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BACKGROUND@#Liver cancer is largely resistant to chemotherapy. This study aimed to identify the effective chemotherapeutics for β-catenin-activated liver cancer which is caused by gain-of-function mutation of catenin beta 1 ( CTNNB1 ), the most frequently altered proto-oncogene in hepatic neoplasms.@*METHODS@#Constitutive β-catenin-activated mouse embryonic fibroblasts (MEFs) were established by deleting exon 3 ( β-catenin Δ(ex3)/+ ), the most common mutation site in CTNNB1 gene. A screening of 12 widely used chemotherapy drugs was conducted for the ones that selectively inhibited β-catenin Δ(ex3)/+ but not for wild-type MEFs. Untargeted metabolomics was carried out to examine the alterations of metabolites in nucleotide synthesis. The efficacy and selectivity of methotrexate (MTX) on β-catenin-activated human liver cancer cells were determined in vitro . Immuno-deficient nude mice subcutaneously inoculated with β-catenin wild-type or mutant liver cancer cells and hepatitis B virus ( HBV ); β-catenin lox(ex3)/+ mice were used, respectively, to evaluate the efficacy of MTX in the treatment of β-catenin mutant liver cancer.@*RESULTS@#MTX was identified and validated as a preferential agent against the proliferation and tumor formation of β-catenin-activated cells. Boosted nucleotide synthesis was the major metabolic aberration in β-catenin-active cells, and this alteration was also the target of MTX. Moreover, MTX abrogated hepatocarcinogenesis of HBV ; β-catenin lox(ex3)/+ mice, which stimulated concurrent Ctnnb1- activated mutation and HBV infection in liver cancer.@*CONCLUSION@#MTX is a promising chemotherapeutic agent for β-catenin hyperactive liver cancer. Since repurposing MTX has the advantages of lower risk, shorter timelines, and less investment in drug discovery and development, a clinical trial is warranted to test its efficacy in the treatment of β-catenin mutant liver cancer.

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Fragile X syndrome(FXS)is caused by abnormal duplication and amplification of the FMR1 gene CGG.This article reports a pair of brothers diagnosed with FXS by genetic testing.Two patients,aged 15 and 14 years old respectively,both had clinical manifestations such as language disorders,intellectual disabilities,attention deficit disorder,autism spectrum disorder,and FXS's characteristic facial features.The proband had a rare late-onset epileptic seizure,which was well treated with levetiracetam,while his younger brother had no electroencephalogram abnormalities after repeated follow-up.This pair of cases suggests that the clinical phenotype of FXS has diversity and heterogeneity.

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Objective:To explore the clinical and CT imaging features of immune checkpoint inhibitor-associated pneumonia (CIP) and to improve the early diagnostic ability of CIP.Methods:From June 1, 2020 to October 31, 2021, the clinical data and chest CT images of 2 067 patients with advanced malignant tumor treated with immune checkpoint inhibitor (ICI) in the First Medical Center, Chinese PLA General Hospital were retrospectively analyzed. Patients with CIP were enrolled according to the guidelines for CIP diagnosis, and the incidence, time from the start of medication to the onset of CIP, medication cycle, imaging features, imaging patterns, CT grade and outcomes were analyzed. χ 2 test was used to compare the incidence of CIP in patients with or without basic lung disease. Results:Among 2 067 patients with malignant tumors treated with ICI, 67 patients developed CIP, the incidence of CIP was 3.2%. The incidence of CIP was significantly different between 386 patients with basic lung disease (7.00%, 27/386) and 1 681 patients without basic lung disease (2.4%, 40/1 681) (χ 2=21.32, P<0.001). The time from the start of medication to the onset of CIP was 7-367 d (median 52 days), and the duration of medication was 1-12 cycles (median 2 cycles). The imaging features of CIP presented as ground glass opacities in 54 cases (80.6%), solid nodules in 26 cases (38.8%), consolidations in 25 cases (37.3%) and irregular reticular opacities in 24 cases (35.8%). The main radiologic pattern was organizing pneumonia (OP, 34 cases, 50.7%), and followed by diffuse alveolar damage (DAD) pattern (14 cases, 20.9%). According to CT grading, there were 26 cases in low risk grade, 17 cases in moderate risk grade and 24 cases in high risk grade. Of 43 low-and medium-risk grade cases, 25 were OP pattern, accounting for 58.1%, and among 24 high-risk grade patients, 13 were DAD pattern, accounting for 54.2%. Forty-three of the 52 patients were initially untreated, of which 23 patients progressed, 17 had lesion shrinkage, and 3 had resolution, and relapsed in 8 cases after resolution or drug withdrawal. Conclusions:The imaging manifestations of CIP are mainly ground glass opacities, nodules, consolidations, and irregular reticular opacities. The radiologic patterns are mainly OP and DAD. OP is the most common pattern in low-moderate risk grade CIP and DAD is the most common pattern in high risk grade CIP. Patients with basic lung disease are more likely to get CIP.

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Objective:To investigate the CT features of lepidic predominant adenocarcinoma (LPA) and other pathological subtypes in early-stage invasive pulmonary adenocarcinoma appearing as ground glass nodule (GGN); and to provide imaging-derived information for the clinical management of GGN.Methods:The clinical and CT data of patients with early-stage invasive pulmonary adenocarcinoma in the First Medical Center of PLA General Hospital from January to December 2019 were retrospectively reviewed. All patients presented with pure GGNs or mixed GGNs with a consolidation-to-tumor ratio (CTR)<0.5, with the pathological results confirmed by surgery. GGNs were divided into LPA and non-LPA (n-LPA) groups according to pathological subtypes. Univariate analysis was used to compare the clinical data and CT characteristics between the two groups. The multivariate analysis was performed for the indicators with statistically significant differences and a multivariate model was generated using the reverse elimination method. The area under the ROC curve (AUC) was used to evaluate the discriminatory power of this model for differentiation of LPA from n-LPA.Results:A total of 630 GGNs from 589 patients were analyzed, with 367 GGNs in LPA group and 263 GGNs in n-LPA group. In univariate analysis, the diameter [(14±5) mm], CT value [(-566±98) HU], and CTR [13.9% (0, 27.3%)] in the LPA group were significantly smaller than those in the n-LPA group [(15±5) mm, (-499±111) HU, 27.8%(7.7%, 40%)], respectively, P<0.05]. The frequency of mGGN, deep lobulation sign, burrs, vascular changes, bronchial changes, and clear tumor-lung interface were significantly higher in the n-LPA group than those in the LPA group ( P<0.05). Multivariate analysis results showed that mean CT values, CTR, deep lobulation sign, burr, vascular changes, and bronchial changes were independent predictors for predicting n-LPA ( P<0.05), which were included in the logistic model. Using the optimal cutoff value of 3.958, the logistic regression model for differentiate LPA from n-LPA had a sensitivity of 76.4%, a specificity of 78.7%, and an area under the curve of 0.840. Conclusion:The CT features are helpful for differentiating lepidic predominant subtype from other subtypes in early-stage invasive pulmonary adenocarcinoma presenting as a GGN.

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Objective To investigate the effect of hyperbaric oxygen therapy on serum procalcitonin (PCT), white blood cells(WBC) and high-sensitivity C-reactive protein (hs-CRP) levels in children with necrotising fasciitis (NF) and its efficacy, and explore the diagnosis value of above indicators.Methods From March 2011 to June 2014,50 cases children with acute necrotic fasciitis treated in Children's Hospital of Hebei Province as study group,which were randomly divided into hyperbaric oxygen group (n =25) and routine group (n =25) .The routine group received the routine therapy of incision and drainage to clear the lesion, hyperbaric oxygen group received hyperbaric oxygen therapy on the basis of routine group,while 50 healthy children were selected as control group.The serum PCT, WBC, hs-CRP levels, efficacy, complications, death and hospitalization time were observed and compared.Results The serum PCT, WBC and hs-CRP levels pre-treatment in study group were higher than those in control group(P<0.05).The area under the ROC curve of PCT and hs-CRP was 1.000,respectively, and WBC was 0.804, there were significant difference between PCT and WBC (Z=5.250,P=0.000), between hs-CRP and WBC (Z=5.037,P=0.000).After treatment, the wounds of 23 case patients (92.00%) were cured in hyperbaric oxygen group, and 21 cases in routine group (84.00%) , there were no significant difference in cure rate between two groups.There were six cases(24.00%) of complications and one case (4.00%) of death in hyperbaric oxygen group,while nine cases (36.00%) of complications and two cases (8.00%) of death, there were no significant difference in complications rate and death rate between two groups.The hospitalization time in hyperbaric oxygen group was (39.17 ±6.73) d, which was significantly lower than (52.13 ±4.28) d in routine group(P<0.05).Conclusion PCT and hs-CRP have certain value in diagnosis of children with acute necrotizing fasciitis; incision and drainage combined with hyperbaric oxygen therapy has a better clinical effect in the treatment of children with acute necrotizing fasciitis.

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Objective To investigate the etiology,clinical features and prognosis of neonatal gastric perforation.Methods The medical records of 18 patients with neonatal gastric perforation with respect to sex,age,birth-weight,course of disease,clinical presentations,and prognosis were retrospectively analyzed.Results There were 13 boys and 5 girls,8 of whom were full term infants and 10 preterm infants.The most common initial manifestations were poor activity,abdominal distension,and respiratory distress.All neonates were treated by surgical operation,the overall survival rate was 11/18.Prematurity,birth-weight,course of disease,were the statistically significant risk factors (P ＜ 0.05).Conclusions Neonatal gastric perfora tion is mainly caused by congenital gastric wall defect,and associated with high mortality,particularly in premature infants.It is necessary to early diagnosis,surgical operation in time,nurse intensively,and observe the condition closely.

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NKX3.1, a prostate-specific homeobox gene, plays an important role in prostate cancer and usually functions as tumor suppressor gene. Previously we have demonstrated that forced expression of NKX3.1 reduced cell growth and invasion in prostate cancer cell line PC-3. Presently, we investigated the effect of NKX3.1 on the sensitivity of the prostate cancer cells to apoptosis inducer tumor necrosis factor-alpha (TNF-alpha) and cycloheximide (CHX). PC-3 cells were transfected with NKX3.1 expression plasmid (pcDNA3.1-NKX3.1) and LNCaP cells were transfected with siRNA expression plasmid (pRNAT-RNAi1) targeting NKX3.1. The cell morphology and apoptotic rate were analyzed by Hoechst 33342 staining and Flow Cytometry in absence or presence of TNF-alpha and CHX. The activity of caspase-3 was determined using DEVD-pNA as substrate. Simultaneously, the effect of NKX3.1 on caspase-3 expression was detected using RT-PCR and Western blot. The results showed that ectopic expression of NKX3.1 promoted TNF-alpha/CHX-induced apoptosis in PC-3 cells, whereas knockdown of NKX3.1 protected LNCaP cells from apoptosis induced by TNF-alpha/CHX. The pro-apoptosis activity of NKX3.1 might partially contribute to its elevation of caspase-3 expression and activity. Manipulating NKX3.1 expression should be a promising therapeutic strategy for treating both androgen-dependent and androgen-independent prostate cancer.

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Insufficient intracellular fat oxidation is an important contributor to aging-related insulin resistance, while the precise mechanism underlying is unclear. AMP-activated protein kinase (AMPK) is an important regulator of intracellular fat oxidation and was evidenced to play a key role in high-glucose and high-fat induced glucose intolerance. In the present study, we investigated whether altered AMPK expression or activity was also involved in aging-related insulin resistance. Insulin sensitivity of rats' skeletal muscles was evaluated using in-vitro glucose uptake assay. Activity of alpha subunit of AMPK (AMPKalpha) was evaluated by measuring the phosphorylation of both AMPKalpha (P-AMPKalpha) and acetyl-CoA carboxylase (P-ACC), while expression of AMPKalpha was assessed by determining the mRNA levels of AMPKalpha1 and AMPKalpha2, and protein contents of AMPKalpha. Compared with 4-month old rats, 24-month old rats exhibited obviously impaired insulin sensitivity. At the same time, AMPKalpha activity significantly decreased, while AMPKalpha expression did not alter during aging. Glucose transporter 4 expression also decreased in old rats. Compared with 24-month old rats, administration of the specific activator of AMPK, 5-aminoimidazole-4-carboxamide riboside (AICAR), significantly elevated AMPKalpha activity and GluT4 expression. Also, aging-related insulin resistance was significantly ameliorated by AICAR treatment. In conclusion, aging-related insulin resistance is associated with impaired AMPKalpha activity and could be ameliorated by AICAR, thus indicating a possible role of AMPK in aging-induced insulin resistance.

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BACKGROUND: NKX3.1 is a prostate-specific homeobox gene related strongly to prostate development and prostate cancer. To study its transcriptional regulation, a 1,040 bp promoter of human NKX3.1 gene was cloned into the upstream of the luciferase reporter gene in pGL3-basic plasmid and a 180 bp region extending from -391 to -212 in the upstream of NKX3.1 gene was identified presenting an inhibitory regulation for NKX3.1 expression in our previous experiments. In this work, it is aimed to identify precisely the functional cis-element within the 180 bp region involved in the inhibitory regulation of the NKX3.1 promoter and to identify its specific binding protein. METHODS: 5'-deletion mutation, substitution mutation, and electrophoresis mobility shift assay (EMSA) were carried out to identify precisely the functional cis-element contributing to the inhibitory regulation of NKX3.1 and its binding ability to nuclear extracts from prostate cancer cell line LNCaP. RESULTS: A 20-bp inhibitory element located between -362 and -343 in the upstream of NKX3.1 gene was identified by deletion and substitution mutation analysis and proven to be a functional inhibitory cis-element by EMSA. CONCLUSIONS: We have identified a functional inhibitory cis-element between -362 and -343 in the upstream of NKX3.1 gene; it is likely to play an important role in downregulating NKX3.1 gene transcription.

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Prostate cancer is a disease involving complicated multiple-gene alterations. Both NKX3.1 and p53 are related to prostate cancer and play crucial roles in prostate cancer progression. However, little is known about the relationships and interactions between p53 and NKX3.1 in prostate cancer. We found that NKX3.1 expression is down-regulated by over-expression of wild type (wt) p53 in prostate cancer LNCaP cells. NKX3.1 is down-regulated at both the mRNA and protein levels by p53 over- expression due to either transient transfection of exogenous p53 or induction of endogenous p53. p53 over-expression represses androgen-induced transactivation of NKX3.1 by inhibiting the promoter of the androgen acceptor (AR) gene and by blocking AR-DNA binding activity. In addition, transfection with the p21 expression vector (pPSA-p21) showed that p21 does not reduce NKX3.1 expression, indicating that NKX3.1 expression is not the result of nonspecific effects of cell growth arrest. Our results provide biochemical and cellular biologic evidence that NKX3.1 is down-regulated by p53 over-expression in prostate cancer cells.

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AIM: To study the basic mechanism of transcriptional regulation, NKX3.1 gene promoter was cloned and its promoter activities in prostate cancer cell lines and other cancer cell lines were tested. METHODS: 1.04 kb - promoter fragment of NKX3. 1 gene was obtained by PCR and cloned into pGL3 - basic and pEGFP - 1 that are promoter - less reporter vectors to examine its promoter activity driving the reporter gene transcription. The promoter activity was determined by dual -luciferase reporter assay and the expression of GFP reporter observed under fluorescence micro scope. RESULTS: The sequence of the cloned 1.04 kb promoter proved to be correct by DNA sequencing. The dual - lu ciferase reporter assay (M1/M2) showed that the promoter activity in LNCaP cell transfected with pGL3 - 1.04 kb promoter was about 1.5 - fold higher than that of pGL3 - control transfection and 50 - fold higher than that of pGL3 - basic transfec tion. To investigate the 1.04 kb - promoter activity in different tumor cell lines, the constructed pGL3 - 1.04 kb promoter and pEGFP - 1.04 kb promoter were transfected into several cell lines, respectively. The results showed that the activity of 1.04 kb promoterin LNCaP was highest among the tested cell lines. Multiple consensus sequence elements have been iden tified within the 1.04 kb fragment using TRANSFAC database. Further experiments will be done to determine their founc tions. CONCLUSION: Cloned 1.04 kb fragment upstream of NKX3.1 gene presented a strong promoter activity and its activity was highest in LNCaP cell among the tested tumor cell lines.

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AIM: To observe the effect of exogenous androgen responsive element decoy on the promoter of prostate specific antigen (PSA) and the growth of LNCaP cells for searching the possibility of gene therapy for prostate cancer. METHODS: Firstly, pGL3 - PSA luciferase expression vector containing 640bp - promoter fragment of PSA gene was constructed. Then, a 23 -mer phosphorothioated ARE decoy based on the deduced ARE sequence at the promoter region of PSA gene was synthesized. pGL3 - PSA and ARE decoy DNA were cotransfected into PC3 - M cell by lipofectamineTM 2000. Through detecting the activity of luciferase, the effect of ARE decoy on the promoter of PSA was studied. Then the ARE decoy DNA was transfected into LNCaP cells. The effect of decoy DNA on the proliferation of LNCaP cells was examined by using MTT assay. The effects of apoptosis were detected by phase contrast microscopy, DNA agrose gel electrophoresis and flow cytometry. Meanwhile, the nuclear extract was prepared from LNCaP cells and DNA - protein interactions were examined by electrophoretic mobility shift assay (EMSA). RESULTS: The reporter assay showed that the aetivity of luciferase was significantly reduced in the ARE decoy - transfected cells, bnt not in the cells transfected with the control decoy. EMSA demonstrated specific binding of the ARE decoy to androgen receptor. The growth of LNCaP was remarkably inhibited and apoptotic morphological changes as well as DNA fragmentation were observed in the ARE decoy- transfected cells. The rate of apoptosis was 22.4% detected by FCM. CONCLUSION: The ARE decoy is capable of inhibiting the promoter of PSA gene and inducing the apoptosis in prostate cancer cells. It may become a potential therapeutic tool for prostate cancers.

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AIM: To observe the effect of E2F decoy DNA on proliferation and apoptosis of androgen-independent prostate cancer cell line PC-3M.METHODS: E2F decoy DNA,ARE decoy DNA and control decoy DNA were transfected into PC-3M cells with lipofectamine,respectively.Their effects on cell proliferation were detected by MTT assay.The changes of cell morphology were observed by inverted phase contrast microscope.The cell apoptotic rate was determined by flow cytometry(FCM) analysis and chromosome DNA ladder was detected by DNA gel electrophoresis.The expression of c-Myc mRNA and cyclin D1 mRNA was detected by RT-PCR.The protein levels of c-Myc and cyclin D1 were detected by Western blotting.RESULTS: The growth of PC-3M cells was inhibited after transfection.The transfected PC-3M cells displayed typical apoptotic morphological changes.The apoptotic rate was 26.35% and DNA ladder was observed after transfection.The expression of c-Myc and cyclin D1 were inhibited.CONCLUSION: These results indicate that E2F decoy DNA induces apoptosis of androgen-independent prostate cancer cell lines PC-3M and inhibits cell proliferation via inhibiting expression of c-Myc and cyclin D1.

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AIM: To study the basic mechanism of transcriptional regulation,NKX3.1 gene promoter was cloned and its promoter activities in prostate cancer cell lines and other cancer cell lines were tested.METHODS:(1.04 kb)-promoter fragment of NKX3.1 gene was obtained by PCR and cloned into pGL_3-basic and pEGFP-1 that are promoter-less reporter vectors to examine its promoter activity driving the reporter gene transcription.The promoter activity was determined by dual-luciferase reporter assay and the expression of GFP reporter observed under fluorescence microscope.RESULTS: The sequence of the cloned(1.04 kb) promoter proved to be correct by DNA sequencing.The dual-luciferase reporter assay(M1/M2) showed that the promoter activity in LNCaP cell transfected with pGL_3-(1.04 kb) promoter was about 1.5-fold higher than that of pGL_3-control transfection and 50-fold higher than that of pGL_3-basic transfection.To investigate the 1.04 kb-promoter activity in different tumor cell lines,the constructed pGL_3-(1.04 kb) promoter and pEGFP-1.04 kb promoter were transfected into several cell lines,respectively.The results showed that the activity of(1.04 kb) promoter in LNCaP was highest among the tested cell lines.Multiple consensus sequence elements have been identified within the(1.04 kb) fragment using TRANSFAC database.Further experiments will be done to determine their founctions.CONCLUSION: Cloned 1.04 kb fragment upstream of NKX3.1 gene presented a strong promoter activity and its activity was highest in LNCaP cell among the tested tumor cell lines.