5-Fluorouracil dose escalation generated desensitized colorectal cancer cells with reduced expression of protein methyltransferases and no epithelial-to-mesenchymal transition potential

Background:Colorectal cancer(CRC)is one of the most frequently diagnosed cancers.In many cases,the poor prognosis of advanced CRC is associated with resistance to treatment with chemotherapeutic drugs such as 5-Fluorouracil(5-FU).The epithelial-to-mesenchymal transition(EMT)and dysregulation in protein methylation are two mechanisms associated with chemoresistance in many cancers.This study looked into the effect of 5-FU dose escalation on EMT and protein methylation in CRC.Materials and Methods:HCT-116,Caco-2,and DLD-1 CRC cell lines were exposed to dose escalation treatment of 5-FU.The motility and invasive potentials of the cells before and after treatment with 5-FU were investigated through wound healing and invasion assays.This was followed by aWestern blot which analyzed the protein expressions of the epithelial marker E-cadherin,mesenchymal marker vimentin,and the EMT transcription factor(EMTTF),the snail family transcriptional repressor 1(Snail)in the parental and desensitized cells.Western blotting was also conducted to study the protein expressions of the protein methyltransferases(PMTs),Euchromatic histone lysine methyltransferase 2(EHMT2/G9A),protein arginine methyltransferase(PRMT5),and SET domain containing 7/9(SETD7/9)along with the global lysine and arginine methylation profiles.Results:The dose escalation method generated 5-FU desensitized CRC cells with distinct morphological features and increased tolerance to high doses of 5-FU.The 5-FU desensitized cells experienced a decrease in migration and invasion when compared to the parental cells.This was reflected in the observed reduction in E-cadherin,vimentin,and Snail in the desensitized cell lines.Additionally,the protein expressions of EHMT2/G9A,PRMT5,and SETD7/9 also decreased in the desensitized cells and global protein lysine and arginine methylation became dysregulated with 5-FU treatment.Conclusion:This study showed that continuous,dose-escalation treatment of 5-FU in CRC cells generated 5-FU desensitized cancer cells that seemed to be less aggressive than parental cells.

Role of epithelial-to-mesenchymal transition in the pulmonary fi brosis induced by paraquat in rats

BACKGROUND:This study aims to explore the characteristics of the epithelial-to-mesenchymal transition(EMT)process and its underlying molecular mechanisms in the period of paraquat(PQ)-induced pulmonary fi brosis(PF).METHODS:Picrosirius red staining and collagen volume fraction were utilized to evaluate the pathological changes of PQ-induced PF in rats.Immunohistochemistry,Western blot,and real-time reverse transcriptase-polymerase chain reaction(RT-PCR)were used to measure the protein and gene expression of EMT markers,EMT-associated transcription factors,and regulators of EMT-related pathways,respectively.RESULTS:The collagen deposition in the alveolar septum and increased PF markers were characteristics of pathological changes in PQ-induced PF,reached a peak on day 14 after PQ poisoning,and then decreased on day 21.The protein and gene expression of the fibrosis marker,EMT markers,transcription factors,and regulators of EMT-related signaling pathways signifi cantly increased at diff erent time points after PQ poisoning compared with corresponding controls(P<0.05),and most of them reached a peak on day 14,followed by a decrease on day 21.The gene expression of EMT markers was significantly correlated with PF markers,transcription factors,and regulators of EMT-related signaling pathways(P<0.05).The mRNA expression of transcription factors was signifi cantly correlated with that of TGF-β1 and Smad2(P<0.05 or P<0.01),instead of Wnt2 andβ-catenin(P>0.05).CONCLUSIONS:EMT process plays a role in the PQ-induced PF,in which most PF and EMT markers have a peak phenomenon,and its underlying molecular mechanisms might be determined by further studies.

Cross-talk between microRNA-let7c and transforming growth factor-β2 during epithelial-to-mesenchymal transition of retinal pigment epithelial cells

AIM: To explore the roles of microRNA-let7 c(miR-let7 c) and transforming growth factor-β2(TGF-β2) and cellular signaling during epithelial-to-mesenchymal transition(EMT) of retinal pigment epithelial cells. METHODS: Retinal pigment epithelial(ARPE-19) cells were cultured with no serum for 12 h, and then with recombinant human TGF-β2 for different lengths of time. ARPE-19 cells were transfected with 1×106 TU/mL miR-let7 c mimcs(miR-let7 cM), miR-let7 c mimcs negative control(miR-let7cMNC) and miR-let7 c inhibitor(miR-let7 cI) using the transfection reagent. The expression of keratin-18, vimentin, N-cadherin, IKB alpha, p65 were detected by Western blot, quantitative polymerase chain reaction and immunofluorescence. RESULTS: The expression of miR-let7c was dramatically reduced and the nuclear factor-kappa B(NF-κB) signaling pathway was activated after induction by TGF-β2(P<0.05). In turn, overexpressed miR-let7 c significantly inhibited TGF-β2-induced EMT(P<0.05). However, miR-let7 c was unable to inhibit TGF-β2-induced EMT when the NF-κB signaling pathway was inhibited by BAY11-7082(P<0.01). CONCLUSION: The miR-let7 c regulates TGF-β2-induced EMT through the NF-κB signaling pathway in ARPE-19 cells.

Epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma: Characterization in a 3D-cell culture model

AIM: To analyze the effect of three-dimensional(3D)-arrangement on the expression of epithelial-tomesenchymal transition markers in pancreatic adenocarcinoma(PDAC) cells.METHODS: HPAF-Ⅱ, HPAC, and PL45 PDAC cells were cultured in either 2D-monolayers or 3D-spheroids. Ultrastructure was analyzed by transmission electron microscopy. The expression of E-cadherin, β-catenin, N-cadherin, collagen type Ⅰ(COL-I), vimentin, α-smooth muscle actin(αSMA), and podoplanin was assayed by confocal microscopy in cells cultured on 12-mm diameter round coverslips and in 3D-spheroids. Gene expression for E-cadherin, Snail, Slug, Twist, Zeb1, and Zeb2 was quantified by real-time PCR. E-cadherin protein level and its electrophoretic pattern were studied by Western blot in cell lysates obtained from cells grown in 2D-monolayers and 3D-spheroids. RESULTS: The E-cadherin/β-catenin complex was expressed in a similar way in plasma membrane cell boundaries in both 2D-monolayers and 3D-spheroids. E-cadherin increased in lysates obtained from 3D-spheroids, while cleavage fragments were more evident in 2D-monolayers. N-cadherin expression was observed in very few PDAC cells grown in 2D-monolayers, but was more evident in 3D-spheroids. Some cells expressing COL-I were observed in 3D-spheroids. Podoplanin, expressed in collectively migrating cells, and αSMA were similarly expressed in both experimental conditions. The concomitant maintenance of the E-cadherin/β-catenin complex at cell boundaries supports the hypothesis of a collective migration for these cells, which is consistent with podoplanin expression.CONCLUSION: We show that a 3D-cell culture model could provide deeper insight into understanding the biology of PDAC and allow for the detection of marked differences in the phenotype of PDAC cells grown in 3D-spheroids.

Unleashing the pathological role of epithelial-to-mesenchymal transition in diabetic nephropathy: The intricate connection with multifaceted mechanism

Renal epithelial-to-mesenchymal transition(EMT)is a process in which epithelial cells undergo biochemical changes and transform into mesenchymal-like cells,resulting in renal abnormalities,including fibrosis.EMT can cause diabetic neph-ropathy through triggering kidney fibrosis,inflammation,and functional impair-ment.The diverse molecular pathways that drive EMT-mediated renal fibrosis are not utterly known.Targeting key signaling pathways involved in EMT may help ameliorate diabetic nephropathy and improve renal function.In such settings,un-derstanding precisely the complicated signaling networks is critical for develo-ping customized therapies to intervene in EMT-mediated diabetic nephropathy.

Epithelial-mesenchymal transition- activating transcription factors- multifunctional regulators in cancer

The process of epithelial to mesenchymal transition(EMT), first noted during embryogenesis, has also been reported in tumor formation and leads to the development of metastatic growth. It is a naturally occurring process that drives the transformation of adhesive,non-mobile epithelial like cells into mobile cells with a mesenchymal phenotype that have ability to migrate to distant anatomical sites. Activating complex network of embryonic signaling pathways, including Wnt, Notch,hedgehog and transforming growth factor-β pathways,lead to the upregulation of EMT activating transcription factors, crucial for normal tissue development and maintenance. However, deregulation of tightly regulated pathways affecting the process of EMT has been recently investigated in various human cancers. Given the critical role of EMT in metastatic tumor formation,better understanding of the mechanistic regulation provides new opportunities for the development of potential therapeutic targets of clinical importance.

p38 MAPK is Crucial for Wnt1- and LiCl-Induced Epithelial Mesenchymal Transition

Idiopathic pulmonary fibrosis （IPF） is characterized by myofibroblast loci in lung parenchyma. Myofibroblasts are thought to originate from epithelial-to-mesenchymal transition （EMT）. Wntl and lithium chloride （LiCl） induce EMT in alveolar epithelial cells （AECs）, but the mechanisms are unclear. AECs were treated with Wntl and LiC1, respectively; morphological change and molecular changes of EMT, including E-cadherin, fibronectin, and vimentin, were observed. SB203580 was administrated to test the role of p38 MAPK signaling in EMT. Then AECs were treated with siRNAs targeting p38 MAPK to further test the effects of p38 MAPK, and the role was further confirmed by re-expression of p38 MAPK. At last β-catenin siRNA was used to test the role of β-catenin in the EMT process and relationship of β-catenin and p38 MAPK was concluded. Exposure of AECs to Wntl and LiC1 resulted in upregulation of vimentin and fibronectin with subsequent downregulation of E-cadherin. Wntl and LiC1 stimulated the p38 MAPK signaling pathways. Perturbing the p38 MAPK pathway either by SB203580 or through p38 MAPK siRNA blocked EMT and inhibited fibronetin synthesis, which were reversed by transfection of p38 MAPK expression plasmid. β-catenin siRNA attenuated the EMT process and decreased p38 MAPK phosphorylation, indicating that β-catenin is involved in the EMT- related changes through regulation of p38 MAPK phosphorylation. These findings suggest that p38 MAPK participates in the pathogenesis of EMT through Wnt pathway and that p38 MAPK may be a novel target for IPF therapy.

Roles of Rho/Rock Signaling Pathway in Silica-induced Epithelial-mesenchymal Transition in Human Bronchial Epithelial Cells

Objective To investigate the roles of Rho/Rock signaling pathway in silica-induced Epithelial-mesenchymal transition （EMT） in human bronchial epithelial cells （BEC） in vitro. Methods Human BEC were incubated with silica with various concentrations for indicated times. Cell viability was assayed by MTT test. Morphologic Changes were observed by microscope. Mesenchymal marker a-smooth muscle actin （a-SMA）, vimentin （Vim）, and epithelial marker E-cadherin （E-cad） were analyzed by Western Blot. The pull-down assay was used to measure Rho activity. In the prevention experiments, the specific inhibitor for Rho effector ROCK （Y27632） was used to inhibit the activity of Rho. Results Human BEC stimulated with silica were converted from a ＂cobblestone＂ epithelial structure into an elongated fibroblast-like shape structure. Incubation of human BEC with silica induced de novo expression of a-SMA and Vim, and loss of E-cad. Also, silica treatment resulted in Rho activation in human BEC. Y27632 up-regulated the E-cad expression but attenuated a-SMA and Vim expression in silica-stimulated cells. Conclusion The activation of Rho/ROCK signaling pathways is most likely involved in Silica-induced EMT in human bronchial epithelial cells.

Re-evaluating the role of epithelial-mesenchymal-transition in cancer progression

Epithelial-mesenchymal transition（EMT） and mesenchymal-epithelial transition（MET） are essential for embryonic development and also important in cancer progression. In a conventional model, epithelial-like cancer cells transit to mesenchymal-like tumor cells with great motility via EMT transcription factors; these mesenchymallike cells migrate through the circulation system, relocate to a suitable site and then convert back to an epithelial-like phenotype to regenerate the tumor. However, recent findings challenge this conventional model and support the existence of a stable hybrid epithelial/mesenchymal（E/M） tumor population. Hybrid E/M tumor cells exhibit both epithelial and mesenchymal properties, possess great metastatic and tumorigenic capacity and are associated with poorer patient prognosis. The hybrid E/M model and associated regulatory networks represent a conceptual change regarding tumor metastasis and organ colonization. It may lead to the development of novel treatment strategies to ultimately stop cancer progression and improve disease-free survival.

Epithelial-mesenchymal transition status of circulating tumor cells in breast cancer and its clinical relevance

Objective:Circulating tumor cells(CTCs)play a critical role in cancer metastasis,but their prevalence and significance remain unclear.This study attempted to track the epithelial-mesenchymal transition(EMT)status of CTCs in breast cancer patients and investigate their clinical relevance.Methods:In this study,the established negFACS-IF:E/M platform was applied to isolate rare CTCs and characterize their EMT status in breast cancer.A total of 89 breast cancer patients were recruited,including stage 0–III(n=60)and late stage(n=29)cases.Results:Using the negFACS-IF:E/M platform,it was found that in human epidermal growth factor receptor 2(HER2)+patients,mesenchymal CTCs usually exhibited a high percentage of HER2+cells.Stage IV breast cancer patients had considerably more CTCs than stage 0–III patients.Among stage 0–III breast cancers,the HER2 subtype included a significantly higher percentage of mesenchymal and biphenotypic(epithelial and mesenchymal)CTCs than the luminal A or B subtypes.Among stage IV patients,CTCs were predominantly epithelial in cases with local recurrence and were more mesenchymal in cases with distant metastasis.By applying a support vector machine(SVM)algorithm,the EMT status of CTCs could distinguish between breast cancer cases with metastasis/local recurrence and those without recurrence.Conclusions:The negFACS-IF:E/M platform provides a flexible and generally acceptable method for the highly sensitive and specific detection of CTCs and their EMT traits in breast cancer.This study demonstrated that the EMT status of CTCs had high clinical relevance in breast cancer,especially in predicting the distant metastasis or local recurrence of breast cancer.

Transcriptional Factor Snail Mediates Epithelial-Mesenchymal Transition in Human Bronchial Epithelial Cells Induced by Silica

Epithelial-mesenchymal transition （EMT） plays an important role in fibrotic diseases. We have previously showed that silica induces EMT in human bronchial epithelial cells （BECs）; however, the underlying mechanism of silica-induced EMT is poorly understood. In the present study, we investigated the role of Snail in silica-induced EMT in human BECs in vitro. Human BECs were treated with silica at various concentrations and incubation times. Then MTr assay, western blot, electrophoretic mobility shift assay （EMSA）, and small interfering RNA （siRNA） transfection were performed. We found that silica increased the expression and DNA binding activity of Snail in human BECs. SNAI silica-induced expression siRNA upregulated the siRNA inhibited the of Snail. Moreover, SNAI expression of epithelial marker E-cadherin, but attenuated the expression of mesenchymal marker a-smooth muscle actin and vimentin in silica-stimulated cells. These results suggest that Snail mediates the silica-induced EMT in human BECs.

Circulating tumor cells with epithelial-mesenchymal transition markers as potential biomarkers for the diagnosis of lung cancer

BACKGROUND Circulating tumor cells(CTCs) can be clustered into three subtypes according to epithelial-mesenchymal transition(EMT) markers: CTCs with epithelial markers(E-CTCs), CTCs with mesenchymal markers(M-CTCs), and CTCs with both markers(E&M-CTCs). CTC detection has clinical implications in the diagnosis of lung cancer(LC).AIM To clarify the diagnostic value of CTCs categorized by EMT markers in LC.METHODS The study included 106 patients with lung adenocarcinoma, including 42 groundglass opacities(GGO) and 64 solid lesions, who underwent surgery between July 2015 and December 2019. Eleven patients with benign tumors and seventeen healthy controls were included. CTCs in peripheral blood and associated EMT markers were detected preoperatively using the CanPatrol TM technique. The diagnostic power of CTCs for discriminating LC cases from controls was analyzed by the receiver operating characteristic(ROC) curve. The CytoploRare technique was used in 20 cases and 18 controls for validation, and Kappa values were calculated to evaluate consistency between techniques.RESULTS Of the 106 LC cases, 94(89.6%) had at least one CTC. CTCs were detectable in 35(83.3%) of 42 GGO cases. Total CTCs and E&M-CTCs were significantly more frequent in LC cases than in benign or healthy controls. The proportion of MCTCs plus E&M-CTCs increased gradually from healthy controls, to benign controls, to LC cases. The area under the ROC curve of total CTCs and E&M-CTCs was > 0.8 and > 10.75, respectively. The combined sensitivity of total-CTCs and E&M-CTCs was 85.85% for LC patients(80.95% for GGO patients) and the specificity was 78.57%.The Kappa value was 0.415,indicating relative consistency between CanPatrol TM and CytoploRare.CONCLUSION CTC detection is valuable for distinguishing LC from controls,and particularly E&M-CTC detection warrants further study.

MiR-663a Inhibits Radiation-Induced Epithelium-to-Mesenchymal Transition by Targeting TGF-β1

Objective miR-663 a has been reported to be downregulated by X-ray irradiation and participates in radiation-induced bystander effect via TGF-β1.The goal of this study was to explore the role of mi R-663 a during radiation-induced Epithelium-to-mesenchymal transition(EMT).Methods TGF-β1 or IR was used to induce EMT.After mi R-663 a transfection,cell migration and cell morphological changes were detected and the expression levels of mi R-663 a,TGF-β1,and EMT-related factors were quantified.Results Enhancement of cell migration and promotion of mesenchymal changes induced by either TGF-β1 or radiation were suppressed by mi R-663 a.Furthermore,both X-ray and carbon ion irradiation resulted in the upregulation of TGF-β1 and downregulation of mi R-663 a,while the silencing of TGF-β1 by mi R-663 a reversed the EMT process after radiation.Conclusion Our findings demonstrate an EMT-suppressing effect by mi R-663 a via TGF-β1 in radiationinduced EMT.

Silencing Neuropilin 1 gene reverses TGF-β1-induced epithelial mesenchymal transition in HGC-27 gastric cancer cell line

Objective The aim of this study was to determine Neuropilin 1(NRP1)contribution to transforming growth factorβ1(TGF-β1)-induced epithelial mesenchymal transition(EMT)of HGC-27 gastric cancer cells and study its mechanism.Methods In this study,TGF-β1 was used to induce EMT in HGC-27 cells.Further,these cells were stably transfected with siRNA targeting NRP1.Wound healing and transwell assays were used to measure cell migration and invasion,respectively.NRP1 and EMT markers were measured using quantitative real time reverse transcription polymerase chain reaction and western blotting.Results Exposure of TGF-β1 conferred a fibroblastic-like shape to cancer cells and significantly increased the expression of NRP1 in HGC-27 cells.TGF-β1 subsequently promoted migration and invasion of HGC-27 cells.Furthermore,silencing NRP1 inhibited the invasion and migration of TGF-β1-induced cells undergoing EMT.Conclusion Silencing NRP1 can inhibit cell migration,invasion,and metastasis and reverse the TGF-β1-induced EMT process of gastric cancer.

Effect of Rapamycin on TGF-β_1-induced epithelial-mesenchymal transition in LoVo colonic adenocarcinoma cells

Objective： To investigate the effect of Rapamycin on epithelial-mesenchymal transition（EMT） of LoVo colonic adenocarcinoma cells in vitro. Methods：Cultured LoVo colonic adenocarcinoma cells were divided into three groups： negative control group, EMT-inducing group（TGF-β1） and EMT-interfering group（TGF-β1 plus Rapamycin）. E-cadherin expression in LoVo cells was detected by Western Blot, while the expression of vimentin was evaluated through immunocytochemistry. The Snail mRNA in LoVo cells was examined by RT- PCR. Results：TGF-β1 induced LoVo cell switching from polygonal to spindle-shaped. TGF-β1 enhanced the expression of vimentin, but lowered the level of E-cadhefin. In contrast, Rapamycin impaired the transition induced by TGF-β1. Rapamycin dramatically abrogated TGF-β1-induced vimentin expression and restored E-cadherin expression in LoVo cells. Rapamycin significantly repressed the upregulation of Snail mRNA expression induced by TGF-β1. Conclusion：Rapamycin dramatically abrogated TGF-β1 induced Snail mRNA expression in LoVo cells, hence inhibiting EMT of these cells in vitro.

Effect of Peroxiredoxin 1 on the biological function of airway epithelial cells and epithelial-mesenchymal transition

Peroxiredoxin 1(PRDX1)participates in tumor cell proliferation,apoptosis,migration,invasion,and the epithelial-to-mesenchymal transition(EMT).This study aimed to investigate the effect of PRDX1 on the EMT of airway epithelial cells stimulated with lipopolysaccharide(LPS)and transforming growth factor-beta 1(TGF-β1).PRDX1 overexpression significantly increased the proliferation and migration of human bronchial epithelial(BEAS-2B)cells,reduced cell apoptosis(p<0.01),and induced EMT and collagen deposition by upregulating the expression of the matrix metallopeptidase(MMP)2,MMP9,α-smooth muscle actin(α-SMA),N-cadherin,vimentin and twist proteins and inhibiting E-cadherin expression(p<0.05).PRDX1 overexpression promoted TGF-β1-mediated inhibition of cell proliferation and migration and significantly enhanced the TGF-β1-induced EMT and collagen synthesis(p<0.05).Knockdown of PRDX1 inhibited cell proliferation,migration,EMT,and collagen synthesis(p<0.01),reversed LPS-mediated inhibition of cell proliferation and migration,and significantly suppressed LPS-induced EMT and collagen synthesis(p<0.01).The result indicating that PRDX1 may be involved in LPS/TGF-1-induced EMT and collagen synthesis in human bronchial epithelial cells.

Strategies for Synchronous and Multiple Metastatic Liver Tumors Designed from Epithelial-Mesenchymal Transition Concept

At some point in the natural course of colorectal cancer up to 50% of patients will develop metastasis to the liver and it is one of the most critical effects for patient prognosis. The incidence of synchronous liver metastasis has been detected at around 20% - 25%, but the optimal timing of surgical resection remains controversial. Neoadjuvant chemotherapy has also been found to be beneficial not only for initially unresectable but also resectable synchronous metastases. Then, traditional surgical strategies of hepatic resection in accordance with past chemotherapeutic regimens have been used decreasingly over the past several years. This review will primarily discuss treatments in association with the recent developed chemotherapeutic regimens and surgical procedure from the clinical data and the concept for epithetlial-mesenchymal transition, which has recently been studied to elucidate mechanisms of the liver metastatic process.

白藜芦醇通过Hippo-YAP信号通路在TGF-β1诱导胃癌细胞EMT过程中的作用及其机制

目的探讨白藜芦醇(RSVL)通过Hippo-YAP信号通路在TGF-β1诱导胃癌细胞上皮间充质转化(EMT)过程中的作用及其机制。方法选取胃癌细胞SGC-7901为研究对象,首先将其随机分为空白组、RSVL低剂量组(5μM)、RSVL中剂量组(10μM)、RSVL高剂量组(20μM),通过细胞增殖(MTT)实验确定RSVL浓度,然后对细胞进行转染,分为si-NC组、TGF-β1+10μM RSVL组、TGF-β1+si-YAP组、TGF-β1+pcDNA3.1-YAP组、TGF-β1+10μM RSVL+pcDNA3.1-YAP组。采用MTT、细胞侵袭(Transwell)、划痕实验,分别检测细胞的增殖、侵袭和迁移;采用蛋白质印迹法(WB)和荧光定量PCR检测E-钙粘连蛋白(E-cadherin)、神经型钙黏附蛋白(N-cadherin)、波形蛋白(Vimentin)、Snali 1、HIP-PO/Yes相关蛋白(YAP)和mRNA。其次,选取24只裸鼠,将其分为模型组、RSVL组(10μM)、RSVL+pcDNA3.1组、RSVL+pcDNA3.1-YAP组,每组各六只。计算小鼠肿瘤的重量和体积;采用免疫组化检测Ki67蛋白。结果RSVL对细胞增殖有抑制作用(P<0.05);与si-NC组相比,其余各组侵袭细胞数、迁移率较低(P<0.05);与si-NC组相比,其余各组E-cadherin蛋白及mRNA表达较高,N-cadherin、Vimentin、Snali 1蛋白及mRNA以及YAP蛋白表达较低(P<0.05);与Model组相比,其余各组小鼠肿瘤的重量和体积均较低(P<0.05);与Model组比较,其余各组Ki67染色强度均显著减弱。结论RSVL可以通过抑制YAP相关通路的激活,来发挥抑制肿瘤细胞SGC-7901上皮间充质转化、增殖、迁移、侵袭的作用。

INHBA-AS1通过c-Myc/SCD通路调控宫颈癌HeLa细胞的鸟氨酸代谢和EMT进程

目的:探讨抑制素β亚基A反义RNA1(INHBA-AS1)对宫颈癌HeLa细胞EMT和鸟氨酸代谢途径的影响及其机制。方法:体外常规培养HeLa细胞,实验分为10组:对照组、阴性对照(NC)组、sh-INHBA-AS1组、PluriSIn 1[硬脂酰辅酶A去饱和酶(stearyl CoA desaturase,SCD)抑制剂]组、NC+PluriSIn 1组、sh-INHBA-AS1+PluriSIn 1组、10058-F4(c-Myc抑制剂)组、NC+10058-F4组、sh-INHBA-AS1+10058-F4组、sh-INHBA-AS1+OE-c-Myc组。平板克隆实验检测各组细胞的增殖能力,FCM检测各组细胞的凋亡情况,Transwell小室实验检测各组细胞的侵袭、迁移能力,qPCR法检测各组细胞中INHBA-AS1、c-Myc、SCD和EMT相关基因(N-cadherin、TGF-β、ZEB1)mRNA的表达,WB法检测各组细胞中c-Myc、SCD、EMT相关(N-cadherin、TGF-β、ZEB1)、S-腺苷-甲硫氨酸脱羧酶(SAMDC)和亚精胺/精胺N1-乙酰转移酶(SSAT)蛋白的表达,ELISA检测各组细胞上清液中鸟氨酸脱羧酶(ODC)的含量。结果:敲减INHBA-AS1表达使HeLa细胞的增殖、侵袭和迁移能力显著降低(均P<0.05)而细胞凋亡率显著升高(P<0.05),q PCR、WB法检测结果显示,敲减INHBA-AS1均可显著抑制HeLa细胞中c-Myc、SCD、N-cadherin、TGF-β、ZEB1和SAMDC的表达(均P<0.05),而促进SSAT的表达(P<0.05),并降低HeLa细胞上清液中ODC的含量(P<0.05)。与c-Myc抑制剂和SCD抑制剂单独处理相比,其联合敲减INHBA-AS1后上述作用更加显著(均P<0.05);与sh-INHBA-AS1组相比,进一步过表达c-Myc后HeLa细胞的增殖能力显著升高(P<0.05)、SCD和N-cadherin蛋白表达水平显著升高(P<0.05)、细胞上清液中ODC含量显著升高(P<0.05)。结论:INHBA-AS1可通过c-Myc调控SCD的表达,从而影响HeLa细胞鸟氨酸代谢和EMT进程,进而促进HeLa细胞的增殖、侵袭和迁移能力。