Long non‐coding RNA LINC00673 silencing inhibits proliferation and drug resistance of prostate cancer cells via decreasing KLF4 promoter methylation

Abstract Prostate cancer is one of the major causes of cancer‐related mortality in men across the world. Recently, long non‐coding RNAs (lncRNAs) and Kruppel‐like factor 4 (KLF4) have been reported to participate in the biology of multiple cancers including prostate cancer. Here, this study aimed to explore the possible role of LINC00673 in prostate cancer via KLF4 gene promoter methylation. Microarray‐based gene expression profiling of prostate cancer was employed to identify differentially expressed lncRNAs and genes, after which the expression of LINC00673 and KLF4 in prostate cancer tissues was determined using RT‐qPCR. Next, the relationship between LINC00673 and KLF4 was evaluated using in silico analysis. Further, the effect of LINC00673 and KLF4 on cell proliferation and drug resistance of transfected cells was examined with gain‐ and loss‐of‐function experimentation. It was found that LINC00673 was highly expressed, while KLF4 was poorly expressed in prostate cancer tissues. Additionally, LINC00673 could bind to KLF4 gene promoter region and recruit methyltransferase to the KLF4 gene promoter region. Moreover, LINC00673 silencing was demonstrated to reduce methylation of the KLF4 gene promoter to elevate the expression of KLF4, thus suppressing the proliferation and drug resistance of prostate cancer cells. In summary, LINC00673 silencing could drive demethylation of the KLF4 gene promoter and thus inhibit the proliferation and drug resistance of prostate cancer cells, suggesting that silencing of LINC00673 and elevation of KLF4 could serve as tumour suppressors in prostate cancer.

to inhibit drug resistance. 4 Moreover, owing to androgen deprivation treatment therapy and increased doses of radiation, great advances have been achieved in the prognosis of prostate cancer patients; however, numerous patients still suffer from recurrence. 5 Hence, the importance of developing new biological targets to improve the treatment outcomes of prostate cancer cannot be overestimated.
Long non-coding RNAs (lncRNAs) are a family of transcripts that are 200 nucleotides long, and their differential expressions are closely implicated in various cellular processes of malignancies such as invasion, apoptosis or proliferation. 6 Microarray-based gene expression profiling analyses in the present study revealed that LINC00673 was differentially expressed in prostate cancer. Furthermore, differential expressions of LINC00673 have been documented in numerous cancers. For example, LINC00673 is overexpressed in both hepatocellular carcinoma and tongue squamous cell carcinoma (TSCC) and also associated with poor prognoses of these carcinomas. 7,8 In addition, Ba et al suggested that LINC00673 could potentiate the progression of gastric cancer, partly owing to its suppressive roles in the expression of Kruppel-like factor 4 (KLF4). 9 KLF4 belongs to the KLFs class of transcriptional mediators which can bind to DNA and are widely expressed in human cancers serving as oncogenes or tumour suppressors. 10 KLF4 is dysregulated in various cancers including prostate cancer, and its enforced expression has been confirmed to attenuate cell metastasis and growth, thus functioning as an inhibitor of prostate cancer. 11 Additionally, the expression of KLF4 stimulated by lysophosphatidic acid has been revealed to be involved in the proliferation and migration abilities of prostate cancer cells. 12 DNA methylation is closely linked to numerous human cancers and KLF4, as readers of DNA methylation specific to sequence, also requires methylated cytosine-phosphate-guanine (CpG). 13,14 Based on these findings, we hypothesized that LINC00673 could participate in the progression of prostate cancer via interaction with KLF4. Therefore, the current study aims to investigate the underlying mechanism by which LINC00673 regulates the development of prostate cancer in regard with KLF4 involvement.

| Ethics statement
The current study was approved by the Ethics Committee and Experimental Animal Ethics Committee of The First Hospital of China Medical University. Signed informed consents were obtained from all participants or their legal guardians prior to the experiment.
All animal experimentation strictly adhered to principles aiming to minimize the number, suffering and discomfort of the included animals.

| Microarray-based gene expression profiling
Prostate cancer-related microarray data were retrieved from the Gene Expression Omnibus (GEO) database (https ://www.ncbi.nlm. nih.gov/geo/). The obtained data were subjected to standardized pretreatment with the "Affy" package of R Language Programming. 15 Next, the "Limma" package was employed to screen the differentially expressed genes (DEGs) 16 with |log2FC|> 1.5 and adj.P Val (P value after correction) <.05 serving as the threshold. Subsequently, a heat map of the obtained DEGs was plotted. All the included patients were aged between 55 and 84 years old with an average age of 69 years and did not undergo drug therapy and radiotherapy prior to the experiment. Among these patients, 15

| Study subjects
patients were at the T1 stage, 15 at the T2 stage and 18 at the T3 stage. A portion of the prostate cancer tissues and adjacent normal tissues were cryopreserved at −80°C, and others were fixed using 10% formalin, dehydrated, paraffin-embedded and stored for subsequent experimentation.

| In situ hybridization
Tissue sections were attached to slides pre-treated with 10% polylysine to perform in situ hybridization in accordance with the instructions of the kits (BOSTER Biological Technology Co., Ltd.). Next, the sections were hybridized with digoxin-labelled LINC00673 probe (Exiqon) at a constant temperature of 52°C for 16 hours, warmbathed with biotinylated mouse anti-digoxin at 37°C for 60 minutes and incubated with streptavidin biotin peroxidase complex (SABC), followed by diaminobenzidine (DAB) developing. The obtained results were independently scored by two pathologists. The cells presenting with tan-stained nuclei were regarded as the positive cells. A total of five visual fields were randomly selected from each section under a 200-fold microscope to calculate the percentage of positive cells. The percentage of the positive cells <5% was indicative of negative cells, while that ≥5% was indicative of positive cells.

| Cell culture and treatment
A total of 10 μg lentiviral vector Pcdh of target plasmid, 7.5 μg helper plasmid PAX and 5 μg helper plasmid Pmd2G were, respectively, diluted with 750 μL of opti-MEM (Gibco) and allowed to stand for 5 minutes. Separately, 112.5 μg PEI was diluted with 750 μL opti-MEM and allowed to stand at room temperature for 5 minutes. Subsequently, the two aforementioned solutions were mixed uniformly. After 20 minutes, the mixture was added to the corresponding cell culture dishes and cultured with 5% CO 2 in air at 37°C with the medium renewed after 6 hours. After 48 hours, the cell supernatant was collected. Following 24-hour culture with 8 mL of complete medium, the cell supernatant was collected. A total of 1 × 10 5 cells were treated with lentivirus and cultured with the medium for 24 hours.
Subsequently, the fluorescence intensity was detected using a fluorescence microscope. Next, the cells were selected for monoclonal cultivation to obtain stable cell lines for xenograft tumour in nude mice. All the following plasmids were purchased from Dharmacon: small interfering RNA (si)-negative control (NC), si-LINC00673, pcDNA-NC, pcDNA-LINC00673 and pcDNA-KLF4.

| Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA content extraction from the cells was performed with the Trizol method (15596026; Invitrogen). The integrity of the extracted RNA was then identified using 1% agarose gel electrophoresis, and RNA concentration and purity were measured using a NanoDrop ND-1000 spectrophotometer. Subsequently, the RNA was reverse transcribed into complementary DNA (cDNA) according to the instructions of the PrimeScript RT reagent kits (RR047A; Takara). All the primers (Table 1) were synthesized by Beijing Genomics Institute Biotech Co., Ltd. β-actin was regarded as the internal control for LINC00673 and mRNA. The fold changes were calculated by means of relative quantification (the 2 −ΔΔC t method).

| Western blot analysis
The cells in each group were lysed with 500 μL of radioimmunoprecipitation assay (RIPA; Pierce). Protein concentration was then measured using bicinchoninic acid (BCA) protein assay kits (BCA1-1KT; Sigma-Aldrich). Afterwards, the total proteins extracted from the cells were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the proteins were transferred onto membranes, which were blocked with 5% skimmed milk-Tris-buffered saline Tween-20 (TBST) for 1 hour. With glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as the internal control, the membrane was incubated overnight at 4°C with rabbit polyclonal antibodies against KLF4 (dilution ratio of 1:1000, ab106629), Cyclin D2 (dilution ratio of 1:1000, ab207604) and Cyclin B1 (dilution ratio of 1:50 000, ab32053). All the aforementioned antibodies were purchased from Abcam Inc. Following incubation with the corresponding secondary antibody immunoglobulin G (IgG; A21020; Abbkine) at 37°C for 45 minutes, the membranes were rinsed with TBST for 45 minutes and allowed to react with the enhanced chemiluminescence reagent (ECL; ECL808-25; Biomiga) for 1 minute. The protein grey values were analysed using the Gel-Pro Analyzer 4.0 (Media Cybernetics) to determine the protein expression.

| 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay
After 48 hours of conventional treatment, the cells were cultured with Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% foetal bovine serum (FBS) and then dispersed into a cell suspension. After counting, the cells were inoculated in a 96-well plate at a density of 8 × 10 3 cells/well. After incubation at 37°C with 5% CO 2 in air for 24 hours, the cells were administered paclitaxel until the final concentrations reached specific parameters of 0, 20, 40 and 60 nmol/L. After 24 hours, cells in each well were added with 10 μL MTT solution (5 mg/mL) and incubated at 37°C for 4 hours. Afterwards, the cells were added with 100 μL dimethyl sulphoxide (DMSO) and oscillated for 10 minutes to uniformly dissolve the crystals. The optical density (OD) value was measured in each well at a wavelength of 570 nm using a microplate reader to calculate the cell inhibition rate (IR; %). Subsequently, the IR curve was plotted, and the half maximal inhibitory concentration (IC50) was calculated.

| Fluorescence in situ hybridization (FISH)
The subcellular localization of LINC00673 in DU145 cells was identified using FISH kits according to the instructions of the Ribo™ lncRNA FISH probe Mix (RiboBio Company). In brief, DU145 cells were inoculated onto the cover glass in a 6-well plate at a density of 6 × 10 4 cells/ well. When cell confluence approximately reached 80% after culture for 1 day, the cover glass was rinsed with phosphate buffered saline (PBS), fixed with 1 mL 4% paraformaldehyde at room temperature and treated with protease K (2 μg/mL), glycine and acetylation reagent. (300 ng/mL), the cover glass was rinsed with phosphate buffered saline with Tween-20 (PBST), and the nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:800) diluted with PBST. Subsequently, the glass was added to the 24-well plate for staining and then rinsed with PBST. Finally, the cover glass was mounted with the anti-fluorescence TA B L E 1 Primer sequences for reverse transcription quantitative polymerase chain reaction quenching agent. Images were acquired from five randomly selected visual fields under a fluorescence microscope (Olympus).

| Dual-luciferase reporter gene assay
Firstly, we designed the dual-luciferase reporter plasmid of KLF4 target gene promoter containing KLF4 wild-type (KLF4-Wt) and KLF4 mutated at the putative LINC00673 binding sites (KLF4mutant type [Mut]). The sequence of KLF4-Wt was TGAGG G G TG G G G G G CG C TAG G G G TG TG G AG AG G AG G C AG TG C CCAGCACTGTGCAGCGTGAACTGGGAGCCTCAAG and that of KLF4-Mut was CTCTGGAGTTTGGTACTGAGAAACATAGAGAGGCGTGA G AC T T TAG C A ATC C AC ATAG TG C C G T TG C TC C C G G G C G .
Subsequently, KLF4-Wt and KLF4-Mut were co-transfected with overexpressing LINC00673 and NC plasmids into DU145 cells, respectively. After 24 hours of treatment, the cells were lysed and centrifuged at 12 000 r/min for 1 minute, after which, the supernatant was collected. The luciferase activity was then detected using a Dual-Luciferase ® Reporter Assay System (E1910; Promega). Each cell sample was added with 100 μL of firefly luciferase reagent to detect the firefly luciferase. The relative luciferase activity was expressed as the ratio of firefly luciferase to renilla luciferase.

| Chromatin immunoprecipitation (ChIP)
ChIP kits (Millipore Inc) were employed in order to determine the enrichment of DNA methyltransferase (DNMT)1, DNMT3a and DNMT3b in the KLF4 gene promoter region. When cell confluence approximately reached 70%-80%, the cells were fixed with 1% formaldehyde at room temperature for 10 minutes to cross-link the intracellular DNA and protein. Next, the cross-linked DNA and protein were randomly cracked into fragments, which were centrifuged at 13 000 r/min at 4°C. The ). Subsequently, protein agarose/sepharose was applied to precipitate the endogenous DNA-protein complex. After centrifugation, the supernatant was discarded, and the non-specific complex was washed and de-cross-linked overnight at 65°C. The obtained DNA fragments were extracted and purified using phenol/chloroform. The binding of KLF4 gene promoter region to DNMT1, DNMT3a and DNMT3b was finally detected using specific primers of the KLF4 gene promoter region.

| RNA-binding protein immunoprecipitation (RIP) assay
A RIP assay was performed in accordance with the instructions of the Magna RIP RNA-Binding Protein Immunoprecipitation kits (Millipore Inc). Briefly, the cells were rinsed with pre-cooled PBS, lysed for 30 minutes with 100 μL lysis buffer supplemented with protease inhibitor and ribonuclease inhibitor, and then centrifuged at 12 000 r/min at 4°C for 3 minutes. A small amount of supernatant was taken as the input positive control and added with 1 μg of the corresponding antibody.
The remaining supernatant was added with NC antibody IgG from normal cells, specific rabbit antibodies of target protein (DNMT1 [Abcam Inc, ab13537], DNMT3a [Abcam Inc, ab2850], DNMT3b [Abcam Inc, ab2851]) and 10-50 μL protein A/G-beads. Next, the supernatant was incubated at 4°C overnight, centrifuged at 3000 r/min for 5 minutes, washed with 1 mL lysis buffer and precipitated using protein A/Gbeads, followed by centrifugation at 1000 r/min at 4°C for 1 minute.
After being added with 15 μL of 2 × SDS loading buffer, the supernatant was heated for 10 minutes. RNA content was extracted and purified from the precipitate using a RNA extraction method. Finally, RT-qPCR was conducted using specific primers of LINC00673 to verify the interaction between LINC00673 and DNMT1, DNMT3a and DNMT3b.

| 5-ethynyl-2′-deoxyuridine (EdU) assay
Cells were inoculated in a 96-well plate at a density of 1.6 × 10 5 cells/ well and cultured for 48 hours. After culture, EdU assay was performed in accordance with the protocols of EdU kits (C10310; Guangzhou RiboBio Co., Ltd.). Cells in each well were cultured with 100 μL of 50 μmol/L EdU at 37°C for 4 hours, fixed with 4% formaldehyde at room temperature for 15 minutes and treated with 0.5% Triton X-100 at room temperature for 20 minutes for permeabilization. After that, cells in each well were incubated with 100 μL of Apollo ® compound (C10338-2; RiboBio Company) at room temperature for 30 minutes, stained using 100 μL of hoechst33342 (Ribobio Company) for 30 minutes and observed under a fluorescence microscope (Olympus). The

number of EdU positive cells (red blood cells) was calculated using the
Image-Pro Plus 6.0 software (Media Cybernetics). 17

| Flow cytometry
Cells undergoing different treatments were detached with 0.25% trypsin and dispersed into a single cell suspension. After being rinsed with PBS, the cells were centrifuged, fixed with 70% pre-cooled ethanol at overnight 4°C and resuspended. Following centrifugation and pre-cooled PBS rinsing, the cells were resuspended in 100 µL PBS, added with RNase until the final concentration reached 50-70 µg/mL, and water-bathed at 37°C for 30 minutes. When the final concentration reached 50 µg/mL after the addition of propidium iodide (PI), the cells were stained avoiding exposure to light at 4°C for 40 minutes and rinsed with PBS. The DNA content was then calculated using a 575 nm bandpass filter to calculate the percentage of cell cycle.

| Xenograft tumour in nude mice
Cells undergone different treatments were washed with normal saline, detached with 1 mL 0.25% trypsin and incubated at 37°C. Center, Chinese Academy of Sciences). The tumour volume was measured and recorded after a week of inoculation. When the mean volume reached 100 mm 3 at the 3rd week, the mice were intraperitoneally injected with paclitaxel (50 μg/kg; 0.01 mL/g) once every 2 days for a total of seven times. Finally, the tumour volumes were measured twice a week.

| Statistical analysis
Statistical analyses were performed using the SPSS21.0 software (IBM Corp). Measurement data were presented as mean ± standard deviation (SD). Data between two groups were analysed using the independent sample t test and corrected using Welch. The normality test of data among multiple groups was analysed using the Shapiro-Wilk method. Data conforming to normal distribution were analysed using one-way analysis of variance (ANOVA). Pairwise comparisons of data among multiple data were conducted using the least significant difference (LSD) t test. Data presenting with skew distribution were compared using the non-parametric Kruskal-Wallis test.

Proliferative activities and tumour volumes at different time-points
were analysed using repeated measures ANOVA. A value of P < .05 indicated statistical significance.

| LINC00673 is highly expressed while KLF4 is poorly expressed in prostate cancer
Initially, we performed a differential gene expression analysis on the prostate cancer-related expression data set, GSE45016. A heat map illustrating the most differentially expressed genes is shown in Figure 1A. Subsequently, higher expressions of LINC00673 were found in prostate cancer tissues relative to adjacent normal tissues (P < .05). Ensuing results from the BLAST website (https ://blast.ncbi. nlm.nih.gov/Blast.cgi?PROGR AM=blast n&PAGE_TYPE=Blast Searc h&BLAST_SPEC=&LINK_LOC=blast tab&LAST_PAGE=tblastn) further revealed that the promoters of LINC00673 and KLF4 possess the same base sequence ( Figure 1B). Additionally, the expression of LINC00673 was detected in prostate cancer tissues using FISH, and the results revealed that LINC00673 was highly expressed in prostate cancer tissues as compared with adjacent normal tissues (P < .05; Figure 1C). Furthermore, RT-qPCR detected that the expression of LINC00673 was higher while that of KLF4 was lower in prostate cancer tissues when compared with adjacent normal tissues (both P < .05; Figure 1D). In addition, RT-qPCR was applied to  Figure 1E). Correlation analysis results further revealed that KLF4 was negatively correlated with LINC00673 ( Figure 1F). In addition, as shown in Table 2, LINC00673 expression was found to be associated with tumour size, tumour node metastasis (TNM) stage and lymph node metastasis (LNM), while no correlations were found in regard to the age of patients with prostate cancer. All these results verified that prostate cancer exhibits high expression of LINC00673 and low expression of KLF4.

| LINC00673 silencing suppresses proliferation in prostate cancer cells
After elucidating the expression patterns of LINC00673 and LINC00673, we treated the prostate cancer cell lines DU145 and PC3 with si-NC and si-LINC00673 to conduct a series of assays to verify the effects of LINC00673 on prostate cancer cell proliferation. Initially, we attenuated LINC00673 in the two aforementioned cell lines and selected the most efficient sequence for subsequent experimentation (Figure 2A). EdU assay was then performed to detect cell proliferation which revealed that EdU positive cells were stained with red colouration (Figure 2B,C). Compared with cells without treatment, proliferative rate was reduced in DU145 cells treated with si-LINC00673 (P < .05), but no significant differences were detected in DU145 cells treated with si-NC (P > .05). Subsequently, cell cycle was measured using flow cytometry, and the results revealed that si-LINC00673-treated DU145 cells exhibited increased proportion arrested at the G0/G1 phase but decreased proportion arrested at the S phase when compared with the DU145 cells without treatment (P < .05); however, the cell cycle did not differ in si-NCtreated cells (P > .05; Figure 2D). Next, the effects of LINC00673 on the mRNA and protein expression of Cyclin D2 and Cyclin B1 were

| LINC00673 silencing inhibits drug resistance of prostate cancer cells
After LINC00673 silencing was found to be able to inhibit prostate cancer cell proliferation, the focus of the current study was shifted to determine the effects of LINC00673 on drug resistance of prostate Similar trends were also observed in PC3 cells confirming our findings (P < .05; Figure 3C). Moreover, we detected IC50 values in DU145 and PC3 cells treated with docetaxel at various concentrations using MTT assay and observed a reduced IC50 value in si-LINC00673treated DU145 and PC3 docetaxel-resistant cells (P < .05; Figure 3D).
All in all, these results indicated that knockdown of LINC00673 could repress paclitaxel and docetaxel resistance in prostate cancer cells. determined by RT-qPCR. F, Correlation analysis of KLF4 and LINC00673 expression. * P < .05, vs adjacent normal tissues/RWPE-1 cell line; # P < .05, vs DU145/pr cell. Data (mean ± SD) between two groups were compared using the unpaired t test, and data among multiple groups were compared using one-way ANOVA. n = 48

| LINC00673 silencing reduces methylation of the KLF4 gene promoter to up-regulate KLF4 expression
Methylation specific PCR was performed to identify methylation of the KLF4 gene promoter in prostate cancer tissues, and it was found that KLF4 was highly methylated in prostate cancer tissues compared with adjacent normal tissues (P < .05; Figure 4A). In order to further verify the changes in the KLF4 gene promoter, the CpG islands in the KLF4 gene promoter were predicted using an online tool EMBOSS CpGPlot available at http://emboss.bioin forma tics.nl/ cgi-bin/embos s/cpgplot ( Figure 4B). Moreover, methylation of CpG islands in the KLF4 gene promoter was detected using MSP and BSP, which revealed that CpG islands in the KLF4 gene promoter were highly methylated in DU145 cells treated with pcDNA-LINC00673, whereas poor methylation was observed in DU145 cells treated with si-LINC00673 ( Figure 4C,D), suggesting that methylation of CpG islands in the KLF4 gene promoter was closely associated with the expression of LINC00673.
Moreover, FISH results revealed that LINC00673 was primarily located in the nucleus ( Figure 4E). In addition, findings from F I G U R E 2 LINC00673 knockdown represses prostate cancer cell proliferation. A, Silencing efficiency of LINC00673 in DU145 and PC3 cell lines detected using RT-qPCR. B, C, Cell proliferation in DU145 and PC3 cells following different treatments detected by EdU assay (×400). D, Cell cycle of DU145 and PC3 cells following different treatments determined by flow cytometry. E, mRNA expression of Cyclin D2 and Cyclin B1 in DU145 and PC3 cells following different treatments determined by RT-qPCR. F and G, Western blot analysis of Cyclin D2 and Cyclin B1 proteins in DU145 and PC3 cells following different treatments. * P < .05, vs DU145 and PC3 cells without treatment. Data (mean ± SD) among multiple groups were compared using one-way ANOVA. The experiment was repeated in triplicate to obtain the mean value dual-luciferase reporter gene assay showed that the luciferase activity was attenuated in response to the co-treatment of KLF4-Wt and pcDNA-LINC00673 (P < .05), but did not differ after co-treatment with KLF4-Mut and pcDNA-LINC00673 (P > .05; Figure 4F).
This finding suggested that LINC00673 could bind to KLF4 gene promoter region, which was consistent with the results predicted

| LINC00673 silencing diminishes methylation of KLF4 gene promoter to suppress prostate cancer cell proliferation
Having identified the correlation between LINC00673 and KLF4, and Cyclin B1 did not differ in response to pcDNA-NC treatment and co-treatment of pcDNA-LINC00673 and pcDNA-KLF4 (both P > .05; Figure 5F-H). Taken together, these findings suggested that LINC00673 knockdown could repress prostate cancer cell proliferation by attenuating methylation of the KLF4 gene promoter.

| LINC00673 silencing reduces the methylation of KLF4 gene promoter to suppress drug resistance in prostate cancer cells
After determining the role of LINC00673 in prostate cancer cell prolif- with those from DU145 cell lines ( Figure 6B,D). In summary, these findings solidified that LINC00673 could inhibit drug resistance in prostate cancer cells by reducing methylation of the KLF4 gene promoter.

| LINC00673 silencing curtails methylation of KLF4 gene promoter to suppress the development of prostate cancer in vivo
After treatment of pcDNA-KLF4, pcDNA-LINC00673 or both, DU145

| D ISCUSS I ON
Prostate cancer is a highly prevalent cancer accompanied by high fatality rates and accounts for a substantial global burden on medical facilities and patients. 18 Due to its variable natural history, researchers are confronted with various challenges in terms of optimum  gene promoter could be considered as markers to predict the early recurrence of cancer. 33 Drug resistance forms an enormous challenge for the management of advanced prostate cancer, and specifically, paclitaxel resistance in prostate cancer can potentially enhance cell migration via enforced extracellular matrix, leading to more invasive and aggressive phenotypes of this unfavourable cancer. 34 Notably, KLF4 is also poorly expressed in breast cancer and has been further suggested as a possible protocol to reduce the resistance of breast cancer cells to tamoxifen. 35 Similarly, lncRNA PCAT-1 silencing attenuates drug resistance and invasion of colorectal cancer cells by reducing the expression of c-Myc. 36 Similarly, depletion of lncRNA plasmacytoma variant translocation 1, which was elevated in gastric cancer tissues and cells conferring resistance to cisplatin, could suppress the progression of multidrug resistance. 37 Moreover, lncRNA MALAT1 down-regulation F I G U R E 7 LINC00673 knockdown reduces methylation of KLF4 gene promoter and consequently retards the development of prostate cancer in vivo. A, B, xenograft tumours and quantitative analysis of tumour volume and weight after pcDNA-KLF4, pcDNA-LINC00673 or both treatments. C, LINC00673 expression and mRNA expression of KLF4 in tumour tissues after pcDNA-KLF4, pcDNA-LINC00673 or both treatments detected by RT-qPCR. D, Western blot analysis of KLF4 protein in tumour tissues after pcDNA-KLF4, pcDNA-LINC00673 or both treatments. * P < .05, vs DU145 cells without treatment. Data (mean ± SD) among multiple groups were compared using one-way ANOVA. N = 8 F I G U R E 8 Regulatory mechanism of LINC00673 mediating proliferation and drug resistance of prostate cancer cells by regulating methylation of the KLF4 gene promoter. LINC00673 silencing decreases methylation of KLF4 gene promoter to upregulate the expression of KLF4, thus suppressing proliferation and drug resistance of prostate cancer cells was revealed to suppress cell proliferation and docetaxel chemoresistance in prostate cancer. 38 Taken together, our findings demonstrated that LINC00673 silencing could disrupt the methylation of the KLF4 gene promoter and consequently suppress cell proliferation and drug resistance in prostate cancer (Figure 8), illuminating potential for future therapeutic strategies for prostate cancer. For better management of prostate cancer, more efforts are warranted in order to uncover the molecular mechanism underlying the functions of LINC00673 in the development and progression of prostate cancer.

ACK N OWLED G EM ENTS
The authors would like to extend their sincere gratitude to the reviewers.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

AUTH O R CO NTR I B UTI O N S
Zhenming Jiang, Yuxi Zhang, Xi Chen, Pingeng Wu and Dong Chen designed the study. Zhenming Jiang, Xi Chen, Pingeng Wu, Dong Chen collated the data. Yuxi Zhang conceived and performed the experiments. Zhenming Jiang and Yuxi Zhang analysed the data and produced the initial draft of the manuscript. All authors contributed to the revision and approved the final submitted manuscript.

E TH I C S S TATEM ENT
The current study was approved by the Ethics Committee and Experimental Animal Ethics Committee of The First Hospital of China Medical University. Signed informed consents were obtained from all participants or their legal guardians prior to the experiment.
All animal experimentation strictly adhered to principles aiming to minimize the number, suffering and discomfort of the included animals.

CO N S E NT FO R PU B LI C ATI O N
Consent for publication was obtained from the participants.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets generated/analysed during the current study are available.