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MiR‐129‐5p promotes docetaxel resistance in prostate cancer by down‐regulating CAMK2N1 expression

Abstract This study focuses on the effect of miR‐129‐5p on docetaxel‐resistant (DR) prostate cancer (PCa) cells invasion, migration and apoptosis. In our study, the expression of CAMK2N1 was assessed by qRT‐PCR in PCa patient tissues and cell lines including PC‐3 and PC‐3‐DR. Cells transfected with miR‐129‐5p mimics, inhibitor, CAMK2N1 or negative controls (NC) were used to interrogate their effects on DR cell invasions, migrations and apoptosis during docetaxel (DTX) treatments. The apoptosis rate of the PCa cells was validated by flow cytometry. Relationships between miR‐129‐5p and CAMK2N1 levels were identified by qRT‐PCR and dual‐luciferase reporter assay. CAMK2N1 was found to be down‐expressed in DR PCa tissue sample, and low levels of CAMK2N1 were correlated with high docetaxel resistance and clinical prediction of poor survival. CAMK2N1 levels were decreased in DR PCa cells treated with DXT. We further explored that up‐regulation of miR‐129‐5p could promote DR PCa cells viability, invasion and migration but demote apoptosis. Involved molecular mechanism studies revealed that miR‐129‐5p reduced downstream CAMK2N1 expression to further impact on chemoresistance to docetaxel of PCa cells, indicating its vital role in PCa docetaxel resistance. Our findings revealed that miR‐129‐5p contributed to the resistance of PC‐3‐DR cells to docetaxel through suppressing CAMK2N1 expression, and thus targeting miR‐129‐5p may provide a novel therapeutic approach in sensitizing PCa to future docetaxel treatment.

presence of the drug. Resistance can be formed through a variety of mechanisms, both intrinsic and extrinsic, but the details remain largely unknown. 3 MicroRNAs (miRNAs) are small 19-25 nucleotide-long, singlestranded non-coding RNAs that silence target genes by cleaving mRNA molecules or inhibiting translation. 4 Recently, studies have shown that miRNAs are frequently misregulated in many types of human cancers. For instance, some may act as potent oncogene promoting tumour growth and migration but demoting apoptosis, while others may act as tumour suppressor genes by targeting downstream genes to inhibit migration and invasion of tumour cells. 5 Among various miRNAs, miR-129 has been shown to play a key role in tumourigenesis, tumour progression, chemotherapy resistance and cell proliferation. 6 For instance, miR-129 was initially confirmed to be decreased in undifferentiated gastric cancer tissue, colorectal, gastric and liver cancer. 7 In oesophageal neoplasms, there have been conflicting studies concerning miR-129 expression, with some groups indicating it was down-regulated compared to normal tissue, 8,9 while others claiming it was increased in tumour tissue. 10 Besides, miR-129-3p was reported to be a novel metastatic microRNA in PCa cells. 11 Collectively, these reports suggested that the function of miR-129 is highly tumour specific.
Besides, researchers found miR-129-3p inhibited docetaxel-induced apoptosis of breast cancer cells by down-regulation of the CP110 protein. 12 However, the involvement of miR-129 in the chemoresistance of cancer, especially in PCa, is largely unknown. Hence, a thoroughgoing understanding of these miscellaneous functions would be indispensable for further steps at developing promising therapies.
CaMKII, which belongs to the calcium/calmodulin-dependent protein kinase II family, is a serine/threonine-specific protein kinase and can phosphorylate nearly 40 distinctive proteins, among which are kinases, ion channels and transcription factors. 13,14 By activating MEK/ERK, CAMKII cascade enhances the phosphorylation of p27 Kip1 to control cell-cycle. 13 There are two potent and specific inhibitors of CAMKII, which have been characterized in human, including CAMK2N1 (also known as CANK2Nα) and CAMK2Nβ genes. The CAMK2Nβ was the first discovered human CAMKII inhibitor and was cloned from human dendritic cells, with an inhibitory effect on the growth of colon adenocarcinoma LoVo cells. 14 The CAMK2N1 is composed of 78-amino acids and was initially identified in the cell junction and synapse. [13][14][15] Additionally, CAMK2N1-mediated inhibition of CaMKII activity controls the progress of cell cycle in colon cancers through deactivation of MEK/ERK kinase activity and p27 protein accumulation. 13,14 In a recent study, genome-wide gene expression analysis uncovered that CAMK2N1 regulated the expression of pivotal genes related to cell-cycle control and apoptosis. 15 Researchers also showed that CAMK2N1 has a vital role to affect tumourigenesis and tumour development. 13,14 Previous study revealed that its expression was down-regulated in PCa, and reintroduction of CAMK2N1 remarkably impaired human PCa cell proliferation and in vivo tumour growth. 15 Furthermore, researchers found that CAMK2N1 played a suppressive role in castration-resistance PCa via suppressing androgen receptor mRNA expression and its regulator. 15 Together, these data indicate that CAMK2N1 plays a pivotal role in the progression of PCa. However, for CAMK2N1, its complete and detailed molecular mechanisms, such as upstream pathway and functions and whether it related to other types of drug resistance in PCa are still unclear.
Here, we analysed microarray data and screened out CAMK2N1 as one of the most down-regulated mRNAs in docetaxel-resistant (DR) PCa cells. The biological function of CAMK2N1 was comprehensively investigated in vitro, exhibiting that CAMK2N1 can effectively inhibit docetaxel resistance in PCa cells. We further employed in silico analysis and molecular techniques to confirm that CAMK2N1 is the target of miR-129-5p, which significantly rescued miR-129-5p promoted PCa docetaxel resistance.

| Patient samples
Thirty-six PCa tissues were obtained from docetaxel-free PCa patients (n = 18) and DR PCa patients (n = 18) by radical prostatectomy at the First Affiliated Hospital of Nanjing Medical University in Jiangsu, China. All the patients were confirmed by exhaustive diagnosis and the tissue samples were independently interrogated by three experienced pathologists. All samples were collected with the informed consent of the patients and the study was approved by the local ethical committee.

| Microarray
Affymetrix microarray platform GPL570 and microarray data GSE33455 used for validation were obtained from Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/). This dataset included 12 prostate tissues samples, three DU-145 cell lines, three DR DU-145 cell lines, three PC-3 cell lines and three PC-3-DR cell lines. The threshold used to screen up-regulated and down-regulated mRNA was log 2 (FC) >1 and log 2 (FC) <−1 (P < 0.05) respectively.

| RNA extraction and qRT-PCR
Trizol Reagent (Invitrogen) was used to extract total RNA including miRNA. Next, 1 μg of total RNA per sample was converted to cDNA by accurate primers using the PrimeScript ™ RT-PCR Kit (TAKARA, Japan) for the detection of CAM2NK1. cDNAs were amplified using PrimeSTAR® HS DNA Polymerase (TAKARA). Quantitative reverse transcription PCR (qRT-PCR) was conducted using the THUNDERBIRD SYBR ® qPCR Mix (Toyobo, Japan). The relative quantification value of mRNAs was normalized to control and calculated through the 2 −ΔΔCt method.
The GADPH and U6 were used as internal control genes for mRNA and miRNA respectively. Primer sequences are given in (Table 1).

| Western blot analysis
Protein was extracted from tissues using RIPA buffer. After denaturing at 95°C for 10 minutes, protein was separated on a 12% SDSpolyacrylamide gel and then transferred to a polyvinylidene fluoride membrane. After blocking with TBST buffer containing 5% non-fat milk for 1 hour at room temperature, the membrane was co-incu-

| Cell transfection
Twenty-four hours before transfection, 1 × 10 6 cells/well logarithmic growth phase PD-3 or PC-3-DR cells were cultured until reaching 80%-90% confluency in six-well plates. The miR-129-5p mimics, inhibitors and negative control (NC) were synthesized by Sangon Biotech. MiRNA-129-5p mimics, inhibitors and NC were transfected into PC-3 and PC-3-DR cells employing Lipofectamine ™ 3000 (Invitrogen) according to the manufacturer's protocol. The final concentration of all constructs in the transfection system was optimized to 20 μmol/L. CAMK2N1 was amplified by PCR using PC-3 cell lines as templates. The constructs were transfected into PC-3 or PC-3-DR cells using Lipofectamine ™ 3000. After 24, 48 or 72 hours, the cells were harvested for following analyses.

| Transwell assay
Matrigel gel (BD, USA) was used to coat 24-well Transwell plates  Firefly luciferase activity was normalized to renilla luciferase activity.

| Statistical analysis
All quantitative values were presented as the mean ± SD of at least three repeated individual experiments for each group and they were statistically analysed with one-way ANOVA. All of the statistical analyses were made using GraphPad Prism v6.0 (GraphPad Software, Inc.) and SPSS (version 21.0). A value of P < 0.05 was considered statistically significant.

| CAMK2N1 is down-regulated in PCa DR tumour tissues and cell lines
Based on microarray platform GPL570 and microarray data GSE33455, we used a t test (P < 0.05) combined with fold change

| The docetaxel-resistance of PC-3-DR is significantly stronger than PC-3
We next studied the cell viability and apoptosis between PC-3 cells and experimentally generated DR sublines (PC-3-DR). CCK-8 was

| Expression of CAMK2N1 attenuated docetaxel resistance PC-3-DR cells
We next explored the role of CAMK2N1 in the docetaxel resistance

| CAMK2N1 is a target of miR-129-5p in PCa cells
Next, we were eager to find upstream regulator candidates that could mediate the CAMK2N1-induced docetaxel-sensitive to docetaxel in PC-3-DR cells. We included four miRNA databases including miRDB, miRBase, RNA22v2.0 and TargetScan Human 7.1 (Table S1) to explore the potential miRNA which may regulate CAMK2N1-induced docetaxel resistance. We predicted that miR-129-5p may potentially target on four sites of 3′-UTR of CAMK2N1 ( Figure 4B). To ascertain whether CAMK2N1 is fine-tuned by miR- In HEK293T cells, ectopically expressed miR-129-5p could only impair the normalized luciferase activity with the wild-type 3′UTR of CAMK2N1, but not with the mutant-type ( Figure 4C). These data revealed that miR-129-5p can directly target CAMK2N1 expression in vitro.

| MiR-129-5p strengthens cell viability and inhibits apoptosis of PCa cells via down-regulating CAMK2N1
To resistance while co-expression of CAMK2N1 significantly rescued this phenotype. We thus provided evidence that CAMK2N1 was the target of miR-129-5p, which has potential to become a promising therapeutic target for the treatment of DR PCa.
The overexpression of CAMK2N1 had been reduced in the progression of medullary thyroid cancer, 16 colon cancers 17 and PCa. 15,18 In the current report, we found the low expression level of CAMK2N1 in both DR PCa patient tissues and PC-3-DR cells, which was consistent with previous studies. 15,18 Moreover, our finding indicated that in DR PCa cells, ectopic expression of CAMK2N1 largely reduced its proliferation, survival and growth while remarkably enhanced its docetaxel-induced apoptosis. Our data were compatible with previous researches which revealed CAMK2N1 has a suppressive role in CRPC. 15,18 These results substantiated that CAMK2N1 played an important role in regulating tumour growth and also mediating various drug-resistance in PCa.
In previous studies, researchers found miR-129 down-regulated in gastric cancer, colorectal cancer, gastric cancer, and liver cancer. [19][20][21][22][23] Whereas, previous study proved that high expression level of miR-129-5p led to development of oesophageal cancer. 10 In addition, Xiao et al illustrated that norcantharidin increased Beclin-1 by regulating miR-129-5p, which in turn trigger autophagic cell death in PCa cells. 24 Li et al showed that lowly expressed miR-129-5p inhibits growth and induces apoptosis in laryngeal carcinoma by targeting adenomatous polyposis coli. 25 Here, we verified that miR-129-5p acted on  20 The discrepancy of the conflicted role of miR-129 in cancer during chemotherapy could contribute to the differences of drugs as the cytotoxic activity of docetaxel is exerted by improving microtubule assembly and preventing microtube disassembly while fluorouracil acts as thymidylate synthase inhibitor blocking synthesis of the pyrimidine thymidine which is required for DNA replication. 26,27 Therefore, the promoting function of miR-129 in chemoresistance could be highly drug-specific, which needs further studies.
MiR-129-5p is a miRNA that has been rarely studied, particularly in PCa. In the current study, we discovered that CAMK2N1 as a target of miR-129-5p and uncovered a novel function of miR-129-5p in promoting proliferation and metastasis of PC-3-DR cells. Nevertheless, some limitations also existed in this report which should be taken into consideration. For instance, though, we identified miR-129-5p as a direct regulator of CAMK2N1 protein translation in vitro, in vivo assays are still needed to further confirm the biological function of miR-129-5p/CAMK2N1 axis. Besides, we found that miR-129-3p also had a targeting relationship with CAMK2N1 and that their targeting regions were conserved too. So, miR-129-3p was also a factor with significant research value, and we would further study its effects on PCa. Nevertheless, conclusion can be drawn from our studies that the miR-129-5p/CAMK2N1 axis has crucial molecular and cellular functions in DR PCa cells, which provide a new therapeutic strategy for future researches.
Furthermore, CAMK2N1 caused down-regulation of MEK/ERK activity and up-regulation of p27 protein, which regulates the cell cycle progression of colon cancer cells, 13,14 and induced apoptosis regulatory kinases including Bax/Bcl2, caspase4, caspase7. 15 These studies suggested how CAMK2N1 inhibited tumourigenesis via regulating cell cycle and improving cell apoptosis.
In conclusion, CAMK2N1 was down-regulated in PC-3-DR cells, as a modulator for docetaxel sensitivity. MiR-129-5p stimulated proliferation and progression of PC-3-DR cells during docetaxel treatment through targeting CAMK2N1. Our finding suggested miR-129-5p might provide a potential therapy target to CRPC in the future.

CONFLI CT OF INTEREST
The authors declare that they have no competing interests.

ETHICS APPROVAL
The study was approved by the Ethics Boards of the First Affiliated Hospital of Nanjing Medical University.