CYPB upregulates and promotes cell proliferation in endometrial carcinoma CURRENT STATUS: POSTED

BACKGROUND The molecular pathogenesis of endometrial cancer is not yet completely understood, preventing the development of successful therapies. Here, we determine the effect of CYPB on the growth of endometrial cancer. In this study, we examined the expression of CYPB in endometrial cancer tissues using immunohistochemistry. CYPB silencing in the human cell line HEC-1-B was used to evaluate the role of CYPB in the malignant phenotype of endometrial tumor cells, while CCK-8 and colony formation assays were performed to assess its effect on tumor cell proliferation. Furthermore, microarray analysis was carried out to compare the global mRNA expression profile between the normal and CYPB-knockdown cell. Gene ontology and KEGG pathway enrichment analysis were performed to determine the potential function of different expressed genes related to CYPB. We found that CYPB was upregulated in endometrial cancer, while cells with suppressed expression of CYPB exhibited markedly reduced migration. We identified 1536 differentially expressed genes (onefold change, p< 0.05), among which 652 genes were upregulated and 884 genes were downregulated, and most of them were enriched in cell cycle, glycosphingolipid biosynthesis, adherens junctions, and metabolism pathways. The results of our study suggest that CYPB may serve as a novel regulator of endometrial cell proliferation, thus representing a novel target for gene-targeted endometrial therapy. assessed by qRT-PCR using SYBR Premix Dimer Eraser (Perfect Real Time, TaKaRa) assay kits. Relative fold changes in expression were calculated using the comparative Ct (2 −∆∆Ct ) method. Purified RNA samples were submitted to Phalanx Biotech (Hsinchu, Taiwan) for microarray analysis. We used the Phalanx Human OneArray Plus Gene Expression Profiling IL, USA). The chi-square test was used to determine the differences in age and tumor grades between high and low expressed CYPB groups. Differences between two groups were analyzed using Student’s t-test for comparison of two groups or by one-way analysis of variance for comparison of more than two groups. P values of < 0.05 were considered statistically significant.


Introduction
Endometrial cancer represents a group of epithelial malignant tumors occurring in the endometrium and is one of the three most common malignant tumors of the female genital tract. Seventy-five percent of the patients are diagnosed in an early phase, and the 5-year survival rate was 65-92% [1,2]. Molecular pathogenesis of endometrial cancer involves abnormalities in many genes and signaling pathways. For example, mutations in P53, increased microsatellite instability, mutations in PTEN, and abnormalities in the Notch signaling pathway all lead to uncontrolled cell proliferation and apoptosis, which in turn lead to the occurrence and development of endometrial cancer [3][4][5][6]. At present, the molecular pathogenesis of endometrial cancer is not entirely understood, and further research is warranted in order to develop successful therapies.
Cyclophilins are highly conserved proteins that are ubiquitously expressed intracellularly. They were first recognized as host cell receptors for the potent immunosuppressive drug cyclosporin A [7,8].
They act as molecular chaperones that fold, translocate, and process newly synthesized proteins.
Hence, to determine the effect of CYPB on the growth of endometrial cancer, we aimed to assess its expression in endometrial tissues and CYPB-silenced HEC-1-B cells and measure the relative gene expression with microarray analysis.

Cell Proliferation And Clone Formation
Cell proliferation was determined using the Cell Counting Kit-8 assay (CCK-8; Beyotime Biotechnology, Shanghai, China) per the manufacturer's instructions. Cells in the logarithmic growth phase (1 × 10 4 cells/mL per well) were grown in 96-well plates in medium containing 10% FBS in an incubator with 5% CO 2 at 37 °C for 72 h. Afterwards, 10 mL of CCK-8 solution was added to each well, and the plates were incubated for an additional 4 h. The absorbance in each well was measured at a wavelength of 450 nm with a microplate reader.
For clone formation, HEC-1-B cells were transfected with CYPB-siRNA for 48 h and were collected and seeded in triplicate into 6-well plates at a density of 1,000 cells/mL per well. The cells were incubated for 10 days at 37 °C in a 5% CO 2 atmosphere. They were then fixed with 4% paraformaldehyde for 30 min and stained with Giemsa (Beyotime Biotechnology) for 20 min. After washing with doubledistilled H 2 O several times, images of the cell plates were taken (Canon, Inc., Tokyo, Japan).

RNA extraction, reverse transcription, qRT-PCR and microarray analysis
Total RNA was extracted using TRIzol, and cDNA was synthesized with PrimeScript RT reagent Kit (TaKaRa, Dalian, China). Gene expression was assessed by qRT-PCR using SYBR Premix Dimer Eraser (Perfect Real Time, TaKaRa) assay kits. Relative fold changes in expression were calculated using the comparative Ct (2 −∆∆Ct ) method. Purified RNA samples were submitted to Phalanx Biotech (Hsinchu, Taiwan) for microarray analysis. We used the Phalanx Human OneArray Plus Gene Expression Profiling platform 6.1 to analyze the CYPB-mediated alterations of mRNA expression.

Immunohistochemistry (ihc)
Sections (4 µm) were cut from the constructed TMA blocks, deparaffinized, and rehydrated. Heatinduced epitope retrieval was performed onboard of the Leica Bond RX platform at 100 ℃ using EDTA buffer (pH 9.0, Leica) for 20 minutes, followed by 15 min of incubation with anti-CYPB antibody (#43603, Cell Signaling Technology, Danvers, MA, USA) or anti-β-catenin antibody (#8480, Cell Signaling Technology) at room temperature and with Bond™ Polymer Refine Detection kit (Leica Biosystems, Buffalo Grove, IL, USA) for 8 min. The reaction was visualized using 3,3′-diaminobenzidine tetrahydrochloride for 10 min and with hematoxylin as a counterstain. Scoring was performed by pathologists (MK, PR) using Nikon Eclipse microscope on TMA glass slides at 20 × magnification.
Tissues were scored for CYPB expression, and the scoring system reflected the extent and intensity of staining: the intensity was assigned a score of 0, 1, 2, or 3, representing negative, weak, moderate, or strong expression, respectively; while the extent was assigned a score of 0, 1, 2, 3 or 4, representing < 5%, 6-25%, 26-50%, 51-75% and > 75% of cells stained. The overall quantitation of the score was obtained by multiplying the average intensity and score of five different high-power fields (at 400 × magnification). The samples were divided into two groups based on final staining scores, which ranged from 0 to 7: the high expression group (scores of ≥ 4) and the low expression group (scores of Gene Ontology Functional And Pathway Enrichment Analysis GO and KEGG pathway enrichment analysis were used for differentially expressed genes (DEGs) using the DAVID database. FDR values of < 0.05 were set as the cut-off criterion for the two analyses.

Statistical analysis
Statistical analysis was performed using SPSS software, version 18.0 (SPSS, Chicago, IL, USA). The chi-square test was used to determine the differences in age and tumor grades between high and low expressed CYPB groups. Differences between two groups were analyzed using Student's t-test for comparison of two groups or by one-way analysis of variance for comparison of more than two groups. P values of < 0.05 were considered statistically significant.

CYPB is over expressed in endometrial cancer
Seventy-four control tissue samples, consisting of 24 normal endometrium tissue and 50 of atypical hyperplasic endometria, and 96 endometrial cancer tissues were used to validate protein expression by immunohistochemistry. CYPB expression was significantly higher in endometrial cancer tissues compared with non-cancer tissues, and it was higher in intrauterine dysplasia than in the normal endometrium ( Fig. 1), suggesting that higher expression of CYPB is associated with the incidence of endometrial cancer. Furthermore, no significant association was observed between CYPB expression and patient age or tumor grades (Table 1).  GO functional and pathway enrichment analysis GO and pathway enrichment analysis showed that the DEGs were significantly enriched in cell cycle, glycosphingolipid biosynthesis, adherens junctions, and metabolism pathways. Important genes and pathways involved in this process are shown in Figure 5.

Validation of differentially expressed RNA by qRT-PCR
To evaluate the reliability of microarray data, we verified the expression of five differentially expressed mRNA in HEC-1-B cells by qRT-PCR assay. As shown in Figure 6, the mRNA expression of SREBF1, SPP1, PPIB and PLAT were significantly downregulation in the CypB-knockdown cells. While the CDKN1A mRNA shown a much higher expression in the CypB-knockdown cells. All these results were consistent with the microarray data.

Discussion
Endometrial carcinoma remains one of the leading causes of death among women, and, therefore, the discovery of novel molecular targets for its diagnosis, prognosis, and treatment is required for improving the clinical strategy and outcome for this disease.  [29], and that it might play a role in apoptosis through the activation of caspases and apoptosis-inducing factor. A study with a two-dimensional gel electrophoresis and MALDI-Q-TOF MS/MS-based proteomics approach found that overexpression of CYPA is significantly correlated with a low degree of cancer differentiation, and its overexpression was associated with decreased survival in endometrial carcinoma [30].
On the other hand, the structurally similar CYPB, which is s is found in the endoplasmic reticulum, has been implicated in STAT3 activation and in the generation of reactive oxygen species in cancer cells [21]. CYPB facilitates the transcriptional activity of STAT5 by inducing the release of the repressor PIAS3, resulting in significantly enhanced STAT5-mediated gene expression [11,15]. At the cell surface, CYPB also serves as a ligand for the CD147 receptor [31], which regulates MAPK activation, motility, calcium transport [31][32][33], and the expression of the pro-apoptotic protein BIM [34]. Gene expression studies revealed that CYPB is highly upregulated in malignancies. Ablation of CYPB expression in glioblastoma multiforme cells suppresses several canonical oncogenic signaling pathways, including mutant P53, MYC, and CHK1.

Conclusions
In this study, we demonstrated that patients with endometrial cancer exhibit a significantly higher expression of CYPB, suggesting that CYPB expression can be considered an effective indicator for the clinical outcome of endometrial cancer. Furthermore, our results demonstrate that CYPB acts as an oncogene in endometrial cancer and that silencing its expression affects the expression of many genes involved in the cell cycle, glycosphingolipid biosynthesis, adherens junctions, and metabolism pathways.

Acknowledgments
We are grateful to the patients who participated in this study and the funds from the Natural Science Foundation of Shandong Province (ZR2016HL39) that supported this study.

Authors' contributions
JL, YZ and HKW contributed to the conception and design. GMQ, XS contributed to the acquisition, analysis, and interpretation of data. XS and ZHL contributed to drafting the work. TGZ, ZHZ and HKW contributed to revising it critically. All authors approved the final version to be published and agreed to be accountable for all aspects of the work.

Ethics approval and consent to participate
The study was approved by the Ethics Committee of Yantai Yuhuangding Hospital in November 27, 2017 (registration number: YLYLLS [2018] 008). Written informed consent was obtained from all participants.

Funding
This research was supported by the Natural Science Foundation of Shandong Province (ZR2016HL39).

Availability of data and materials
The datasets generated and/or analyzed during the current study is available from the corresponding author on reasonable request.

Consent for publication
The consent for publication is not applicable because this study does not include any individual details, images, or videos.          The expression of real-time qPCR was in accordance with microarray data Figure 6 The expression of real-time qPCR was in accordance with microarray data