1.Long-term survival outcomes and prognostic factors following radical resection of pancreatic body and tail cancer:a retrospective analysis of 992 patients
Dong XU ; Yang WU ; Kai ZHANG ; Nan LYU ; Qianqian WANG ; Pengfei WU ; Jie YIN ; Baobao CAI ; Guodong SHI ; Jianzhen LIN ; Yazhou WANG ; Lingdi YIN ; Zipeng LU ; Min TU ; Jianmin CHEN ; Feng GUO ; Jishu WEI ; Junli WU ; Wentao GAO ; Cuncai DAI ; Yi MIAO ; Kuirong JIANG
Chinese Journal of Surgery 2026;64(1):46-54
Objective:To investigate the survival outcomes and prognostic factors in patients undergoing radical resection for pancreatic body and tail cancer.Methods:A retrospective case series study was conducted on 992 patients who underwent radical resection for pancreatic body and tail cancer at the Pancreatic Center of the First Affiliated Hospital of Nanjing Medical University from January 2016 to June 2024. In this study, 577 (58.2%) were male and 415 (41.8%) were female,with an age of (65±9) years (range: 26 to 86 years). Follow-up continued until June 2024. Survival rates were estimated using the Kaplan-Meier method,and prognostic factors were identified using univariate and multivariate Cox proportional hazards models.Results:Among 992 patients,open surgery was the predominant approach (89.1%, 884/992), and radical antegrade modular pancreatosplenectomy (RAMPS) was performed in 317 patients (32.0%). Combined organ resection,venous resection,and arterial resection were performed in 23.5%, 9.3%,and 11.2% of patients,respectively. The rates of R0, R1-1 mm, and R1-direct resections were 49.8% (494/992),41.5% (412/992), and 8.7% (86/992),respectively. Stage ⅡB was the most common TNM stage (32.2%,319/992). A total of 801 patients (80.8%) received adjuvant chemotherapy. The median follow-up period was 32.0(8.8) months(range:3.2 to 105.3 months),during which 508 patients (51.2%) died. The overall median survival (OS) was 26.4 months,with 1-,3-, and 5-year survival rates of 79.0%,40.0%, and 29.0%, respectively. In the recent five years (from 2020 to 2024), the median OS improved significantly to 34.1 months compared to 20.0 months from 2016 to 2019 ( P<0.01). Histological subtype analysis showed that the median OS time was 26.7 months for pancreatic ductal adenocarcinoma (PDAC, n=855),58.9 months for invasive intraductal papillary mucinous carcinoma (IPMC, n=32),and 15.7 months for adenosquamous carcinoma of pancreas (ASCP, n=73) ( P=0.001). Among PDAC patients, adjuvant chemotherapy significantly improved survival (29.1 months vs. 14.4 months, P<0.01);in IPMC patients, adjuvant chemotherapy also extended survival (65.7 months vs. 58.9 months, P=0.047). Although ASCP patients receiving chemotherapy had a longer median OS time than those without (18.8 months vs. 8.9 months),the difference was not statistically significant ( P=0.151). Multivariate Cox regression analysis in PDAC patients indicated that adjuvant chemotherapy, R0 resection, T stage,N stage,and tumor differentiation were independent prognostic factors ( P<0.01). The median OS time by TNM stage was:not reached for stage ⅠA, 51.6 months for ⅠB, 25.5 months for ⅡA, 23.7 months for ⅡB, 23.0 months for Ⅲ, and 14.4 months for Ⅳ. The median OS time for R0,R1-1 mm,and R1-direct resections was 34.1,24.7,and 15.7 months,respectively ( P<0.01). Conclusion:Adjuvant chemotherapy,R0 resection,tumor stage,and differentiation are independent prognostic factors for pancreatic body and tail cancer.
2.Long-term survival outcomes and prognostic factors following radical resection of pancreatic body and tail cancer:a retrospective analysis of 992 patients
Dong XU ; Yang WU ; Kai ZHANG ; Nan LYU ; Qianqian WANG ; Pengfei WU ; Jie YIN ; Baobao CAI ; Guodong SHI ; Jianzhen LIN ; Yazhou WANG ; Lingdi YIN ; Zipeng LU ; Min TU ; Jianmin CHEN ; Feng GUO ; Jishu WEI ; Junli WU ; Wentao GAO ; Cuncai DAI ; Yi MIAO ; Kuirong JIANG
Chinese Journal of Surgery 2026;64(1):46-54
Objective:To investigate the survival outcomes and prognostic factors in patients undergoing radical resection for pancreatic body and tail cancer.Methods:A retrospective case series study was conducted on 992 patients who underwent radical resection for pancreatic body and tail cancer at the Pancreatic Center of the First Affiliated Hospital of Nanjing Medical University from January 2016 to June 2024. In this study, 577 (58.2%) were male and 415 (41.8%) were female,with an age of (65±9) years (range: 26 to 86 years). Follow-up continued until June 2024. Survival rates were estimated using the Kaplan-Meier method,and prognostic factors were identified using univariate and multivariate Cox proportional hazards models.Results:Among 992 patients,open surgery was the predominant approach (89.1%, 884/992), and radical antegrade modular pancreatosplenectomy (RAMPS) was performed in 317 patients (32.0%). Combined organ resection,venous resection,and arterial resection were performed in 23.5%, 9.3%,and 11.2% of patients,respectively. The rates of R0, R1-1 mm, and R1-direct resections were 49.8% (494/992),41.5% (412/992), and 8.7% (86/992),respectively. Stage ⅡB was the most common TNM stage (32.2%,319/992). A total of 801 patients (80.8%) received adjuvant chemotherapy. The median follow-up period was 32.0(8.8) months(range:3.2 to 105.3 months),during which 508 patients (51.2%) died. The overall median survival (OS) was 26.4 months,with 1-,3-, and 5-year survival rates of 79.0%,40.0%, and 29.0%, respectively. In the recent five years (from 2020 to 2024), the median OS improved significantly to 34.1 months compared to 20.0 months from 2016 to 2019 ( P<0.01). Histological subtype analysis showed that the median OS time was 26.7 months for pancreatic ductal adenocarcinoma (PDAC, n=855),58.9 months for invasive intraductal papillary mucinous carcinoma (IPMC, n=32),and 15.7 months for adenosquamous carcinoma of pancreas (ASCP, n=73) ( P=0.001). Among PDAC patients, adjuvant chemotherapy significantly improved survival (29.1 months vs. 14.4 months, P<0.01);in IPMC patients, adjuvant chemotherapy also extended survival (65.7 months vs. 58.9 months, P=0.047). Although ASCP patients receiving chemotherapy had a longer median OS time than those without (18.8 months vs. 8.9 months),the difference was not statistically significant ( P=0.151). Multivariate Cox regression analysis in PDAC patients indicated that adjuvant chemotherapy, R0 resection, T stage,N stage,and tumor differentiation were independent prognostic factors ( P<0.01). The median OS time by TNM stage was:not reached for stage ⅠA, 51.6 months for ⅠB, 25.5 months for ⅡA, 23.7 months for ⅡB, 23.0 months for Ⅲ, and 14.4 months for Ⅳ. The median OS time for R0,R1-1 mm,and R1-direct resections was 34.1,24.7,and 15.7 months,respectively ( P<0.01). Conclusion:Adjuvant chemotherapy,R0 resection,tumor stage,and differentiation are independent prognostic factors for pancreatic body and tail cancer.
3.Molecular Mechanism of Liuwei Dihuangwan Regulating GPNMB Expression and Enhancing Autophagy in Prevention and Treatment of Alzheimer's Disease
Yuxi LIU ; Zhongkang ZHU ; Songnan WANG ; Jiali LIU ; Ye YIN ; Jiarui MIAO ; Shunuo HE ; Danyu ZHAO
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(13):1-10
ObjectiveThis study aims to investigate the effect of Liuwei Dihuangwan on the autophagy function in the hippocampus of senescence-accelerated mouse prone 8 (SAMP8) by regulating the expression of glycoprotein non-metastatic melanoma protein B (GPNMB). Furthermore, it is designed to explore the mechanism of the method of tonifying the kidneys and replenishing essence in the treatment of Alzheimer's disease (AD). MethodsIn experiment 1, 24 5-month-old SAMP8 mice were randomly and equally divided into the model group, and the low-, middle- and high-dose(0.59,1.18,2.36 g·kg-1) Liuwei Dihuangwan groups. At the same time, six 5-month-old senescence accelerated mouse resistant 1 (SAMR1) mice were used as the control group. The learning and memory ability was evaluated through novel object recognition experiment. Serum cortisol (Cort), adrenocorticotropic hormone (ACTH) and urine 17-hydroxycorticosteroid (17-OHCS) levels were detected by enzyme-linked immunosorbent assay (ELISA). The ultrastructure of hippocampal neurons was observed by transmission electron microscope (TEM), and the expression levels of hippocampal GPNMB, a disintegrin and metalloproteinase 10 (ADAM10) and autophagy-related proteins were detected by Western blot. In experiment 2, 18 SAMP8 mice were randomly and equally divided into the model group, vector control group (Vector), and GPNMB overexpression group (GPNMBOE). Lentiviral vectors were stereotactically injected into the brain (2 μL per side in the GPNMBOE group). Western blot was used to detect the expression of the above target proteins in the hippocampus; In Experiment 3, 24 SAMP8 mice were randomly and equally divided into the model group, Liuwei Dihuangwan group, Liuwei Dihuangwan+negative control (NC) group, and Liuwei Dihuangwan+GPNMB silencing group (shGPNMB). Before drug treatment, the Liuwei Dihuangwan+NC group and the Liuwei Dihuangwan+shGPNMB group were injected with negative control and GPNMB silencing lentivirus, respectively. Western blot was used to detect the expression of the above target proteins in the hippocampus. ResultsThe novel object discrimination index of mice in the model group was significantly lower than that of mice in the control group (P<0.01). The novel object discrimination index of mice in the medium- and high-dose Liuwei Dihuangwan groups was significantly higher than that of mice in the model group (P<0.01). Aggregated autolysosomes were observed in the normal hippocampus tissue by TEM. In the model group, mitochondria were dominant, and no typical characteristic autophagosomes were observed. In the low- and medium-dose Liuwei Dihuangwan groups, a small number of autolysosomes and autophagosomes with double-membrane structures were observed. In the high-dose Liuwei Dihuangwan group, the number of autophagosomes and autolysosomes was greater than that in the low- and medium-dose groups. The results of ELISA and Western blot showed that compared with the control group, the levels of serum Cort, ACTH, and urine 17-OHCS in the model group were substantially increased, while the expression of hippocampal ADAM10, Beclin1, and microtubule associated-protein light chain 3-Ⅱ/Ⅰ (LC3 Ⅱ/Ⅰ) was significantly decreased. The expression of GPNMB and ubiquitin binding protein p62 was significantly increased (P<0.05, P<0.01). Compared with the model group, the serum Cort and ACTH levels in the low-, medium-, and high-dose Liuwei Dihuangwan groups were significantly reduced, while only the urine 17-OHCS level in the high-dose group was significantly reduced. The hippocampal GPNMB, ADAM10, Beclin1, and LC3 Ⅱ/Ⅰ expression levels in the medium-, and high-dose groups of Liuwei Dihuangwan were significantly increased compared to the model group, whereas the expression of p62 was significantly reduced (P<0.01). The above indicators showed a progressive trend among the three groups. Compared with the model group, the GPNMBOE group showed a significant increase in GPNMB, ADAM10, Beclin1, LC3 Ⅱ/Ⅰ expression, and a significant decrease in p62 expression (P<0.01). Compared with the model group, the expression of GPNMB, ADAM10, Beclin1, and LC3 Ⅱ/Ⅰ in the hippocampus of the Liuwei Dihuangwan group significantly increased, while the expression of p62 significantly decreased (P<0.01). Compared with the Liuwei Dihuangwan group, the Liuwei Dihuangwan+shGPNMB group showed a significant decrease in GPNMB, ADAM10, Beclin1, LC3 Ⅱ/Ⅰ, and a significant increase in p62 expression (P<0.01). ConclusionLiuwei Dihuangwan can enhance hippocampal autophagy function and improve AD by upregulating GPNMB expression.
4.Research progress on pathogenesis and exercise intervention of adolescent with upper cross syndrome
YIN Ding, WANG Hui, ZHAI Suo, CHEN Miao
Chinese Journal of School Health 2025;46(1):144-147
Abstract
Upper cross syndrome (UCS) is an abnormal body posture phenomenon. The detection rate of UCS in adolescents is high in China, which has become a serious public health problem threatening the health of adolescents. Exercise therapy has a positive effect on the prevention and treatment of UCS.The article reviews the pathological mechanisms of muscle imbalance, muscle interaction inhibition, and neural control related to the occurrence and development of UCS both domestically and internationally in recent years.And it elucidates the effects and corresponding mechanisms of exercise interventions such as gentle exercise, stretching exercise and physical function training on UCS, so as to provide help for the correction of UCS.
5.Analysis on epidemiological characteristics of severe congenital heart disease in Chongqing city during 2007-2023
Fen WANG ; Guanghui YIN ; Jie ZHANG ; Miao FENG ; Jun LIU
Chongqing Medicine 2025;54(2):505-511
Objective To understand the epidemiological characteristics of severe congenital heart dis-ease in Chongqing City during 2007-2023 to provide a basis for its comprehensive prevention and control measures.Methods Based on hospital monitoring data,453 children patients with severe congenital heart dis-ease monitored by the birth defects monitoring institutions in Chongqing city from January 2007 to December 2023 were included in the study.They were grouped by year,perinatal infants gender,maternal permanent res-idence(urban/rural),maternal age,different regions and other categories.The χ2 test was used to analyze the difference in the incidence rate of different categories of severe congenital heart diseases,and the Joinpoint re-gression model was used to analyze the change trend.Results A total of 1 468 005 perinatal neonates were monitored in Chongqing City during 2007-2023 and 453 cases of severe congenital heart disease were found,with an incidence rate of 3.09/10 000,in which the incidence rate of atrioventricular septal defect was 2.16/10 000,the incidence rate of tetralogy of fallot was 0.66/10 000 and the incidence rate of transposition of great ar-teries was 0.27/10 000.The total incidence rate of cities and towns was higher than that in the countryside(χ2=64.08,P<0.001),the urban area was higher than the Chongqing southeast and Chongqing northeast towns cluster(χ2=49.34,P<0.001),the female was higher than the male(χ2=5.63,P=0.018).The inci-dence rates in different ages groups showed the U shape distribution(χ2=31.63,P<0.001).The incidence rate of the pregnant women<20 years old group and pregnant women≥35 years old group was higher,which of the 25-29 years old group was lower.The incidence rate of severe congenital heart disease in Chongqing City during 2007-2023 appeared the turning point,which during 2007-2016 was gradually increased(APC=-15.95),and which during 2016-2023 was gradually decreased(APC=-15.36).Conclusion The incidence rate of severe congenital heart disease in Chongqing city during 2007-2023 was increased first and then de-creased,moreover there were differences in time,region and population.
6.Change Law in Material Basis of Rhei Radix et Rhizoma During Nine-time Repeating Steaming and Sun-drying Processing Based on Characteristic Chromatogram and Chemometrics
Xuan AO ; Taotao LIU ; Miao YU ; Lan WANG ; Xiaojie YIN ; Jingyu ZUO ; Li LI
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(15):191-198
ObjectiveTo characterize the changes in the overall chemical profile and key index components during nine-time repeating steaming and sun-drying processing of Rhei Radix et Rhizoma, and to reveal the change law of its material basis. MethodsHigh performance liquid chromatography(HPLC) was used to analyze the changes in the overall chemical profile of Rhei Radix et Rhizoma decoction pieces, and the contents of 15 main active components such as chrysophanol-8-O-β-D-glucoside, chrysophanol and gallic acid in the process of nine-time repeating steaming and sun-drying were determined. Combined with chemometrics, the contents and quantity ratio relationships of the glycosides, aglycones and tannins during the processing of Rhei Radix et Rhizoma were analyzed, and the partial least squares-discriminant analysis(PLS-DA) and cluster analysis of the main components in different steaming times were conducted, the statistically significant differential markers were selected with the variable importance in the projection(VIP) value>1. ResultsIn the nine-time repeating steaming and sun-drying process of Rhei Radix et Rhizoma, there were certain regularity in the number and peak area of characteristic peaks and the steaming and sun-drying times, the anthraquinone glycosides and aglycones could be roughly divided into three stages, including rapid change stage, fluctuation change stage and stable stage, and the total amount of tannins showed a decreasing trend. However, the ratios between the three components mentioned above tended to stabilize after five rounds of steaming and sun-drying. The results of PLS-DA and cluster heatmap showed that the content of each component in Rhei Radix et Rhizoma fluctuated greatly during the 1-4 steaming and sun-drying processes, while the content of each component was relatively close during the 5-9 steaming and sun-drying processes. After screening, it was found that chrysophanol, emodin, chrysophanol-8-O-β-D-glucoside, rhein, physcion and emodin-8-O-β-D-glucoside could be used as the index components for distinguishing the processed products of Rhei Radix et Rhizoma with different steaming and sun-drying times. ConclusionThe changes in the properties and efficacy of Rhei Radix et Rhizoma caused by the processing of nine-time repeating steaming and sun-drying are due to the changes in the composition and ratio of various glycosides and complex tannins in this herb, which is also the key to the formation of its characteristic of "purgation with supplement". This study can provide a basis for the research on the processing mechanism of Rhei Radix et Rhizoma and the establishment of processing specifications.
7.Assoication between 24 hour activity time allocation and active health levels among college students in Yinchuan City
CHEN Miao, ZHAI Suo, DING Wenqing, YIN Ding
Chinese Journal of School Health 2025;46(7):950-955
Objective:
To explore the potential classification of 24 hour activity time allocation among college students in Yinchuan and its association with active health levels, so as to provide references for optimizing activity time allocation to enhance active health levels.
Methods:
From November 18 to December 6, 2024, a stratified cluster random sampling method was used to select 2 422 first and second year college students from full time undergraduate institutions in Yinchuan. The Chinese College Students 24 hour Movement Behaviors Questionnaire (24 h MBQ) and Active Health Behavior Scale were used to assess 24 hour activity time allocation and evaluate active health levels. Latent profile analysis (LPA) was employed to categorize activity types, and a binary Logistic regression analysis was conducted to analyze the relationship between active health levels and activity types.
Results:
A total of 1 087 students (44.9%) were found of meeting active health standards, and significant statistical differences were found in active health levels across different genders, grades, academic qualities, sources of origin and academic categories ( χ 2= 22.03 , 7.65, 25.50, 10.12, 43.44, all P <0.01). Moreover, significant statistical differences could also be found among college students 24 hour activity time across different genders, ages, grades, sources of origin, academic qualities, and academic categories ( t/Z/H/F=-5.70-111.39, P <0.05).The 24 hour activity time allocation was classified into four types:academic high ( 6.9 %), low activity rest (8.8%), light activity (67.8%), and high activity dynamic (16.4%). Significant statistical differences were observed in activity time allocation categories across different ages, academic qualities and academic categories ( χ 2=15.52-108.46, all P <0.05). Using the high activity dynamic type as a reference, the light activity type ( OR=0.39, 95%CI =0.31-0.50), low activity rest type ( OR=0.10, 95%CI =0.06-0.15), and academic high type ( OR=0.03, 95%CI =0.02-0.07) had lower active health levels among college students (all P <0.01).
Conclusion
There is a significant difference in 24 hour activity time allocation among college students in Yinchuan, and different activity types are associated with active health levels.
8.Association between plant-based dietary patterns and gestational weight gain among pregnant women with gestational diabetes mellitus
SHI Meiqi ; YIN Yuhua ; WANG Xuhong ; FU Yuanqing ; MIAO Zelei ; HU Wensheng
Journal of Preventive Medicine 2025;37(5):503-506
Objective:
To investigate the association between plant-based dietary patterns and gestational weight gain (GWG) among pregnant women with gestational diabetes mellitus (GDM), so as to provide the evidence for guiding the reasonable diet during pregnancy.
Methods:
GDM pregnant women who participated in the WeBirth project in Hangzhou Obstetrics and Gynecology Hospital were selected. Maternal age and pre-pregnancy body mass index (BMI) were collected. The Chinese version of Pregnancy Physical Activity questionnaire was used to assess the daily activity equivalent. The food frequency questionnaire was used to collect the frequency and amount of food intake in the last month before enrollment. The overall plant-based diet index (PDI), healthy plant-based diet index (HPDI), and unhealthy plant-based diet index (UPDI) were constructed based on food intake and grouped by quartiles. Multiple linear regression models were used to analyze the association between plant-based dietary patterns and GWG.
Results:
A total of 1 943 pregnant women with GDM, with a median age of 30.91 (interquartile range, 4.92) years. The median BMI of pre-pregnancy was 21.51 (interquartile range, 4.06) kg/m2. The medians of PDI, HPDI and UPDI were 32.42 (interquartile range, 4.60), 32.48 (interquartile range, 4.41) and 32.40 (interquartile range, 5.36), respectively. The median of GWG was 11.30 (interquartile range, 4.52) kg. Multiple linear regression analysis showed that PDI (Q3 group, β=0.674, 95%CI: 0.064-1.285; Q4 group, β=0.702, 95%CI: 0.098-1.306), UPDI (Q3 group, β=1.332, 95%CI: 0.771-1.894; Q4 group, β=1.115, 95%CI: 0.550-1.681) were positively associated with GWG after adjusting for age, pre-pregnancy BMI, daily activity equivalent and daily energy intake. No significant association was found between HPDI and GWG (all P>0.05).
Conclusion
UPDI was associated with a higher risk of GWG in pregnant women with GDM.
9.METTL3 regulates ferroptosis and malignant progression of cervical cancer cells through mediating TRPM7 methylation
Miao FU ; Peng LIU ; Wen TIAN ; Sha WANG ; Xiaomei YIN ; Hao LIU ; Donghai WANG
Basic & Clinical Medicine 2025;45(10):1318-1325
Objective Methyltransferase 3(METTL3)mediated N6-methyladenosine(m6A)methylation modifica-tion of transient receptor potential cation channel subfamily M member 7(TRPM7)regulates ferroptosis and malig-nant progression in cervical cancer(CESC).Methods Totally 40 patients with cervical cancer were collected.Cer-vical cancer tissues and adjacent normal tissues(≥3 cm from the edge of the tumor tissue)were sampled at opera-tion and then divided into experimental group and control group,respectively.RT-qPCR and Western blot were used to detect the differences in TRPM7 mRNA and protein expression between the two groups.TRPM7-interfering cell lines were constructed to investigate the effects of TRPM7 on CESC cells.Cell proliferation and apoptosis were assessed using 5-ethynyl-2'-deoxyuridine(EdU)assay and flow cytometry,respectively.Transwell chamber assays were employed to evaluate cell invasion and migration capabilities.The levels of ferroptosis in CESC cells were measured using kits for reactive oxygen species(ROS),malondialdehyde(MDA),glutathione(GSH),and Fe2+.Bioinformatics tools were utilized to predict methyltransferases associated with TRPM7.The interaction between METTL3 and TRPM7 was examined through RNA immunoprecipitation(RIP)and methylated RNA immunoprecip-itation quantitative PCR(Me-RIP qPCR).The effect of METTL3 on the stability of TRPM7 expression was assessed using actinomycin D assay.Results TRPM7 was highly expressed in CESC tissue and cells.Knockdown of TRPM7 significantly inhibited cell proliferation,promoted cell apoptosis,suppressed cell migration and invasion capabilities,and enhanced ferroptosis levels(P<0.05).Bioinformatics predictions suggested that METTL3 might act as a methyltransferase for TRPM7.Interference with METTL3 gene expression significantly reduced TRPM7 pro-tein levels,decreased TRPM7 m6A modification levels,and impaired TRPM7 gene stability(P<0.05).Conclusions METTL3 regulates CESC proliferation,apoptosis,migration,invasion,and ferroptosis by m6A meth-ylation modification of the TRPM7 gene.
10.Effect of miR-185-5p targeted negative regulation of TM9SF1 on proliferation,migration and autophagy in lung adenocarcinoma cells
Xiao-Na WANG ; Xiu-Ying GONG ; Miao-Miao ZHAO ; Qing-Hua LIU ; Yong LI ; Kun WANG ; Chong-Gao YIN ; Hong-Li LI
Medical Journal of Chinese People's Liberation Army 2025;50(5):566-574
Objective To investigate the effect of miR-185-5p-mediated targeted negative regulation of transmembrane 9 superfamily member 1(TM9SF1)on proliferation,migration and autophagy in lung adenocarcinoma cells.Methods The expression of miR-185-5p in lung adenocarcinoma tissues was analyzed using dataset GSE51853 downloaded from the Gene Expression Omnibus(GEO)database.Potential target proteins of miR-185-5p were predicted using online databases(miRTargetLink,miRTarbase,and DIANA-microT-CD),and autophagy-related proteins were obtained from HADb.The intersected results from these four databases was identified,and survival curves of vascular endothelial growth factor A(VEGFA)and TM9SF1 within the overlapping candidates were analyzed using the StarBase database.TM9SF1 3'UTR wild-type(WT)or TM9SF1 3'UTR mutant(MUT)reporter plasmids were separately co-transfected with miR-185-5p control plasmid(CON)or miR-185-5p overexpression plasmid(over-miR-185-5p)into HEK-293T cells.A dual-luciferase reporter gene assay was employed to assess the binding interaction between miR-185-5p and TM9SF1 and quantify the subsequent luciferase activity.Western blotting was used to assess TM9SF1 protein expression levels in A549 cells transfected with over-miR-185-5p.A549 cells were divided into three groups:(1)CON+NC group,co-transfected with miR-185-5p control plasmid and TM9SF1 control plasmid;(2)over-miR-185-5p+NC group,co-transfected with over-miR-185-5p and TM9SF1 control plasmid;(3)over-miR-185-5p+over-TM9SF1 group,co-transfected with both miR-185-5p and TM9SF1 overexpression plasmids.EdU cell proliferation assay,wound healing assay,and Transwell migration assay were performed to validate the effects of miR-185-5p targeted binding to TM9SF1 on proliferation and migration capacities in lung adenocarcinoma.Changes in autophagic flux and mitochondrial membrane potential(MMP)of lung adenocarcinoma cells were detected using stubRFP-sensGFP-LC3 lentivirus and JC-1 assays,respectively.Results In the GSE51853 dataset,miR-185-5p expression level was significantly lower in lung adenocarcinoma tissues compared with normal lung tissues(P<0.01).qRT-PCR analysis revealed that miR-185-5p expression was downregulated in lung adenocarcinoma cell lines NCI-H1299 and A549 compared with normal lung epithelial cells BEAS-2B(P<0.01).Bioinformatics predictions using miRTargetLink,miRTarbase,DIANA-microT-CD,and HADb databases indicated that miR-185-5p could target and regulate the autophagy-related protein TM9SF1.Dual-luciferase reporter assays and Western blotting demonstrated that miR-185-5p directly bound to the 3'UTR region of TM9SF1 mRNA,and overexpression of miR-185-5p significantly reduced the expression of target protein TM9SF1(P<0.05).EdU cell proliferation,wound healing,and Transwell migration assays demonstrated that miR-185-5p overexpression inhibited proliferation and migration capacities of lung adenocarcinoma cells,whereas TM9SF1 overexpression could attenuate this inhibition effect(P<0.05).Results of stubRFP-sensGFP-LC3 for autophagic flux analysis demonstrated that overexpression of miR-185-5p enhanced autophagic flux in A549 cells,whereas co-overexpression of miR-185-5p and TM9SF1 suppressed autophagic flux.JC-1 assays showed a decreased MMP level in A549 cells after miR-185-5p overexpression,with higher MMP level observed when miR-185-5p and TM9SF1 were co-overexpressed.Conclusion miR-185-5p may suppress proliferation,migration,and autophagy capacities in lung adenocarcinoma cells by targeting TM9SF1 through negative regulation.


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