The Effect And Mechanism Of ?-carotene On Esophageal Squamous Cell Carcinoma By Regulating MiR-375

Esophageal cancer is the eighth most common cancer in the world and the sixth most common cause of cancer death.There are two main types of esophageal cancer:esophageal adenocarcinoma and esophageal squamous cell carcinoma?ESCC?.Among them,ESCC is a major subtype of esophageal cancer in China.It is a malignant tumor produced by esophageal epithelial cells and has a poor prognosis,easy metastasis,high invasiveness,and high mortality.Although there has been some progress in the basic and clinical research of ESCC in recent years,the molecular mechanism and genetic basis of ESCC has not yet been revealed.At present,therapies based on fluorouracil and platinum drugs are still used clinically.The median survival time of patients after treatment failure is less than 1 year,and the prognosis is extremely poor.At present,the common treatment plan for natural anti-cancer compounds synergistic drugs has been recognized by people.Therefore,finding effective natural compounds for anti-tumor research has become one of the focuses of cancer therapy research.?-carotene,a natural plant pigment,is widely present in various fruits and vegetables.Studies have shown that in daily diets,people who regularly supplement carotenoids or radish hormones have a significantly lower risk of developing various cancers.Studies have shown that?-carotene can inhibit the proliferation of a variety of tumors,including breast cancer,colon cancer,melanoma,leukemia and so on.The results of the previous laboratory study showed that?-carotene can inhibit the proliferation of ESCC cells,promote its apoptosis,arrest its cell cycle in G0/G1 phase,and play an effective anti-tumor effect in vivo.However,the anti-tumor effect of?-carotene is a complex process involving multiple genes and proteins,and the mechanism of action remains to be further revealed.MicroRNAs?miRNAs?are non-coding RNAs of 18-22 bp in length that are involved in important physiological processes in the body and can be used as oncogenes or tumor suppressor genes to regulate the expression of related proteins through positive or negative regulation.The development of tumors.It has been found that the occurrence of tumors is usually accompanied by the abnormal expression of miRNAs.Studying the mechanism of miRNAs in tumors will help reveal the molecular mechanisms and genetic basis of tumors.Studies have shown that miRNA plays an important role in the anti-tumor effects of drugs.For example,curcumin,a natural compound,can upregulate the expression of Let-7a and regulate the Notch pathway,thereby inhibiting ESCC proliferation and promoting apoptosis.It is not clear whether?-carotene can exert an anti-tumor effect by regulating miRNA.ObjectThis study aims to investigate the role of?-carotene in the specific anti-tumor effects of miRNAs in ESCC.Methods1.Screening for differentially expressed miRNAsqRT-PCR was used to screen differentially expressed miRNAs in ESCC after treatment with?-carotene.Western blot was used to detect the expression changes of target proteins with differentially expressed miRNAs.2.In vitro studiesmiR-375 inhibitor and mimic were transfected into ESCC TE1 and EC1 cells by Lipofectamine₂000_Reagl to construct ESCC cells with knockdown or overexpression of miR-375.The proliferation of ESCC cells was detected by CCK-8assay,and AnnexinV-FITC/PI staining was used.The changes of cell apoptosis and cell cycle were detected by flow cytometry.The migration ability of ESCC cells was detected by Transwell chamber.The changes of miRNA target protein,?-catenin,apoptosis-related protein and E-cadherin were detected by Western blot.Quantitative PCR detects the expression changes of target protein and downstream protein mRNA.The expression of intracellular reactive oxygen species?ROS?was detected by chemical fluorescent probe method.3.In vivo studiesThe nude mice were injected subcutaneously with EC1 cells,EC1 cells transfected with miR-375 inhibitor,and EC1 cells transfected with miR-375 mimic to establish a nude mouse model of human ESCC xenografts.Group and?-carotene treatment groups,nude mice were divided into six groups,control group,?-carotene treatment group,transfection inhibitor control group,transfection inhibitor+?-carotene treatment group,transfection mimic control group,transfection mimic+?-carotene treatment group.When the control group tumor volume increased to about 100-150mm³ being dosing treatment,once every other day,continuous administration for three weeks,before each dose weight and tumor volume measurement and record,analyze and process the data and observe the judgment the difference between volume and tumor mass between groups.Fluorescence spectrophotometry was used to detect the changes of ROS in each group.TUNEL was used to detect the apoptosis of EC1 cells in each tumor tissue.Immunohistochemistry was used to detect the expression of apoptosis proteins Bcl-2,Bax and caspase-3.ResultsPart I Screening Differentially Expressed miRNAs in ESCC Cells after?-carotene TreatmentAfter?-carotene was applied to TE1 cells,qRT-PCR was performed on 20miRNAs reported in the literature to play an important role in the development of ESCC.It was found that miR-375 was up-regulated about 4 fold in TE1 cells after?-carotene treatment.TE1 and EC1 cells were treated with different concentrations of?-carotene.The expression of miR-375 was gradually increased with?-carotene concentration and detected by Western blot.After treatment with?-carotene,the expression of target protein in miR-375 was significantly reduced?P<0.05?,demonstrating that?-carotene may directly or indirectly regulate miR-375 to exert its anti-tumor effectPart II In vitro study of the role of miR-375 in the anti-tumor process of ESCC with?-carotene1.Effect of miR-375 on anti-tumor effect of?-carotene in vitro miR-375 inhibitor or mimic was transfected into ESCC cells.The qRT-PCR results showed that miR-375 expression could be significantly inhibited or up-regulated.Different concentrations of?-carotene were used to treat TE1 and EC1cells before and after transfection.The results indicated that the down-regulation of miR-375 resulted in the inhibition of?-carotene on the proliferation and migration of ESCC cells,the induction of apoptosis,and the effect on cells.Blocking of the cycle was significantly attenuated,and over-expression of miR-375 could promote the effects of?-carotene on proliferation,apoptosis,migration,and cycle of ESCC cells.In addition,?-carotene can upregulate the expression of ROS in cells,increase the content of MDA,and cause the decrease of mitochondrial membrane potential.After miR-375 inhibitor,it will inhibit the effect of?-carotene on intracellular redox levels.2.?-carotene exerts anti-tumor effects through miR-375 regulation of?-catenin signaling pathwayWestern blot analysis showed that?-catenin expression was significantly up-regulated in ESCC cells after?-carotene treatment.After miR-375 inhibitor was transfected into ESCC cells,the expression levels of miR-375 target protein FZD8,JAK2 and PDK1 were significantly up-regulated,while the expression of?-catenin was down-regulated,and the expression of?-catenin was detected in cells transfected with?-carotene.No significant change has occurred;and transfection of miR-375mimic can promote the effect of?-carotene on?-catenin expression.The above results indicate that?-carotene can regulate?-catenin signaling pathway through miR-375,thus exerting anti-tumor effects in ESCC.Part III The Effect of miR-375 on the Anti-tumor Effect of?-carotene in vivoTumor-bearing studies in nude mice have shown that?-carotene can significantly inhibit the growth of transplanted tumors in nude mice,promote the apoptosis of ESCC cells,and up-regulate the expression of miR-375 in tumor.There was no significant difference in the tumor volume of nude mice transfected with inhibitor control group and transfected with inhibitor+?-carotene treatment group.The tumor volume of transfected mimic control group was significantly higher than that of mimic+?-carotene transfected group.Decrease,the difference is significant.TUNEL assay and Western blot showed that compared with the?-carotene treatment group,the pro-apoptotic ability of?-carotene in the transfected inhibitor+?-carotene treatment group was significantly reduced.Conclusion1.?-carotene significantly up-regulated the expression of miR-375 in ESCC cells,and with the increase of?-carotene concentration,the expression of miR-375increased gradually;2.In vitro studies showed that after the down-regulation of miR-375 expression in ESCC cells,the role of?-carotene about inhibited the proliferation and migration of ESCC cells,promoted apoptosis and repressed the cell cycle was significantly weakened.The expression of E-catenin is no longer regulated by?-carotene,suggesting that?-carotene can exert its anti-tumor effects through miR-375 regulation of?-catenin signaling pathway.3.The co-action of?-carotene and miR-375 mimic can effectively enhance the inhibitory effect of?-carotene on the proliferation and migration of ESCC cells,the pro-apoptotic effect and the blockage of the cycle.4.In vivo studies showed that?-carotene could inhibit the growth of ESCC xenografts and up-regulate the expression of miR-375 in cells,but had no significant effect on the growth of ESCC xenografts with miR-375 down-regulation...