Human ovarian tissue specimens

Totally 10 pairs of high-grade serous ovarian carcinoma tissues and adjacent non-tumor tissues were obtained from patients undergoing surgery at Ruijin Hospital, Shanghai Jiaotong University School of Medicine from 2022 to 2024. The fresh tissue samples were collected immediately after surgery and stored in preservation buffer at -80℃. Patients didn’t receive chemotherapy or radiotherapy before operation. Informed consent was obtained from each patient, and the use of clinical samples in this study was approved by the Ruijin Hospital, Shanghai Jiaotong University School of Medicine institutional review board.

Cell lines and cell culture

Human ovarian cancer cell lines SK-OV-3 and A2780 cells, human immortalized ovarian surface epithelial cell line IOSE80, and human embryonic kidney 293 T (HEK293T) cell line were obtained from the American Type Culture Collection (ATCC, Manassas, USA). Human ovarian cancer cell line HO8010 was purchased from the Type Culture Collection Centre of Chinese Academic of Science (Shanghai, China). Cells were maintained in high glucose DMEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% (v/v) Fetal bovine serum (FBS, Life Technologies, Inc., Grand Island, NY) and 1% (v/v) penicillin–streptomycin solution (Beyotime Biotechnology, Shanghai, China) in a cell incubator (Thermo Scientific) at 37 °C with 5% CO2 as described previously [8, 20,21,22].

Oligonucleotides, plasmids, viruses and infections

The primers for quantitative Real Time PCR (qRT-PCR) were as follows:

human GPR137-F-5’-ACCTGGGGAACAAAGGCTAC-3’;

human GPR137-R-5’-TAGGACCGAGAGGCAAAGAC-3’;

human RAB8A-F-5’-CTACGACATCACCAACGAGAAG-3’;

human RAB8A-R-5’-CATCACACTTGTTCCCGAGTAT-3’;

human GLI1-F-5’-CCACGGGGAGCGGAAGGAG-3’;

human GLI1-R-5’-ACTGGCATTGCTGAAGGCTTTACTG-3’;

human PTCH1-F-5’-ACAAACTCCTGGTGCAAACC-3’;

human PTCH1-R-5’-CTTTGTCGTGGACCCATTCT-3’;

human GAPDH-F-5’-CCTCAACTACATGGTTTACATGTTCC-3’;

human GAPDH-R-5’-GAAGATGGTGATGGGATTTCCATTG-3’.

The human GPR137 and RAB8A lentiviral expression vectors, pCDH-CMV-GPR137-EF1-Puro and pCDH-CMV-RAB8A-EF1-Puro, and GPR137-shRNA and RAB8A-shRNA-expressing lentiviral vectors, pLV3-U6-GPR137(human)-shRNA-Puro and pLV3-U6-RAB8A(human)-shRNA-Puro, were constructed by Mr. Qiang Xu, and the pCDH-CMV-MCS-EF1-Puro empty vector and the pLV3-U6-shRNA-Puro vector containing a scrambled shRNA sequence were used as a control, respectively. Lentiviruses were generated as described previously [23], and the lentiviruses-containing supernatants with the titers greater than 1 × 106 cfu/ml was applied for infection of human SK-OV-3, A2780, or HO8910 cells in the presence of 8 μg/ml polybrene (Sigma, St. Louis, MO, USA).

Antibodies and chemicals

GPR137 (bs-16270R, 1:1000), RAB8A (bs-6176R, 1:1000), GLI2 (bs-11564R, 1:1000), and PCNA (bs-2007R, 1:1000) antibodies were from Bioss (Beijing, China). Flag (M185-3, 1:10000) antibody was from MBL (Beijing, China), and antibodies for GLI1 (sc-20687, 1:1000), GLI3 (sc-74478, 1:1000), SuFu (sc-137014, 1:1000), IFT88 (sc-84318, 1:1000), KIF3A (sc-376680, 1:1000), MMP-2 (sc-13595, 1:1000), MMP-9 (sc-21733, 1:1000), GFP (sc-9996, 1:5000), EZRIN (sc-58758, 1:1000), GAPDH (sc-32233, 1:2000) and normal mouse IgG (sc-2025) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PTCH1 (#2468, 1:1000) antibody, and normal rabbit IgG (#2729) were purchased from Cell Signaling Technology (Danvers, MA, USA). Ki67 antibody (ER1706-46, 1:200) was from Huabio (Hangzhou, China), and αTubulin antibody (AF5012, 1:1000) was from Beyotime Institute of Biotechnology (Shanghai, China). Alexa555-conjugated secondary antibody was from Life Technology. DAPI (2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, #C1002) was obtained from Beyotime Institute of Biotechnology (Shanghai, China). Recombinant human SHH protein (N-Shh, C24II, N-Terminus) was from R&D Systems (#1845-SH). Actinomycin D, SAG, GDC-0449, and GANT61 were purchased from Selleckchem (Shanghai, China).

RNA isolation, reverse transcription and qRT-PCR

Total RNA was isolated from SK-OV-3, A2780, HO8910, and IOSE80 cells, or from fresh clinical tissues by using a TRIzol reagent (Takara Biotechnology Co., Ltd., Dalian, China) as per the manufacturer’s instructions. Briefly, a total of 5 μg RNA in a volume of 20 μl was reversely transcribed with a SuperScript III reagent (Life Technologies) and the oligo-(deoxythymidine) primer with incubation at 42 °C for 1 h. After cDNA synthesis, each reaction mixture was diluted with a volume of 80 μl Tris–EDTA buffer. qRT-PCR was subsequently conducted to examine the mRNA expression levels of human GPR137, RAB8A, GLI1 and PTCH1 using corresponding paired primers, respectively. The relative amounts of the target mRNA levels were normalized to the GAPDH levels, respectively, and the relative difference in mRNA levels was consequently calculated by 2−∆∆Ct method as described previously [8]. Data were displayed as the relative expression of target genes in each group to the corresponding control group (vehicle, control, or scramble shRNA group) that was set as “1”.

RNA stability assay

RNA stability assay was carried out as previously described [23]. Briefly, 5 × 105 GPR137-shRNA-expressing or control scrambled sequence-expressing SK-OV-3 or A2780 cells were seeded in 3.5-cm cell culture plates and were treated with 10 μg/ml actinomycin D (AcD). Cells were subsequently harvested at time 0 h, 2 h, 4 h, and 8 h time points, and RNA was isolated and subjected to qRT-PCR as described above, with equal amount of RNA from the four time points provided to each RT reaction. In each group, RNA expression at different time points was normalized against that at the corresponding 0 h time point to calculate relative fold-enrichment.

RNA immunoprecipitation (RIP) assay

RIP assay was conducted as described previously [23]. In brief, an actively growing SK-OV-3 cell monolayer was cross-linked with 1% formaldehyde, and then cross-linking was stopped by 0.25 M glycine. Cells were lysed in immunoprecipitation assay buffer RIPA containing 50 mM Tris (pH 7.5), 1% NP-40, 0.5% sodium deoxycholate, 0.05% SDS (sodium dodecyl sulfate), 1 mM EDTA, 150 mM NaCl, 40 units RNAsin, and protease inhibitors. Lysates were then sonicated, and insoluble material was removed by centrifugation and precleared with protein A Sepharose, followed by immunoprecipitation experiment with GPR137 antibody or corresponding IgG. The harvested immunoprecipitates were subsequently washed five times in high-stringency RIPA buffer containing 50 mM Tris (pH 7.5), 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 M NaCl, 1 M urea, 40 units RNAsin and protease inhibitors. Protein was then eluted with elution buffer containing 50 mM Tris (pH = 7), 5 mM EDTA, 10 mM DTT, 1% SDS, and cross-links were reversed at 70 °C for 45 min. RNA was extracted by TRIzol reagent (Takara Biotechnology Co., Ltd., Dalian, China) and treated with DNase I (DNA-free kit, Ambion), and bound RAB8A RNAs were consequently analyzed by qRT-PCR as described above.

Transient transfection and GLI-luciferase (GLI-Luc) reporter assay

Transient transfections in cells were carried out by using Lipofectamine 2000 reagent (Invitrogen, Thermo Fisher Scientific) as per the manufacturer’s instructions. GLI-Luc reporter (containing 8 × GLI-binding sites) activities were determined by using the dual luciferase reporter assay (Promega) as per the manufacturer’s protocol as described previously [17]. A pRL/TK-luciferase reporter plasmid purchased from Promega was utilized as a second reporter for normalizing the results. The data were obtained by analyzing triplicated samples.

Nucleo-cytoplasmic separation assay, membrane-cytoplasmic separation assay, western blot and co-immunoprecipitation (co-IP)

For nucleo-cytoplasmic separation assay, the nuclear and cytosolic fractions of HO8910 cells were prepared by using the nuclear and cytosol protein extraction kit from Beyotime Institute of Biotechnology (#P0027, Shanghai, China) according to the manufacturer's protocol as described previously [23]. For membrane-cytoplasmic separation assay, the membrane and cytosolic fractions of SK-OV-3 and A2780 cells were obtained by using the membrane and cytosol protein extraction kit from Beyotime Institute of Biotechnology (#P0033, Shanghai, China) according to the manufacturer's protocol as described previously [24]. Western blots were performed using standard protocols as described previously [9]. Briefly, total protein extracts were prepared, and protein concentrations were determined by using a standard Bradford assay. After that, a total of 50 μg protein was subjected to SDS-PAGE followed by a transfer onto PVDF (Polyvinylidene fluoride) membranes (Millipore, Bedford, MA). Membranes were incubated with target primary antibodies at 4 °C overnight followed by incubation in corresponding secondary antibodies (Beyotime Institute of Biotechnology) at room temperature for 2 h. The intensity of protein bands-derived signals was subsequently quantified using a NIH ImageJ software (ImageJ, http://rsb.info.nih.gov/ij/). Co-immunoprecipitation was conducted as described previously [25]. In brief, SK-OV-3, A2780 or HO8910 cells were harvested and lysed in IP lysis buffer (containing 100 mM NaCl, 50 mM Tris–HCl (pH 8.0), 5 mM EDTA, 1% Brij35, 2 mM Na3VO4, 10 mM NaF, 2 mM β-glycerophosphate and 2 mM PMSF), and were incubated with different antibodies or protein A/G plus agarose (sc-2003, Santa Cruz). The beads were next washed five times with the IP lysis buffer, and the immune complex was eluted with western blot sample buffer. Lysates and immunoprecipitates were subjected to western blot as described above.

Immunofluorescence (IF) staining

IF staining was carried out using chamber slides (Nalge Nunc International, Naperville, IL) as described previously [23]. N-SHH-rp-treated SK-OV-3 cells were fixed in ice-cold methanol for 10 min and were permeabilized next with 0.1% TritonX-100 in PBS (PBST) for 10 min. After blocking with BSA solution, SK-OV-3 cell samples were incubated with a GPR137 primary antibody and subsequently with the fluorescent Alexa 555-conjugated secondary antibody. Nuclei were counterstained with DAPI at room temperature for 5 min. Sample slides were then analyzed by a laser scanning microscope (Zeiss, Germany).

Cell proliferation by cell counting Kit-8 (CCK-8) assay

CCK-8 assay was conducted as per the manufacturer’s protocol (Yeasen, Shanghai, China) as described previously [8]. After treatment, SK-OV-3, A2780, or HO8910 cells were incubated with 10 μl CCK-8 reagent per well (96-well plate) for 2 h at 37 °C in the dark and the absorbance was subsequently measured at a wavelength of 450 nm using a micro-plate reader (Spark, Switzerland). Data were displayed as fold changes in each group relative to the corresponding control at 0 h.

Wound healing assay

Wound healing assay was carried out as previously described [26]. In brief, SK-OV-3, A2780, or HO8910 cells (4 × 105 cells) were seeded in six-well plate and were transfected with indicated overexpressing- and/or shRNA sequence-carrying plasmids. 24 h after transfection, cells were subjected to serum starvation for another 12 h. After rinsed with medium to remove unattached cells, the confluent layer of cells was scratched with a sterile 10 μl-tip to create an artificial wound. Cell migration to the wounded gap was subsequently monitored by microscopy after 48 h and the wound closure was analyzed using ImageJ software.

Matrigel invasion assay

Invasive capacity of cells was measured using Matrigel (BD Biosciences, NJ)-coated Transwell inserts (6.5 μm, Costar, Cambridge) containing polycarbonate filters with 8 μm pores as detailed previously [8]. In brief, the mixture of Matrigel and medium with a volume ratio of 1:2 (totally 50 µl) was enclosed by each Transwell membrane. After transfected with indicated overexpressing- and/or shRNA sequence-carrying plasmids and cultured for 48 h, SK-OV-3, A2780, or HO8910 cells (2 × 105) in a total of 200 µl volume of serum-free medium were seeded in the upper chamber, whereas 600 µl volume of medium with 10% FBS was added into the lower well. After 24 h-incubation, the non-invading cells that remained on the upper surface of the filter were removed slightly, and the cells that had passed through the filter and attached to the bottom of the membrane were then fixed in methanol at room temperature for 10 min and stained with 0.2% crystal violet for 5 min. Numbers of the invasive cells were counted randomly in six selected fields from triplicate chambers in each experiment. The migrated cells were counted under a light microscope, and data were displayed as fold changes in each group relative to the control.

Colony formation assay

Colony formation assay was carried out as previously described [27, 28]. In brief, SK-OV-3, A2780, or HO8910 cells were infected with indicated lentiviruses that carrying overexpressing- and/or shRNA sequence. After 24 h, the cells were seeded in six-well plates at a density of 1000 cells/well in 2 ml culture medium per well. After two week-culture, colonies were fixed in methanol at room temperature for 10 min and then stained with 0.5% crystal violet in methanol or 0.25% Coomassie blue in methanol. Colonies were subsequently counted and photographed, and the colony numbers were consequently statistically analyzed.

Xenografts assay

Female Balb/c-nu/nu mice (6-week-old) were rested for a week and were bred under pathogen-free conditions (temperature, 18–22 °C; humidity, 50–60%; 12/12-h light/dark cycle). Tumor-bearing mice model was established by subcutaneous inoculation with xenografts of human HO8910 cells (5 × 106 cells in 100 µl volume of PBS per site) infected with control lentiviruses or RAB8A-overexpressing lentiviruses, or with human SK-OV-3 cells (5 × 106 cells in 100 µl volume of PBS per site) infected with control lentiviruses carrying a scrambled sequence or lentiviruses carrying GPR137-shRNA sequence into the left armpit as described previously [20]. The body weights, and volumes (V) of the xenograft tumors were measured every 3 d in SK-OV-3 cell infected nude mice as follows: V = 0.5 × a × b2, where “a” indicates the long axis and “b” indicates the short axis. When the volume of one tumor reaches 800 mm3 at day 18 (18 d) after inoculation, all the mice were sacrificed, and the subcutaneous tumors were collected, weighed and used for further analysis. All animal care and handling procedures were approved by the Institutional Animal Care and Use Committee of Zhejiang University, and were performed according to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85–23, revised 1996).

In situtransplantation tumor model of ovarian cancer in nude mice

Female Balb/c-nu/nu mice (6–8 week-old, 18–20 g of body weight) were rested for a week and were bred under pathogen-free conditions (temperature, 18–22 °C; humidity, 50–60%; 12/12-h light/dark cycle). The experimental nude mice were fixed after anesthesia, the back skin was prepared and disinfected, a longitudinal incision of one cm was made from the center of the back to the right, and an incision of one cm was made on the upper edge of the iliac crest. A volume of 0.01 ml (3.0 × 106 cells) of RAB8A-shRNA-expressing or control lentiviruses infected-A2780 cell suspension was injected into the deep part of the left ovary, compressed for one minute, and the injection hole was sealed with biological protein gel. The body weights of the mice were measured every 4 d, and when the volume of one tumor reaches 800 mm3 at day 28 (28 d) after inoculation, all the mice were sacrificed, and the ovarian tumors were collected, weighed and used for further analysis. All animal care and handling procedures were approved by the Institutional Animal Care and Use Committee of Zhejiang University.

Immunohistochemistry (IHC) staining

IHC staining was conducted by using the Histostain-Plus Kit (Kangwei Reagents, Beijing, China) according to the manufacturer’s instructions as detailed previously [20]. In brief, paraffin-embedded xenografts tumor sections or human clinical OC sections of 4 μm-thickness were deparaffinized and rehydrated in xylene and a graded series of ethanol. After antigen retrieval in solution containing 10 mM sodium citrate and 10 mM citric acid, tissue sections were next incubated with 3% H2O2 in methanol to quench endogenous peroxidase followed by sequential incubation including with normal serum for 30 min at room temperature, with primary antibodies against GPR137, RAB8A, Ki67, or IgG at 4 °C overnight, and subsequently with HRP-labeled secondary antibody (Life Technologies) at room temperature for 30 min. The diaminobenzidine (DAB) solution was used for development of color, and the sections were counterstained with hematoxylin. The sections were consequently sealed with neutral resin and observed under the microscope. GPR137 expression and RAB8A expression was scored as previously described [9] based on the proportion of cells showing GPR137 or RAB8A IHC staining across three non-adjacent fields in each sample with the following criteria: 0 (no cell positive); 1 (< 50% cells weakly positive); 2 (< 50% cells intensely staining); 3 (≥ 50% cells weakly positive); 4 (≥ 50% cells intensely staining).

RNA-Sequence (Seq) analysis

A minimum of 3 μg of total RNA from RAB8A-overexpressing HO8910 cells was oligo (dT) selected using the Dynabeads mRNA purification kit (Invitrogen). Then, the mRNA was isolated from total RNA and was next fragmented into short fragments with a fragmentation buffer (Ambion), and double-stranded cDNA was subsequently synthesized with these obtained short fragments as templates. Next, the generated cDNA was end-repaired, ligated to Illumina adapters, size selected on agarose gel (approximately 250 bp) and PCR amplified. The cDNA library was then sequenced on an Illumina HiSeq 2000 sequencing platform (Berry Genomics). The Stringtie software was applied for predicting the transcripts of all samples, and the RSEM software was used to call bowtie2 for comparison results. The number of reads aligned to each transcript for each sample was obtained, and the gene expression levels for each transcript were estimated as the number of reads per kilo-base of exon model per million mapped reads (RPKM) using only uniquely mapped reads in exonic regions. After a comprehensive analysis of gene expression levels, differential gene analysis between sample groups was performed. A gene is considered significantly differentially expressed if its expression differs between samples from the two groups, control group and RAB8A-overexpression group, with the log2(Fold Change) > 1 or < -1, and the p value < 0.05 as calculated by Cufflinks. Volcano plots and cluster heatmaps were drawn for visually displaying the differentially expressed genes between the two groups, followed by Gene Ontology (GO, http://www.geneontology.org/) enrichment analysis of GO terms with false discovery rate (FDR) ≤ 0.05 using hyper-geometric distribution and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of cell pathways with FDR ≤ 0.05.

Bioinformatics analysis

The expression of GPR137 [log2(TPM + 1)] and RAB8A [log2(TPM + 1)] in ovarian cancer (OV, T, n = 426) tissues and normal ovarian tissues (OV, N, n = 88) was assessed using the OV dataset in accordance with GEPIA (Gene Expression Profiling Interactive Analysis) database (http://gepia.cancer-pku.cn). In addition, GEPIA online tool was used to produce Kaplan–Meier survival plots (Disease Free Survival) with median in group cutoff (high-50% and low-50%) and 95% confidence interval, and the correlations between OV patient GPR137 and RAB8A expression, GPR137 and PTCH1 expression as well as RAB8A and PTCH1 expression with auto best cutoff.

Statistics analyses

The results are shown as mean ± SDs. The GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA, USA) and Excel were used for statistical analysis. Statistical significance of the data was analyzed by unpaired Student’s t-test between two groups or with one-way ANOVA among multiple groups, which was assessed at p < 0.05 (* or #) and p < 0.01 (** or ##). Experiments were independently triplicated and representative experiments are shown.