Objective:To investigate the expression of serum human epididymal protein 4 (HE4) in colorectal cancer patients and its relationship with recurrence and metastasis.Methods:A prospective study was conducted to collect 100 patients with colorectal cancer admitted to the First Affiliated Hospital of Hebei North University from January to December 2018 as the observation group, and 50 healthy volunteers as the control group. All patients underwent radical surgery for colorectal cancer, and serum samples were collected before surgery. Enzyme linked immunosorbent assay (ELISA) was used to detect serum HE4 levels, and the relationship between serum HE4 levels and clinical pathological characteristics in the observation group was analyzed. The relationship between preoperative serum HE4 levels and postoperative recurrence and metastasis of colorectal cancer in the observation group was also analyzed. The COX proportional risk model was used to analyze the influencing factors of postoperative recurrence and metastasis of colorectal cancer, and the receiver operating characteristic (ROC) curve was drawn to analyze the predictive value of preoperative serum HE4 levels in the observation group for postoperative recurrence and metastasis of colorectal cancer.Results:The serum HE4 level in the observation group was significantly higher than that in the control group, and the difference was statistically significant ( P<0.05). The serum HE4 levels in patients with low differentiation were higher than those in patients with medium and high differentiation, while those with lymph node metastasis had higher serum HE4 levels than those without lymph node metastasis (all P<0.05). The postoperative recurrence and metastasis rate of colorectal cancer in patients with low serum HE4 levels was significantly lower than that in patients with high levels ( P<0.05). The COX proportional risk model showed that differentiation degree, lymph node metastasis, and serum HE4 levels were influencing factors for postoperative recurrence and metastasis of colorectal cancer (all P<0.05). The sensitivity and specificity of preoperative serum HE4 levels in predicting postoperative recurrence and metastasis of colorectal cancer were 72.7% and 73.1%, respectively. Conclusions:Serum HE4 is highly expressed in colorectal cancer and is closely related to postoperative recurrence and metastasis of colorectal cancer.

Objective:To investigate the effects of orlistat on the viability of human gall-bladder cancer (GBC) cells.Methods:The experimental study was conducted. The human GBC NOZ cells with high expression of FSAN was screened out through in vitro cultivating human GBC-SD, SGC-996 and NOZ cells. The cell proliferation assay, clone formation assay and protein detection experiment were used to analysis of the effects of orlistat on the viability of human GBC cells. Cell grouping: NOZ cells cultured with medium were set as the control group, cultured with medium + 10 μmol/L orlistat were set as the low-dose orlistat group, cultured with medium + 100 μmol/L orlistat were set as the high-dose orlistat group, respectively. Observation indicators: (1) expression of FASN protein in human GBC cells; (2) effects of orlistat on the proliferation of human GBC NOZ cells; (3) effects of orlistat on apoptosis of human GBC NOZ cells. Measurement data with normal distribution were represented as Mean± SD, the ANOVA test was used for comparison between groups and the least significant difference method was used for pairwise comparison. Results:(1) Expression of FASN protein in human GBC cells. Results of western blot showed that the relative expression of FASN protein in human GBC NOZ, GBC-SD and SGC-996 cells was 0.57±0.06, 0.12±0.04 and 0.10±0.02, respectively, showing a significant difference among them ( F=115.67, P<0.05). There were significant differences between the NOZ cells and the GBC-SD or the SGC-996 cells ( P<0.05), and there was no significant difference between the GBC-SD cells and the SGC-996 cells ( P>0.05). (2) Effects of orlistat on the proliferation of human GBC NOZ cells. ① Results of cell proliferation assay showed that the absorbance value of NOZ cells was 2.34±0.12, 1.57±0.08 and 1.07±0.13 in the control group, low-dose orlistat group and high-dose orlistat group, respectively, showing a significant difference among them ( F=205.88, P<0.05). ② Results of clone formation assay showed that the number of NOZ cells clones was 257±23, 153±11 and 83±11 in the control group, low-dose orlistat group and high-dose orlistat group, respectively, showing a significant difference among them ( F=92.64, P<0.05). ③Results of western blot showed that the relative expression of Cyclin-D1 protein of NOZ cells was 2.31±0.10, 1.52±0.05 and 1.23±0.11 in the control group, low-dose orlistat group and high-dose orlistat group, respectively, showing a significant difference among them ( F=120.73, P<0.05). The relative expression of CDK-4 protein of NOZ cells was 1.58±0.04, 1.21±0.02 and 1.19±0.04 in the control group, low-dose orlistat group and high-dose orlistat group, respectively, showing a signifi-cant difference among them ( F=110.45, P<0.05). (3) Effects of orlistat on apoptosis of human GBC NOZ cells. Results of western blot showed that the relative expression of Bcl-2 protein of NOZ cells was 1.07±0.03, 0.36±0.03 and 0.15±0.02 in the control group, low-dose orlistat group and high-dose orlistat group, respectively, showing a significant difference among them ( F=1 242.93, P<0.05). The relative expression of Bax protein of NOZ cells was 0.51±0.03, 0.38±0.05 and 1.38±0.04 in the control group, low-dose orlistat group and high-dose orlistat group, respectively, showing a signifi-cant difference among them ( F=583.51, P<0.05). Conclusion:Orlistat can inhibit the growth of human GBC NOZ cells and promote their apoptosis.

Cadmium (Cd) is a common heavy metal in the environment. Cd²⁺ may penetrate the blood-brain barrier and produce neurotoxicity, thus inducing various neurodegenerative diseases. Celastrol is an effective component of Tripterygium wilfordii Hook. F., which has many pharmacological effects such as anti-cancer and anti-inflammatory. Here we explored the effect of celastrol on the corresponding neurotoxicity induced by Cd²⁺. Cell proliferation test, cell membrane integrity test, and cell morphology were observed to analyze the effect of Cd²⁺ on the viability of HMC3. The neurotoxicity of Cd²⁺ and the effect of celastrol on the corresponding neurotoxicity induced by Cd²⁺ were analyzed by nitric oxide (NO) test, lipid peroxidation (MDA) test, and Western blotting. When the concentration of Cd²⁺ reached 40 μmol/L, the inhibition rate of HMC3 cell proliferation was (57.17±8.23)% (P < 0.01, n=5), compared with the control group. The cell activity continued to reduce when the Cd²⁺ concentration further increased. When the concentration of Cd²⁺ was higher than 40 μmol/L, the cell membrane of HMC3 was significantly damaged, and the damage was dose-dependent. Upon increasing the Cd²⁺ concentration, the cell morphology began to change and the adhesion also became worse. Cd²⁺ significantly increased the amount of NO released by HMC3 cells, while celastrol effectively inhibited the NO release of HMC3 cells induced by Cd²⁺. Cd²⁺ greatly increased the release of MDA in HMC3 cells, and the level of MDA decreased rapidly upon the addition of 10^-7 mol/L celastrol. Cd²⁺ increased the expression of p-PI3K protein, and the levels of p-PI3K protein and p-AKT protein were inhibited by the addition of celastrol (10^‒7 mol/L, 10^‒6 mol/L), thus preventing cell apoptosis. In conclusion, celastrol inhibits Cd²⁺ induced microglial cytotoxicity and plays a neuroprotective role.
Anti-Inflammatory Agents/pharmacology* ; Apoptosis ; Cadmium/toxicity* ; Nitric Oxide/pharmacology* ; Pentacyclic Triterpenes ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins c-akt/metabolism* ; Triterpenes/pharmacology*

Objective:To investigate the effect of coix seed oil on chemosensitivity of colon cancer cells.Methods:HT29 cell line was cultured in vitro. Different concentrations of coix seed oil (1, 2, 4, 8 mg/ml) and 30 μg/ml 5-fluorouracil (5-FU) were incubated with HT29 cells for 24 hours to simulate chemotherapy. The cell proliferation inhibition rate, apoptosis rate and cell cycle ratio were measured by methyl thiazolyl tetrazolium (MTT) method and flow cytometry, and the protein expression of cleaved caspase-3 was measured by Western blot. Results:The inhibition rate of cell proliferation in 1, 2, 4, 8 mg/ml coix oil + 5-FU group was significantly higher than that in 5-FU group and coix oil group, and the inhibition rate in 2, 4, 8 mg/ml coix oil + 5-FU group was significantly higher than that in 1 mg/ml coix oil + 5-FU group ( P<0.05). The apoptosis rate in 5-FU group, coix oil group and 1, 2, 4, 8 mg/ml coix oil + 5-FU group was higher than that in the blank control group ( P<0.05). The apoptosis rate in 1, 2, 4, 8 mg/ml coix oil + 5-FU group was significantly higher than that in 5-FU group and coix oil group ( P<0.05). The apoptosis rate of 2, 4, 8 mg/ml coix oil + 5-FU group was significantly higher than that of 1 mg/ml coix oil + 5-FU group ( P<0.05). The expression of cleaved caspase-3 in each group was basically in line with the apoptosis rate of flow cytometry. The percentage of G1/M phase cells in 5-FU group, coix oil group and 1, 2, 4, 8 mg/ml coix oil + 5-FU group was significantly higher than that in the blank control, and the percentage of S phase cells was lower comparing with blank control ( P<0.05). Besides, the percentage of G1/M phase cells in 1, 2, 4, 8 mg/ml coix oil + 5-FU group was significantly higher than that in 5-FU group and coix oil group, and the percentage of S phase cells was significantly lower than that in 5-FU group and coix oil group ( P<0.05). The percentage of G1/M phase cells in 2, 4, 8 mg/ml coix oil + 5-FU group was significantly higher than that in 1 mg/ml coix oil + 5-FU group, and the percentage of S phase cells was significantly lower than that in 1 mg/ml coix oil + 5-FU group ( P<0.05). Conclusions:Coix seed oil may enhance the chemosensitivity of colon cancer cells by inducing cell cycle arrest and apoptosis.

Objective:To investigate the changes of porphyrin metabolites in urine of patients with colorectal cancer before and after operation and their correlation with prognosis.Methods:One hundred patients with colorectal cancer were collected in First Affiliated Hospital of Hebei North University from June 2016 to December 2016, urine was collected before operation, 1 week after operation, 1 year after operation and before recurrence. The contents of urinary porphyrin metabolites of uroporphyrinogenI (UP Ⅰ) and coproporphyrinogen Ⅲ(CP Ⅲ) were detected by high performance liquid chromatography. Toanalyse the changes of UPⅠ and CPⅢ levels before and after operaction of colorectal cancer and their correlation with clinicopathologicalcharacteristics,and the recurrence and metastasis after operation.Results:The levels of UPⅠ and CPⅢ in urine of patients with colorectal cancer after operation were significantly lower than those before operation [(66.80 ± 17.62) μmol/g vs. (35.58 ± 9.32) μmol/g, (20.14 ± 3.14) μmol/g vs. (10.38 ± 0.85) μmol/g] ( P<0.05). The levels of UP Ⅰ and CP Ⅲ in urine of patients with Dukes C/D stage were significantly higher than those with Dukes A/B stage [(45.26 ± 5.26) μmol/g vs. (28.56 ± 3.45) μmol/g, (86.57 ± 6.58) μmol/g vs. (52.48 ± 3.36) μmol/g], the levels of UP Ⅰand CPⅢ in urine of patients with lymph node metastasis were significantly higher than those without lymph node metastasis [(45.44 ± 5.46) μmol/g vs. (30.27 ± 6.07) μmol/g, (86.67 ± 6.87) μmol/g vs. (56.10 ± 11.08) μmol/g], there were significant differences ( P<0.05). Urinary levels of UPⅠ and CPⅢ were independent risk factors for recurrence and metastasis of colorectal cancer after operation ( OR=1.149 and 1.065, P<0.05). Conclusions:Porphyrin metabolites (UPⅠ and CPⅢ) in urine may serve as a new marker for assessing colorectal cancer.

Objective:To explore the effect and mechanism of 5-Aminolevulinic Acid-Photodynamic Therapy (5-ALA-PDT) on the apoptosis of the human colonic carcinoma HT-29 cells.Methods:HT-29 cells were cultured in vivio and divided into four groups: blank control group, 5-ALA group, PDT group and 5-ALA-PDT group.The control group was not given photosensitizer and light treatment; 5-ALA group was given photosensitizer ; PDT group was given light treatment; 5-ALA-PDT group was given photosensitizer and light treatment at the same time. Flow cytometry was used to observe the apoptosis of HT-29 cells. Reverse transcription polymerase chain reaction (RT-PCR) was used to observe the expression of B-type lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in HT-29 cells. Ultraviolet spectrophotometry was used to detect the expression of Caspase-3. Results:The apoptotic rate of 5-ALA-PDT group was significantly higher than that of blank control group, 5-ALA group and PDT group ( P<0.05). Compared with the blank control group, 5-ALA-PDT group and PDT group, the expression of Bcl-2 in the 5-ALA-PDT group was statistically significant ( P<0.05), but there was no significant difference in Bax expression among the four groups ( P>0.05). The expression of Bax/Bcl-2 in 5-ALA-PDT group was significantly higher than that in blank control group, 5-ALA group and PDT group ( P<0.05). The expression of Caspase-3 in 5-ALA-PDT group was significantly higher than that in blank control group, 5-ALA group and PDT group ( P<0.05). Conclusions:5-ALA-PDT can induce apoptosis of HT-29 cells, and its mechanism may be related to the induction of apoptosis through Bax/Bcl-2 pathway.

Objective:To investigate the accumulation of porphyrin metabolites [uroporphyrinogen (UP) Ⅰ and coproporphyrinogen (CP) Ⅲ] induced by 5-aminolevulinic acid (5-ALA) in the urine of rats with colorectal cancer.Methods:The rat model of colorectal cancer was established by dimethylhydrazine (DMH). Urine samples were collected from 30 colorectal cancer rats (colorectal cancer group) and 30 normal rats (normal group). Each animal was given 5-ALA (50 mg/kg) by gavage, and urine was collected after 2, 4, 6 and 8 h. The contents of urinary porphyromogen Ⅰ and porphyromogen faecalis Ⅲ in urine were detected by high performance liquid chromatography (HPLC).Results:There was no significant difference in the contents of UP Ⅰ and CP Ⅲ in urine between colorectal cancer group and normal group before oral administration of 5-ALA ( P>0.05). After oral administration of 5-ALA, the contents of UP Ⅰ and CP Ⅲ in urine of colorectal cancer group were significantly higher than those of normal group ( P<0.05). The contents of UP Ⅰ and CP Ⅲ in urine of colorectal cancer group reached the highest value at 4 hours. According to the receiver operating characteristic (ROC) curve drawn from 4-hour test results, the threshold value of UP Ⅰ for colorectal cancer diagnosis was 50.43 μmol/g, with corresponding sensitivity 96.7%, and the specificity 63.3%, respectively. The threshold value of CP Ⅲ for colorectal cancer diagnosis was 108.85 μmol/g, with corresponding sensitivity 66.7%, and the specificity 86.7%, respectively. Conclusions:The accumulation of porphyrin metabolites induced by 5-ALA in the urine of rats with colorectal cancer is significant. The porphyrin metabolites in urine may be a new tumor marker of colorectal cancer, which provides an experimental basis for the diagnosis of colorectal cancer.

Objective: To study the application of urinary 5-aminolevulinic acid (5-ALA)detection in screening and identification of colorectal cancer and adenomatous polyps. Methods: The clinical data of 500 high-risk patients(including 22 cases with colorectal cancer, 134 cases with adenomatous polyps, and 344 cases with other patients) at the First Affiliated Hospital of Hebei North University from January 2018 to October 2018 were collected. High performance liquid chromatography(HPLC) was used to detect urinary 5-ALA and fecal occult blood test was used to detect faeces. Sensitivity and specificity of two methods was compared. At the same time, urine samples of 431 cases(including 22 cases with colorectal cancer, 134 cases with adenomatous polyps and 275 cases with colorectal normal mucosa)were collected, and the difference of the content of urinary 5-ALA among three groups was compared. Results: The sensitivity of urinary 5-ALA for the colorectal cancer screening was74.9%, and the specificity was 72.5%. The sensitivity of urinary 5-ALA for the adenomatous polyps screening was 70.1%, and the specificity was75.0%. The sensitivity of fecal occult blood test for the colorectal cancer screening was 63.6%, and the specificity was 62.1%. The sensitivity of fecal occult blood test for the adenomatous polyps screening was 42.3%, and the specificity was 62.5%. The content of urinary 5-ALA of the colorectal cancer group [(9.35 ± 0.46) μmol/g] was significantly higher than that of the adenomatous polyps group [(7.24 ± 0.64) μmol/g] (P < 0.05) and normal colorectal mucosa group [(3.12 ± 0.24) μmol/g] (P < 0.05), and the content of urinary 5-ALA of the adenomatous polyps group was significantly higher than that of colorectal normal mucosa group (P < 0.05). Conclusions For screening of colorectal cancer and adenomatous polyps, the content of urinary 5-ALA by HPLC is better than fecal occult blood test, and this approach can do great help to identify colorectal cancer, adenomatous polyps and normal colorectal mucosa.

Objective: To investigate the effect of miR-124 on the invasion and migration of esophageal cancer KYSE170 cells by regulating autophagy. Methods: miR-124 mimic was transfected into esophageal cancer KYSE170 cells. Transwell assay was used to detect the change of invasion and migration ability of cells. Dual luciferase reporter gene assay was used to verify the targeted regulation of BECN1 (Beclin1) by miR-124, and Western blotting was used to analyze the expressions of BECN1, P62 and LC3 protein. siRNA targeting BECN1 was transfeted into KYSE170 cells, and then the cell invasion and migration ability was calculated by Transwell assay. The expressions of BECN1, P62 and LC3 protein were detected by Western blotting. miR-124 mimic and BECN1 over-expression plasmid were co-transfected into KYSE170 cells, and then Transwell assay was used to detect the changes of cell invasion and migration ability, and Western blotting to examine the expression levels of autophagy-related gene. Results: The invasion and migration ability of KYSE170 cells were significantly inhibited after transfection with miR-124 mimic (All P<0.05). The expression of autophagyrelated protein P62 was increased, and the expression of BECN1 and LC3 was significantly decreased (All P<0.01); in addition, the activity of luciferase reporter gene was also significantly reduced (P<0.01). Silencing BECN1 expression inhibited the invasion and migration of esophageal cancer KYSE170 cells (P<0.01). However, after co-transfection with BECN1 over-expression plasmids, the effects of miR-124 mimic on the autophagy, invasion and migration of esophageal carcinoma KYSE170 cells were significantly weakened (P<0.01), it was also accompanied with lower P62 expression, and higher LC3 expression (P<0.01). Conclusion: miR-124 mimic can inhibit the invasion and migration of esophageal carcinoma cells. The mechanism may be related to the autophagy-related gene BECN1 expression.

CCCTC-binding factor (CTCF) is a zinc-finger protein, serving an important part in the genome architecture as well as some biochemical processes. Over 70,000 CTCF binding DNA sites have been detected genome-wide, and most anchors of chromatin loops are demarcated with the CTCF binding. Various protein or RNA molecules interact with DNA-bound CTCF to conduct different biological functions, and potentially the interfaces between CTCF and its cofactors can be targets for drug development. Here we identify the effective region of CTCF in DNA recognition, which defines the exposed CTCF surface feature for the interaction of cofactors. While the zinc-finger region contributes the most in DNA association, its binding affinity varies based on different DNA sequences. To investigate the effectiveness of individual zinc-fingers, the key residues are mutated to inactivate the DNA binding ability, while the finger configuration and the spacing between fingers are preserved. The strategy is proved to be successful, while clear differences are observed in the DNA binding affinities among the 11 finger mutants and the result is consistent to previous studies in general. With the help of inactivated finger mutants, we identify the ineffective fingers and the dominant effective fingers, which form distinctive patterns on different DNA targets.