RETRACTED ARTICLE: MCM3AP-AS1/miR-876-5p/WNT5A axis regulates the proliferation of prostate cancer cells

Background Although the fact that long non-coding RNA MCM3AP antisense RNA 1 (MCM3AP-AS1) is oncogenic in several cancers is well documented, very few researchers investigate its expression and function in prostate cancer. Methods Paired prostate cancer samples were selected, and expressions of MCM3AP-AS1, miR-876-5p and WNT5A were examined by qRT-PCR. MCM3AP-AS1 shRNA was transfected into LNCaP and PC-3 cell lines, and then the proliferative activity and apoptosis of cancer cells were detected by CCK-8 assay, EdU assay and flow cytometry analysis, respectively. qRT-PCR and Western blot were used to analyze the changes of miR-876-5p and WNT5A. Luciferase reporter gene assay was employed to determine the regulatory relationship between miR-876-5p and MCM3AP-AS1, miR-876-5p and WNT5A. Results MCM3AP-AS1 was significantly up-regulated in cancerous tissues of prostate cancer samples, positively correlated with the expression of WNT5A, while negatively related with miR-876-5p. After transfection of MCM3AP-AS1 shRNA into prostate cancer cells, the proliferative ability of cancer cells was signally inhibited, but the apoptosis of cancer cells was increased. MCM3AP-AS1 shRNA could reduce the expression of WNT5A on both mRNA and protein levels. Besides, MCM3AP-AS1 was identified as a sponge of miR-876-5p. WNT5A was validated as a target gene of miR- 876-5p. Conclusion MCM3AP-AS1 is abnormally up-regulated in prostate cancer tissues and can modulate the proliferation and apoptosis of prostate cancer cells, which has the potential to be the “ceRNA” to regulate the expression of WNT5A by targeting miR-876-5p.


Introduction
Prostate cancer (PCa) is labeled as one of the most prevailing tumors among male patients, with the morbidity and mortality of PCa worldwide accounting for 7.1/10 5 and 3.8/10 5 in 2018, respectively [1]. PCa often infiltrates adjacent tissues, accompanying with lymph node metastasis and hematogeneous metastasis, which poses a serious threaten to the life quality and survival time of patients [2][3][4].
Long non-coding RNA (lncRNA), a group of non-coding RNA (ncRNA), whose length is over 200 bp, lacks open reading frame and does not have complete protein coding function [5]. Though lncRNAs cannot be translated into proteins, they participate in various biological processes, including cell growth and death, cell differentiation, inflammatory response, immunity and tumorigenesis [6,7]. Accumulating research validates that lncRNAs can affect the progression of many tumors [8,9]. LncRNA micro-chromosome maintenance protein 3-associated protein antisense RNA 1 (MCM3AP-AS1) is reported to play an important role in the progression of several kinds of cancers. For example, up-regulation of MCM3AP-AS1 expression in hepatocellular carcinoma and glioblastoma can promote malignant phenotypes of cancer cells [10,11]. However, the detailed function and mechanism of MCM3AP-AS1 in PCa are still unclear.
Belonging to ncRNA as well, microRNAs (miRNAs) regulate a series of physiological and pathological processes, including proliferation, differentiation, apoptotic signal transduction and organ development [12,13]. In recent years, numerous research has shown that miRNAs are involved in the progression of many tumors, and the abnormal expression profile of miRNAs is also related to PCa [14,15]. MiR-876-5p origins from the precursor RNA transcribed from human chromosome 9p21.1, whose expression in breast cancer tissues and gastric cancer tissues is markedly down-regulated, and it functions as a tumor suppressor in these cancers [16,17]. However, the role of miR-876-5p in the progression of PCa needs further study.
As a member of Wnt protein family, WNT5A can regulate several biological processes, including cell differentiation, proliferation, migration, apoptosis and so on [18,19]. Recent scientific reports demonstrate that WNT5A facilitates malignant phenotypes of cancer cells [20][21][22]. In PCa, it boosts the bone metastasis of cancer cells [21,22]. It is indicated that WNT5A is involved in the progression of PCa, but its upstream regulatory mechanism needs to be further explored.
Interestingly, bioinformatics analysis suggested that there were potential binding sites between MCM3AP-AS1 and miR-876-5p, miR-876-5p and the 3′UTR of WNT5A. These computational predictions suggested that there exited a possible regulatory mechanism among MCM3AP-AS1, miR-876-5p and WNT5A. In the study, we confirmed the expression of MCM3AP-AS1 was upregulated in PCa tissues and that MCM3AP-AS1 could facilitate PCa cell proliferation and inhibit apoptosis through regulating miR-876-5p/WNT5A axis. This work furnished a new theoretical basis for diagnosis and treatment of PCa.

Clinical tissue specimens
Thirty patients with prostate cancer (who underwent prostatectomy in the Affiliated Hospital of Qingdao University from April 2017 to March 2018) were enrolled, and PCa tissues were obtained during surgery. In the control group, the specimens were from adjacent tissues of the same patient (at least 3 cm away from the cancerous tissues), and no cancer cells were found by pathological examination after operation. All specimens were immediately removed and stored in liquid nitrogen at − 196 ℃ for the following experiments. Equipped with the informed consents of all patients involved, our study was endorsed by the Research Ethics Committee of the Affiliated Hospital of Qingdao University.

Cell culture and transfection
Human prostate cancer cell lines (C4-2, PC-3, LNCaP, DU145, and 22Rv1) were available from the Institute of Basic Medicine, Peking Union Medical College. Noncancerous stromal cell line of the prostate PrSC cell was purchased from the China Center for Type Culture Collection (Wuhan, China). All of the cells were cultured with Dulbecco's Modified Eagle's Medium (DMEM, Invitrogene, Carlsbad, CA, US) containing 10% fetal bovine serum (FBS, Invitrogene, Carlsbad, CA, US) in incubator at 37 ℃ in 5% CO 2 and saturated humidity.

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
100 mg tissue or 1 × 10 6 cells were collected, and cells were lysed with TRIzol reagenet (Invitrogene, Carlsbad, CA, US) and total RNA was extracted. After modulating the concentration of RNA to 300-500 ng/μL, RNA was reversely transcribed by reverse transcription kit (Invitrogene, Carlsbad, CA, US) to produce cDNA. Next, RT-PCR was performed using SYBR Green Premix Ex Taq II (TaKaRa, Dalian, China) in accordance with the manufacturer's protocol. GAPDH was regarded as the standard internal reference of MCM3AP-AS1 and WNT5A. Besides, U6 was used as the standard internal parameter of miR-876-5p. Relative quantitative method 2( −ΔΔCt ) was utilized to calculate the results. The experiment was repeated three times independently. The specific primer sequence information was listed in Table 1.

Cell counting Kit-8 (CCK-8) assay
LNCaP and PC-3 cells transfected for 24 h were inoculated in 96-well plates, and the cell concentration was regulated to 5 × 10 3 /mL. After the cells were cultured for 1, 2, 3 and 4 d, respectively, 10 μL enhanced CCK-8 kit (Dojindo, Tokyo, Honshu, Japan) was added, with which the cells were incubated for 2 h at 37 ℃. Ultimately, the absorbance of the cells at 450 nm was measured using a microplate reader (Thermo-Fisher Scientific, Massachusetts, US). The experiment was repeated three times independently.

Ethynyl-deoxyuridine (EdU) assay
PCa cells were inoculated into 24-well plates at the density of 5 × 10 4 cells per well, and cultured for 24 h. Next, 200 μL Edu solution (50 μM) was added to each well and incubated at 37 ℃ for 2 h. Then cells were fixed with 4% paraformaldehyde for 30 min, and after adding 0.5% Triton X-100, the cells were incubated for 20 min. Moreover, cells were stained with Apollo643 and Hoechst 33342 for 30 min, respectively. At last, the pictures were captured by fluorescence microscopy (Olympus, Tokyo, Japan). The experiment was repeated three times independently.

Flow cytometry analysis
FITC-annexinV/PI double stain method was used to detect the apoptosis of PCa cells. FITC-AnnexinV/Apoptosis Kit (Invitrogen, Waltham, MA, USA) was used in this experiment. In brief, LNCaP and PC-3 cells in the logarithmic growth phase were harvested and then resuspended in annexin-binding buffer and stained with FITC-annexinV solution and PI solution for 15 min at room temperature in dark. Then, after diluted in Annexin binding buffer, the stained cells were detected using a flow cytometer AccuriC6 (BD Biosciences, Franklin Lakes, NJ, USA). Flowjo software (TreeStar, SanCarlos, CA, USA) was applied to analyze the percentage of apoptotic cells. The experiment was repeated three times independently.

Luciferase reporter assay
The binding relationships were detected by dual luciferase reporter assay system (Promega, Madison, WI, USA). The target fragments of wild type (wt) MCM3AP-AS1 and mutant (mut) MCM3AP-AS1 were constructed and integrated into pGL3 vector (Promega, Madison, WI, USA) to obtain pGL3-MCM3AP-AS1-wild type and pGL3-MCM3AP-AS1-mutant reporter vectors. HEK-293T cells were then inoculated on 24-well plates with 10 5 cells per well. MiR-876-5p mimics or negative controls were co-transfected into the cells with wt-reporter or mut-reporter vectors, respectively. The culture was continued at 37 ℃ for 48 h prior to the measurement of luciferase activity in each group. The experiment was repeated three times independently.

Western blot
The cells were lysed on ice for 30 min with RIPA lysate (Beyotime Biotechnology, Shanghai, China). The supernatant was collected after centrifugation for 14,000 rpm for 15 min at 4 ℃ in a cryogenic centrifuge. BCA kit (Thermo, Shanghai, China) was used to detect the protein concentration. Moreover, the protein loading was modulated according to the protein quantification results, and the protein samples were electrophoresed by 10% SDS-PAGE. Next, the proteins were transferred to the polyvinylidene fluoride (PVDF) membrane, which was then blocked with 5% skim milk for 2 h. After membranes were washed with TBST buffer, primary anti-WNT5A antibody (ab174963, abcam, 1:1000), anti-Bcl-2 antibody (ab196495, abcam, 1:1000), and anti-Bax antibody (ab53154, abcam, 1:1000) were added into the membrane respectively, and incubated for 8 h at 4 ℃. Then the PVDF membranes were washed with TBST, and were incubated at room temperature for 1 h with the secondary antibody. After the membranes were rinsed again, color rendering was performed using hypersensitive ECL (Hubei Biossci Biotechnology, Wuhan, China). The experiment was repeated three times independently.

Statistical analysis
Statistical analysis was performed employing SPSS13.0 statistical software (SPSS Inc., Chicago, IL, USA), and the data were shown as mean ± standard deviation (x ± s). Whether the data are normally distributed was examined by One-Sample Kolmogorov-Smirnov test. For normally distributed data, independent sample t test was used to make the comparison between 2 groups. One-way ANOVA test was used to make the  comparison among 3 or more groups. If there was significant difference, Newman-Keuls analysis was used to make the comparison between 2 groups. For skewed distributed data, paired sample Wilcoxon signed rank test was used to make comparison between 2 groups. A significant difference was considered when P < 0.05.

MCM3AP-AS1 was up-regulated in PCa and predicted a poor prognosis
First of all, we examined the expressions of MCM3AP-AS1 in PCa tissues and PCa cell lines by qRT-PCR. The results indicated that the expressions of MCM3AP-AS1 in PCa tissues (fold change = 1.63) and cell lines were significantly higher than that of control group (Fig. 1a, b). Consistently, in The Cancer Genome Atlas (TCGA) data, the expression of MCM3AP-AS1 in PCa was also higher than normal tissue according to the GEPIA database (The red column is the cancerous tissue, and the gray column is the adjacent tissue) (Additional file 1: Figure S1). In addition, in order to detect the relationship between MCM3AP-AS1 expression and prognosis of the patients, we conducted Kaplan-Meier survival analysis with the PCa patients from TCGA. The results unveiled that the disease-free survival of MCM3AP-AS1 high expression group was shorter than that of MCM3AP-AS1 low expression group (Fig. 1c), suggesting that the dysfunction of MCM3AP-AS1 could take part in the progression of PCa.

Knockdown of MCM3AP-AS1 inhibited PCa cell proliferation and promoted apoptosis
To explore the role of MCM3AP-AS1 in PCa, we knocked down MCM3AP-AS1 in LNCaP and PC-3 cell lines (Fig. 2a, b). CCK-8 and EdU assay experiments showed that the proliferation of LNCaP and PC-3 cells decreased significantly after knocking down MCM3AP-AS1 (Fig. 2c-e, Additional file 3). Flow cytometry analysis indicated that knocking down MCM3AP-AS1 promoted cell apoptosis compared with the sh-NC group (Fig. 2f ). Consistently, compared with the control group, Bax expression in MCM3AP-AS1 knockdown group increased markedly, while the expression of Bcl-2 decreased (Fig. 2g), suggesting that MCM3AP-AS1 was involved in the apoptosis of PCa cells. These data further indicated that MCM3AP-AS1 was related to the malignant phenotypes of PCa cells.

Knockdown of MCM3AP-AS1 down-regulated WNT5A in prostate cancer
Previous research authenticates that Wnt5a, as an oncogene, promotes the progression of prostate cancer [21,22]. Then, we explored the regulatory function of MCM3AP-AS1 on Wnt5a in prostate cancer. We firstly detected the expression of WNT5A in PCa tissues and cell lines. As illustrated (Fig. 3a, b), WNT5A mRNA expressions in PCa tissues (fold change = 2.24) and cell lines were significantly up-regulated compared with in adjacent tissues and PrSC cellS. qRT-PCR results showed that the expression MCM3AP-AS1 was positively correlated with that of WNT5A in PCa samples (Fig. 3c). Consistently, Western blot and qRT-PCR indicated that knockdown of MCM3AP-AS1 could decrease the c Disease-free survival period of patients with high and low expression of MCM3AP-AS1 was compared by Kaplan-Meier analysis. P < 0.01 and P < 0.001 were denoted by ** and *** respectively expression of WNT5A on both protein level and mRNA level (Fig. 3d, e). These results implied that MCM3AP-AS1 could promote the progression of PCa by modulating WNT5A.

MCM3AP-AS1 targeted miR-876-5p in prostate cancer
RNA transcripts are reported to function by competitively binding miRNAs [23]. To find out whether MCM3AP-AS1 has interactions with miRNAs, we searched in the LncBase Predicted v.2 and StarBase, and found the miR-876-5p was predicted as a potential target of MCM3AP-AS1 in both tools (Fig. 4a). It was noteworthy that the expression of miR-876-5p in PCa tissues (fold change = 2.85) and cell lines was significantly downregulated (Fig. 4b, c). We then determined the correlation coefficient of miR-876-5p with MCM3AP and validated P < 0.05, P < 0.01, and P < 0.001 were denoted by *, **, and *** respectively the negative relationship between them in PCa samples (Fig. 4d). Moreover, dual luciferase reporter assay showed that the activity of luciferase reporter consisting of MCM3AP-wt could be reduced by miR-876-5p mimics, but the luciferase activity of MCM3AP-mut vector was not affected by the co-transfection of miR-876-5p (Fig. 4e, f ), which validated the targeting relationship between MCM3AP-AS1 and miR-876-5p. Additionally, qRT-PCR showed that down-regulation of MCM3AP in LNCaP and PC-3 cell lines significantly increased the expression of miR-876-5p (Fig. 4g, h). Collectively, all of the data indicated the fact that miR-876-5p was a target of MCM3AP-AS1, and could be negatively regulated by it.

Up-regulation of miR-876-5p inhibited proliferative ability of PCa cells and induced apoptosis
To explore the function of miR-876-5p in PCa, we constructed LNCaP and PC-3 cell lines transfected with miR-876-5p mimics (Fig. 5a, b). CCK-8 and EdU assays displayed that compared with in the control group, the proliferation of LNCaP and PC-3 cells decreased significantly after transfection of miR-876-5p mimics ( Fig. 5c-e). After investigating the effect of miR-876-5p on the proliferation of PCa cells, we studied the regulatory role of miR-876-5p on the apoptosis of PCa cells. Flow cytometry analysis indicated that miR-876-5p mimics promoted the apoptosis of PCa cells compared with the miR-control group (Fig. 5f ). What's more, compared with in the miR-control group, the Bax level in the miR-876-5p mimics group increased markedly, while the Bcl-2 level decreased observably (Fig. 5g). These results clarified that as a tumor suppressor, miR-876-5p was associated with the malignant phenotypes of PCa.

Discussion
An increasing number of research demonstrated that abnormal expression of lncRNA may affect the progression of tumors. For instance, lncRNA GLCC1 promotes colorectal cancer progression and glucose metabolism by regulating c-Myc [24]. Additionally, lncRNA MIAT promotes malignant phenotype of papillary thyroid cancer cells by regulating LASP1 [25]. Intriguingly, lncR-NAs exert an important role in the progression of PCa. For instance, the up-regulation of lncRNA FEZF1-AS1 expression in PCa can promote the proliferation and metastasis of prostate cancer cells through Notch signaling pathway [26]. Besides, LncRNA SNHG12 can boost the development of PCa and is associated with poor prognosis [27]. Functioning as an oncogenic lncRNA, MCM3AP-AS1 is up-regulated in papillary thyroid cancer [28], glioblastoma [11], hepatocellular carcinoma [10]. A recent study reports that the expression of MCM3AP-AS1 was up-regulated in PCa; MCM3AP-AS1 silencing can inhibit proliferation and facilitate the apoptosis of PCa cells by disrupting methylation of the NPY1R promoter to inactivate the MAPK pathway [29]. In this work, we also investigated the role of MCM3AP-AS1 in PCa. Consistently, we demonstrated that MCM3AP-AS1 was up-regulated in PCa tissues and cells; it was found that high expression of MCM3AP-AS1 was correlated with worse prognosis of patients, and MCM3AP-AS1 could regulate the proliferation and apoptosis of PCa cells.
A considerable existing studies find that miRNAs can function as a tumor suppressors or oncogenes. For instance, miR-136 regulates JNK signaling pathway by targeting MAP2K4, thus playing an anti-cancer role in gallbladder cancer [30]. The abnormal expression of miRNAs is also associated with the progression of PCa. MiR-141 regulates the stemness of PCa cells via modulating Rho GTPase family members and stem cell molecules [31]. MiR-498 facilitates proliferation, migration and invasion of PCa cells, and reduces radiosensitivity by targeting PTEN [32]. MiR-487a-3p serves as a tumor suppressor in prostate cancer by targeting CCND1 (cyclin D1, CCND1) [33]. MiR-876-5p has been validate as a tumor suppressor in multiple tumors including breast cancer, gastric cancer, colorectal cancer, head and neck squamous cell carcinoma, liver cancer and so on [16,17,[34][35][36], and its downstream targets includes TFAP2A, MITF, RASAL2, vimentin, DNMT3A and so on [16,17,[34][35][36]. In this study, for the first time, we found that miR-876-5p was remarkably down-regulated in PCa tissues and cells, and could inhibit the proliferation of PCa cells.  6 miR-876-5p targeted WNT5A in prostate cancer samples. a TargteScan datebase predicted WNT5A was as a candidate downstream gene of miR-876-5p. b Dual luciferase reporter assay was used to validate the targeting relationship between miR-876-5p and WNT5A. c The correlation between WNT5A and miR-876-5p in PCa tissues was analyzed. d Western blot was used to detect WNT5A levels in PC-3 and LNCaP cells transfected with mimics and inhibitors of miR-876-5p. P < 0.001 was denoted by ** *  Fig. 7 In LNCaP cells, MCM3AP-AS1 could regulate proliferation and apoptosis by regulating miR-876-5p/WNT5A. c qRT-PCR was used to detect the expression level of MCM3AP-AS1 (a), miR-876-5p (b) and WNT5A (c) in the cells co-transfected with MCM3AP-AS1 plasmid and miR-876-5p mimics. d The proliferation of LNCaP cells was detected by CCK-8 assay. e The proliferation of LNCaP cells was detected by EdU method. f Flow cytometry analysis was used to detect the apoptosis of PCa cells after transfection. g Western blot was used to detect the expression levels of Bcl-2 and Bax. h Graphic Abstract: MCM3AP-AS1 could up-regulate WNT5A via sponging miR-876-5p, thus promoting the proliferation of prostate cancer cells. P < 0.05 was denoted by *, P < 0.01 was denoted by **, and P < 0.001 was denoted by *** WNT5A expression is up-regulated in lung cancer, ovarian cancer, pancreatic cancer, PCa and gastric cancer, and the overexpression of WNT5A is associated with invasiveness, progression and poor prognosis of these tumors [17,[20][21][22]37]. In our study, we confirmed that WNT5A was up-regulated in PCa tissues and cells, which is consistent with previous report on WNT5A expression in PCa [22]. We further demonstrated that miR-876-5p could negatively regulate it, and this targeting relationship has also been reported in gastric cancer [17]. LncRNAs may contain one or more miRNA response elements and can act as endogenous miRNA sponges, which contributes to the downregulation of intracellular miRNA [38,39]. This is also called "competing endogenous RNA (ceRNA)" mechanism. ceRNA mechanism plays crucial roles in cancer progression. For example, in oral squamous cell carcinoma, lncRNA GAS5 up-regulates PTEN by targeting miR-21 and activates AKT signaling pathway to inhibit cancer progression [40]. MCM3AP-AS1 promote the progression of papillary thyroid cancer by regulating the miR-211-5p/SPARC axis [28]. In PCa, lncRNA SNHG7 promotes PCa progression by regulating the miR-324-3p/WNT2B axis [41]. In this study, we explored the interactions among MCM3AP-AS1, miR-876-5p and WNT5A in PCa. We demonstrated that MCM3AP-AS1 targeted miR-876-5p and negatively regulated it, and in turn increased the expression of WNT5A. Our work partly partly explained the mechanism by which WNT5A was dysregulated in PCa.
This study has several limitations. First of all, in vivo experiments are required to demonstrate the following work. Whether other malignant phenotypes of PCa cells such as metastasis are modulated by MCM3AP-AS1 deserves further investigation due to the crucial role WNT5A plays in prostate cancer metastasis [21,22]. Besides, other downstream targets of MCM3AP-AS1 need to be identified. Nonetheless, our study manifest that MCM3AP-AS1 is up-regulated in PCa tissues and cells, and functional experiments demonstrate that MCM3AP-AS1 can promote PCa cell proliferation and inhibit cell apoptosis by controlling the expression of miR-876-5p/WNT5A. MCM3AP-AS1 may be applied as a novel therapy target for treating PCa in the future.