The Expression Of MiR-301a-3p In Esophageal Squamous Cell Carcinoma,and Its Fuction And Mechanisms Of Regulating Cell Proliferation

Malignant tumor is one of the three major diseases that are dangerous to human health.The incidence of esophageal cancer ranks sixth among malignant tumors worldwide,and the mortality rate is the fourth.The incidence of esophageal cancer in our country accounts for more than 50%of the world.In 2015,the incidence of esophageal cancer in China was 246,000,with an incidence rate of 17.87 per 100,000;188,000 deaths and a mortality rate of 13.68 per 100,000.The pathological types of esophageal cancer mainly include squamous cell carcinoma and adenocarcinoma.Contrary to European and American countries,more than 90%of esophageal cancer is squamous cell carcinoma in China.Its incidence reveals obvious familial and geographical clustering,which extremely important to study the pathogenesis of esophageal cancer.It has been validated that the combined effects of environment,dietary habits,geographical location and other factors have led to the occurrence of esophageal cancer.Genetic studies demonstrate that the occurrence of tumor is caused by the imbalance in the expression of tumor-promoting genes and tumor suppressor genes.MicroRNA(MiRNA)is a type of non-coding,endogenous small RNA composed of 2l-25 nucleotides,which regulates the basic physiological processes of many cells.Studies have shown that it has both anti-tumor effects and cancer-promoting effects.Previous studies have confirmed that a variety of miRNAs are dysregulated in esophageal squamous cell carcinoma tissues and cells,and their expression changes are related to the prognosis of esophageal squamous cell carcinoma.MiR-301a-3p is a member of the miR-301 family.It is widely expressed in various organs of the human body.It has important biological functions and is related to the occurrence of various tumors.However,the role of miR-301a-3p in the development of esophageal cancer is still unclear.Therefore,in order to study its role in esophageal squamous cell carcinoma,this article first compares the expression levels of miR-301a-3p between esophageal cancer tissues and paracancerous tissues,esophageal squamous carcinoma cells and normal mucosal cells.And the relationship between its expression level and clinical characteristics and postoperative pathological stages was analyzed.We verified the effect of miR-301a-3p on the proliferation activity of esophageal cancer cells through cell function tests.After verifying the downstream target genes of miR-301a-3p through dual luciferase test and Western Blot,we further studied the mechanism of miR-301a-3p regulating the proliferation activity of esophageal cancer cells.This article clarified the influence of miR-301a-3p on the biological characteristics of esophageal cancer cells and the mechanism of action and laid the foundation for exploring new therapeutic targets for esophageal cancer.Part one Expression of miR-301a-3p in esophageal squamous cancer tissues and cell lines and the correlation with the clinicopathological features.Objective:The expression of miR-301a-3p is dysregulated in many tumors.This study aims to look at the expression changes of miR-301a-3p in esophageal squamous cell carcinoma tissues and cells,and its relationship with the clinicopathological characteristics of patients with esophageal squamous cell carcinoma.Methods:The esophageal squamous carcinoma and matched adjacent tissue samples were collected from the patients who underwent esophagectomy for squamous cell carcinoma.Esophageal squamous carcinoma cell lines and normal esophageal mucosal epithelial cell lines were cultured.Real-time PCR was used to detect miR-301a-3p in tissues and cells.Clinical data of patients were collected and the correlation between miR-301a-3p expression and clinical characteristics and pathological features were analyzed.Results:The expression of miR-301a-3p in esophageal squamous carcinoma tissues was significantly higher than that of its adjacent tissues(P<0.01).Compared with normal esophageal mucosal epithelial cell Het-1A,miR-301a-3p expression was increased in Eca-109 and TE-1 cells(P<0.01).The high expression of MiR-301a-3p was significantly correlated with lymph node metastasis(P=0.017),tumor length exceeding 4cm(P=0.03),and later TNM stage(P=0.009).Conclusions:The expression of miR-301a-3p is up-regulated in esophageal squamous cell carcinoma tissues and Eca109 cell lines.The relative level of miR-301a-3p was significantly correlated with lymph node metastasis,tumor size and TNM stage.MiR-301a-3p may be involved in the development of esophageal squamous cell carcinoma.Part two Effects of miR-301a-3p on the proliferation and clone formation in esophageal squamous carcinoma cell line Eca109.Objective:MiR-301a-3p has pro-tumor effect in many cancers,and the mechanism in different cancers is different.This part mainly studies the effect of miR-301a-3p on the proliferation and cloning of esophageal squamous cell carcinoma by regulating the PI3K/AKT pathway.Methods:The effect of miR-301a-3p on the biological behavior of cancer cells was determined by overexpressing and inhibiting the expression of miR-301a-3p by transfection with miR-301a-3p mimics or miR-301a-3p inhibitors in esophageal squamous carcinoma cell lines Eca109.The Real time-PCR method was used to verify the expression of miR-301a-3p.The MTT assay was used to detect cell proliferation activity,and the colony formation assay was used to detect the ability of cell clone.Western Blot was used to detect the expression levels of p-AKT/AKT in the PI3K/AKT pathway,as well as the downstream genes BCL-2 and BAX.In rescue experiment,we detected the expression of p-AKT/AKT,as well as BCL-2 and BAX,after using LY294002to inhibit the PI3K/AKT pathway.Results:1.Eca109 cells were used to establish an in vitro cell model.The expression of miR-301a-3p was detected by Real time-PCR.The expression levels of miR-301a-3p in miR-301a-3p mimics group and the NC group were:3.96±0.39,1±0.08 respectively(P<0.01).The expressions of the miR-301a-3p in miR-301a-3p inhibitor group and the NC group were 0.34±0.08,1±0.13(P<0.05).These results indicated that the transfection experiment was successful and subsequent experiments could be carried out.2.The MTT experiment showed that 48 hours after transfection,the OD values of the Eca109 cell line in miR-301a-3p mimic group and the negative control group were 0.765±0.050 and 0.560±0.049,respectively(P<0.01).In contrst,the OD values of the Eca109 cell line in miR-301a-3p inhibitor group and the NC group were 58±6 and 116±12(P<0.05).Therefore,compared with the NC group,the proliferation ability of Eca109 cells transfected with miR-301a-3p mimics was significantly enhanced,and the proliferation ability of Eca109 cells transfected with miR-301a-3p inhibitor was significantly decreased.In the cell cloning experiment,the number of colony formation of the miR-301a-3p mimic group and the NC group were 201±15 and 138±12(P<0.05).By contrast,it was 58±6 and 116±12 in the miR-301a-3p inhibitor group and the control group(P<0.05).Thus,the cloning ability of Eca109cells transfected with miR-301a-3p mimics was significantly enhanced,and decrease in those transfected with miR-301a-3p inhibitor,compared with the control group.3.The expression of p-AKT/AKT and BCL-2 was increased,the expression of BAX was decreased in Eca109 cell after transfected with miR-301a-3p mimics(P<0.05).Conversely,the expression of p-AKT/AKT and BCL-2 was decreased,the expression of BAX was increased in Eca109cell after transfected with miR-301a-3p inhibitor(P<0.05).4.Eca-109 cells were transfected with miR-301a-3p mimic for 48 h,followed by treatment with LY294002 for 24h to determine whether miR-301a-3p regulated the expression of BCL-2 and BAX via the activation of PI3K/AKT.The expression of BCL-2 was higher,and BAX was lower in miR-301a-3p mimics group than in miR-301a-3p mimics+LY294002 group(P<0.05).The results showed that the effects of miR-301-3p on the expression of BCL-2 and BAX were rescued by LY294002 treatment.Conclusions:Our study indicates that miR-301a-3p activates the PI3K/Ak T pathway to enhance the ability of esophageal squamous cell carcinoma cell proliferation and clone formation,and promote tumor growth.Part three Screening and verifying miR-301a-3p target genes in esophageal squamous carcinoma cells.Objective:In the second part of the study,it was observed that miR-301a-3p can enhance the proliferation and cloning ability of esophageal squamous cell carcinoma cells by activating the PI3K/AKT pathway,but the specific target genes are still unclear.Most current studies have shown that miRNAs inhibit the translation of target genes and regulate their expression by binding to the3’UTR of target gene m RNA.In this part of the study miR-301a-3p target genes were screened and verified to clarify its mechanism of action.Methods:MiRBase software(http://www.mirbase.org)was used to predict miR-301a-3p target genes,and found the protein PTEN whose expression was negatively regulated by miR-301a-3p and played a role in the PI3K/AKT pathway.The expression of PTEN in esophageal squamous cell carcinoma tumor tissues and adjacent normal tissues was detected by Western Blot,and miR-301a-3p by Real time-PCR.The correlation between miR-301a-3p and PTEN was verified.The expression level of PTEN in the cells was detected,after transfecting miR-301a-3p mimics or miR-301a-3p inhibitor in Eca109cells for 48 hours.The dual luciferase reporter gene experiment verified that miR-301a-3p directly binds with the 3’UTR region of PTEN m RNA to regulate its expression.Rescue experiments were done for the following 4groups:NC mimics/inhibitor+NC PTEN/NC si-PTEN group,miR-301a-3p mimics/inhibitor+NC PTEN/si-PTEN group,NC mimics/inhibitor+PTEN/si-PTEN group and miR-301a-3p mimics/inhibitor+PTEN/si-PTEN,and the proliferation and clone formation of the above groups were detected in the Eca109 cell line.Western Blot was used to detect the expression of p-AKT/AKT,BCL-2 and BAX to verify whether PTEN chould reverse the effect of miR-301a-3p or not.Results:1.The online search for miRbase found that miR-301a-3p and PTEN3’UTR had complementary binding sites at 398-418 nt.Dual-luciferase reporter gene analysis showed that the fluorescent activity of wild-type PTEN in Eca109 cells was significantly inhibited by miR-301a-3p,but miR-301a-3p did not affect the luciferase activity of mutant PTEN in Eca109 cells.It is suggested that the PTEN gene was the target gene in esophageal squamous carcinoma cells.2.Compared with tissues adjacent to cancer,the level of PTEN protein in ESCC tissue was reduced(P<0.01),and negatively correlated with the expression of miR-301a-3p in Eca109 cells(R~2=0.5945,P<0.01).After transfection of miR-301a-3p mimic in Eca109 cell line,the expression of PTEN decreased significantly(P<0.05)and increased after transfection of miR-301a-3p inhibitor(P<0.01).3.After 48 hours of transfection the proliferation and cloning ability of the cells in each groups were tested after co-transfection.First,we used the miR-301a-3p mimics+PTEN group and its related control group for experiments.After a certain period of transfection,we observed the proliferation and clone formation in each group.The cell proliferation and cloning ability of miR-301a-3p mimics+PTEN group was significantly lower than that of miR-301a-3p mimics+NC PTEN group(P<0.05).It shows that PTEN could reverse the effect of miR-301a-3p on cells after simultaneous transfection.The same experiments were performed with the miR-301a-3p inhibitor+si-PTEN group and the related control group.The results showed that the cell proliferation and cloning ability of the miR-301a-3p inhibitor+si-PTEN group was higher than that of miR-301a-3p inhibitor+NC si-PTEN group(P<0.05).It is proved that si-PTEN can reverse the inhibitory effect of miR-301a-3p inhibitor on cell proliferation after transfection.4.Western Blot was used to detect the expression of p-AKT/AKT,BAX and BCL-2 in Eca109 cells.Compared with the miR-301a-3p mimics+NC PTEN group,the expressions of p-AKT/AKT and BCL-2 were decreased in miR-301a-3p mimics+PTEN group,and the expression of BAX increased.The results indicated that miR-301a-3p could increase the expression level of p-AKT/AKT,BCL-2,and decrease the expression of BAX,while PTEN could reverse the effect of miR-301a-3p on the above proteins.Compared with the miR-301a-3p inhibitor+NC si-PTEN group,the expression of p-AKT/AKT and BCL-2 were increased in miR-301a-3p inhibitor+si-PTEN group,and the expression of BAX was decreased.The results indicated that si-PTEN reversed the effect of miR-301a-3p inhibitor on the PI3K/AKT pathway and proteins.Conclusions:MiR-301a-3p can regulate the PI3K/AKT pathway by targeting PTEN to promote the proliferation of Eca109 cells.