TMED3 Promotes Breast Cancer Cells Proliferation And Invasion Via Wnt/?-catenin Signaling Pathway

Objecttive:TMED?transmembrane emp24 domain-containing protein?,a family of?24kDa type-I transmembrane proteins contain a GOLD?Golgi dynamics?domain,is involved in transporting the vesicular trafficking of proteins through endoplasmic reticlulum-Golgi system.TMED plays an important role in early embryonic development and immune response.The ten members of TMED protein family in mammalian have been discovered until now,which the most common six members,namely TMED1,TMED2,TMED3,TMED7,TMED9 and TMED10.Notably,recent studies have reported that TMED3 also serves as oncogenes or metastatic suppressors implicated in various cancers.Previous studies showed that TMED3 overexpression was associated with poor prognosis for clear cell renal cell carcinoma and hepatocellular carcinoma.TMED3 was overexpressed in colon cancer,however,knockdown of TMED3 could induce xenograft lung metastases and promote metastatic cancer cells proliferation,so is a metastatic suppressor in colon cancer.However,whether TMED3 also be implicated in breast cancer progression needs to be explored.Wnt/?-catenin signaling pathway activation is proved to be related to various human cancers.Wnt/?-catenin signaling is implicated in different stages of mammary gland development and is also important for mammary tumorigenesis when aberrantly activated.In addition,activation of Wnt/?-catenin signaling pathway involved in breast cancer progression could promote cells invasion and metastasis.During the activation of Wnt/?-catenin signaling pathway,?-catenin is released from APC/Axin/GSK-3?/?-catenin complex,so results in the accumulation of?-catenin in the cytoplasm.Then,?-catenin translocate into the nucleus where it binds the transcription factors LEF1/TCF to activate a series of downstream target genes?c-myc,c-jun,cyclinD1 and MMP7?transcription of Wnt signaling pathway.In flies and mammals,TMED3 protein involves in Wnt transporting and controls Wnt secretion.Therefore,wherther is also related to the Wnt/?-catenin signaling pathway about the effect of TMED3 on the tumor progression,which deserves to explore it.In the present study,we will examine the relationship between the expression of TMED3 in breast cancer and the clinical pathological factors and prognosis.We will explore the possible molecular mechanisms of the biological effects TMED3 on breast cancer cells proliferation,migration and invasion.Methods:1.Tissue specimens.This study included 182 paraffin-embedded primary breast cancer tissues and 60 paired noncancerous tissues that had been collected between January 2010 and October 2018.Tissues were randomly selected from the archives of the Institute of Pathology,the Dalian Municipal Central Hospital Affiliated of Dalian Medical University.In addition,fresh primary breast cancer tissues and surrounding adjacent noncancerous tissues were obtained from 29 patients with newly diagnosed breast cancer who underwent surgical resection.Fresh samples were snap-frozen and stored at-80?until analysis.The use of patient material was approved by the Ethics Committee of the Dalian Municipal Central Hospital Affiliated of Dalian Medical University?YN2019-054-01?.2.Tissue microarray construction and immunohistochemistry analysis.For TMA,histological sections were evaluated,and representative areas of tumor tissue that were free from necrosis or hemorrhage were pre-marked on paraffin embedded donor blocks.Two 2.0mm cores were punched out from each tumor sample,and all cores were embedded in a separate paraffin block.The TMA blocks were sectioned at a thickness of 4?m.Immunostaining was performed according to the Eli Vision kit instructions.The primary antibody for TMED3 antibody diluted 1:50 was incubated overnight at 4?.Then,HRP conjugated polymers were applied to the slides for 30minutes.DAB visualization was performed,and the slides were counterstained with hematoxylin.The immunostaining of TMED3 was scored by two independent observers.Five fields were randomly examined at 400×magnification per slide.3.Cell culture and transfection procedure.The breast cancer cell lines MCF-7,T47D and MDA-MB-231 and human breast epithelial cells MCF-10A were cultured in DMEM.The medium was supplemented with 10%fetal bovine serum?FBS?,100IU/m L penicillin and 100ug/ml streptomycin and the cells were cultured in a humidified cell incubator at 37? with an atmosphere of 5%CO₂.TMED3 plasmid and si RNA sequences were synthesized by Shanghai Genepharma Co.,Ltd.Cell transfections were performed using the Lipofectamine 3000 reagent according to the manufacturer's instructions.In addition,siRNA-?-catenin and siRNA-Axin2 was usedforinterferingWnt/?-catenin signaling in cells.Cells were incubated in transfection media for 4 h,which was then replaced with standard media.Empty vector was used as a negative control.4.RNA extraction and quantitative real-time polymerase chain reaction?qRT-PCR?.Total RNA was extracted from frozen tissues or cell lines using TRIzol reagent,according to the manufacturer's instructions.The primers used for amplifying TMED3 and endogenous control?-actin were synthesized by Sangon Biotech.qRT-PCR was performed using SYBR Green master mix kit from TianGen Biotech.The fold change in the expression of target genes was calculated by the 2^-??CT method.5.Western blot analysis.Proteins were separated by SDS-PAGE,transferred to polyvinylidene difluoride?PVDF?membranes.Membrane was blocked in 5%fetal calf serum and incubated at 4?overnight with different primary antibody.After incubation with second antibody at 37?for 2h.Target protein bands were detected with enhanced chemiluminescence reagent.A gel imaging system was used to analyze band density,which were compared against an internal control.6.Immunofluorescence analysis.Cells grown on coverslips were fixed with 4%paraformaldehyde in PBS on ice for 15 min.Coverslips were washed twice with PBS,permeabilized by incubation with 0.2%Triton X-100 for 10 min,washed twice with PBS,and incubated in 5%horse serum for 30 min.Coverslips were placed in ahumidified chamber and incubated with Axin2 and?-catenin antibodies at 4?overnight.Subsequently,coverslips were washed twice with PBS,incubated with TRITC-conjugated anti-rabbit secondary antibody at 37? for 30 min,washed with PBS,and incubated with 0.1%DAPI at 37? for 30 min.Coverslips were mounted on a glass slide with 25?l Slow Fade.Slides were analyzed with an Olympus fluorescence microscope and images were evaluated using Axiovision software v4.6.7.CCK-8 and colony formation assay to detect cells proliferation.Cell proliferation was assessed using the Cell Counting Kit 8,according to the manufacturer's instructions.Cells were seeded into the wells of a microtitre plate.Each wellcontained 2000 cells,100?l DMEM with 10%FBS,and 10?l CCK-8.The plate wascultured at 37? for 2 hours.Absorbance of each well was measured at 450 nm,daily,for 5 days.The experiment was repeated 3 times.Cell proliferation was further investigated using the colony formation assay.Transfected cells were seeded in 6-well plates?approx.500 cells?,incubated for 3 weeks,fixed with 4%paraformaldehyde for15 min,washed with PBS,and stained with 0.2%crystal violet for 10min.The number of colonies?>50 cells?was counted under a microscope.The experiment was performed in duplicate and repeated at least twice.8.Flow cytometry assay to detect cell cycle.Cell cycles were analyzed by flow cytometry.Cells were harvested at 48h post-transfection by trypsinization,fixed in500?l of 70%cold ethanol at 4? overnight,stained with 100?l RNase A and 400?lpropidium iodide at 37? for 30 min.10000 cells per sample were analyzed on EPICS XL flow cytometer with Modfit software.9.Wound healing assay.For the wound healing assay,5×10⁵ cells were seeded in6-well plates and incubated until they reached 100%confluence.The confluence plates were scratched using a sterile pipette tip,washed twice with PBS to remove the detached cells,and photographed under a microscope at 0 and 24h.Cell migration was measured by monitoring the width of the scratch over time.10.Transwell assay.The cell invasion assay was performed using a 24-well Transwell chamber coated with Matrigel basement membrane matrix.Cells suspended in serum-free media were placed in the upper Matrigel chamber.Medium supplemented with 10%FBS was placed in the lower chamber.Cells that passed through the membrane after 24 hours were visualized with crystal violet staining.11.Statistical analysis.SPSS version 17.0 for Windows was used for all statistical analyses.A?2 test was used to analyze possible correlations between TMED3expression and clinicopathologic factors.Student's t test was used to compare the differences between groups.The calculated results with P<0.05 was considered as statistical significance.Results:1.Overexpression of TMED3 in breast cancer associated with clinicopathologic factors and prognosis.Negative/weak staining was observed in most normal breast tissues.In breast cancer tissues,TMED3 showed moderate to strong cytoplasmic,nuclear and membranous expression in 62.1%?113/182?of cases.TMED3 protein expression was significantly elevated in 82.8%?24/29?of fresh breast cancer tissue samples compared to adjacent non-cancerous tissue samples.79.3%?23/29?of cases the m RNA expression of TMED3 was significantly increased in breast cancer tissue samples compared with matched normal tissues.TMED3overexpression was significantly associated with estrogen receptor?ER,P=0.028?,progesterone receptor?PR,P=0.032?,human epidermal growth factor receptor-2?Her-2,P=0.040?negative status,as well as nodal metastases?P=0.002?.Kaplan–Meier survival analysis revealed that patients with elevated TMED3 expression had shorter overall survival than those with low TMED3 expression.2.TMED3promotes cells proliferation,invasion and cell cycle progression in breast cancer.CCK-8 showed that TMED3 overexpression remarkably increased the proliferation ability of MCF-7 cells,while its interference significantly decreased the proliferation ability of T47D cells.Colony formation ability demonstrated that TMED3overexpression significantly upregulated the colony formation ability of MCF-7 cells,while its interference attenuated the colony formation ability of T47D cells.Flow cytometry assay results revealed that TMED3 overexpression increased the S phase percentage and G2 phase was decreased in MCF-7 cells compared with control groups,while its knockdown decreased the S phase percentage and increased G2phase in T47D cells.The effect of TMED3 expression on relevant cell cycle proteins were measured by Western blot analysis.The results showed that TMED3overexpression significantly upregulated CDK2,CDK4,CDK6,cylinD1 and cyclinE expression levels in MCF-7 cells,whereas knockdown of TMED3 significantly downregulated these protein expression levels in T47D cells.Wound healing assay results showed that TMED3 overexpression significantly increased migratory distance in MCF-7 cells,while its interference decreased migratory distance in T47D cells.Transwell test results showed that TMED3 overexpression markedly increased the number of invading cells in MCF-7 cells,while its interference decreased the number of invading cells in T47D cells.Furthermore,the related migrating proteins expression levels of MMP2,MMP7,MMP9,RHOA,RHOB,RHOC,Rock1 were examined by Western blot analysis after TMED3 overexpression or knockdown,respectively.The results revealed that overexpression of TMED3 markedly increased RHOA,RHOB,RHOC,ROCK1,MMP2,MMP7 and MMP9 expression levels in MCF-7 cells.In contrast,knockdown of TMED3 reduced expression levels of RHOA,RHOB,RHOC,ROCK1,MMP2,MMP7 and MMP9 in T47D cells.3.TMED3regulated target proteins expression in breast cancer cells via activating Wnt/?-catenin signaling.Immunofluorescence staining analysis results showed that the overexpression of TMED3 in MCF-7 cells resulted in substantial cytoplasmic and nuclear accumulation of?-catenin in these cells,especially in nuclear,and expression of Axin2 in cytoplasm was also increased.TMED3 overexpression increased the levels of?-catenin and Axin2 proteins in MCF-7 cells,wheras knowdown of TMED3decreased the levels of?-catenin and Axin2 proteins in T47D cells.In MCF-7 cells,?-catenin and Axin2 expression levels significantly increased in cells with TMED3overexpression,accordingly,also increased the levels of cyclinD1,cyclinE,c-myc,MMP7 and TCF4 proteins expression.However,silencing?-catenin or Axin2significantly reversed TMED3-induced cyclinD1,cyclinE,c-myc,MMP7 and TCF4upregulated effects.No significant decrease of MMP2 was revealed.This is further evidence that TMED3 promoted proliferation and migration in breast cancer cells via the influence of?-catenin and Axin2 levels.Conclusion:1.TMED3 protein and mRNA expression levels were significantly increased in breast cancer samples.TMED3 overexpression significantly correlated with ER,PR,Her-2 status and lymph nodal metastases.High TMED3 expression of breast cancer patients had shorter overall survival than those with low TMED3expression.2.TMED3 promoted breast cancer cells proliferation,migration and cell cycle progression.3.TMED3 promoted breast cancer cells proliferation and invasion via Wnt/?-catenin signaling pathway.