression was downregulated by DNMT1-mediated DNA methylation, which reversely induced 44 DNMT1 expression through the AKT or ERK signaling pathway. The results of in vitro and in vivo 45 experiments demonstrated that CAMK2N1 inhibited PCa cell invasion, migration and proliferation and 46 these effects were reversed by DNMT1. 47 Conclusions 48 DNMT1-mediated hypermethylation of CAMK2N1 not only downregulates gene expression but also 49 promotes the progression of PCa, which could be served as a potential predictive biomarker. 50 Keywords: prostate cancer, CAMK2N1, DNA methylation, DNMT1, 5-Aza-2'-deoxycytidine 51 Background 52 Prostate cancer (PCa) is the most common malignancy among older males in Western countries, has 53 the second highest mortality rate, and has become a serious global public health problem [1]. The 54 tumorigenesis of PCa is a complex multifactorial and multistep process involving the changes of 55 signaling pathways, oncogenes and tumor suppressor genes [2,3]. It is currently believed that the 56 abnormal expression of genes caused by epigenetic modifications, such as DNA methylation, histone 57 acetylation and noncoding RNAs, plays an important role in the occurrence and development of PCa 58 [4,5]. 59 Calcium/calmodulin-dependent protein kinase II inhibitor I (CAMK2N1) is an endogenous CaMKII 60 suppressor gene. Recent studies have shown that Ca 2+ /CaMKII signaling pathway plays an important 61 role in tumorigenesis and that inhibition of CaMKII blocks tumor cell growth [6,7]. CAMK2N1 62 induces apoptotic cell death and inhibits cell proliferation, cell cycle progression and androgen receptor 63 (AR) expression in PCa [8,9]. The expression of CAMK2N1 is increased by the activation of 64 PI3K/AKT signaling and decreased in response to androgen signaling [9-11]. Our previous research 65 confirms that CAMK2N1 is significantly downregulated in PCa and negatively correlates with the 66 degree of malignancy [8,9]. However, the molecular mechanism of CAMK2N1 downregulation in PCa 67 is still unclear. 68 DNA methylation abnormalities, including hypomethylation or hypermethylation of specific gene, 69 are significant epigenetic changes in PCa [12]. Hypermethylation of CpG islands in promoter region 70 results in downregulated or even inactivated expression of genes such as AR, GSTP1 Epigentek, USA). The genomic DNA was isolated and bound to strip wells in this assay. The 142 methylated fraction of DNA was detected using antibodies and then quantified colorimetrically by 143 reading the absorbance in a microplate spectrophotometer. The sequences of all primers are shown in 144 Additional file 3: Table S3. 145

RNA isolation and quantitative reverse transcription PCR (qRT-PCR) 146
Total RNA was isolated from cultured cells using TRIzol reagent (Invitrogen) according to the 147 established protocol [20]. Total RNA (1 μg) was reversely transcribed to cDNA using PrimeScript RT 148 Master Mix (Takara, China). Then, qRT-PCR was carried out in a Roche Light Cycler 480 system with 149 SYBR Premix Ex TaqTM (Takara). The primer sequences are shown in Additional file 3: Table S3. 150 Relative gene expression levels were calculated by normalization to GAPDH and quantification via 151 the 2 -△△ Ct method. 152

Western blot 153
Total protein was isolated from cultured cells with RIPA lysis buffer (Beyotime, China) and 1 mM 154 PMSF buffer (Beyotime). Then, the protein concentration was determined using a BCA protein assay 155 kit (Beyotime). Samples containing equal amounts of protein were loaded into SDS-PAGE gels. After 156 electrophoresis, proteins in the gels were transferred onto PVDF membranes (Millipore, USA). After 157 blocking with 5% bovine serum albumin at room temperature for 2 h, membranes were incubated with 158 the primary antibodies listed above overnight at 4°C. After several TBST washes and incubation with 159 HRP-conjugated secondary antibodies, bound proteins were detected with ECL regents (Bio-Rad, 160 USA) in a chemiluminescence detection system (Syngene, USA). The bands intensity was quantified 161 with ImageJ software and normalized to that of GAPDH. The relative band intensity in the control 162 group was set to 1. 163

Immunofluorescence staining 164
After washing with phosphate-buffered saline (PBS), cells were fixed with 4% paraformaldehyde for 165 20 min and permeabilized with 0.1% Triton X-100 for 20 min at room temperature. of the IgG control sample to 1. The sequences of CAMK2N1 are shown in Additional file 3: Table S3. 178

Cell invasion and migration assays 179
For the invasion assay, Matrigel-coated Transwell inserts (Corning, USA) were pre-treated with serum-180 free RPMI-1640 medium at 37°C for 2 h. After removing the medium, we added 750 μl of RPMI-1640 181 medium supplemented with 10% FBS as a chemoattractant to each lower chamber, added 5 × 10 4 cells 182 to each upper chamber, and incubated the plates at 37°C for 24 h. Then, the inserts were removed, and 183 noninvaded cells on the upper surface of the membranes were removed with a cotton swab. The invaded 184 cells on the lower surface of the membranes were then fixed with 100% methanol for 15 min and 185 stained with 1% crystal violet. Cells in three microscopic fields were photographed and counted. For 186 the migration assay, a procedure similar to that used in the invasion assay was used, but the Matrigel 187 coating was omitted. Three independent experiments were performed. 188

Wound healing assay 189
Cells were seeded in a 6-well culture plate, and wounds were made with 1-ml pipette tips in the middle 190 of the six-well plates. Then, cells were cultured with serum-free RPMI-1640 medium. This experiment was conducted as described previously [8,9]. Primary antibodies were used at the 205 appropriate dilutions in the experiments. Sections (4 μm) were prepared from FFPE DU145 tumor 206 tissues harvested from nude mice. 207

Statistical analysis 208
All results are presented as the mean ± standard deviation (SD) values, and data were analyzed using 209 GraphPad Prism software. At least three repeated experiments were carried out. The statistical 210 significance of differences between two groups was assessed by using Student's t-test. Differences 211 among multiple groups were analyzed using one-way analysis of variance (ANOVA) followed by 212 Tukey's multiple comparison test. The two-tailed Pearson correlation coefficient was used for 213 correlation analysis. The log-rank test was used to compare survival distributions. Statistical 214 significance was assumed for P < 0.05. 215

Results 216
The promoter of CAMK2N1 is hypermethylated in prostate cancer cells compared to normal 217 prostate epithelial cells 218 We first analyzed the mRNA expression of CAMK2N1 in normal prostate epithelial cells and PCa 219 cells using qRT-PCR. The results showed that CAMK2N1 mRNA expression was markedly decreased 220 in LNCaP, DU145 and PC-3 cells compared to RWPE-1 cells, especially in AR-negative DU145 and 221 PC-3 cells (Fig. 1A islands likely to be hypermethylated in the promoter region and first exon of CAMK2N1 (Fig. 1B). 226 We determined the DNA methylation percentage of CAMK2N1 in RWPE-1, LNCaP, DU145 and PC-227 3 cells through BS. We identified that in the first amplicon the DNA methylation percentage at these 228 22 CG sites was significantly higher than that in other amplicons not only in PCa cells but also in 229 normal prostate epithelial cells, indicating that the CG sequences in this region are the key site of 230 methylation for regulating gene expression (Fig. 1C). The results also showed that the average DNA 231 methylation percentage of CAMK2N1 was 6.8% in RWPE-1 cells but was 56.3% in LNCaP cells, 232 22.3% in DU145 cells and 19.1% in PC-3 cells (Fig. 1C). Based on the above results, we performed 233 the BS analysis again (Additional file 4: Fig. S1) and quantified the DNA methylation percentage in 234 twenty sequencing clones. We confirmed again that the CG sequences in the CAMK2N1 gene 235 promoter was hypermethylated in prostate cancer cells compared to normal prostate epithelial cells 236 (Fig. 1D). Interestingly, AR-positive LNCaP cells had a higher DNA methylation percentage than AR-237 negative DU145 cells and PC-3 cells (Fig. 1E). 238

DNA hypermethylation of CAMK2N1 is identified in prostate cancer tissues and is associated 239
with clinicopathological characteristics 240 To investigate whether there is DNA hypermethylation of CAMK2N1 in PCa tissues, we analyzed data 241 from the TCGA database. The results revealed that CAMK2N1 expression was reduced in PCa tissues 242 compared to normal prostate tissues ( Fig. 2A). Correspondingly, the DNA methylation level of 243 CAMK2N1 in PCa samples was higher than that in normal prostate samples (Fig. 2B). The cg loci 244 from cg14477205 to cg24294857 are located upstream of TSS, while other cg loci are located 245 downstream (Fig. 2C). From the methylation levels of these arrays, compared with the downstream, 246 there was indeed hypermethylation in the upstream of CAMK2N1 TSS, which was consistent with our 247 results in prostate cells shown in Fig. 1C (Fig. 2C). Moreover, CAMK2N1 gene expression was 248 negatively correlated with the average DNA methylation level for the all cg loci (Fig. 2D). Although 249 CAMK2N1 hypermethylation did not affect the T or N stages, Gleason scores, prostate-specific antigen 250 ( To further support our hypothesis, we collected FFPE prostate tissues to measure the DNA 254 methylation level of CAMK2N1 between benign samples from 16 BPH patients and tumor samples 255 from 52 PCa patients. According to the location of CAMK2N1 DNA hypermethylation determined in 256 PCa cells, as shown in Fig. 1, we elected to use sites 4 to 8 among in the first amplicon from BS as the 257 key methylation sites and performed pyrosequencing. The results indicated that at site 4, the DNA 258 methylation percentage of CAMK2N1 in PCa tissues was 7 times higher than that in BPH tissues (Fig.  259 2H). At other sites, although there were no significant differences, the DNA methylation percentage in 260 PCa tissues was higher than that in BPH tissues ( Interestingly, we found out that site 4 is cg22942704 locus that was analyzed in TCGA dataset. 267 However, TCGA analysis demonstrated that there was no significant difference of methylation level 268 at cg22942704 locus among PCa patients with different pathological characteristics (Additional file 5: 269 Fig. S2E). Nevertheless, T4 tumor or Gleason score 10 tumor still has a slight higher methylation level 270 of CAMK2N1 (Additional file 5: Fig. S2E). increased the expression of not only CAMK2N1 but also AR, accompanied by the decreased 280 expression of DNMT1 (Fig. 3G-I). To further clarify the relationships between the expression of 281 CAMK2N1 and its DNA methylation status, the DNA methylation level in PCa cells treated with 5-282 Aza-CdR were analyzed by BS. The results indicated that the DNA methylation percentage was 283 decreased after 5-Aza-CdR treatment from 14.5% to 9.5% in DU145 cells, from 16.8% to 13.6% in 284 PC-3 cells and from 69.1% to 45% in LNCaP cells (Fig. 3J). 285

DNMT1 interacts with the promoter of CAMK2N1 and inhibits the expression of CAMK2N1 via 286
DNA methylation 287 When we inhibited DNA methyltransferase activity with 5-Aza-CdR, we observed that the expression 288 of DNMT1 was decreased, whereas CAMK2N1 expression was increased. Thus, we hypothesized that 289 DNA methyltransferases, especially DNMT1, may bind to the gene sequence of CAMK2N1 to exert 290 biological effects. To verify this hypothesis, a ChIP assay was performed in 5-Aza-CdR-treated DU145 291 cells. The amplicon of CAMK2N1 that binds to DNMT1 in DU145 cells can be detected (Fig. 4A), 292 indicating that DNMT1 was able to interact with the promoter of CAMK2N1. This conclusion was 293 also supported by the result that mRNA expression of CAMK2N1 combined with DNMT1 was reduced 294 in 5-Aza-CdR-treated DU145 cells due to the inhibition of DNMT1 (Fig. 4B). To investigate the 295 specific relationships between DNMT1 and CAMK2N1, we knocked down and overexpressed 296 DNMT1 in DU145 and LNCaP cells. qRT-PCR and western blot analyses showed that suppression of 297 DNMT1 expression increased the mRNA and protein expression levels of CAMK2N1 ( Fig. 4C-H), 298 while overexpression of DNMT1 decreased CAMK2N1 expression (Fig. 4I-N) Fig. S4A and B). To further prove that CAMK2N1 regulates the expression of 312 DNMT1 through the AKT or ERK pathway, we used the AKT inhibitor AKTi and the ERK inhibitor 313 U0126. The results indicated that in DU145 and LNCaP cells, knockdown of CAMK2N1 elevated the 314 expression of DNMT1, whereas the addition of the AKT inhibitor (Fig. 5C-E and Additional file 7: 315 Fig. S4C and D) and ERK inhibitor (Fig. 5F-H and Additional file 7: Fig. S4E and F) abrogated the 316 effects of CAMK2N1 knockdown on DNMT1 expression not only at the mRNA level but also at the 317 protein level. Although CAMK2N1 can regulate the expression of DNMT1, it cannot affect the 318 genome-wide DNA methylation level by changing DNMT1 expression (Additional file 7: Fig. S4G). 319

Silencing CAMK2N1 promotes tumor progression by inducing DNMT1 in vitro and in vivo 320
To investigate the potential biological effects of DNMT1 and CAMK2N1 on tumor progression, 321 DU145 cell lines with stable knockdown of DNMT1 and CAMK2N1 were established. The wound 322 healing assay revealed that knockdown of CAMK2N1 promoted the migration of DU145 cells, but 323 knockdown of DNMT1 not only abolished this effect but also inhibited cell migration (Fig. 6A and B). 324 Accordingly, knockdown of CAMK2N1 significantly induced PCa cell migration and invasion in 325 Transwell migration and Matrigel invasion assays, in contrast to the effects of DNMT1 knockdown 326 ( Fig. 6C and D). These cells were injected subcutaneously into BALB/c nude mice. The tumor volume 327 and weight were significantly increased in the CAMK2N1 knockdown group compared with the 328 control group, while knockdown of DNMT1 reversed these effects (Fig. 6E-H). Immunohistochemistry 329 analysis was applied to detect the protein expression of DNMT1 and CAMK2N1 in xenograft tumors 330 of each group. The expression of CAMK2N1 was obviously increased when DNMT1 expression was 331 markedly reduced in tumor tissues (Fig. 6I). We also found that knockdown of CAMK2N1 increased 332 the expression of DNMT1, consistent with the results described above. 333   clone. There is a total of twenty clones per prostate cell lines. Data are presented as mean ± SD, one-554 way ANOVA test was applied, *P < 0.05 and ***P < 0.001. 555 in BPH and PCa tissues. PCa patients were divided into TNM stage 2 and stage 3-4 groups, Gleason 565 score 6, 7 and 8-10 groups, PSA value < 10 ng/ml, 10-20 ng/ml and > 20 ng/ml groups (n=16-52). L-566

Discussion
O Pyrosequencing results at site 5 (n=16-52). Data are presented as mean ± SD, unpaired t tests, two-567 tailed Pearson correlation coefficient, one-way ANOVA and log rank test were used, *P < 0.05, **P < 568 0.01 and ***P < 0.001. 569 with or without 20 μM 5-Aza-CdR treatment for 96h. Black and white points represent methylated and 578 unmethylated CG site respectively. Data are presented as mean ± SD, one-way ANOVA test was used, 579 *P < 0.05, **P < 0.01 and ***P < 0.001, compared to the DMSO group, GAPDH was used as a control. 580 The representative agarose gel of CAMK2N1 methylated and unmethylated amplicons. After 2 days 591 treatment of transfection, MSP was used for DNA methylation analysis. M means methylated amplicon 592 and U means unmethylated amplicon. Data are presented as mean ± SD, t test and one-way ANOVA 593 test were used, *P < 0.05, **P < 0.01 and ***P < 0.001, compared to the control group. 594 as a control; n=3). C-E CAMK2N1 knockdown DU145 cells were treated with 10 μM AKT signaling 600 pathway inhibitor AKTi for 1 day. The expression of DNMT1, p-AKT and t-AKT was analyzed by 601 qRT-PCR and western blot (GAPDH was used as a control; n=3). F-H CAMK2N1 knockdown DU145 602 cells were treated with 10 μM ERK signaling pathway inhibitor U0126 for 1 day. The expression of 603 DNMT1, p-ERK1/2 and t-ERK1/2 was analyzed by qRT-PCR and western blot (GAPDH was used as 604 a control; n=3). Data are presented as mean ± SD, t test and one-way ANOVA test were used, *P < 605 0.05, **P < 0.01 and ***P < 0.001. 606 The DNA methylation level of CAMK2N1 in PCa cells and normal prostate epithelial cells. A The mRNA expression of CAMK2N1 was determined in RWPE-1, LNCaP, DU145 and PC-3 cells by using qRT-PCR (n=3). B The CpG islands distribution of CAMK2N1 in the promoter region and rst 546 exon by searching the online tool (https://www.ebi.ac.uk/Tools/seqstats/emboss_newcpgreport/). Four pairs of BS primers were designed to cover these CpG islands of CAMK2N1 in the promoter region and rst exon. C BS analysis of RWPE-1, LNCaP, DU145 and PC-3 cells. One point represents one CG site, in which black point represents methylated CG site and white point represents unmethylated CG site. One horizontal row represents one clone and one vertical row represents one CG site. Finally, ten clones were selected randomly. D, E The quanti cation of BS analysis at the rst amplicon region in normal prostate cells and PCa cells. One point represents the DNA methylation percentage of one clone. There is a total of twenty clones per prostate cell lines. Data are presented as mean ± SD, one-way ANOVA test was applied, *P < 0.05 and ***P < 0.001.

Figure 2
Correlations between DNA methylation, CAMK2N1 expression and clinical outcomes in PCa tissues. A The paired study of CAMK2N1 expression in normal prostate tissues and PCa tissues from TCGA data. B The DNA methylation level of CAMK2N1 in normal prostate tissues and PCa tissues from TCGA paired data. C The methylation level of CAMK2N1 at all cg loci that were analyzed in TCGA data (plot represents 5-95 percentile). D The correlation between the mean value of methylation level of all cg loci in CAMK2N1 gene and gene expression from TCGA data. E The overall survival of patients with CAMK2N1 hypermethylation and patients with CAMK2N1 hypomethylation. F, G The progression-free survival of PCa patients with different CAMK2N1 methylation levels in cg00980178 locus and cg08898653 locus. H-K The quanti cation of pyrosequencing results at site 4 in BPH and PCa tissues. PCa patients were divided into TNM stage 2 and stage 3-4 groups, Gleason score 6, 7 and 8-10 groups, PSA value < 10 ng/ml, 10-20 ng/ml and > 20 ng/ml groups (n=16-52). L-O Pyrosequencing results at site 5 (n=16-52). Data are presented as mean ± SD, unpaired t tests, two-tailed Pearson correlation coe cient, one-way ANOVA and log rank test were used, *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 3
The expression and DNA methylation percentage of CAMK2N1 after 5-Aza-CdR treatment in PCa cells.
qRT-PCR analysis was used to determine the effects of 5 μM, 10 μM and 20 μM 5-Aza-CdR on the mRNA expression of DNMT1 and CAMK2N1 in A DU145 and D PC-3 cells (n=3).
Western blot analysis of CAMK2N1 and DNMT1 expression in B, C DU145 and E, F PC-3 PCa cells after continuously treating with 10 μM and 20 μM 5-Aza-CdR for 96 h (n=3). G The mRNA expression of DNMT1, CAMK2N1 and AR was determined by qRT-PCR in LNCaP cells after 5-Aza-CdR treatment (n=3). H, I The protein expression of CAMK2N1, AR and DNMT1 was analyzed by western blot in LNCaP cells after 5-Aza-CdR treatment (n=3). J BS analysis in DU145, PC-3 and LNCaP cells with or without 20 μM 5-Aza-CdR treatment for 96h. Black and white points represent methylated and unmethylated CG site respectively. Data are presented as mean ± SD, one-way ANOVA test was used, *P < 0.05, **P < 0.01 and ***P < 0.001, compared to the DMSO group, GAPDH was used as a control. DNMT1 suppresses the expression of CAMK2N1 through DNA hypermethylation in PCa cells. A The representative agarose gel and B mRNA expression of CAMK2N1. ChIP analysis was used to con rm the binding of DNMT1 to the promoter of CAMK2N1 in DMSO-treated and 5-Aza-CdR-treated DU145 cells. Data were normalized to input DNA and displayed as fold enrichment relative to IgG group. IgG antibody, input DNA and blank control were used as controls (The amplicon size of CAMK2N1 is 131 bp; n=3). C-E DU145 and F-H LNCaP cells were transfected with DNMT1 siRNA. After 2 or 3 days, cells were harvested for CAMK2N1 and DNMT1 expression analysis by qRT-PCR and western blot (GAPDH was used as a control; n=3). I-K DU145 and L-N LNCaP cells were transfected with DNMT1 cDNA clones. After 2 or 3 days, cells were harvested for CAMK2N1 and DNMT1 expression analysis by qRT-PCR and western blot (GAPDH was used as a control; n=3). O The representative agarose gel of CAMK2N1 methylated and unmethylated amplicons. After 2 days treatment of transfection, MSP was used for DNA methylation analysis. M means methylated amplicon and U means unmethylated amplicon. Data are presented as mean ± SD, t test and one-way ANOVA test were used, *P < 0.05, **P < 0.01 and ***P < 0.001, compared to the control group. The CAMK2N1-mediated regulation of DNMT1 via the AKT or ERK signaling pathway in PCa cells. A DU145 cells were transfected with CAMK2N1 siRNA. After 2 days, the mRNA expression of DNMT1 and CAMK2N1 was analyzed by qRT-PCR (GAPDH was used as a control; n=3). B The expression of CAMK2N1, DNMT1, p-AKT, t-AKT, p-MEK1, t-MEK1, p-ERK1/2, t-ERK1/2 was determined by western blot with CAMK2N1 knockdown (si-CA) in DU145 cells (GAPDH was used as a control; n=3). C-E CAMK2N1 knockdown DU145 cells were treated with 10 μM AKT signaling pathway inhibitor AKTi for 1 day. The expression of DNMT1, p-AKT and t-AKT was analyzed by qRT-PCR and western blot (GAPDH was used as a control; n=3). F-H CAMK2N1 knockdown DU145 cells were treated with 10 μM ERK signaling pathway inhibitor U0126 for 1 day. The expression of DNMT1, p-ERK1/2 and t-ERK1/2 was analyzed by qRT-PCR and western blot (GAPDH was used as a control; n=3). Data are presented as mean ± SD, t test and oneway ANOVA test were used, *P < 0.05, **P < 0.01 and ***P < 0.001. presented as mean ± SD, t test and one-way ANOVA test were used, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

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