1.Clinical features and genetic analysis of three patients with Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome due to variants of FOXP3 gene
Cuifang ZHENG ; Yingying MENG ; Zhaohui DENG ; Jing LIU ; Gangfeng YAN ; Ying HUANG
Chinese Journal of Medical Genetics 2024;41(2):181-186
Objective:To analyze the clinical characteristics of three patients with Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome.Methods:Three patients with IPEX syndrome diagnosed at the Children′s Hospital of Fudan University from January 24, 2013 to July 29, 2019 were selected as the study subjects. Their clinical features, laboratory investigations and results of genetic testing were summarized. Treatment and prognosis were also explored.Results:All of the three children had developed the disorder during infancy. One child had initial features including diabetes and diabetic ketoacidosis, whilst the other two had initiated by diarrhea. All patients had gastrointestinal involvement, and one was diagnosed as very early onset inflammatory bowel disease by colonoscopy and biopsy. Two children also had endocrine glands involvement. One child had manifested type 1 diabetes and positivity for thyroglobulin and thyroid peroxidase antibodies, though his thyroid function had remained normal. Another one had hypothyroidism and was treated by levothyroxine. Genetic testing revealed that all children had harbored missense variants of the FOXP3 gene, including c. 1222G>A (p.V408M), c. 767T>C (p.M256T) and c. 1021A>G (p.T341A). The clinical symptoms of one patient were alleviated following allogeneic hematopoietic stem cell transplantation. One patient was stable after treatment with infliximab plus insulin, and one child had died of refractory septic shock and multiple organ dysfunction syndrome at 3 months old. Conclusion:FOXP3 gene variant-associated IPEX syndrome may have very early onset and diverse clinical manifestations. For male patients with infantile onset chronic diarrhea, multiple endocrine or multiple system involvement, genetic testing is recommended, which may facilitate early diagnosis, treatment and genetic counseling.
2.A Case Report of Multidisciplinary Diagnosis and Treatment of a Patient with Tuberous Sclerosis Complex and Multi-Organ Involvement
Hua ZHENG ; Yunfei ZHI ; Lujing YING ; Lan ZHU ; Mingliang JI ; Ze LIANG ; Jiangshan WANG ; Haifeng SHI ; Weihong ZHANG ; Mengsu XIAO ; Yushi ZHANG ; Kaifeng XU ; Zhaohui LU ; Yaping LIU ; Ruiyi XU ; Huijuan ZHU ; Li WEN ; Yan ZHANG ; Gang CHEN ; Limeng CHEN
JOURNAL OF RARE DISEASES 2024;3(1):79-86
Tuberous sclerosis complex(TSC)is a rare genetic disease that can lead to benign dysplasia in multiple organs such as the skin, brain, eyes, oral cavity, heart, lungs, kidneys, liver, and bones. Its main symptoms include epilepsy, intellectual disabilities, skin depigmentation, and facial angiofibromas, whilst incidence is approximately 1 in 10 000 to 1 in 6000 newborns. This case presents a middle-aged woman who initially manifested with epilepsy and nodular depigmentation. Later, she developed a lower abdominal mass, elevated creatinine, and severe anemia. Based on clinical features and whole exome sequencing, the primary diagnosis was confirmed as TSC. Laboratory and imaging examinations revealed that the lower abdominal mass originated from the uterus. CT-guided biopsy pathology and surgical pathology suggested a combination of leiomyoma and abscess. With the involvement of multiple organs and various complications beyond the main diagnosis, the diagnostic and therapeutic process for this patient highlights the importance of rigorous clinical thinking and multidisciplinary collaboration in the diagnosis and treatment of rare and challenging diseases.
3.Evaluation of the Effect for National Centralized Drug Procurement Policy of PPIs in 33 Hospitals in Wuhan
Zhijuan LIN ; Li LIU ; Wenjuan HE ; Zhiliang ZHANG ; Zhaohui GUO ; Ping LIU ; Quan LEI ; Ying XU
Chinese Journal of Modern Applied Pharmacy 2024;41(12):1723-1728
OBJECTIVE
To analyze the use status and development trend of proton pump inhibitors(PPIs) in 33 hospitals in Wuhan, Hubei Province after the implementation of the national centralized drug procurement(NCDP) policy, and to provide reference for promoting the subsequent rational use of NCDP drugs and improving related policies.
METHODS
To make statistics and analysis of purchasing amount of PPIs, defined daily dose system(DDDs), defined daily dose consumption(DDDc) and utilization rate of 33 hospitals in Wuhan in 2019 and 2022.
RESULTS
After the implementation of the NCDP policy, the total purchasing amount of PPIs decreased by 53.6%, DDDs decreased by 15.4%, DDDc decreased by 45.2%, and the utilization rate of PPIs injectable dosage forms decreased by 12.6%. After NCDP, the highest growth rate of oral dosage forms was omeprazole(5.7%), followed by rabeprazole(5.0%), while injectable dosage forms showed a significant difference in utilization rate, with a significant decline in NCDP varieties and a significant increase in non-NCDP varieties. The overall NCDP utilization rate of PPIs in Wuhan was 64.9%, with little difference among hospitals of different grades.
CONCLUSION
The NCDP policy achieves the purpose of reducing the drug cost of patients and improving the accessibility of drugs, and is more optimized in the selection of dosage forms, which is in line with the policy expectations overall; but the quantity and price of PPIs in Wuhan decreased after NCDP, and highlighted a certain tendency in the selection of varieties. In the future, we still need to optimize measures to guide clinical priority in the selection of NCDP drugs, to ensure and improve the implementation of NCDP policy.
4.Effect of over-expression of NDRG1 on resistance of castration-resistant prostate cancer resistant cell line C4-2/ENZA and its mechanism
Ying ZHANG ; Zhaohui WAN ; Xianxun JIANG
Journal of Jilin University(Medicine Edition) 2024;50(3):708-717
Objective:To discuss the effect of N-myc downstream-regulated gene 1(NDRG1)on the enzalutamide(ENZA)resistance in the castration-resistant prostate cancer(CRPC),and to clarify its mechanism.Methods:The human CRPC C4-2 cells and ENZA-resistant strain C4-2/ENZA cells were cultured in vitro.The expression levels of NDRG1 mRNA in the C4-2/ENZA cells and their parental C4-2 cells were detected by real-time fluorescence quantitative PCR(RT-qPCR)method.The expression levels of NDRG1,androgen receptor(AR),and prostate-specific antigen(PSA)proteins in the cells were detected by Western blotting method to verify the transfection efficiency of the cells.The C4-2/ENZA cells were divided into blank group(normally cultured without treatment),negative control lentivirus(Lv-NC)group(transfected with Lv-NC),Lv-NDRG1 group(transfected with Lv-NDRG1),Lv-NC+ENZA group(transfected with Lv-NC followed by ENZA treatment),Lv-NDRG1+ENZA group(transfected with Lv-NDRG1 followed by ENZA treatment),Lv-NDRG1+epidermal growth factor(EGF)group(transfected with Lv-NDRG1 followed by EGF treatment),and Lv-NDRG1+EGF+ENZA group(transfected with Lv-NDRG1 followed by EGF and ENZA treatment).The half-maximal inhibitory concentration(IC50),resistance index(RI),and proliferation activity of the cells were detected by MTT assay;the apoptotic rates of the cells in various groups were detected by flow cytometry;RT-qPCR method was used to detect the expression levels of NDRG1 mRNA in the cells in various groups;Western blotting method was used to detect the expression levels of NDRG1,AR,phosphorylated AR at serine213(p-ARSer213),phosphorylated AR at serine81(p-ARSer81),and PSA proteins in the cells in various groups.Results:Compared with C4-2 cells,the expression levels of NDRG1 mRNA and protein in the C4-2/ENZA cells were significantly decreased(P<0.01)and the expression levels of AR and PSA proteins were increased(P<0.01),indicating low expression of NDRG1 in the ENZA-resistant C4-2/ENZA strain.Compared with Lv-NC group,the expression levels of NDRG1 mRNA and protein in the cells in Lv-NDRG1 group were significantly increased(P<0.01),indicating the successful construction of an NDRG1 gene over-expression strain of C4-2/ENZA resistant cells.The MTT assay results showed that compared with the C4-2 cells,the IC50 of the C4-2/ENZA cells was increased(P<0.01)and the RI was 17.78;compared with Lv-NC group,the IC50 of the C4-2/ENZA cells in Lv-NDRG1 group was decreased(P<0.01).After 24 h of EGF treatment,compared with Lv-NC group,the IC50 of the C4-2/ENZA cells in Lv-NC+EGF group was significantly increased(P<0.01);compared with Lv-NDRG1 group,the IC50 of the C4-2/ENZA cells in Lv-NDRG1+EGF group was increased(P<0.01).Compared with before ENZA treatment,after 24 h of ENZA treatment,the proliferation activities of C4-2 and C4-2/ENZA cells were gradually decreased(F=223.80,P<0.01;F=81.46,P<0.01).Compared with Lv-NC group,the proliferation activity in the C4-2/ENZA cells in Lv-NDRG1 group after 24 h of ENZA treatment was significantly decreased(P<0.01).After 24 h of EGF treatment,compared with Lv-NC group,the proliferation activity of the C4-2/ENZA cells in Lv-NC+EGF group was significantly increased(P<0.01),while the the proliferation activity of the C4-2/ENZA cells in Lv-NDRG1+EGF group was significantly decreased(P<0.01).The chosen concentration and treatment duration for further testing were 10 000 μmol·L-1 ENZA and the intervention time was 24 h.The flow cytometry results showed that after 24 h of ENZA treatment,compared with Lv-NC group,the apoptotic rate of the cells in Lv-NDRG1 group was significantly increased(P<0.01);compared with Lv-NC+ENZA group,the apoptotic rate of the cells in Lv-NDRG1+ENZA group was significantly increased(P<0.01).After 24 h of EGF treatment,compared with Lv-NDRG1 group,the apoptotic rate of the cells in Lv-NDRG1+EGF group was significantly decreased(P<0.01),while the apoptotic rate of the cells in Lv-NDRG1+ENZA group was significantly increased(P<0.01);compared with Lv-NDRG1+ENZA group,the apoptotic rate of the cells in Lv-NDRG1+EGF+ENZA group was significantly decreased(P<0.01).The Western blotting results showed that after 24 h of ENZA treatment,compared with Lv-NC group,the expression levels of AR and PSA proteins and the ratio of p-ARSer213/AR and p-ARSer81/AR in the cells in Lv-NDRG1 group were significantly decreased(P<0.05 or P<0.01).After 24 h of EGF treatment,compared with Lv-NC group,the expression levels of AR and PSA proteins and the ratio of p-ARSer213/AR and p-ARSer81/AR in the cells in Lv-NC+EGF group were significantly increased(P<0.05 or P<0.01);compared with Lv-NDRG1 group,the expression levels of AR and PSA proteins and the ratio of p-ARSer213/AR and p-ARSer81/AR in the cells in Lv-NDRG1+EGF group were significantly increased(P<0.01).Conclusion:Over-expression of NDRG1 can reduce the resistance of CRPC to ENZA,and its mechanism may be related to the inhibition of AR signaling.
5.Analysis of the characteristics of patients with amyotrophic lateral sclerosis with neuromuscular junction dysfunction prior to motor neuron degeneration
Jinghong ZHANG ; Zhaohui CHEN ; Li LING ; Hongmei CHENG ; Ying ZHANG ; Jiarui ZHAO ; Xusheng HUANG
Chinese Journal of Internal Medicine 2024;63(7):660-665
Objective:To investigate the clinical and electrophysiological characteristics of patients with amyotrophic lateral sclerosis (ALS) with positive repetitive nerve stimulation (RNS) test results on the accessory nerve and negative needle electromyography (EMG) test results on the sternocleidomastoid with the goal to enrich the knowledge of disease progression in patients with ALS.Methods:The clinical data of 612 patients diagnosed with ALS at the Neurology Department of the First Medical Center, Chinese PLA General Hospital from June 2016 to August 2022 were collected. In total, 267 cases had undergone EMG tests on the sternocleidomastoid following a positive 3 Hz RNS test result on the accessory nerve, who were selected as the study subjects. The differences in clinical indicators were compared between RNS (+)/EMG (-) group and RNS (+)/EMG (+) group. A binomial distribution model with multiple variables was built to quantitatively analyze the major factors and their effects.Results:At the initial visit, 15.8% of patients with ALS were 3 Hz RNS (+) on the accessory nerve and EMG (-) on the ipsilateral sternocleidomastoid, accounting for 36.3% of RNS (+) patients. The decremental range of the 3 Hz RNS test delivered to the accessory nerve in these patients [-14% (-19%, -12%)] was lower than that in patients with RNS (+)/EMG (+) [-17% (-23%, -13%)] ( P<0.05), while the ratio of upper limb onset (64.9%) and non-definite diagnosis (28.9%) were higher [54.7% and 13.5% for patients with RNS (+)/EMG (+), P<0.05]. Furthermore, the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) score [40 (37, 42)], body mass index (BMI) [23.8 (22.0, 25.4) kg/m 2] and forced vital capacity (FVC) [92.8% (76.6%, 103.8%)] were higher in patients with RNS(+)/EMG(+) ( P<0.05). The multivariate model suggested that, in patients with RNS (+)/EMG (-), the ratio of upper limb onset to lower limb onset was 1.04, while that of upper limb onset to bulbar onset was 2.02, and that of lower limb onset to bulbar onset was 1.94. The ratio of non-definite ALS to definite ALS was 1.13. The ALSFRS-R score, BMI, and FVC had a protective contribution to the electrophysiological function of the motor neurons. The ratio of the effect size of the ALSFRS-R or BMI to that of FVC was 3.37 and 1.14, respectively. Conclusions:Patients with ALS that were 3 Hz RNS (+) on the accessory nerve and EMG (-) on the ipsilateral sternocleidomastoid had a smaller decremental range of the compound muscle action potential amplitude, and a higher proportion of upper limb onset and non-definite ALS. A higher ALSFRS-R score, BMI, and FVC have a protective effect to the electrophysiological function of motor neurons. The effect size of the ALSFRS-R score is the largest, followed by BMI and FVC.
6.Association between umbilical artery ultrasound parameters and birth outcomes in gestational diabetes mellitus
Ying Pan ; Li Zhou ; Lianjie Dou ; Jijun Gu ; Dan Huang ; Zhaohui Huang ; Anhui Zhang ; Hong Tao ; Li Zhang ; Jiahu Hao
Acta Universitatis Medicinalis Anhui 2023;58(7):1184-1188
Objective :
To investigate the effects of gestational diabetes mellitus (GDM) on birth outcome and umbilical artery (UA) blood flow parameters in the third trimester, and to analyze the role of UA blood flow parameters in GDM and birth outcome.
Methods :
Based on the birth cohort from Wuhu , Anhui , China , 189 pregnant women with GDM were collected as the case group. The non⁃GDM pregnant women were matched 1 ∶ 1 according to age and pre⁃pregnancy body mass index , and 189 normal pregnant women were selected as the control group. Pregnant women with GDM were divided into poorly controlled group and well controlled group according to fasting blood
glucose in the third trimester. The UA blood flow parameters and fetal birth outcomes in the third trimester were tracked.
Results :
Compared with the control group , UA parameters in poorly controlled and well controlled groups
significantly increased (F = 6. 63 , P < 0. 05 ; F = 4. 43 , P < 0. 05 ; F = 5. 57 , P < 0. 05) . Poor glycemic control of GDM was associated with increased birth weight and risk of larger than gestational age. The multi⁃factor linear regression model showed that the Z score of the peak systolic velocity/end diastolic velocity (S/D) in the poorly controlled group was negatively correlated with birth weight (β = - 209. 78 , 95% CI: - 301. 48 - 118. 07) . S/D index Z score mediated the relationship between poor blood glucose control and birth weight. The intermediate effect value was - 58. 41 (95% CI: - 106. 40 ~ - 19. 65) , accounting for 25. 98% of the total effect.
Conclusion
Poor glycemic control in GDM is a risk factor for fetal weight gain , and UA function plays a partial mediating role in influencing neonatal birth weight. GDM pregnant women should strictly control blood glucose level to better protect maternal and infant health.
7.Application of argatroban in patients receiving extracorporeal membrane oxygenation support: a case-control study
Zixuan ZHANG ; Xiao TANG ; Rui WANG ; Xuyan LI ; Ying LI ; Zhaohui TONG ; Bing SUN
Chinese Critical Care Medicine 2022;34(12):1305-1310
Objective:To evaluate the safety and efficacy of argatroban applied as alternative anticoagulant in critical illness patients underwent extracorporeal membrane oxygenation (ECMO) with contraindications of unfractionated heparin (UFH), and to further explore the effective dose of argatroban.Methods:From July 1, 2013 to February 28, 2022, there were 14 patients who admitted in the respiratory intensive care unit (RICU) of Beijing Chao-Yang Hospital received ECMO and used argatroban for anticoagulation (argatroban group). Two of them received argatroban as the initial anticoagulant. The remaining 12 patients used UFH at first, and then switched to argatroban. UFH group included 28 patients who received UFH for anticoagulation after matching the demographic characteristics. Primary endpoint was the prevalence of ECMO-related thrombotic events. Secondary endpoints included the type of thrombotic events, prevalence of ECMO-related major bleeding events, bleeding sites, ICU mortality, mortality during ECMO, liver and kidney function, thrombelastogram, blood transfusion, dosage of argatroban, the dynamic changes of coagulation variables 4 days before and 7 days after argatroban treatment.Results:In argatroban group, there were 8 patients received veno-venous ECMO (VV-ECMO), 2 patients with veno-arterial ECMO (VA-ECMO), and 4 patients with veno-arterio-venous ECMO (VAV-ECMO). In UFH group, VV-ECMO was applied in 23 patients, VA-ECMO and VAV ECMO was established in 3 patients and 2 patients, respectively. In endpoint events, the incidence of ECMO related thrombotic events in argatroban group was slightly higher than that in UFH group (28.6% vs. 21.4%). The ECMO running time in argatroban group was slightly longer than that in UFH group [days: 16 (7, 21) vs. 13 (8, 17)]. The incidence of ECMO-related bleeding events (28.6% vs. 32.1%) and mortality during ECMO (35.7% vs. 46.4%) in argatroban group were slightly lower than those in UFH group. However, the differences were not statistically significant (all P < 0.05). The platelet transfusion in argatroban group was significantly higher than that in UFH group [U: 7.7 (0, 10.0) vs. 0.8 (0, 1.0)]. The coagulation reaction time (R value) in thrombelastography in argatroban group was significantly longer than that in UFH group [minutes: 9.3 (7.2, 10.8) vs. 8.8 (6.3, 9.7)]. The maximum width value [MA value, mm: 48.4 (40.7, 57.9) vs. 52.6 (45.4, 61.5)] and blood clot generation rate [α-Angle (deg): 54.1 (45.4, 62.0) vs. 57.9 (50.2, 69.0)] in the argatroban group were significantly lower than those in the UFH group (all P < 0.05). The activated partial thromboplastin time (APTT) was prolonged after changing from UFH to argatroban in the argatroban group [seconds: 63.5 (58.4, 70.6) vs. 56.7 (53.1, 60.9)]. The PLT level showed a decreasing trend during UFH anticoagulation therapy, and gradually increased after changing to argatroban. D-dimer level was 19.1 (7.0, 28.7) mg/L after switching to argatroban, and then no longer showed an increasing trend. The level of fibrinogen (FIB) showed a decreasing trend during the anticoagulant therapy of UFH (the lowest was 23.6 g/L), and fluctuated between 16.8 and 26.2 g/L after changing to argatroban. The median initial dose of argatroban was 0.049 (0.029, 0.103) μg·kg -1·min -1, which the highest dose was in VV-ECMO patients of [0.092 (0.049, 0.165) μg·kg -1·min -1]. The initial dose of VAV-ECMO was the lowest [0.026 (0.013, 0.041) μg·kg -1·min -1], but without significant difference ( P > 0.05). The maintenance dose of argatroban was 0.033 (0.014, 0.090) μg·kg -1·min -1, VV-ECMO patients was significantly higher than those in VA-ECMO and VAV-ECMO patients [μg·kg -1·min -1: 0.102 (0.059, 0.127) vs. 0.036 (0.026, 0.060), 0.013 (0.004, 0.022), both P < 0.05]. Conclusion:Argatroban appears to be a feasible, effective and safety alternative anticoagulant for patients with contraindications to UFH who undergoing ECMO support.
8.Mechanism study of neurofibromin 1 in gallbladder cancer
Lingxiao ZHANG ; Zhaohui JIN ; Xuya CUI ; Shimei QIU ; Ping DONG ; Ying HUANG ; Yingbin LIU
Chinese Journal of Digestive Surgery 2022;21(7):941-948
Objective:To investigate the mechanism of neurofibromin 1 (NF1) in gallbla-dder cancer.Methods:The experimental study was conducted. Human gallbladder cancer cell lines, including GBC-SD, NOZ, SGC996, EH-GB1, ZJU0428, human embryonic kidneys cell line 293T and human cervical cancer cell line HELA, were cultured. The recombinant plasmids (mRFP-YAP1 FL-FLAG and eGFP-MYC-NF1 2650?2750-HA) were constructed for co-immunoprecipitation experiment. The truncated Yes associated protein 1(YAP1) and NF1 recombinant proteins were purified in vitro. The interaction between NF1 and YAP1 in vitro or in vivo were verified by isothermal titration calori-metry (ITC) assay, GST pull-down experiment, co-immunoprecipitation, immunofluorescence, laser confocal microscopy, and the expression of NF1 protein in different gallbladder cancer cell lines was verified by Western blot experiments. Observation indicators: (1) interaction between NF1 and YAP1 in vitro; (2) interaction between NF1 and YAP1 in cells; (3) expression of NF1 protein in different human gallbladder cancer cell lines. The dissociation constants were exported from ITC 200 software and represented as Mean± SD. Count data were represented as absolute numbers. Results:(1) Interaction between NF1 and YAP1 in vitro. ① Results of ITC assay showed that there was interac-tion between PPQY and YAP1-WW1, between PPQY and YAP1 (Amino acid residues 162?275), and the dissociation constants between PPQY and YAP1-WW1, between PPQY and YAP1(Amino acid residues 162?275) were (0.42±0.06)mmol/L, (0.69±0.14)mmol/L, respectively. ② GST pull-down results indicated that the target protein His-Sumo-YAP1 WW1 was obviouly observed in protein lane of reaction system between GST-PPQY recombinant protein and His-Sumo-YAP1 WW1, relative to the reaction system between GST protein and His-Sumo-YAP1 WW1. The target protein His-Sumo-YAP1 WW2 was obviouly observed in protein lane of reaction system between GST-PPQY recombinant protein and His-Sumo-YAP1 WW2, relative to the reaction system between GST protein and His-Sumo-YAP1 WW2. (2) Interaction between NF1 and YAP1 in cells. ① Co-immunoprecipitation results indica-ted that NF1 protein was observed in cell lysis solution which was incubated by FLAG gel beads and cotransfected with mRFP-YAP1 FL-FLAG and eGFP-MYC-NF1 2650?2750-HA. ② Immuno-fluorescence and laser confocal microscopy results indicated that YAP1 and NF1 with obvious fluorescence were co-localized in the cytoplasm of human gallbladder cancer NOZ cells. However, YAP1 with obvious fluorescence was localized in the nucleus of human gallbladder SGC996 cells and NF1 showed weak fluorescence. (3) Expression of NF1 protein in different human gallbladder cancer cell lines. Western blot results showed that with the expression level of NF1 protein in HELA cell line as the standard, the relative expression levels of NF1 protein in EH-GB1, GBC-SD, NOZ, SGC996, ZJU0428 cell lines were 1.28, 0, 1.01, 0, 0, respectively. Conclusion:NF1 affects the gallbladder cancer by directly acting on YAP1 protein.
9.Effects of K-ras gene silence on the expression of oncogenes in HBE cells treated with PM 2.5
Runbing LI ; Juning PU ; Boru LI ; Ying CAI ; Zhaohui ZHANG ; Xinyun XU
Chinese Journal of Industrial Hygiene and Occupational Diseases 2021;39(11):825-830
Objective:To explore the effects of K-ras gene on the expressions of oncogenes and cancer suppressor genes in human bronchial epithelial (HBE) cells which were exposed to PM 2.5. Methods:According to the mRNA sequence of K-ras gene provided by GenBank in September 2019, interference sequences were designed and synthesized, and the recombinant lentiviral vector was transfected into HBE cell to construct the K-ras gene-silenced cells. HBE cells and K-ras gene-silenced cells were exposed to 10 μg/ml, 50 μg/ml PM 2.5 suspension and 10 μmol/L Cr 6+. Real-time fluorescent quantitative PCR was used to detect the mRNA expression levels of c-myc, c-fos, N-ras, cyclin-D1, p16 and p53 genes, the expression levels of p53 and c-myc proteins were detected by Western blot. Results:In K-ras silenced cell group, K-ras mRNA expression level decreased (80.5%±3.6%) and K-ras protein level decreased (58.9%±4.7%) when compared with the control group ( P<0.01) . Compared with the correspoding cell control group without exposure, the mRNA expression levels of c-myc, c-fos, N-ras and cyclin-D1 genes in HBE cell group exposed to different concentrations of PM 2.5, K-ras silenced cell group exposed to different concentrations of PM 2.5, HBE cell group exposed to 10 μmol/L Cr 6+ and K-ras silenced cell group exposed to 10 μmol/L Cr 6+ were increased, the mRNA expressions of p16 and p53 genes were decreased ( P<0.01) . Compared with HBE cell group exposed to 10 μg/ml PM 2.5, the mRNA expressions of c-myc, c-fos and p16 genes in K-ras silenced cells exposed to 10 μg/ml PM 2.5 were decreased, and the p53 mRNA level was increased ( P<0.01) . Compared with HBE cell group exposed to 50 μg/ml PM 2.5, the mRNA expression levels of c-fos, N-ras, cyclin-D1, p16 and p53 genes in K-ras silenced cell group exposed to 50 μg/ml PM 2.5 were decreased ( P<0.01) . Compared with the HBE cell group without exposure, c-myc protein increased and p53 protein decreased in HBE cells exposed to 50 μg/ml PM 2.5 ( P<0.05) . Compared with the K-ras silenced cell group without exposure, c-myc protein increased in K-ras silenced cells exposed to 50 μg/ml PM 2.5 ( P<0.05) . Conclusion:PM 2.5 can increase the expression levels of oncogenes in HBE cells, and K-ras gene silencing can inhibit the expression levels of oncogenes in HBE cells treated with PM 2.5.
10.Effects of K-ras gene silence on the expression of oncogenes in HBE cells treated with PM 2.5
Runbing LI ; Juning PU ; Boru LI ; Ying CAI ; Zhaohui ZHANG ; Xinyun XU
Chinese Journal of Industrial Hygiene and Occupational Diseases 2021;39(11):825-830
Objective:To explore the effects of K-ras gene on the expressions of oncogenes and cancer suppressor genes in human bronchial epithelial (HBE) cells which were exposed to PM 2.5. Methods:According to the mRNA sequence of K-ras gene provided by GenBank in September 2019, interference sequences were designed and synthesized, and the recombinant lentiviral vector was transfected into HBE cell to construct the K-ras gene-silenced cells. HBE cells and K-ras gene-silenced cells were exposed to 10 μg/ml, 50 μg/ml PM 2.5 suspension and 10 μmol/L Cr 6+. Real-time fluorescent quantitative PCR was used to detect the mRNA expression levels of c-myc, c-fos, N-ras, cyclin-D1, p16 and p53 genes, the expression levels of p53 and c-myc proteins were detected by Western blot. Results:In K-ras silenced cell group, K-ras mRNA expression level decreased (80.5%±3.6%) and K-ras protein level decreased (58.9%±4.7%) when compared with the control group ( P<0.01) . Compared with the correspoding cell control group without exposure, the mRNA expression levels of c-myc, c-fos, N-ras and cyclin-D1 genes in HBE cell group exposed to different concentrations of PM 2.5, K-ras silenced cell group exposed to different concentrations of PM 2.5, HBE cell group exposed to 10 μmol/L Cr 6+ and K-ras silenced cell group exposed to 10 μmol/L Cr 6+ were increased, the mRNA expressions of p16 and p53 genes were decreased ( P<0.01) . Compared with HBE cell group exposed to 10 μg/ml PM 2.5, the mRNA expressions of c-myc, c-fos and p16 genes in K-ras silenced cells exposed to 10 μg/ml PM 2.5 were decreased, and the p53 mRNA level was increased ( P<0.01) . Compared with HBE cell group exposed to 50 μg/ml PM 2.5, the mRNA expression levels of c-fos, N-ras, cyclin-D1, p16 and p53 genes in K-ras silenced cell group exposed to 50 μg/ml PM 2.5 were decreased ( P<0.01) . Compared with the HBE cell group without exposure, c-myc protein increased and p53 protein decreased in HBE cells exposed to 50 μg/ml PM 2.5 ( P<0.05) . Compared with the K-ras silenced cell group without exposure, c-myc protein increased in K-ras silenced cells exposed to 50 μg/ml PM 2.5 ( P<0.05) . Conclusion:PM 2.5 can increase the expression levels of oncogenes in HBE cells, and K-ras gene silencing can inhibit the expression levels of oncogenes in HBE cells treated with PM 2.5.


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