Effect and Mechanism of LncRNA-PBC11 in Regulating EMT Progression of Prostate Cancer Cells

The invasion and metastasis of prostate cancer is the main reason for its poor prognosis. Up to now, ADT (androgen deprivation therapy, ADT), whether surgical or drug therapy, is the basic method for the treatment of prostate cancer. However, ADT therapy is generally not recommended for the clinical treatment of metastatic prostate cancer. EMT (epithelial-to-mesenchymal transition, EMT) of tumor cells is the internal driving factor of tumor invasion and metastasis. Therefore, in-depth study on the regulation mechanism of EMT in prostate cancer cells will help us develop the new prevention and treatment plan for prostate cancer. Here, we will demonstrate and elaborate an lncRNA-PBC11 closely related to EMT of prostate cancer cells. The research data show that the content of PBC11 in metastatic prostate tumors increases signi�cantly compared to non-metastatic, and can competitively bind hsa-miR-137 with 3'UTR of Twist1 in the form of miRNA sponge, thereby weakening the antitumor activity of hsa-miR-137. Our functional experiments con�rmed that PBC11 knockdown is an important means for the normal operation of EMT inhibitory pathway hsa-miR-137/Twist1 in prostate cancer cells, and PBC11 knockdown combined with hsa-miR-137 overexpression showed excellent resistance to EMT in prostate cancer cells induced by TGF-β1. In a word, our study shows that PBC11 up-regulates the expression of Twist1 protein by blocking hsa-miR-137/ Twist1 pathway and promotes the progress of EMT in prostate cancer cells, overexpression of PBC11 can effectively inhibit EMT in prostate cancer cells by restoring the negative regulation of Twist1 by hsa-miR-137.


Introduction
In recent years, prostate cancer has the second highest incidence of the global male population rate in all tumor types.Clinical statistics show that the incidence rate of prostate cancer is increasing rapidly among men over 50 years old, and the mortality rate is signi cantly higher in patients over 85 years old.
Thanks to the improvement of early screening and the development of treatment methods, the overall survival rate of prostate cancer patients has been signi cantly improved [1][2][3].Even so, the treatment of MPC (metastatic prostate cancer, MPC) is a major challenge facing the medical community, which is more severe due to the aging of the population and the improvement of the diagnosis rate.At present, the treatment of prostate cancer is mainly through inhibiting androgen receptor activity.The standard treatment for hormone sensitive prostate cancer is ADT (androgen deprivation therapy, ADT) [4][5].
Although more than 80% of prostate cancer patients respond to ADT, a large number of patients are prone to side effects.Although a variety of new therapies and drugs have been used for the treatment of advanced and metastatic prostate cancer, but it will develop into CRPC (castration resistant prostate cancer, CRPC) after an average of 24 months [6].Serum PSA can be used as a response index for prostate cancer treatment, but it cannot predict the development of CRPC according to the serum PSA value [7].Therefore, the identi cation of new potential biomarkers and therapeutic targets is very important to improve and replace the existing clinical therapies for prostate cancer.Here, we report for the rst time that the lncRNA-PBC11 has a positive correlation with prostate cancer metastasis.The mechanism study shows that PBC11 can inhibit the tumor suppressor pathway hsa-miR-137/Twist1 in the form of miRNA sponge, and its knockdown can effectively inhibit EMT of prostate cancer cells induced by TGF-β1 by restoring the function of hsa-miR-137.Hsa-miR137 overexpression combined with PBC11 knockdown has more advantages than PBC11 knockdown in combating EMT progression of prostate cancer cells.We believe that this study will not only further enrich the molecular mechanism of prostate cancer malignancy, but also provide a strong theoretical support for hsa-miR137 and PBC11 as the new biological targets for prostate cancer patients.

Cell culture
Prostate cancer cells LNCaP and PC3 purchased from National Collection of Authenticated Cell Cultures (Shanghai, China) were maintained in F-12K medium (Thermo Fisher, CA, USA) supplemented with 10% fetal calf serum (FBS, Thermo Fisher).The lentiviral packaging cell line, 293T purchased from American Type Culture Collection (VA, USA) were maintained in Dulbecco's modi ed Eagle's DMEM medium (Dulbecco's modi ed Eagle's medium, DMEM, Thermo Fisher) supplemented with 10% FBS.All adherent cells were passaged by digestion with 0.25% trypsin (Thermo Fisher) once the cells reached a density of 70%, and were cultured in an atmosphere with saturated humidity at 37°C containing 5% CO2.
Tumor tissues 24 pairs of metastatic and non-metastatic prostate tumors with their precancerous lesions were collected from 24 prostate patients (12 cases of each group) in the Department of Jinshan Hospital of Fudan University.There was no age limit for enrollment.Informed consent was obtained from all patients in accordance with the standards set by the Ethics Committee of the Jinshan Hospital of Fudan University.Clinicopathological information pertaining to the patients and tumor classi cation is provided in Table 1.
Total RNA extracted from the tissues was used for the detection of PBC11, hsa-miR-137 and Twist1 mRNA levels by using RT-qPCR (realtime quantitative PCR RT-qPCR).Total protein was extracted from the tissues and used to detect the proteins of Twist1 E-cadherin and Vimentin by western blotting.The detection indexes were used for correlation analysis and evaluation of EMT of tumor cells.
Lentivirus mediated hsa-miR-137 overexpression and PBC11 silencing in prostate cancer cells Human genomic DNA was extracted and used for ampli cation of the precursor of has-miR-137(270bp) with the primers 5′-TGCTACCTTGGCAACCACGGGCG-3′ and 5′-GGGCGGGCTCAGCGAGCAGCAA-3′.The PCR product was digested and ligated to expression vector to construct pCDH-miR-137.An siRNA (5'-GCTACTCTCACAGCAGCAC-3') sequence complementary to PBC11 was chosen, and the corresponding shRNA oligonucleotide DNA synthesized were annealed and cloned to construct pSIH1-shRNA-PBC11.An siRNA with a scrambled sequence (5'-ACGACCCGCTTAGCCATAC -3') was used as a NC (negative control, NC) to construct pSIH1-NC.The recombinant vectors were con rmed by DNA sequencing and endotoxin free DNA was prepared in all cases.
A total of 1 × 10 6 293T cells in logarithmic growth phase were plated in 10-cm dishes in 10 ml DMEM supplemented with 10% FBS and cultured overnight under normal conditions.Recombinant vectors (2μg each of pCDH-miR-137, pSIH1-shRNA-PBC11 or pSIH1-NC) and 10 μg of the pPACK packaging plasmids (System Biosciences, CA, USA) were co-transfected into 293T using Lipofectamine 2000 transfection reagent (Thermo Fisher).The culture medium was completely replaced with DMEM containing 1% FBS prior to transfection.After 48 h of transfection, the supernatant was harvested and cleared by centrifugation at 5,000 × g for 10 min at 4°C and then passed through a 0.45 µm polyvinylidene di uoride membrane (Millipore, MI, USA).The viral titer was determined using a gradient dilution method.The recombinant lentiviruses were named Lv-miR-137, Lv-shRNA-PBC11 and Lv-NC, respectively.
LNCaP and PC3 cells in logarithmic phase were plated in 6-well plates at a density of 2×10 5 cells/well.One day later, lentiviruses (Lv-NC, Lv-miR-137 and Lv-PBC11) were added to the cells at an MOI (multiplicity of infection, MOI) of 10.The infection e ciency was evaluated by observing the uorescence of GFP (green uorescent protein, GFP) 72 hour after infection.Total RNA were isolated and subjected to RT-qPCR to analyze the levels of hsa-miR-137 and PBC11.

Cell proliferative, invasion and migration assay
LNCaP and PC3 cells were used for cell viability assay by using a cell counting kit-8 assay kit (CCK-8, Dojindo, Japan).LNCaP cells were used for invasion assay by using the QCMTM 24-well Fluorimetric Cell Invasion Assay kit (Millipore) according to the manufacturer's instructions.Migration ability of PC3 cells was assessed using wound healing assay.The cells used in the experiments were infected with the lentiviruses (Lv-NC or Lv-miR-137 or Lv-shRNA-PBC11 or Lv-miR-137+ Lv-shRNA-PBC11) for 72huors,then EMT model was established by adding TGF-β1(5ng/ml) for 48 hours.

RIP (RNA Binding Protein Immunoprecipitation, RIP)
We identi ed the binding of Twist1 to PBC11 in LNCaP cells by RIP experiment.Twist1 protein in cells was precipitated by IP and its bound RNA was obtained from eluent.Then, the target product (PBC11) in the puri ed product was qualitatively detected by RT-PCR.The speci c scheme of RIP refers to Ahmad m Khalil's research [8].The 2μg Twist1 primary antibody (Abcam, Cambridge, UK) was used for IP, and the PCR primer sequence used for target product identi cation is 5'-AACCACGTGCCCCACAGCGG-3' (forward) and 5'-CTCTGGTGGATGCTGGTTATT-3'(reverse).

Statistical analysis
The data are shown as the means ± standard deviation (SD) of three independent experiments.All statistical data were analyzed using SPSS GradPack version 20.0 statistical software (IBM Corp., Armonk, NY, USA) and GraphPad Prism 7.0 (GraphPad Software, Inc., La Jolla, CA, USA).Comparisons between groups were performed using a two-tailed Student's t-test or one-way ANOVA with a post-hoc Tukey test.Differences were considered statistically signi cant when p < 0.05.

Data availability
The data that support the ndings of this study are available from the corresponding author upon reasonable request.

Results
Twist1 protein is highly expressed in metastatic prostate tumors which is attributed to abnormal posttranscriptional processing RT-qPCR data showed that the mRNA level of Twist1 was higher in tumors than that in its precancerous lesions(P 0.05 vs. precancerous lesions), but there was no signi cant difference between metastatic and non-metastatic tumors (P 0.05 vs. non-metastatic tumors.Fig. 1A).Analysis of western blotting showed that Twist1 was higher in tumors than that in its precancerous lesions(P 0.05 vs. precancerous lesions), and but there was a signi cant difference between metastatic and non-metastatic tumors (P 0.01, vs. non-metastatic tumors.Fig. 1B).Bioinformatics analysis identi ed a seven-base hsa-miR-137 seed sequence 5'-GCAAUAA-3' in the 3'-UTR of Twist1 mRNA (Fig. 1C).Luciferase experimental data showed that both pGL3-wt-twist1 and pGL3-mt-twist1 could signi cantly up regulate the luciferase activity in 293T cells 48 hours after transfection (P< 0.01, vs 293T cells).Hsa-miR-137 overexpression and inhibition (via transfection of hsa-miR-137 mimics or inhibitor) can only inhibit the increase of luciferase in 293T cells caused by pGL3-wt-twist1(P< 0.01, vs 293T cells transfected with pGL3-wt-Twist1), but have no signi cant effect on the increase of luciferase in 293 cells caused by pGL3-mt-Twist1(P 0.05, vs 293T cells transfected with pGL3-mt-Twist1).Co-transfection of hsa-miR-137 NC had no effect on the luciferase activity of either of the two vectors (p > 0.05, vs. 293T cells transfected with pGL3-wt-Twist1 or pGL3-mt-Twist1) (Fig. 1D).RT-qPCR data also showed that the level of hsa-miR-137 was lower in tumors than that in its precancerous lesions(P 0.05 vs. precancerous lesions), but there was no signi cant difference between metastatic and non-metastatic tumors (P 0.05 vs. non-metastatic tumors.Fig. 1E).Comprehensive analysis data revealed that in the non-metastatic tumors, the hsa-miR-137 expression was negatively correlated with that of Twist1 protein.However, in metastatic tumors, there was no expression correlation between them (Fig. 1F).
Hsa-miR-137 loses its negative regulation on Twist1 is related to the high expression of PBC11 in metastatic prostate tumors RT-qPCR data showed that the level of PBC11 was the highest in metastatic prostate tumors(P 0.01 vs. the other three groups), and there was no signi cant difference between non-metastatic prostate tumors and the two precancerous lesions(P 0.05.Fig. 2A).Comprehensive analysis data revealed that in the nonmetastatic tumors, the PBC11 expression was positively correlated with Twist1 protein in both metastatic and non-metastatic prostate tumors (Fig. 2B).Bioinformatics analysis indicated the existence of six 7base hsa-miR-137 binding sites in the PBC11 (Fig. 2C).The results of the luciferase assay showed that hsa-miR-137 mimics lost their inhibitory effect on luciferase activity caused by of the pGL3-wt-Twist1 in 293T cells following the transfection of pcDH-PBC11 (Fig. 2D).RIP was used for analyze the direct binding of Twist1 to PBC11, and a negative result was obtained (Fig. 2E).
Lentivirus mediated hsa-miR137 overexpression and Twist1 gene silencing in prostate cancer cell lines

LNCaP and PC3
After being infected with lentiviruses for 72 hours, the results of cell counting showed that the majority of cells in the same eld of vision have the GFP expression, indicating that the lentivirus infection e ciency of the two cells was close to 100% (Fig. 3AB, upper).Data of RT-qPCR showed that hsa-miR-137 level signi cantly increased in the Lv-miR-137 group (p < 0.01 vs. cells or Lv-NC group or Lv-shRNA-PBC11 group), and there was no signi cant difference between the cells, Lv-NC and Lv-shRNA-PBC11 groups(P 0.05).Data of RT-qPCR also showed that PBC11 level signi cantly decreased in the Lv-shRNA-PBC11 group (p < 0.01 vs. cells or Lv-NC group or Lv-miR-137 group), and there was no signi cant difference between the cells, Lv-NC and Lv-miR-137 groups (P 0.05) (Fig. 3A B, down).

PBC11 knockdown can ensure the effect of hsa-miR-137 on the inhibiting of EMT in prostate cancer cells induced by TGF-β1
Cell proliferation assay data showed that induction of TGF-β1 signi cantly enhanced the proliferation of LNCaP and PC3 cells with a signi cant difference (P 0.05 vs. cells not treated).The overexpression of hsa-miR-137 slightly suppressed the proliferation of LNCaP and PC3 cells treated with TGF-β1 but there was no signi cant difference between them (P 0.05 vs. cells treated with TGF-β1).The silence of PBC11 suppressed the proliferation of LNCaP and PC3 cells induction by TGF-β1 with a signi cant differences (P 0.05 vs. cells treated with TGF-β1).Compared with overexpression of hsa-miR-137 or silence of PBC11, overexpression of hsa-miR-137 combined silence of PBC11 could signi cantly inhibit the proliferation activity of tumor cells under TGF-β1 induction (P 0.01 vs. cells treated with TGF-β1,Fig.4A).
Data of cell invasion assays showed that the invasive ability signi cantly enhanced in LNCaP cells under TGF-β1 induction(P 0.01 vs. cells not treated).The overexpression of hsa-miR-137 or silence of PBC11 slightly suppressed the invasion of LNCaP cells treated with TGF-β1,but there was no signi cant difference between groups (P 0.05 vs. cells treated with TGF-β1).Compared with overexpression of hsa-miR-137 or silence of PBC11, overexpression of hsa-miR-137 combined silence of PBC11 could signi cantly inhibit the invasion of tumor cells under TGF-β1 induction (P 0.01 vs. cells treated with TGF-β1.P 0.05 vs. cells treated with TGF-β1 with infection of Lv-miR-137 or Lv-shRNA-PBC11.)(Fig. 4B).The cell migration detection data in PC3 cells showed that the change of cell migration in each treatment groups was completely consistent with the invasion of LNCaP cells (Fig. 4C).
The progression of prostate cancer metastasis is related to EMT of tumor cells and PBC11 silencing combined with has-miR-137 overexpression can effectively inhibit TGF-β1 induced EMT in LNCaP cells Western blotting result showed that expression of E-cadherin was low in non-metastatic tumors than that in its precancerous lesions but there was no signi cant difference between them (P 0.05 vs. precancerous lesions ), was lower in metastatic tumors than that in its precancerous lesions with a signi cant difference(P 0.01 vs. precancerous lesions),and was lower in metastatic tumors than that in non-metastatic tumors with a signi cant difference(P 0.01 vs. non-metastatic tumors ).The results of western blot also showed that the expression of Vimentin in each group was completely opposite to that of E-cadherin.Fig. 5A).RT-qPCR results showed that TGF-β1 induction for 48 hours had no signi cant effect on the content of hsa-miR137 in LNCaP cells (P 0.05 vs. cells not treated), while Lv-miR-137 or Lv-miR-137 combined Lv-shRNA-PBC11 infection could signi cantly up-regulate its levels in LNCaP cells induced by TGF-β1 (P 0.01 vs. cells induced by TGF-β1).Induction of TGF-β1 for 48 hours signi cantly increased PBC11 level in LNCaP cells (P 0.05 vs. cells not treated), and hsa-miR-137 overexpression had no signi cant effect on the content of PBC11 in NCaP cells induced by TGF-β1 (P 0.05 vs. cells induced by TGF-β1), hsa-miR-137 decreased signi cantly in Lv-miR-137 combined Lv-shRNA-PBC11 infection and TGF-β1 induced cells (P 0.05 vs. cells induced by TGF-β1 group or cells induced by TGF-β1+Lv-miR-137 infection group)(Fig.5B).The results of western blotting also showed that compared with the cell group, the expression of Twist1 could be signi cantly up-regulated by TGF-β1 induction for 48 hours (P 0.01 vs. cells not treated), while Lv-miR-137 or Lv-NC infection had no effect on Twist1 protein in LNCaP cells induced by TGF-β1(P 0.05 vs. cells induced by TGF-β1).The expression of Twist1 signi cantly decreased in tumor cells with Lv-miR-137 combined Lv-shRNA-PBC11 infection and TGF-β1 induction.The protein detection data also showed that the expression of EMT factors N-cadherin and Akt in the downstream regulated by Ttwsit1 were completely consistent with Twist1, while the expression of EMT inhibitor E-cadherin was opposite to Twist1 (Fig. 5C).

Discussion
Thanks to the medical innovation and new drug development, the mortality rate of prostate cancer has declined in recent years, but its incidence rate is still increasing signi cantly worldwide.A statistic in 2018 shows that prostate cancer has become the most common male cancer in 20 countries and regions around the world, and the mortality of prostate cancer patients in ve regions has risen to the second of tumor types [9][10].Up to now, there is no speci c treatment for prostate cancer.Postoperative metastasis and recurrence is an important reason for the increase of mortality in prostate cancer patients [11].Under normal circumstances, metastasis is common in the middle and late stage of tumor progression, but different from other tumors, some patients with prostate cancer have been found to have tumor metastasis in the early stage of disease progression, and half of them have been found to have lymph node metastasis in the rst diagnosis.Distal bone marrow metastasis is the most likely type of prostate cancer.More than 70% of prostate cancer patients will be accompanied by bone metastasis, and more than 85% of prostate cancer deaths are pathologically found to be complicated with tumor bone metastasis.However, so far, as no breakthrough has been made in the mechanism research and treatment of prostate cancer metastasis, it has become an important research topic in urology to explore new targets for the treatment of prostate cancer, further study the potential molecular mechanism of malignant proliferation and metastasis of prostate cancer, and then provide scienti c basis for the early diagnosis and prognosis of prostate cancer.AR (Androgen receptor signaling pathway, AR) is a key regulatory pathway for the occurrence and progression of prostate cancer.Studies have shown that it is closely related to the malignant progression and drug resistance of prostate cancer.AR and its downstream oncogenes have also been tried to be studied as targets for the treatment of pre prostate cancer.However, at present, drugs targeting these targets (such as bevacizumab) have not made signi cant clinical treatment progress [12][13].
In recent years, the basic research on prostate cancer has made continuous progress.The role of noncoding RNA in the progression of prostate cancer has been paid more and more attention and studied.MiRNAs (MicroRNA, miR) is a small, noncoding endogenous single stranded RNA that regulates gene expression by binding to the 3'-UTR of target genes (mRNAs), and participates in the processes of cell proliferation, differentiation, apoptosis and metabolism [14].The expression of miRNAs is closely related to the occurrence and development of prostate cancer and has been proved to have the potential to diagnose and predict the prognosis of patients.The latest research shows that the contents of miR-215-5p, miR-93, miR-410-3p and miR-191 in prostate cancer tissues are higher than those in adjacent or normal prostate tissues, and their contents are positively correlated with clinical T stage, pelvic lymph node and bone metastasis.Compared with TNM stage, highly expressed miRNAs can provide additional median OS (overall survival, OS) information [15][16][17][18].MiRNA is involved in prostate cancer progression and regulation in a variety of ways, and many miRNAs have been shown to inhibit prostate cancer progression.Bin Yang et al. reported that the level of miR-139-5p decreased in prostate cancer tissues and prostate cancer cell lines.The transfection of miR-139-5p mimics reduced the proliferation and migration of prostate cancer cells.The mechanism results also showed that miR-139-5p could inhibit the EMT progression of prostate cancer cells by inhibiting the expression of Sox5 [19] Bin Yang et al. found that miR-34a was expressed in prostate cancer tissues, while overexpression of miR-34a could inhibit Wnt/β-catenin pathway by regulating the transcriptional activity of Wnt1, which regulates the proliferation and migration of PC3 cells and promotes apoptosis [20].Compared with normal/benign prostate tissues, miR-128 was proved to be signi cantly reduced in prostate cancer.Exogenous miR-128 inhibited tumor regeneration in a variety of prostate cancer xenotransplantation models.Researchers determined the tumor inhibitory function of miR-128 in prostate cancer by limiting the CSC characteristics mediated by BMI-1 and other central stem cell regulators which has potential signi cance for gene therapy of prostate cancer [21] MiR-137 is located on human chromosome 1, previous studies have shown that it is closely related to the development of prostate cancer.MiR-137 has been proved to be able to participate in the EMT regulation of prostate cancer cells exposed to hypoxic by targeting LGR4 [22] Qi Quan Wu et al. have shown that miR-137 can effectively inhibit glycolysis in prostate cancer by down regulating NOX4, so it may be a potential theoretical target for the treatment of prostate cancer [23].In addition, miR-137 was also con rmed to be related to the mechanism of drug resistance in prostate cancer.Prostate cancer is usually treated with hormones, including bicalutamide.Although this treatment was initially effective, the tumor eventually gained resistance, resulting in CRPC recurrence.Preparation studies showed that methyl CpG binding protein 2 (MeCP2) and DNA methyltransferase (DNMT) synergistically promoted the methylation of miR-137 promoter, thereby reducing transcription, resulting in enhanced TRIM24 expression and glutamine metabolism.These ndings describe a new mechanism affecting TRIM24 deregulation in human cancer and provide a molecular link between miR-137, TRIM24 and tumor proliferation in CRPC [24].All these studies indicate that the decrease of hsa-miR-137 is the important reason for the progression of prostate cancer and its drug resistance which is consistent with our research data, and miR-137 has obvious tumor suppressor gene properties in the progression of prostate cancer metastasis.Compared with these studies, we found that hsa-miR-137 has lost its antitumor activity and cannot be attributed to the decrease in its content in metastatic prostate tumors.Speci c research data showed that hsa-miR-137 lost negative regulation of the expression of its target gene Twist1, an prostate tumor metastasis promoting factor in metastatic tumors.So, what causes the functional inactivation of hsa-miR-137 in the progression of prostate cancer?LncRNA (long noncoding RNA, lncRNA) directly targets and regulates DNA, RNA and protein targets by competing with endogenous RNA.Studies have shown that the expression of NEAT1 (nuclear rich transcription factor 1, NEAT1) and SNHG7 (small nucleolar RNA host gene 7, SNHG7) are increased in prostate cancer tissues, which are closely related to GS and TNM stages of prostate cancer, and the highly expressed lncRNA is an independent risk factor for the prognosis of prostate cancer patients [25][26].With the development of transcriptome sequencing analysis, many lncRNAs have been found to be associated with prostate cancer [38,39].PCA3 is a well-de ned lncRNA, which has been approved by FDA for the clinical decision of repeated biopsy of prostate cancer.It is the rst urine detection marker that can be used for the diagnosis of prostate cancer [27][28].Studies have shown that PCA3 is not expressed in normal and malignant non prostate derived tissues, including urogenital organs such as kidney, bladder, eminal vesicle and testis.The expression of PCA3 increased only in patients diagnosed with prostate cancer, rather than BPH (benign prostatic hyperplasia, BPH), PIN (prostatic intraepithelial neoplasia, PIN), ASAP (atypical small acinar hyperplasia, ASAP), in ammation or chronic prostatitis [29][30].These studies show that the functional study of lncRNA plays an important role in the clinical treatment and diagnosis of prostate cancer.
In this study, we found and reported for the rst time that lncRNA-PBC11, which is closely related to the invasion and metastasis of prostate cancer, is located on chromosome 10 of human genome.PBC11 content in prostate cancer tissue is signi cantly higher than that in adjacent tissues, and is signi cantly higher in metastatic prostate cancer tissue than that in non-metastatic tumor tissue.Mechanism studies showed that PBC11 could inhibit the negative regulation of hsa-miR-137 on the expression of its target protein Twist1 in the form of miRNA sponge, which was completely consistent with the results of detection and correlation analysis of the expression of hsa-miR-137 and its target protein Twist1 in clinical samples.The data showed that the content of hsa-miR-137 and the expression of Twist1 protein maintained a negative correlation only in non-metastatic prostate cancer tissues, in metastatic prostate cancer tissues, although the content of hsa-miR-137 did not change signi cantly, the expression of Twist1 was signi cantly up-regulated, and the two of them no longer had a negative correlation, indicating that hsa-miR-137 lost its antitumor activity in metastatic prostate cancer tissues.
In the functional experiment, the EMT model of prostate cancer cell was established by induction of TGFβ 1 in vitro, and the combined treatment scheme for suppression of EMT in prostate cancer cells was discussed with hsa-miR-137 and PBC11 as targets.The results showed that hsa-miR-137 overexpression and PBC11 silencing alone were not enough to effectively inhibit EMT induced by TGF-β1 in prostate cancer cells, but hsa-miR-137 overexpression combined with PBC11 silencing showed a great inhibitory effect on EMT with the speci c manifestation of the inhibition of proliferation, invasion and migration of prostate cancer cells under induced conditions by TGF-β1.This is obviously the result of PBC11 knockdown releasing the inhibition of hsa-miR-137 on Twist1 expression, that is, restoring the operation of hsa-miR-137/Twist1, an inhibition pathway of prostate cancer invasion and metastasis.In short, bene t from knockdown of PBC11, hsa-miR-137 inhibited the expression of Twist1 and downstream proteins N-cadherin and AKT1(EMT promoting proteins), and up-regulated the expression of E-cadherin (EMT inhibitory protein) by the retrieve of anti-cancer function.
Our data also show that TGF-β1 induction had no signi cant effect on the content of hsa-miR-137 in prostate cancer cells, but signi cantly increased the relative content of PBC11, indicating that PBC11 has the potential to become a clinical marker for screening the risk of invasion and metastasis of prostate cancer.Of course, the research on screening markers still needs a lot of attempts and innovation of detection methods, which will be an important direction and future works for us.The prostate cancer staging system adopts the TNM staging system developed by the American Joint Committee on Cancer Staging (8th edition). Figures

Figure 4 Effect
Figure 4

Figure 5 Analysis
Figure 5

Table 1
Clinicopathological features of 24 Prostate patients