| Choriocarinoma is a rare malignant gestational trophoblastic disease that occurs mainly in women of childbearing age and is prone to distant metastases and spread to other organs.Choriocarcinoma can be divided into three types of pregnancy and non-pregnancy choriocarcinoma and unclassified choriocarcinoma.In Asia,the incidence of choriocarcinoma is 3 to 9 times that of Europe and North America.With the improvement of medical technology level,chemotherapy drugs that are commonly used in cancer treatment have caused great pain and even lead to treatment failure because of their non-selective killing effect,multidrug resistance,and severe adverse reactions.As a result,the curability rate is reduced.Therefore,the search for new antitumor drugs and their potential targets has become a hot topic in cancer research.MiR-373 acts as an intracellular regulator and is a non-coding regulatory RNA with a length of 18-25 nt.Its mechanism of action is through the 3’untranslated region(3’ UTR)of the mRNA of the target protein gene.Completely or incompletely complementary binding,thereby inhibiting protein mRNA translation or degradation of downstream products,plays a regulatory role at the post-transcriptional level.The literature reports that miR-373 was first confirmed to be an oncogene miRNA in human testicular germ cell tumors,which can promote tumor cell growth.Further studies showed that miR-373 cells exhibit high expression.It has been reported in the literature that most of the members of the TGFβ-Smads signaling pathway can be targeted by the microRNA molecules to bind directly or indirectly to protein expression levels.MiRNAs can directly regulate the expression levels of TGFβ signaling pathway members and downstream target genes.Other studies have shown that miR-373 and miR-520 c have similar effects in promoting the infiltration and metastasis of breast cancer cells by inhibiting the expression of the common target molecules CD44 and LATS2 mRNA and inhibiting the infiltration and metastasis of tumors.Regulate the role of tumor growth.TGFβ was discovered during the study of tumor virus transformation and was therefore named transforming growth factor β(TGFβ).TGFβ is a multifunctional growth factor that plays an important regulatory role in a variety of cellular processes.Its mechanism of action is through the combination of receptors to achieve signal transmission.Previous research has found that TGFβ-Smads signaling pathway is closely related to the proliferation of choriocarcinoma cells and is a key signaling pathway regulating the proliferation and invasion of tumor cells.Targeted drugs of key molecules are one of the effective treatments for choriocarcinoma.More and more studies have shown that dihydromyricetin has potential therapeutic effects in a variety of malignant tumors and has selective toxic effects.However,the anti-tumor mechanism of action of dihydromyricetin on human choriocarcinoma cell lines remains unclear.Rattan tea is commonly known as the Dragon Boat Festival tea,rattan tea.It belongs to Ampelopsis grossedentata,which is a species endemic to China and mainly distributed in Hubei,Hunan,Guangxi,Jiangxi and Fujian.Rattan tea is widely used in traditional medicine and therefore enjoys the title of "God Tea".Its main ingredient is flavonoids.Dihydromyricetin as the main component in plant vine tea,has similar pharmacological effects to rattan tea.Studies have shown that it has many physiological effects such as anti-inflammatory and anti-bacterial,analgesic and antitussive,anti-hypertension,hypoglycemic,hepatoprotective,and immune-enhancing.With the improvement of modern drug extraction and purification technology,dihydromyricetin has been purified and extracted as an active ingredient in plant flavonoid extracts.Due to its extensive pharmacological effects and low toxicity and high efficiency,dihydromyricetin is used in various studies.Dihydromyricetin has been widely used in basic research in recent years,and many results show that its anti-oxidation,liver protection,anti-hypertension,anti-tumor effects.The literature reports that the anti-tumor activity of dihydromyricetin is mainly involved in the process of tumorigenesis,development,invasion and metastasis,including inhibiting cell proliferation,arresting cell cycle,inducing apoptosis,fighting cell invasion and metastasis,and combating tumor angiogenesis.Our early results showed that miR-373-5p is differentially expressed in human choriocarcinoma tissues and normal tissues,and miR-373-5p is more highly expressed in choriocarcinoma tissues than in normal chorionic tissues.TGFβ-Smads signaling pathway is closely related to the proliferation of choriocarcinoma cells,and it is a key signal pathway to regulate the proliferation and invasion of tumor cells.This shows that both of them are involved in the regulation of the occurrence and development of human choriocarcinoma cells,but the specific mechanism has not yet been clarified.The purpose of this study was to further clarify the role of miR-373 in the proliferation of human choriocarcinoma cells,and to explore the effect of dihydromyricetin on the expression of TGFβRII and miR-3730-5p in human choriocarcinoma cell lines.Purpose:To determine the relative expression changes of microRNAs in different cell lines and to investigate the effect of dihydromyricetin on the relative expression of miR-373-5p and TGFβRII,Smad2,and P-smad2 proteins and mRNA in human choriocarcinoma JAR and JEG-3 cell lines.Transfection technique was used to investigate the effect of miR-373-5p on the relative expression of TGFβRII,Smad2,P-smad2,C-myc protein and mRNA in JAR cell lines.Method:1.Normal human villous epithelial HTR-8 cells and human choriocarcinoma JAR and JEG-3 cells were cultured in vitro.The relative expression changes of miR-373-5p in the three cell lines were detected by RT-qPCR.2.JAR cells were selected for transient transfection and transfected into Negative Control FAM.The fluorescence signal of each group was observed under microscope.In this ratio,Negative Control and hsa-miR-373-5p mimics were transfected into JAR cells.RT-qPCR assay for the relative expression of miR-373-5p in cells after transfection3.Transfection of Negative Control,hsa-miR-373-5p mimics,Negative Control inhibitor,and has-miR-373-5p inhibitor.After a certain period of time,the culture was stopped.Total protein was extracted from each group and western blotting was performed.The relative expression changes of TGFβRII,Smad2,P-smad2,and C-myc proteins were detected in each group.4.In the experimental group,40μg/ml and 80μg/ml DMY were used to treat JAR cells,and the culture was terminated 24 h later.The relative expression of miR-373-5p was detected by RT-qPCR.5.In the experimental group,40μg/ml,60μg/ml,and 80μg/ml DMY were administered to JAR and JEG-3 cells,and a blank control group was established.The control group was added with the same volume as the experimental group.Complete culture medium was terminated after 24 hours.Total protein and total mRNA were extracted from each group.Western blotting was used to detect the relative expression changes of TGFβRII,Smad2 and P-smad2 proteins in JAR and JEG-3 cells.RT-qPCR was used to detect the relative expression changes of TGFβRII and Smad2 mRNA in JAR cells.Result:Compared with human normal nourishing epithelial cell line HTR-8,the relative expression of miR-373-5p in human choriocarcinoma cell lines JAR and JEG-3 was decreased,and the difference was statistically significant(P<0.05);After JAR cells were transfected with Negative Control FAM,the intracellular fluorescence signal was observed to increase with time under fluorescence microscope;intracellular hsa-miR-373-5p mimics were transiently transfected,compared with negative control group,has-miR-373-5p mimics were transferred.The relative expression of miR-373-5p in the choriocarcinoma JAR cell line was increased,the difference was statistically significant(P<0.05),and the transfection efficiency was about 80%.Compared with negative control group,the relative expression of TGFβRII,Smad2,P-smad2,C-myc protein in JAR cells transfected with has-miR-373-5p mimics was decreased,and the difference was statistically significant(P<0.05);Compared with the negative control inhibitor group,the relative expression of TGFβRII,Smad2,P-smad2,and C-myc protein in the JAR cells transfected with the has-miR-373-5p inhibitor increased,and the difference was statistically significant(P<0.05);Compared with control group,40μg/ml and 80μg/ml of DMY in the experimental group acted on JAR cells,and the relative expression of miR-373-5p was increased,the difference was statistically significant(P<0.05);Compared with the blank control group,the relative expression of TGFβRII,Smad2,and P-smad2 protein in the two human choriocarcinoma cells treated with different concentration of DMY 40μg/ml,60μg/ml,and 80μg/ml was decreased and the relative concentration was decreased.The increase was gradually reduced,and the difference was statistically significant(P<0.05).Compared with the blank control group,the relative expression of TGFβRII and Smad2 mRNA in the JAR cell line under different concentrations of DMY 40 μg/ml,60μg/ml,and 80μg/ml was decreased in the experimental group.Decreased,and gradually decreased with increasing concentrations,the difference was statistically significant(P <0.05).Conclusion:1.MiR-373-5p regulates human choriocarcinoma cells through TGFβ-Smads signal pathway and dihydromyricetin through TGFβ-Smads signal pathway and miR-373-5p exerts regulatory effects on human choriocarcinoma cells.2.Dihydromyricetin has anti-tumor effect on human choriocarcinoma cells,comprehensive pre-study results show that dihydromyricetin may regulate the proliferation,differentiation and metastasis of human choriocarcinoma cells through miR-373-5p-TGFβ-Smads signaling pathway,which may provide a kind of potential treatment strategies for choriocarcinoma patients. |
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