[Optimal melanin removal methods for HE staining, immunohistochemistry and molecular detection].

Objective: To seek the optimal melanin-removal method for hematoxylin and eosin (HE) staining, immunohistochemistry and molecular detection. Methods: Thirty-eight paraffin tissue samples of malignant melanoma diagnosed at the Fujian Cancer Hospital, Fuzhou, China between January 2018 and March 2022 were collected and used to make a tissue microarray. Melanin in these cases was removed using warm hydrogen peroxide, double oxidation depigmentation, modified potassium permanganate-oxalic acid or trichloroisocyanuric acid, followed by HE staining. The cases were divided into two cohorts: one was subject to the one of the above four methods to remove melanin first, followed by immunohistochemistry (SOX-10, Ki-67, HMB45 and Melan A), while the other was subject to immunohistochemical staining first and then a melanin removal. Following that, seventeen melanin-rich paraffin tissue samples were collected and depigmented using the methods described above. DNA extraction was then done, followed by assessments of DNA content and quality. Moreover, the completeness of melanin removal, the effect on HE and immunohistochemical staining, and the quality of DNA were compared between the depigmented methods. Results: Regarding the effectiveness of melanin removal, the modified potassium permanganate-oxalic acid and the warm hydrogen peroxide methods were the most effective, and both showed residual melanin in only 5.26% (2/38) of the cases. The trichloroisocyanuric acid method showed residual melanin in 10.53% (4/38) of the cases. The worst was the double oxidation depigmentation method, which showed pigment residue in 15.79% (6/38) of the cases. For HE staining, the percentage of good staining with the warm hydrogen peroxide method was 92.11%, higher than the other three methods. For immunohistochemical staining, the mean staining scores of immunohistochemistry first followed by melanin removal with modified potassium permanganate-oxalic acid, double oxidation and trichloroisocyanuric acid were 20.84, 26.63 and 35.02, respectively. These immunohistochemical staining scores were higher than those of melanin removal first followed by immunohistochemistry (8.70, 15.41 and 21.22, respectively). The mean staining score of melanin removal by warm hydrogen peroxide method followed by immunohistochemistry was 33.57, superior to that of immunohistochemistry followed by the melanin removal (19.96). Moreover, the staining scores of HMB45, MelanA and Ki-67 with immunohistochemical staining followed by trichloroisocyanuric acid method were 36.45, 33.79, and 36.24, respectively, while the staining score of SOX10 with melanin removal by warm hydrogen peroxide followed by immunohistochemistry was 34.39. The DNA was significantly degraded by modified potassium permanganate-oxalic acid, double oxidation depigmentation and trichloroisocyanuric acid, whereas the mean concentration of DNA extracted after melanin removal by hydrogen peroxide method was 59.59 µg/L, substantially higher than that of DNA extracted without melanin removal (30.3 µg/L, P=0.001). The A260/A280 of DNA extracted after melanin removal by hydrogen peroxide was between 1.8 and 2.0 in all cases, and the A260/A230 was above 2.0 in sixteen cases, suggesting high purity of DNA. However, the DNA extracted without removing the melanin showed poor purity, with A260/A280 below 1.8 in eight cases and A260/A230 below 2.0 in sixteen cases. Conclusions: Warm hydrogen peroxide showed the least melanin residue, superior HE staining and a minimal effect on DNA purity/quality compared to the other three methods. It thus appears most suitable for PCR, NGS and other molecular detection. Melanin removal with trichloroisocyanuric acid after immunohistochemical staining has the least melanin residual, and thus could be the most convenient and efficient. However, it is noted that the efficacy of the same depigmentation method varies with different antibodies. Therefore, the optimal depigmentation method should be selected based on the specific markers of interest.

[A retrospective study of postoperative adjuvant therapy following immunotherapy combined with targeted therapy and sequential curative surgical procedures for initially unresectable hepatocellular carcinoma].

Objective: To report the clinical efficacy of adjuvant therapy based on pathological results following immunotherapy combined with targeted therapy and sequential curative surgical procedures in patients with initially unresectable hepatocellular carcinoma. Methods: This is a retrospective case series study. Data from 100 patients who underwent adjuvant therapy based on pathological results following immunotherapy combined with targeted therapy and sequential curative surgical procedures with long-term survival were collected from December 2018 to December 2022 at the Faculty of Hepato-Pancreato-Biliary Surgery, First Medical Center, Chinese People's Liberation Army General Hospital. According to inclusion and exclusion criteria, 47 cases were included, among which patients who met the discontinuation criteria and maintained a drug-free tumor-free status. Thirty-nine male and eight female patients were included, with an age of (54.2±18.8)years(range:38 to 73 years) at initial diagnosis. At the time of initial diagnosis, 43 cases (91.5%) were classified as Barcelona Clinic Liver Cancer stage C. Survival curves were made using Kaplan Meier method. Results: Forty-seven patients underwent R0 resection, all achieved a drug-free tumor-free state through postoperative adjuvant therapy based on pathological examination results. Thirty-six patients(76.6%) maintained a drug-free tumor-free survival status for more than 6 months,28 patients(59.6%) for more than 12 months,and 8 patients(17.0%) for more than 24 months. The longest drug-free tumor-free survival in this cohort reached 48 months. The median follow-up time in this study was 32 months. After diagnosis, the overall survival rates at 1- and 3- years were 97.7%(95%CI:93.4% to 100%) and 90.7%(95%CI:82.5% to 99.8%). The postoperative recurrence-free survival rates at 1- and 3- years were 91.0%(95%CI:83.0% to 99.8%) and 71.3%(95%CI:58.7% to 86.5%). Conclusions: The adjuvant therapy based on pathological results following immunotherapy combined with targeted therapy and sequential curative surgical approach provides long-term survival benefits for patients with initially unresectable hepatocellular carcinoma. Standardized adjuvant therapy maybe sustain long-term tumor-free status,and achieve drug-free tumor-free survival.

[Expression of cation chloride cotransporter (NKCC1/KCC2) in brain tissue of children with focal cortical dysplasia type â ¡].

Objective: To investigate the expression of cation chloride cotransporter (NKCC1/KCC2) in the neurons from cerebral lesions of children with focal cortical dysplasia (FCD) type Ⅱ, to provide a morphological basis for revealing the possible mechanism of epilepsy. Methods: Eight cases of FCD type Ⅱ diagnosed at Beijing Haidian Hospital, Beijing, China and 12 cases diagnosed at Xuanwu Hospital, Capital Medical University, Beijing, China from February 2017 to December 2019 were included. The expression of NKCC1 and KCC2 in FCD type Ⅱa and FCD type Ⅱb was detected using immunohistochemistry and double immunohistochemical stains. The average optical density of NKCC1 in dysmorphic neurons and normal neurons was also determined using immunohistochemical staining in FCD type Ⅱa (10 cases). Results: The patients were all younger than 14 years of age. Ten cases were classified as FCD type IIa, and 10 cases as FCD type Ⅱb. NKCC1 was expressed in the cytoplasm of normal cerebral cortex neurons and KCC2 expressed on cell membranes. In dysmorphic neurons of FCD type Ⅱa, expression of NKCC1 increased, which was statistically higher than that of normal neurons (P<0.01). Aberrant expression of KCC2 in dysmorphic neurons was also noted in the cytoplasm. In the FCD Ⅱb type, the expression pattern of NKCC1/KCC2 in dysmorphic neurons was the same as that of FCD type Ⅱa. The aberrant expression of NKCC1 in balloon cells was negative or weakly positive on the cell membrane, while the aberrant expression of KCC2 was absent. Conclusions: The expression pattern of NKCC1/KCC2 in dysmorphic neurons and balloon cells is completely different from that of normal neurons. The NKCC1/KCC2 protein-expression changes may affect the transmembrane chloride flow of neurons, modify the effect of inhibitory neurotransmitters γ-aminobutyric acid and increase neuronal excitability. These effects may be related to the occurrence of clinical epileptic symptoms.

[Clinical efficacy of transjugular intrahepatic portosystemic shunt for gastrointestinal hemorrhage in patients with idiopathic noncirrhotic portal hypertension].

Objective: To explore the medium-long term efficacy of transjugular intrahepatic portosystemic shunt (TIPS) for gastrointestinal hemorrhage in patients with idiopathic non-cirrhotic portal hypertension (INCPH). Methods: From March 2013 to July 2018, clinical data of 13 INCPH patients, including 5 males, 8 females,with gastrointestinal hemorrhage were retrospectively analyzed, who were diagnosed at the First Affiliated Hospital of Zhengzhou University, Anyang Fifth People' s Hospital and Yuncheng Central Hospital. All patients received TIPS treatment. The general information, postoperative survival rate, the incidence of rebleeding, shunt dysfunction rate, and incidence of hepatic encephalopathy were analyzed. Results: All 13 patients with INCPH completed TIPS successfully with an average age of 45±8 (33 to 59) years. The hepatic venous pressure gradient (HVPG) decreased from 20.0-26.0 (22.6±1.9) mmHg before procedure to 8.0-14.0 (9.4±3.2) mmHg after. The median follow-up time was 44±7 (31 to 53) months. One patient died of liver failure 27 months after TIPS. Hepatic encephalopathy occurred cumulatively in 1 case (1/13), 1 case (1/13) and 1 case (1/13) in 12, 24 and 36 months after TIPS. Stent restenosis occurred cumulatively in 2 cases (2/13), 3 cases (3/13) and 3 cases (3/13) in 12, 24 and 36 months after TIPS. Portal vein thrombosis occurred cumulatively in 2 cases (2/13), and no primary liver cancer developed. Conclusions: TIPS is safe and effective in the treatment of INCPH with gastrointestinal bleeding with favorable medium-long term outcome.

Propofol ameliorates acute postoperative fatigue and promotes glucagon-regulated hepatic gluconeogenesis by activating CREB/PGC-1α and accelerating fatty acids beta-oxidation.

Postoperative fatigue (POF) is the most common and long-lasting complication after surgery, which brings heavy burden to individuals and society. Recently, hastening postoperative recovery receives increasing attention, but unfortunately, the mechanisms underlying POF remain unclear. Propofol is a wildly used general anesthetic in clinic, and inspired by the rapid antidepressant effects induced by ketamine at non-anesthetic dose, the present study was undertaken to investigate the anti-fatigue effects and underlying mechanisms of propofol at a non-anesthetic dose in 70% hepatectomy induced POF model in rats. We first showed here that single administration of propofol at 0.1 mg/kg ameliorated acute POF in hepatectomy induced POF rats. Based on metabonomics analysis, we hypothesized that propofol exerted anti-fatigue activity in POF rats by facilitating free fatty acid (FFA) oxidation and gluconeogenesis. We further confirmed that propofol restored the deficit in FFA oxidation and gluconeogenesis in POF rats, as evidenced by the elevated FFA utilization, acetyl coenzyme A content, pyruvic acid content, phosphoenolpyruvic acid content, hepatic glucose output and glycogen storage. Moreover, propofol stimulated glucagon secretion and up-regulated expression of cAMP-response element binding protein (CREB), phosphorylated CREB, peroxlsome prolifeator-activated receptor-γ coactivator-1α (PGC-1α), phosphoenolpyruvate carboxykinade1 and carnitine palmitoltransferase 1A. In summary, our study suggests for the first time that propofol ameliorates acute POF by promoting glucagon-regulated gluconeogenesis via CREB/PGC-1α signaling and accelerating FFA beta-oxidation.

CircPRKCI regulates proliferation, migration and cycle of lung adenocarcinoma cells by targeting miR-219a-5p-regulated CAMK1D.

OBJECTIVE: Circular ribonucleic acids (circRNAs) are considered as the key regulatory factors for human malignancies in recent years, and lung adenocarcinoma (LUAD) is a common malignancy worldwide, but the molecular mechanism of circRNAs in LUAD has not been completely investigated. Therefore, the mechanism by which circRNA protein kinase C iota (circPRKCI) regulates LUAD cell migration proliferation, and cycle was preliminarily explored in this research, so as to provide new ideas for the treatment of LUAD. PATIENTS AND METHODS: First of all, the circPRKCI expression level in LUAD tissues was tested via quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay, and the relationship between circPRKCI and the patients' prognosis was analyzed. Then, circPRKCI expression was inhibited by small interfering RNA (siRNA), and the influence of circPRKCI on t LUAD cells' ability to proliferate was verified via 5-ethynyl-2'-deoxyuridine (EdU) and cell counting kit-8 (CCK-8) assays. Moreover, the influence of circPRKCI on LUAD cells' ability to migrate was testified by transwell assay, and the regulation of LUAD cell cycle by circPRKCI was confirmed by flow cytometry. The micro RNAs (miRNAs) with binding sites to the 3' untranslated region (UTR) of circPRKCI and the genes binding to miRNAs were discovered using bioinformatics websites, and their associative relation was further explored through Dual-Luciferase reporter gene assay, qRT-PCR assay, Pearson correlation analysis and reverse experiment. RESULTS: It was verified via qRT-PCR assay that circPRKCI was expressed at a remarkably higher level in LUAD tissues relative to that in paracancerous normal tissues. The highly expressed circPRKCI led to poor prognosis of patients. Besides, qRT-PCR assessment results indicated that circPRKCI expression level rose notably in LUAD cell lines, while it was lowered markedly in LUAD cells transfected with si-circPRKCI. According to CCK-8 and EdU assay results, the proliferative ability of LUAD cells was weakened clearly after knocking down circPRKCI. It was manifested in the results of transwell assay that the knockdown of circPRKCI significantly repressed the capacity of LUAD cells to migrate. Furthermore, the results of cell cycle test displayed that inhibiting circPRKCI could induce the arrest of LUAD cell cycle in the G1 phase. It was discovered through bioinformatics websites that miR-219a-5p had binding sites to circPRKCI 3'UTR, and the results of Dual-Luciferase reporter gene assay revealed that circPRKCI was able to bind to miR-219a-5p. It was uncovered by the qRT-PCR assay results that miR-219a-5p was lowly expressed in LUAD tissues, and its relative expression had an inverse relation with that of circPRKCI according to the Pearson correlation analysis. In addition, it was shown in the results of reverse experiment that miR-219a-5p could regulate the influence of circPRKCI on the malignant phenotype of LUAD. It was found by means of bioinformatics websites that calcium/calmodulin dependent protein kinase ID (CAMK1D) was a downstream target gene of miR-219a-5p and could the two conjugated with each other based on the results of Dual-Luciferase reporter gene assay. Moreover, qRT-PCR assay findings illustrated that CAMK1D was evidently highly expressed in LUAD tissues, and the results of Pearson correlation analysis revealed that CAMK1D expression exhibited a negative association with that of miR-219a-5p and a positive correlation with that of circPRKCI. CONCLUSIONS: CircPRKCI is significantly highly expressed in LUAD, and the highly expressed circPRKCI is capable of facilitating LUAD cell migration, proliferation and cycle. CircPRKCI may regulate the malignant phenotype of LUAD via the miR-219a-5p/CAMK1D axis.

Linc00601 upregulation promotes hepatocellular carcinoma development by activating MAPK signaling pathway.

OBJECTIVE: To study the expression of linc00601 in hepatocellular carcinoma (HCC) tissues and cells, and to study the biological function and downstream mechanism of linc00601 in HCC using in vitro experiments. PATIENTS AND METHODS: The expression of linc00601 in HCC was predicted via bioinformatics, and the expression of linc00601 in HCC tissues and cells was detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). After interference with the expression of linc00601, the interference efficiency was determined using qRT-PCR, and the changes in HCC cell proliferation, cycle distribution, and apoptosis were determined through Cell Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. Finally, the expressions of molecular markers in downstream signaling pathway were determined through Western blotting. RESULTS: It was found via bioinformatics that the expression of linc00601 was upregulated in HCC. The results of qRT-PCR revealed that the expression of linc00601 was upregulated in 36 cases of HCC tissues compared with that in para-carcinoma tissues, and it was also upregulated in HCC cells. According to the results of CCK-8 assay, HCC cell proliferation was inhibited after interference with the expression of linc00601. In the si-linc00601 group, the apoptosis rate rose, and the cell cycle was arrested at the G1/G0 phase compared with those in the si-NC group. The results of Western blotting revealed that after the knockdown of linc00601 in HCC cells, the expressions of molecular markers (p-P38, p-ERK) in the downstream mitogen-activated protein kinase (MAPK) signaling pathway were downregulated. CONCLUSIONS: Linc00601 is upregulated in HCC, which promotes the development of HCC via activating the MAPK signaling pathway.

Dexmedetomidine protects against lidocaine-induced neurotoxicity through SIRT1 downregulation-mediated activation of FOXO3a.

Lidocaine, a typical local anesthetic, has been shown to directly induce neurotoxicity in clinical settings. Dexmedetomidine (DEX) is an alpha-2-adrenoreceptor agonist that has been used as anxiolytic, sedative, and analgesic agent which has recently found to protect against lidocaine-induced neurotoxicity. Nicotinamide adenine dinucleotide-dependent deacetylase sirtuin-1 (SIRT1)/forkhead box O3 (FOXO3a) signaling is critical for maintaining neuronal function and regulation of the apoptotic pathway. In the present study, we designed in vitro and in vivo models to investigate the potential effects of lidocaine and DEX on SIRT1 and FOXO3a and to verify whether SIRT1/FOXO3a-mediated regulation of apoptosis is involved in DEX-induced neuroprotective effects against lidocaine. We found that in both PC12 cells and brains of mice, lidocaine decreased SIRT1 level through promoting the degradation of SIRT1 protein. Lidocaine also increased FOXO3a protein level and increased the acetylation of SIRT1 through inhibiting SIRT1. Upregulation of SIRT1 or downregulation of FOXO3a significantly inhibited lidocaine-induced changes in both cell viability and apoptosis. DEX significantly inhibited the lidocaine-induced decrease of SIRT1 protein level and increase of FOXO3a protein level and acetylation of FOXO3a. Downregulation of SIRT1 or upregulation of FOXO3a suppressed DEX-induced neuroprotective effects against lidocaine. The data suggest that SIRT1/FOXO3a is a potential novel target for alleviating lidocaine-induced neurotoxicity and provide more theoretical support for the use of DEX as an effective adjunct to alleviate chronic neurotoxicity induced by lidocaine.

[Etiology and epidemiological characteristics of gastroenteritis virus in food-borne diarrhea from three cities in Shandong Province, 2017].

Objective: To analyze the etiology and epidemiological characteristics of gastroenteritis virus in foodborne diseases from three cities in Shandong. Methods: From January to December 2017, six sentinel hospitals in Jinan, Yantai and Linyi city of Shandong Province were selected as the research sites. Stool samples of 1 397 diarrhea patients were collected, as well as basic information and clinical symptoms. Duplex quantitative RT-PCR was used to detect Norovirus genogroupⅠ (Nov GⅠ) and genogroupⅡ (Nov GⅡ), Sapovirus (SAV) and Human astrovirus (HAstV), respectively, quantitative RT-PCR was used to detect group A Rotavirus (RVA), and quantitative PCR was used to detect Enteric adenovirus (EAdV). The specific gene of the virus were sequenced and typed. It was compared that the gastroenteritis virus rate in cases with different characteristics and the clinical symptoms difference between the virus positive and negative cases. Results: The median age (P(25), P(75)) was 23 (1, 42) , mainly male, 57.48% with 803 cased and children under 5 years old, 36.36% with 508 cases. The positive rate of gastroenteritis virus was 33.93% (474 cases), and that of Jinan, Linyi and Yantai City were 32.03% (147/459), 41.54% (189/455) and 28.57% (138/483), respectively (P<0.001). Nov GⅡ had the highest positive rate, 16.54% (231 cases), which, mainly GⅡ.P16/GⅡ.2 (48.28%, 56/116), peaked in May (24.75%, 50/202) and June (19.59%, 38/194). In patients of gastroenteritis virus positive, 44.51% (211/474) had vomiting symptoms, higher than that of patients of gastroenteritis virus negative (34.13%, 315/923). The difference was statistically significant (P<0.001). Conclusion: In Shandong Province, the majority of gastroenteritis patients were male and children under 5 years old. Nov GⅡ possessed highest epidemic intensity, and peaked in spring and summer. Viral gastroenteritis had atypical clinical symptoms.

[Amplitude of low-frequency fluctuations of resting-state functional MRI in colorectal cancer patients with depression].

Objective: To investigate the alterations of the cerebral resting-state spontaneous neural activity in colorectal cancer patients with depressive symptoms. Methods: Thirty-three colorectal cancer patients (patient group) with depression and 43 healthy subjects (control group) underwent the resting state functional magnetic resonance imaging (rs-fMRI). The amplitude of low-frequency fluctuations (ALFF) and fractional ALFF (fALFF) were calculated. Two independent samples t test were used to compare the ALFF and fALFF values between two groups by DPABI software, and then correlation analysis was performed between ALFF and fALFF with statistical significance and Patient Health Questionnaire (PHQ-9) scores and Generalized Anxiety Disorder (GAD-7) scores. Results: Compared with the control group, the patient group showed significantly lower ALFF and fALFF values in the bilateral precuneus, calcarine gyrus, lingual gyrus, left cuneus, superior, middle, inferior occipital gyrus and right fusiform gyrus (t=-5.730, P<0.05; t=-4.872, P<0.05). There were no significant correlations between the ALFF and fALFF values in these regions and PHQ-9 or GAD-7 scores (P>0.05). Conclusion: Spontaneous decrease of neural activity in occipital and parietal lobes exists in colorectal cancer patients with depression at resting-sate, which may be a potential neurobiological marker.

[Expression of autophagy-related proteins in cortical nodules of tuberous sclerosis complex].

Objective: To investigate the expression of LC3B, p-AMPKα and p27 in cortical tuberous sclerosis complex (TSC). Methods: Nineteen specimens of surgically resected TSC cortical tubers were collected at Xuanwu Hospital, Capital Medical University, from 2014 to 2017. The expression of the three proteins in the lesions and the adjacent relatively normal regions was detected by immunohistochemical staining (EnVision two-step method). Results: LC3B was mainly expressed in the dysmorphic neuron and giant cell in TSC cortical tubers and in the adjacent relatively normal neurons, and the expression was diffuse or perinuclear cytoplasmic. There was no significant difference in the average optical density between abnormal cells and neurons adjacent to the lesions (0.343±0.195 vs. 0.419±0.088, P>0.05). p-AMPKα was localized in the cytoplasm of dysmorphic neurons and giant cell in TSC cortical tubers. The average optical density of abnormal cells in the lesions was significantly higher than that of neurons adjacent to the lesions (0.306±0.123 vs. 0.233±0.654, P<0.05). P27 showed nuclear positivity, mainly expressed in the neurons and glial cells close to TSC cortical tubers, while the positive rate in the abnormal cells in TSC cortical tubers was low (15/19 vs. 7/19, P<0.05). Conclusion: There is no significant decrease in the level of autophagy in dysmorphic neurons and giant cells in TSC cortical tubers, which may be related to the compensatory mechanism of AMPK signaling pathway, but without activation of downstream p27.

Downregulation of lncRNA MEG3 attenuates high glucose-induced cardiomyocytes injury by inhibiting mitochondria-mediated apoptosis pathway.

OBJECTIVE: The aim of the present work was to investigate the effects and mechanisms of long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3) in cardiomyocytes injury and apoptosis induced by high glucose (HG) in vitro. MATERIALS AND METHODS: HG-induced rats' cardiomyocytes with si-MEG3 transfection were constructed. Cell viability and lactate dehydrogenase (LDH) level were examined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and LDH assay kits, respectively. Cardiomyocytes apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining. The expressions of Bcl-2, Bax, cleaved caspase-9 and cleaved caspase-3 proteins were determined by Western blot. The expression of lncRNA MEG3 was measured by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). RESULTS: Our results indicated that the expression of MEG3 was significantly upregulated in HG-treated cardiomyocytes. The downregulation of MEG3 could attenuate cardiomyocytes injury and apoptosis by decreasing the Bax/Bcl-2 ratio, cleaved caspase-9 and cleaved caspase-3 expression. CONCLUSIONS: The downregulation of MEG3 could attenuate cardiomyocytes injury and apoptosis induced by HG. The molecular mechanism was associated with the inhibition of the mitochondria-mediated apoptosis pathway.

TRIM66 promotes malignant progression of hepatocellular carcinoma by inhibiting E-cadherin expression through the EMT pathway.

OBJECTIVE: The aim of this study was to explore the regulatory role of TRIM66 in the development of hepatocellular carcinoma (HCC), and to investigate its underlying mechanism. PATIENTS AND METHODS: A total of 88 pairs of HCC tissues and para-cancerous tissues were surgically resected. The expression of TRIM66 was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The correlation between TRIM66 expression and clinic-pathologic characteristics of HCC patients was analyzed. Follow-up data of enrolled HCC patients were collected for survival analysis. Subsequently, TRIM66 expression in HCC cells was determined by qRT-PCR as well. By constructing si-TRIM66, the biological performances of transfected HCC cells were determined using cell counting kit-8 (CCK-8), colony formation and transwell assay. Western blot was performed to measure the protein expressions of relative genes in epithelial-mesenchymal transition (EMT) pathway. Finally, HCC cells were co-transfected with si-TRIM66 and pcDNA-E-cadherin, followed by detection of invasive and migratory abilities. RESULTS: TRIM66 was highly expressed in HCC tissues compared with that of para-cancerous tissues. High expression of TRIM66 was positively correlated with tumor stage, lymph node metastasis and distant metastasis, whereas not correlated with age and sex of HCC patients. Kaplan-Meier curves revealed that a higher expression of TRIM66 was associated with worse prognosis of HCC. Similarly, TRIM66 was also highly expressed in HCC cells. The knockdown of TRIM66 in HCC cells significantly inhibited the proliferative, invasive and migratory abilities of transfected cells. However, TRIM66 down-regulation significantly induced cell apoptosis. Western blot results showed that TRIM66 knockdown in HCC cells markedly downregulated the protein expressions of E-cadherin, N-cadherin, Vimentin and ß-catenin. The inhibited migration and invasion of HCC cells resulted from TRIM66 knockdown were partially reversed by E-cadherin overexpression. CONCLUSIONS: TRIM66 is highly expressed in HCC, which is positively correlated with tumor stage, lymph node metastasis and distant metastasis of HCC patients. In addition, TRIM66 promotes the malignant progression of HCC by inhibiting E-cadherin through the EMT pathway.

LncRNA 00152 promotes the development of hepatocellular carcinoma by activating JAK2/STAT3 pathway.

OBJECTIVE: The aim of this study was to explore the regulatory role of lncRNA 00152 and JAK2/STAT3 pathway in the pathogenesis of hepatocellular carcinoma (HCC), and to investigate the possible underlying mechanism. PATIENTS AND METHODS: Expression levels of lncRNA 00152 in HCC tissues, matched para-cancerous tissues and normal liver tissues were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), respectively. The correlation between lncRNA 00152 expression and pathological characteristics of HCC patients was analyzed. Meanwhile, the expression level of lncRNA 00152 in HCC cell lines was detected by qRT-PCR. After knockdown or overexpression of lncRNA 00152 in MHCC97 or HB611 cells, the proliferative ability and cell cycle were detected by EdU assay and flow cytometry, respectively. Also, Western blot was conducted to detect the protein expression levels of JAK2 and STAT3 in MHCC97 and HB611 cells. RESULTS: The expression of lncRNA 00152 in HCC tissues was significantly higher than that of matched para-cancerous tissues and normal liver tissues. LncRNA 00152 expression was positively correlated with tumor stage and tumor size, whereas negatively correlated with the overall survival of HCC patients. High expression of lncRNA 00152 might be a potential hallmark for the diagnosis of HCC, with the AUC of 0.8425. Similarly, lncRNA 00152 was highly expressed in HCC cell lines when compared with that of normal liver cells. Knockdown of lncRNA 00152 in MHCC97 cells remarkably decreased the proliferative ability and arrested cell cycle. Overexpression of lncRNA 00152 in HB611 cells significantly promoted cell proliferation and cell cycle. Furthermore, Western blot results showed that lncRNA 00152 knockdown upregulated the protein expression levels of JAK2 and STAT3 in HCC cells. CONCLUSIONS: High expression of lncRNA 00152 promotes the development of HCC by activating the JAK2/STAT3 pathway.

Integrative diagnosis of cancer by combining CTCs and associated peripheral blood cells in liquid biopsy.

Circulating tumor cells (CTCs), as cells shed from solid tumor into the vasculature, play a significant role in tumor metastasis. In the peripheral blood, immune cells and stromal cells can interact with CTCs and influence their biological behaviors of survival, proliferation, dissemination, and immune evasion. These peripheral blood cells can evolve synergistically with CTCs to constitute the liquid microenvironment which is essential for tumor progression. Here, we review the mechanisms of peripheral blood cells interacting with CTCs and uncover their effects on both CTCs and tumor metastasis. Then, we introduce the applications of these CTC-associated peripheral blood cells in the clinical setting. Besides, some peripheral blood cell subsets are of additional clinical values to CTCs in cancer diagnosis and prognosis. To improve the clinical utility of CTCs, an integrative analysis of CTCs and associated peripheral blood cells should be advocated for, which could provide a novel insight into tumor biology and offer comprehensive information in cancer diagnosis, prognosis, and therapy efficacy evaluation.

Effect of ZEB2 silencing on cisplatin resistance in gastric cancer.

OBJECTIVE: To investigate the effect of ZEB2 silencing on cisplatin resistance in gastric cancer. MATERIALS AND METHODS: The resulting cell line, SGC7901/DDP, was transfected with ZEB2 siRNA, non-specific siRNA, or vehicle control. The effectiveness of ZEB2 silencing was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. MTT viability assay was used to determine the cisplatin-sensitivity of cells. Cell apoptosis was measured by flow cytometry. RESULTS: A significant decrease in ZEB2 in mRNA and protein level was seen in cells transfected with ZEB2 siRNA, compared to that in cells transfected with non-specific siRNA or vehicle. Transfection with ZEB2 siRNA in cisplatin-resistant SGC7901/DDP cells resulted in a significant decrease in cell viability in response to the cisplatin treatment, and cell viability decreased with increasing cisplatin concentrations. A higher apoptotic rate was also seen in cells transfected with ZEB2 siRNA under cisplatin treatment. CONCLUSIONS: ZEB2 silencing can effectively make gastric cells sensitive to cisplatin treatment in vitro.

[The change of female progesterone level and blood calcium concentration in perimenopausal women with benign paroxysmal positional vertigo].

Objective: The estrogen level and blood calcium concentration changes were studied on menopausal women with benign paroxysmal positional vertigo (BPPV). Methods: Between January 2015 and January 2016, 70 menopause women with BPPV in outpatient clinics of Department of Otorhinolaryngology, Inner Mongolia Medical University Affiliated Hospital were included in this study as research group, while 30 menopause healthy women who came to hospital for check-up were included as control group. Serum levels of estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH), progesterone (PRO), testosterone (T), serum prolactin (PRL) and the calcium concentration were analysed and comparied between research group and control group. SPSS17.0 statistical software was used to analyze the data. χ(2) test was used to compare the percentage of decreased serum level of sex hormone, and t test was used to compare the serum level of sex hormone and calcium concentration of two groups. Results: In research group, sex hormone decreased proportion of E2 (91%) and PRO (67%) were obviously higher than those in control group (χ(2) value was 8.13, 10.28, respectively, all P<0.05). The E2 and PRO in research group were significantly lower than those in control group ((33.18±31.45) pmol/L vs (64.92 ±31.52) pmol/L, (0.64±0.48) nmol/L vs (1.02±0.60) nmol/L, t value was 6.238, 8.566, respectively, all P<0.05). There was no statistically significant difference of the level of LH, PRL, T, FSH and blood calcium concentration in research group compared with control group ((29.81±13.13) U/L vs (27.21±10.19) U/L, (0.49±0.20) nmol/L vs (0.49±0.15) nmol/L, (0.56±0.42) nmol/L vs (0.73±0.62) nmol/L, (64.25±31.44) U/L vs (60.38±29.97) U/L, (2.28±0.17) mmol/L vs (2.32±0.21) mmol/L, t value was 13.427, 14.876, 7.505, 12.090, 7.532, respectively, all P>0.05). Conclusion: The level of E2 and PRO decrease obviously in postmenopausal women with BPPV, which can cause the inner ear microcirculation disorder , may be one of the risk factors of BPPV.

[Study on apoptosis mechanism of H9c2 cardiomyocytes induced by N, N-dimethylformamide].

Objective: To observe the change levels of nuclear factor-kappa B (NF-κB) p65 protein in cytoplasm and nuclear, phosphorylation of inhibitor of kappa B (p-IκB) protein and cytochrome C (Cyt-c) , cleaved cysteinyl aspartate specific proteinase-3 (Cleaved caspase-3) , B-cell lymphoma/leukemia-2 (Bcl-2) in cytoplasm in the process of N, N-dimethylformamide (DMF) -induced apoptosis in H9c2 cardiomyocytes, and explore the tentative mechanism of apoptosis. Methods: H9c2 cardiomyocytes were exposed to 200 mmol/L DMF. Western blotting was used to detect the protein expression levels of p65 in cytoplasm and nuclear, p-IκB after exposure for 0, 2, 4, 6, 8, 12 h, and the protein expression levels of Cyt-c, Cleaved caspase-3, Bcl-2 in cytoplasm after exposure for 0, 2, 4, 8, 12, 24 h. Immunofluorescencecytochemistry (IFC) was used to observe the location of Cyt-c after 200 mmol/L DMF exposure for different times. Results: The levels of p65 in cytoplasm and nuclear and p-IκB among groups were statistically significant (F were 7.79, 33.11, 90.25, respectively, all P<0.01) . Compared with the control group, the levels of p65 in cytoplasm of 2, 4, 6 h group were significantly decreased (all P<0.01) ; the levels of p65 in nuclear of 2, 4, 6, 8 h were significantly increased (all P<0.01) ; the levels of p-IκB of 2, 4, 6 h group were significantly increased (all P<0.01) . The levels of Cyt-c, Cleaved caspase-3 and Bcl-2 among groups were statistically significant (F were 51.42, 503.68, 73.37, respectively, all P<0.01) . Compared with the control group, the levels of Cyt-c of 8, 12 h group were significantly increased (both P<0.01) ; the levels of Cleaved caspase-3 of 2, 4, 8, 12, 24 h were significantly increased (all P<0.01) ; the levels of Bcl-2 of 2, 4, 8, 12, 24 h group were significantly decreased (all P<0.01) . IFC showed that Cyt-c was released from the mitochondria to the cytoplasm gradually as the extension of the exposure time. Conclusion: NF-κB signaling pathway and mitochondrial pathway are involved in the mechanism of DMF-induced apoptosis in H9c2 cardiomyocytes.