Study On Effects Of Hypoxia And Microvesicles On Cell Fusion

Cell fusion,also known as cell hybridization,refers to the phenomenon that two or more cells form a daughter cell through cell membrane fusion,cytoplasmic mixing,and nuclear fusion.Fused cells have the similar phenotypes of all their parental cells.It is of great significance to the development of tumors.Tumor microenvironment plays an important role in tumor survival and progression.However,the effects of tumor microenvironment and cell fusion have not been specifically confirmed,and the regulation and mechanism are still unclear.The tumor microenvironment is closely related to the progression of tumors.Current studies confirm that hypoxia causes EMT in tumors,which can change the polarity of epithelioid cells,reduce the expression of epithelial specific protein E-cadherin(E-Cad)and increase expression of interstitial specific protein N-cadherin(N-Cad),resulting in decreased binding among cancer cells.Subsequently,cancer cells are easy to shed from the tumor,which largely contributes to their metastasis.Interestingly,this is similar to the state of cell fusion.Some studies have discussed the relationship between EMT and cell fusion,but they lack of relevant experimental data.Extracellular vesicles(EVs)are cystic structures released by cell membrane-derived cells and contain a large number of biologically active substances,such as proteins,RNAs,and lipids,which is crucial in regulating target cells.And EVs can mainly be divided into exosomes(40-100 nm)and microvesicles(100-1000 nm).As a member of the tumor microenvironment,EVs play an important role in the proliferation,invasion,and metastasis of tumor.The mesenchymal cells in the microenvironment can take up tumor cell-derived EVs,thus changing themselves to adapt to the proliferation,invasion,and metastasis of tumor.Endothelial cells are an important type of mesenchymal cells.Hematopoietic metastasis of tumors are usually based on the destruction of the integrity of vascular endothelium,therefore,providing space for tumor cells to grow or metastasize into the blood vessel lumen.On the one hand,the polarity of endothelium is altered and the integrity of endothelium is destroyed through the direct or indirect contact with tumors;on the other hand,this destruction could be directly mediated through the fusion of tumor cells and endothelial cells.Microvesicles released by tumor cells promote endothelial cells to transform into a more suitable state for their fusion with tumor cells.This transformation not only destroys endothelium integrity by increasing the possibility for cell fusion,but also changes the endothelium polarity,both of which provides convenience for tumor metastasis.This study investigated the role of hypoxia and microvesicles in the fusion of oral squamous cell carcinoma cells(OSCC)and mucosal epithelial cells or endothelial cells,and further explored the underlying mechanism of hypoxia in promoting cell fusion.Part I:Preliminary study on the promotion of hypoxia in cell fusion between CAL-27 and HIOECObjective:To explore the function of hypoxia and the role of EMT in regulating the fusion of OSCC and human immortalized flower epithelial cells(HIOEC).Material and methods:The oral squamous cell carcinoma cell line CAL-27 and HIOEC were transfected with green fluorescent protein(GFP-)and red fluorescent protein(RFP-)respectively using a lentivirus empty vector.After transfection for 72 hours,the cell line with relatively stable fluorescence was achieved by puromycin and flow cytometry fluorescence sorting(FACS):CAL-27 was stained with red fluorescence(RFP-CAL-27)and HIOEC(GFP-HIOEC)was stained with green fluorescence;then the same amount of RFP-CAL-27 and GFP-HIOEC were co-cultured,and fluorescence microscope(FM)was used to observe cell fusion and to count;FACS was performed to calculate the cell fusion rate after 3-day co-cultivation between RFP-CAL-27 and GFP-HIOEC.The nuclei were stained with DAPI and the morphology of the fused cells was observed by laser scanning confocal fluorescence microscopy(LSCM).GFP-HIOEC were co-cultured with equal amounts of RFP-CAL-27 in a 1.0%O2,37? incubator for continuous observation.On the first and third days,six visual fields were randomly selected and the number of fused cells in the hypoxic experimental group and the control group was calculated using Artificial Cell Counting.On the third day,FACS was used to calculate the cell fusion rate of the two co-culture systems to investigate the effect of hypoxia on CAL-27 and HIOEC cell fusion.HIOEC was cultured at 1.0%O2 at 37? for 24 h,then proteins were extracted,western blot was performed,and EMT index was observed.GFP-HIOEC was co-cultured with RFP-CAL-27 in normaxia for 3 days before a 24 h culture alone in hypoxia at 1.0%O2 at 37?.The cell fusion rate was detected by artificial cell counting and FACS.Then,DAPT,a blocker of EMT,was used,EMT index was measured again,and the rate of fused cells was measured by artificial cell counting and FACS.Result:1.FM results showed that spontaneous fusion of CAL-27 and HIOEC occurred,and the fused cells exhibited orange fluorescence.In addition,the fluorescence intensity was relatively stable.2.LSCM showed that the fused cell membrane were intact without shrinkage and foaming;the nuclear membrane was intact and the nucleus was not pyknosis.3.The results of manual counting showed that the number of fused cells in the two groups of co-culture system increased in a time-dependent manner,while the number of fused cells in the hypoxic group was more than 1.5-2 times than that of control group(P<0.01).4.The results of FACS showed that the cell fusion rate was 0.85%±0.06%in experimental group and 0.39%± 0.08%in control group.The experimental results of the two groups have statistically significant differences(P<0.01).5.Hypoxic pretreatment led to the EMT of HIOEC.Both artificial counting method and FACS showed that the hypoxic treatment group had higher fusion rate than control group.6.After EMT was partially blocked by DAPT,the fusion rate also decreased significantly.Conclusion:The spontaneously cell fusion between CAL-27 and HIOEC could happen,and it be enhanced by hypoxia via EMT.Part ?:Study on the mechanism of microvesicles in regulating cell fusion between CAL-27 and human umbilical vein endothelial cellsObjective:To investigate the role of microvesicles in promoting the fusion of CAL-27 and human umbilical vein endothelial cells(HUVEC),and to explore the potential role of fusion protein VCAM-1 in promoting the fusion of CAL-27 and HUVEC mediated by CAL-27-derived microvesicles(TMVs).Material and methods:TMVs was performed through differential microcentrifuge extraction.TMVs were added to a co-culture system of RFP-CAL-27 and GFP-HUVEC.After co-cultivation for 3 days,the number of fused cells and the cell fusion rate were counted by manual counting and FACS,respectively.TMVs were co-cultured with HUVECs and the expression of VCAM-1 mRNA was detected by quantitative real-time RT-PCR at 6,12,and 24 h.siRNA was used to interfere with VCAM-1 expression in GFP-HUVEC,and then TMVs were added(or not)to the co-culture system of GFP-HUVEC(or non-interfering GFP-HUVEC)and RFP-CAL-27 after siRNA interference.The number of fused cells was calculated by artificial cell counting on the 1st and 3rd days;and the cell fusion rate on the 3 d was detected by FACS.Result:1.Compared to control group,the number of fused cells and cell fusion rate in TMVs-treated group were significantly increased(p<0.05).2.RT-PCR showed that the expression of VCAM-1 increased after TMVs stimulation and this increase peaked at 6 h.3.After silencing VCAM-1,calculate the number of fused cells by artificial cell counting.The results showed that the number of fused cells in both groups increased with time,but the number of fused cells in the experimental group was significantly lower than that in the control group(P<0.05).FACS results showed that the cell fusion rate in experimental group was 1.29%± 0.05%,which was significantly higher than that in control group(0.94%± 0.15%).Conclusion:TMVs could positively regulate the expression of VCAM-1 which plays an important role in cell fusion between CAL-27 and HUVEC.Part ?:Study on the mechanism of miR-146a-5p in TMVs to cell fusion between CAL-27 and HUVECObjective:To explore the underlying mechanisms of normoxia-derived and hypoxia-derived microvesicles in promoting the cell fusion between CAL-27 and HUVEC,and the effects of miR-146a-5p within tihe micro vesicles on the cell fusion.Material and methods:The expression of miR-146a-5p in HUVEC,CAL-27,and TMVs was detected by RT-PCR.The HUVEC was transfected by miR-146a-5p mimics and inhibitor,and then the cell fusion rates between CAL-27 and the transfected or normal HUVEC were measured.TMVs were isolated from CAL-27 transfected by miR-146a-5p and the expression of miR-146a-5p in the TMVs were detected by RT-PCR.The effects of cell fusion by normal CAL-27 derived TMVs and transfected CAL-27 derived TMVs were compared.Next,the alteration of miR-146a-5p in TMVs isolated from normoxia and hypoxia was measured,as well as the influence on cell infusion.Results:1.The expression of miR-146a-5p could be detected in CAL-27,and the expression level was significantly higher than that in HIOEC,oral keratinocytes(OKC)and HUVEC.2.RT-PCR results displayed that miR-146a-5p mimics and inhibitor were transfected into CAL-27 and HUVEC with high efficiency.3.Compared with HUVEC in the control group,the fusion of HUVEC and CAL-27 transfected with mimics and inhibitors significantly changed.To be specific,the fusion rate of HUVEC transfected with miR-146a-5p mimics was significantly increased,while the fusion rate of HUVEC transfected with miR-146a-5p inhibitor was significantly reduced.4.Hypoxia up-regulated the expression of miR-146a-5p in CAL-27,which promoted the fusion of HUVEC and CAL-27.Moreover,the expression of miR-146a-5p in TMVs increased after hypoxia treatment.The hypoxic treated TMVs promote the fusion of CAL-27 and HUVEC.5.The expression of miR-146a-5p in TMVs from CAL-27 transfected with mimics and inhibitros changed significantly.Moreover,there was a significant difference in cell fusion rate between CAL-27 and HUVEC after HUVEC was stimulated with TMVs(conventional,mimics,inhibitor).Conclusion:Hypoxia could promote the expression of miR-146a-5p in OSCC,and miR-146a-5p can be transmitted to HUVECs through TMVs,making it easier for TMVs-stimulated HUVEC to fuse with CAL-27.In summary,we confirmed that hypoxia and microvesicles could promote cell fusion.And hypoxia,EMT,VCAM-1 and TMVs play important roles in cell fusion.And on one hand,hypoxia promotes EMT,which is beneficial to fuse with CAL-27.On the other hand,hypoxia could enhance cell fusion by increasing the expression of miR-146a-5p in TMV.And those may be a mechanism of hypoxia promoting cell fusion.