Objective:To investigate the effect of miR-1-3p on mitophagy in human esophageal squamous cell carcinoma (ESCC) cells and the related mechanisms.Methods:The differentially expressed miRNAs in ESCC were screened using the GEO database. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure miR-1-3p expression in normal esophageal epithelial cells (HET-1A) and ESCC cell lines (TE1, KYSE30, KYSE150, KYSE410, Eca109). Bioinformatics tools were utilized to predict target genes of miR-1-3p, subcellular localization was confirmed by fluorescence in situ hybridization. The targeting relationship between miR-1-3p and SLC7A11 was validated using dual-luciferase reporter assay. Cell proliferation and apoptosis were detected by CCK8 assay and flow cytometry, respectively. Furthermore, experimental validation demonstrated that overexpression of SLC7A11 rescued the presence of the miR-1-3p/SLC7A11 axis. Confocal microscopy was used to detect changes in mitochondrial autophagic lysosomes, while transmission electron microscopy was employed to observe mitophagy and morphological alterations. Western blot was conducted to evaluate the expression of autophagy-related proteins LC3 and P62. Flow cytometry was used to measure mitochondrial membrane potential and reactive oxygen species (ROS). Immunohistochemistry was applied to assess SLC7A11 expression in 133 ESCC patient tissues and 115 normal esophageal epithelial tissues. The correlation between SLC7A11 expression level and clinicopathological features was analyzed. Survival analysis was performed using the Kaplan-Meier method, and Cox proportional hazard regression models were used for multivariate analysis.Results:The expression of miR-1-3p in ESCC cells was significantly lower than that in HET-1A cells ( P＜0.05). SLC7A11 was a target gene of miR-1-3p. Transfection of miR-1-3p mimic inhibited the proliferation of ESCC cells. CCK-8 assay results showed that the proliferative capacity of KYSE30 and KYSE410 cells in the miR-1-3p mimic group (absorbance values: 2.88±0.24 and 2.88±0.18, respectively) was significantly lower than that in the miRNA mimic negative control (NC) group (3.94±0.27, P＜0.001; 4.20±0.21, P＜0.001). Meanwhile, the proliferative capacity of KYSE30 and KYSE410 cells in the miR-1-3p mimic+SLC7A11-overexpression (OE) group (absorbance values: 3.57±0.15 and 3.60±0.13, respectively) was significantly higher than that in the miR-1-3p mimic +empty vector (EV) group (2.54±0.10, P＜0.001, 2.36±0.16, P＜0.001). Additionally, transfection of miR-1-3p mimic promoted apoptosis. Flow cytometry results demonstrated that the apoptosis rates of KYSE30 and KYSE410 cells in the miR-1-3p mimic group [(9.22±0.05)% and (6.55±0.37)%, respectively] were significantly higher than those in the miRNA mimic NC group [(0.81±0.17)%, P＜0.001); (1.04±0.12)%, P＜0.001]. Conversely, the apoptosis rates of KYSE30 and KYSE410 cells in the miR-1-3p mimic + SLC7A11-OE group [(0.73±0.04)% and (1.19±0.05)%, respectively] were significantly lower than those in the miR-1-3p mimic+EV group [(9.83±0.41)%, P＜0.001); (6.09±0.17)%, P＜0.00)]. MiR-1-3p mimic downregulated SLC7A11 protein expression and the LC3Ⅱ/LC3I ratio ( P＜0.05), upregulated P62 protein expression ( P＜0.05), this phenomenon can be rescued by overexpressing SLC7A11 ( P＜0.05). Additionally, miR-1-3p mimic increased ROS levels and decreased mitochondrial membrane potential (JC-1 aggregate/monomer ratio), this phenomenon can be rescued by overexpressing SLC7A11 ( P＜0.05). SLC7A11 expression was higher in ESCC tissues compared to normal esophageal epithelial tissues ( P＜0.001), and SLC7A11 serves as an independent prognostic factor in ESCC ( HR=2.15, 95% CI: 1.27-3.65, P=0.004). Conclusion:miR-1-3p inhibits mitophagy in esophageal squamous cell carcinoma by targeting SLC7A11.

Objective:To investigate the effect of miR-1-3p on mitophagy in human esophageal squamous cell carcinoma (ESCC) cells and the related mechanisms.Methods:The differentially expressed miRNAs in ESCC were screened using the GEO database. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure miR-1-3p expression in normal esophageal epithelial cells (HET-1A) and ESCC cell lines (TE1, KYSE30, KYSE150, KYSE410, Eca109). Bioinformatics tools were utilized to predict target genes of miR-1-3p, subcellular localization was confirmed by fluorescence in situ hybridization. The targeting relationship between miR-1-3p and SLC7A11 was validated using dual-luciferase reporter assay. Cell proliferation and apoptosis were detected by CCK8 assay and flow cytometry, respectively. Furthermore, experimental validation demonstrated that overexpression of SLC7A11 rescued the presence of the miR-1-3p/SLC7A11 axis. Confocal microscopy was used to detect changes in mitochondrial autophagic lysosomes, while transmission electron microscopy was employed to observe mitophagy and morphological alterations. Western blot was conducted to evaluate the expression of autophagy-related proteins LC3 and P62. Flow cytometry was used to measure mitochondrial membrane potential and reactive oxygen species (ROS). Immunohistochemistry was applied to assess SLC7A11 expression in 133 ESCC patient tissues and 115 normal esophageal epithelial tissues. The correlation between SLC7A11 expression level and clinicopathological features was analyzed. Survival analysis was performed using the Kaplan-Meier method, and Cox proportional hazard regression models were used for multivariate analysis.Results:The expression of miR-1-3p in ESCC cells was significantly lower than that in HET-1A cells ( P＜0.05). SLC7A11 was a target gene of miR-1-3p. Transfection of miR-1-3p mimic inhibited the proliferation of ESCC cells. CCK-8 assay results showed that the proliferative capacity of KYSE30 and KYSE410 cells in the miR-1-3p mimic group (absorbance values: 2.88±0.24 and 2.88±0.18, respectively) was significantly lower than that in the miRNA mimic negative control (NC) group (3.94±0.27, P＜0.001; 4.20±0.21, P＜0.001). Meanwhile, the proliferative capacity of KYSE30 and KYSE410 cells in the miR-1-3p mimic+SLC7A11-overexpression (OE) group (absorbance values: 3.57±0.15 and 3.60±0.13, respectively) was significantly higher than that in the miR-1-3p mimic +empty vector (EV) group (2.54±0.10, P＜0.001, 2.36±0.16, P＜0.001). Additionally, transfection of miR-1-3p mimic promoted apoptosis. Flow cytometry results demonstrated that the apoptosis rates of KYSE30 and KYSE410 cells in the miR-1-3p mimic group [(9.22±0.05)% and (6.55±0.37)%, respectively] were significantly higher than those in the miRNA mimic NC group [(0.81±0.17)%, P＜0.001); (1.04±0.12)%, P＜0.001]. Conversely, the apoptosis rates of KYSE30 and KYSE410 cells in the miR-1-3p mimic + SLC7A11-OE group [(0.73±0.04)% and (1.19±0.05)%, respectively] were significantly lower than those in the miR-1-3p mimic+EV group [(9.83±0.41)%, P＜0.001); (6.09±0.17)%, P＜0.00)]. MiR-1-3p mimic downregulated SLC7A11 protein expression and the LC3Ⅱ/LC3I ratio ( P＜0.05), upregulated P62 protein expression ( P＜0.05), this phenomenon can be rescued by overexpressing SLC7A11 ( P＜0.05). Additionally, miR-1-3p mimic increased ROS levels and decreased mitochondrial membrane potential (JC-1 aggregate/monomer ratio), this phenomenon can be rescued by overexpressing SLC7A11 ( P＜0.05). SLC7A11 expression was higher in ESCC tissues compared to normal esophageal epithelial tissues ( P＜0.001), and SLC7A11 serves as an independent prognostic factor in ESCC ( HR=2.15, 95% CI: 1.27-3.65, P=0.004). Conclusion:miR-1-3p inhibits mitophagy in esophageal squamous cell carcinoma by targeting SLC7A11.

Objective To investigate the survival benefits of simultaneous integrated boost intensity-modulated radiotherapy ( SIB-IMRT ) in the treatment of esophageal squamous cell carcinoma ( ESCC ) . Methods From July 2003 to March 2014,1748 patients with ESCC received 3DCRT or IMRT in a single institution were enrolled in this retrospective study. Among them, 809 patients received conventional fractionated radiotherapy with the standard prescription dose and 110 patients received SIB-IMRT ( SIB-IMRT group).Survival analysis was performed and propensity score matching (PSM 1vs1) was conducted to evaluate and compare the survival benefits between SIB-IMRT and conventional fractionated radiotherapy. Results The baseline characteristics significantly differed between two groups. In the SIB group,the age was significantly younger ( 64 years vs. 66 years, P=0. 001 ) , the percentage of patients with cervical/upper thoracic tumors was considerably higher (53. 6％ vs. 31. 0％,P=0. 000) and the proportion of N2 patients was significantly higher ( 21. 8％ vs. 13. 7％,P=0. 027) compared with those in the other group. Accordingto the PSM of 1:1, 218 patients were successfully matched. After matching, the clinical data did not significantly differ between two groups. Prior to matching,the median survival time in the standard dose and SIB-IMRT groups were 23 and 21 months (P=0. 638).After matching,the median survival time in the SIB-IMRT group was 22 months,significantly longer than 18 months in the standard dose group (P=0. 000). Subgroup analysis demonstrated that patients with large tumors ( GTV volume>40 cm3 ) and middle/lower thoracic tumors obtained more survival benefits from SIB-IMRT. The median survival time of patients in the standard dose group was 14 months, significantly shorter than 21 months in the SIB-IMRT group ( P=0. 001).The median survival time of patients with middle/lower thoracic tumors in the SIB-IMRT group was 17 months,significantly longer than 9 months in the standard dose group (P=0. 000).Multivariate analysis using Cox regression model indicated that age, tumor site and radiotherapy modality were the independent prognostic factors. The HR of SIB-IMRT was 0. 551(P=0. 000),which was a factor for survival benefits. Conclusions SIB-IMRT possesses potential survival benefits for ESCC compared with conventional fractionated radiotherapy. Patients with large tumors and middle/lower thoracic tumors are more prone to obtaining benefits from SIB-IMRT than their counterparts.

Objective To analyze the survival of patients with esophageal squamous cell carcinoma (ESCC)treated by different regimens and different radiation doses and to explore the optimal radiation dose and subgroups with potential clinical benefit. Methods A total of 1387 patients with ESCC who received conformal radiotherapy or intensity-modulated radiotherapy in our hospital from July 2003 to March 2014 were enrolled in this retrospective study. The patients who received different radiation doses in radiotherapy alone or in concurrent chemoradiotherapy were analyzed.The log-rank test and Cox regression analysis were used to explore the optimal radiation dose and the benefited subgroups. Results A total of 780 patients only received radiotherapy. Among them,the median survival of patients receiving radiation dose<60 Gy(n=91),60 Gy(n=429),and>60 Gy(n=260)was 9,20,and 23 months,respectively,suggesting a significant difference(P=0.000).The patients with a radiation dose of 60 Gy had a similar survival curve to the patients with radiation dose>60 Gy,both significantly higher than that in patients with radiation dose<60 Gy (P=0.000,0.000).Totally 302 patients received concurrent chemoradiotherapy. Among them,the median survival of patients receiving radiation dose<60 Gy(n=18),60 Gy(n=224),and>60 Gy(n=60)was 22, 34,and 15 months,respectively,suggesting a significant difference(P=0.004).The survival curve showed no significant difference between the patients with radiation dose<60 Gy and>60 Gy(P=0.952),while the patients with a radiation dose of 60 Gy had a better survival compared with the patients with radiation dose<60 Gy or>60 Gy. The Cox multivariate regression analysis indicated that the ESCC patients receiving radiotherapy alone or concurrent chemoradiotherapy had different prognosis;gross tumor volume(GTV)and radiation dose were two independent prognostic factors in the same treatment model(P=0.045,0.001).In radiotherapy alone,radiation dose ≥60 Gy was a protective factor for the patients' survival(P=0.000).In concurrent chemoradiotherapy,a radiation dose of 60 Gy was a protective factor,while radiation dose<60 Gy or>60 Gy presented no survival benefit(P=0.051). Conclusions The optimal radiation dose is no less than 60 Gy in ESCC patients treated by radiotherapy alone. If the patients receive concurrent chemoradiotherapy,the radiation dose of 60 Gy is recommended.