LINC01213 promotes prostate cancer cell progression through miR-597/ BCL2L1 axis

Background: Majority of cancer related deaths in males are attributed to prostate cancer (PRAD) throughout the world. Recently, the role of long non-coding RNAs (lncRNAs) in the pathogenesis of cancer has been widely explored. In this study, we investigated the role of lncRNA LINC01213 (LINC01213) in tumorigenesis of prostate cancer (PRAD). Methods: PRAD and adjacent tissue samples were collected from cancer patients. Survival rate among these patients was compared by Kaplan–Meier analysis. PRAD cells viability was estimated by CCK-8 method while AnnexinV/PI cytometry assay was used to determine the percent of apoptotic cells. qRT-PCR and western blot assay were used to determine the mRNA and protein expressions, respectively. Interaction between LINC01213 and corresponding miRNA as well as between miRNA and mRNA was con�rmed by dual luciferase reporter gene assay. PRAD cells were also injected subcutaneously in nude mice to support in vitro �ndings. Results: It was observed that LINC01213 was highly expressed in PRAD samples and cell lines. Down-regulation of LINC01213 in PRAD cells decreased cell viability and inhibited proliferation. Luciferase reporter gene assay and RNA pull-down con�rmed that LINC01213 targeted miR-597-3p. Increased expression of miR-597-3p resulted in decreased BCL2L2 expression in vitro. Inhibitory effects of miR-597-3p on PRAD cells’ survival and growth were diminished after LINC01213 overexpression which was also associated with alteration in the protein expression of BCL-xL, BCL-2 as well as caspase 3 and caspase 9. Conclusion: Taken together, our �ndings suggest that LINC01213 plays its role in PRAD tumorigenesis through miR-597-3p/ BCL2L2 dependent pathway with associated modulation of genes involved in cell survival and apoptosis.


Introduction
Prostate cancer (PRAD) is one of the leading causes of cancer related deaths throughout the world and the most diagnosed type of cancer in men [1].It has been shown that almost 1.6 million people are diagnosed with PRAD each year [2].The incidence of PRAD has shown interesting differences across the globe.For instance, African-Americans' have the highest incidence of PRAD while the lowest incidence has reported in Asian men [1,3].Moreover, the odds of PRAD diagnosis in less developed countries are 1 in 47 than 1 in 6 in developed ones [4].This discrepancy may be explained by early detection of prostatespeci c antigen (PSA) in develop countries and the differences in lifestyle.Early detection of PRAD might help in the removal of the localized tumor through radiation and surgical removal but may inversely effect the quality of life [5].
The abnormal expression of lncRNA is closely related to the occurrence and development of tumors.
LncRNAs adsorb miRNA through sponging and regulate the expression of downstream genes, thereby, play important roles in the occurrence and development of tumors [6].For instance, MEG3 has been shown to act as tumor suppressor at human pituitary tumor derived cell line [7].LncRNA ADAMTS9-AS2 has been shown to inhibit glioma cell migration [8].LncRNA PVT1 have been shown to regulate the growth and predictor of prognosis in prostate cancer [9].lncRNA UCA1 has been shown to promote to proliferation and tumor metastasis in gastric cancer [10].LncRNA SNHG16 has been shown to promote tumor growth of pancreatic cancer [11].LncRNA H19 has been shown to promote tumor progression in cervical cancer by targeting miR-138-5p [12].
It has been reported that LINC01213 plays a role in promoting melanoma and breast cancer [13,14].
However, no report is available that investigates the role of LINC01213 in prostate cancer.According to the analysis of TCGA database, the expression of LINC01213 in cancer tissues is generally higher than that in normal tissues.lncBASE.2predicts that LINC01213 can target miR-597-3p, and miR-597 has been shown to be involved in a variety of cancer types such as breast cancer [15] and colon cancer [16].miRDB predicts that BCL2L1 is the target gene of mir-597-3p, and BCL2L1(BCL-XL) is a member of BCl2 family, which participates in the regulation of apoptosis.We took the high expression of LINC01213 in prostate cancer as the breakthrough point, and explored by silencing LINC01213 in prostate cancer cell line.

Methods
Cell culture RWPE-1 cells were grown in Keratinocyte Serum Free Medium (Gibco, Invitrogen, USA) supplemented with bovine pituitary extract and human recombinant epidermal growth factor to make the complete medium.
Collection of clinical samples 60 pairs of tissue samples from PRAD patients were collected at Taikang Tongji (Wuhan) Hospital.Samples were collected after surgical removal of PRAD and adjacent tissue samples which were immediately stored at -80°C until further use.All the clinical procedures were in accordance with the declaration of Helsinki and written informed consents were collected from all the participants of the study.All the protocols were reviewed and approved by the committee for human experimentations, Taikang Tongji (Wuhan) Hospital, China.

Cell viability assay
Cell viability was determined by the cell counting kit 8 assay (CCK-8).Brie y, 6 × 10 3 cells were seeded into 96 well cell culture plates and cultured for an overnight.Different treatments were applied and cells were incubated for 24, 48, and 72 h.At the end of incubation period, 10 µL of CCK-8 (Dojindo, Japan) was added to each well and further for 1h at 37 °C.Finally, the absorbance was measured at 450 nm wavelength using Elx800 absorbance reader (BioTek Instruments).

Colony formation assay
This assay was conducted to investigate the proliferation capacity of the cells.For this purpose, PRAD cells transfected or not were seeded onto 24-well plates with 500 cells per well.Cells were cultured for 10 days under standard laboratory conditions.The, cell were xed with 100% methanol (Sigma, USA) and colonies were visualized under laboratory microscope (Olympus, Japan)after staining with 0.1% crystal violet solution (Solarbio, China).

Western blot assay
Collected cells were lysed in ice cold RIPA buffer (Servicebio, China) with added protease inhibitor cocktail (Sigma, USA).Protein content of samples was determined by using BCA assay kit and proteins were separated by electrophoresis in 4%-20% polyacrylamide gels (GenScript).Afterwards, the proteins were transferred to polyvinylidene uoride membranes (Millipore, USA) followed by saturation in 5% skim milk at room temperature.Then, primary antibody (1:500) was added to the membranes and incubated for an overnight at 4°C.Then, the secondary antibodies (1:2000) and incubated at room temperature for 2 h.Protein blots were detected using Enhanced Chemiluminescence Kit (FDbio Science, China).

Flow cytometery assay
Cells were collected after 72 h of transfection or not and washed three times with PBS.Cells were then incubated in dark with FITC-Annexin V and propidium iodide (Liankebio, China) for 15 min.Flow cytometery was performed using FACSCanto ow cytometer (BD, USA) and FlowJo 10.0 software was used to determine the percent of apoptotic cells.

Subcutaneous tumorigenesis assay in nude mice
BALB/c male nude mice weighing approximately 22-25 g were purchased form the Zhejiang Chinese Medical University (Hangzhou, China).Mice were acclimatized for at least a week under standard laboratory conditions.Afterwards, 2 × 10 6 PRAD cells suspended in 200 cells μL of PBS were subcutaneously injected into the left ank of the mice.Tumor xenograft diameter was measured after each week for a total of 4 weeks for determining the tumor volume using V = π/6 × length × width 2 and tumor weight was also determined at the end of this period.All the experimental protocols were approved by the animal ethical committee of Taikang Tongji (Wuhan) Hospital and were in compliance with the ARRIVE guidelines.
Quantitative reverse transcription polymerase chain reaction (RT-qPCR) TRIzol reagent (Invitrogen, USA) was used for the extraction of total RNA from the samples and the RNA concentrations were determined by using nano-drop spectrophotometer.High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scienti c, USA) was used to reverse transcribe RNA to cDNA.This was followed by PCR for 40 cycles of alternate temperatures of denaturation, annealing and extension.Gene expression was analyzed employing SYBR Green PCR master mix (Thermo Fisher Scienti c, USA) using 2 -ΔΔCt method.Product speci city was analyzed by melt-curve analysis and all the experiments were replicated at-least in triplicate.

Luciferase reporter gene assay
Approximately 5 × 10 4 cells were seeded onto a 96-well plate for 24 h followed by transfection/cotransfection for 48 h with Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's guidelines.Luciferase assay kit (Promega, USA) was used to perform the luciferase activity according to the provided protocols.In brief, to a volume of 100 µL containing cells an equal volume of luciferase assay solution was added and incubated for 20 min at room temperature.Microplate reader (Synergy H4 Hybrid Reader, BioTek, Winooski, USA) was used to measure luciferase activities [17].

RNA pull-down assay
Previously described procedures were used for the RNA pull-down assay [18].This assay was conducted according to the previously described procedures.Biotin-labeled bio-LINC01213 probe was provided by Sangon Biotech (Shanghai, China).Cells were collected and lysed.One part of the lysate was kept to be used for input control.The other part was incubated with magnetic Dynabeads M-280 Streptavidin beads (Invitrogen, Carlsbad, CA, USA) beads for an overnight at 4 °C.qRT-PCR assay was used to determine the miR-597-3p enrichment.

Statistical analysis
Statistical analysis of the results was conducted by the GraphPad prism software version 6. Kaplan-Meier analysis was used to compare the survival difference between patient groups.Student's t test was used for two groups while One-way ANOVA was used for the statistical analysis of more than two groups.P values less than 0.05 were considered signi cant.The data represent the mean ± SD of at-least three independent experiments.

Results
LINC01213 is highly expressed in PRAD tissues and cell lines TGCA database was used to analyze the expression level of LINC01213 in PRAD tissue samples.It was observed that LINC01213 had signi cantly increased (P < 0.05) expression in PRAD tissue than the normal controls (Fig. 1A).To further con rm these ndings, 66 pairs of PRAD clinical samples with normal adjacent tissues were collected.qRT-PCR analysis showed that PRAD tissue samples exhibited signi cantly increased (P < 0.01) expression of LINC01213 (Fig. 1B).The median expression value of LINC01213 in PRAD tissue in Fig. 1B was taken as the cut-off value.On the basis of this value 66 PRAD patients were divided into high and low expression groups of LINC01213, each containing 33 patients.Kaplan-Meier survival curve was used to evaluate the overall survival rate of these two groups.It was observed that high expression of LINC01213 was associated with signi cantly less (P = 0.0345) survival than the low expression (Fig. 1C).Furthermore, the expression level of LINC01213 in PRAD cell lines LNCaP, 22Rv1, C4-2, DU145, PC-3 and normal prostate epithelial cell line RWPE-1 was detected by qRT-PCR.It was observed that tumor cell lines exhibited signi cantly increased (P < 0.01) expression of LINC01213 than the normal cells (Fig. 1D).The relationship between LINC01213 expression and clinical pathological data was analyzed by Chi-square test (Table1).Data suggested that LINC01213 expression was closely related to clinical stage, lymph node metastasis and tumor size (P < 0.05) but was independent of the patient's age and gender.
Silencing LINC01213 inhibited the proliferation of PRAD cells and promoted the apoptosis of PRAD cells Two cell lines with the highest expression of LINC01213 in Fig. 1D i.e.LNCaP and PC-3 were selected to establish a stable LINC01213 knock-down and the silencing e ciency > 50% was con rmed by qRT-PCR (Fig. 2A).It was observed that LINC01213 knock-down resulted in signi cantly decreased (P < 0.01) survival of LNCaP and PC-3 as indicated by OD values during CCK-8 assay (Fig. 2B).In addition, LINC01213 knock-down signi cantly reduced the proliferation ability of LNCaP and PC-3 cells during colony formation assay (Fig. 2C) and increased the rate of apoptosis (Fig. 2D).Interesting, LINC01213 resulted in signi cantly reduced (P < 0.01) subcutaneous tumor growth in nude mice both in terms of volume (Fig. 2E) and weight (Fig. 2F) during 28 days of observation.
LINC01213 promotes PRAD through miR-597-3P/BCL2L1 axis To further unveil the mechanism of LINC01213, pcDNA3.1-LINC01213was used to overexpress the LINC01213 and the e cacy was con rmed by qRT-PCR (Fig. 5A).Henceforth, the expression level of BCL2L1 was detected by qRT-PCR in different groups of LNCaP and PC-3 cells (miR-NC, miR-597-3p mimics, miR-597-3p mimics+pcDNA3.1-LINC01213).The results showed that overexpression of miR-597-3p decreased the expression level of BCL2L1, while co-transfection pcDNA3.1-LINC01213restored the expression of BCL2L1 in these cells (Fig. 5B).Changes in cell viability were determined by CCK-8 assay which showed that overexpression of miR-597-3p reduced PRAD cell viability and this effect was curtailed after LINC01213 overexpression in these cells (Fig. 5C).Similarly, miR-597-3p signi cantly (P < 0.001) reduced the colony formation (Fig. 5D) and increased the rate of apoptosis (Fig. 5E) of LNCaP and PC-3 cells while LINC01213 overexpression reversed these effects.Henceforth, western blot method was employed to investigate the effect of miR-597-3p overexpression on the expression of proteins involved in cell proliferation and apoptosis.It was observed that miR-597-3p overexpression reduced the protein expression of BCL-xL and BCL-2 while the protein expression of cleaved-caspase3 and cleaved-CASPASE9 were signi cantly increased in LNCaP and PC-3 cells (Fig. 5F).In contrast, the protein expression of caspase3, caspase9 as well as GAPDH remained unaffected (Fig. 5F).

Discussion
It was observed that LINC01213 played a crucial in the pathogenesis of PRAD as it was highly expressed on the PRAD clinical samples as well as on cell lines.We used mechanistic approach in this study which revealed that LINC01213 could exert its role on PRAD cells through miR-597-3p and BCL2L1 dependent pathway.Tumor inhibiting properties of miR-597-3p have been previously reported by Wen et al. who have shown that this miRNA could inhibit the proliferation and invasion of thyroid carcinoma SW579 cells by modulating the expression of RAB23 [19].In the current report, miR-597-3p overexpression adversely a affected the survival and proliferation of PRAD cell lines which is in corroboration of the report by Wen et al.
Our results indicate that LINC01213 targeted BCL2L1 protein and also modulated the expression of other proteins involved in cell survival and apoptosis.BCL2L1 gene has been previously implicated in the pathogenesis of various cancers [20,21].For instance, Sillars-Hardebol et al. have reported that BCL2L1 played a functional role in colorectal cancer [22].Yang et al. have shown that lncRNA LINC02595 promoted colorectal cancer progression through the inhibition of miR-203b-3p and thereby upregulating BCL2L1 expression [23].Another study by Ostadrahimi et al. has shown that BCL2L1 gene was upregulated in PRAD tissue samples and cell lines which is also in corroboration of the current ndings [24].BCL2 and BCL-xL are the members of BCL-2 family of proteins that are known to exert anti-apoptotic effects and promote cell survival [25].In contrast, Caspase 3 and caspase 9 belong to the caspase family of cysteine proteases that have been implicated in apoptosis.

Table 1 :
Brentnall et al. have previously reportedthat caspase 3 and caspase 9 play distinct roles during intrinsic apoptosis.Hence, caspase 9 can be involved in initiation of apoptosis while caspase 3 plays a role in execution[26].It was observed that miR-597-3p overexpression in PRAD cell lines decreased the protein expression of anti-apoptotic factors (BCL2 and BCL-xL) and increased the expression of pro-apoptotic agents (caspase 3 and caspase 9), thereby, promoting apoptosis that was manifested by reduced survival and proliferation of PRAD cells lines.In contrast, LINC01213 overexpression in these cells reversed the effects of miR-597-3p overexpression in these cells by increasing the expression of anti-apoptotic factors.A previous study byZhang etal.has shown that miR-608 suppressed the progression of prostate cancer by targeting the BCL2L1/caspase-3 pathway.Another report by Rachel et al. has suggested that caspase-3 protein expression was lost in human prostate cancer which served to promote the prostate tumorigenesis [27].Rodríguez-Berriguete et al. have shown that immunoreactivity to caspase-3 and caspase-9 is frequently lost in human prostate tumors which further strengthen our ndings [28].Taken together, our ndings suggest that LINC01213 modulates the progression of PRAD through the modulation of miR-597-3p/ BCL2L1 dependent pathway.Furthermore, LINC01213 may serve as the biomarker of PRAD and also a prognostic indicator of the disease.We propose LINC01213 as a novel target for future RNA based therapeutic approaches for the treatment of PRAD.Rodriguez-Menocal L, De Guevara RL, et al.Caspase-9, caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis.BMC Cell Biol 2013;14:32.27.Winter RN, Kramer A, Borkowski A, et al.Loss of caspase-1 and caspase-3 protein expression in human prostate cancer.Cancer Res 2001;61:1227-32. 2 .Rodríguez-Berriguete G, Galvis L, Fraile B, et al.Immunoreactivity to caspase-3, caspase-7, caspase-8, and caspase-9 forms is frequently lost in human prostate tumors.Hum Pathol 2012;43:229-37.The relationship between LINC01213 expression and clinical pathological data