OBJECTIVE: To explore the potential mechanism of resistance to axitinib in clear cell renal cell carcinoma (ccRCC), with a view to expanding the understanding of axitinib resistance, facilitating the design of more specific treatment options, and improving the treatment effectiveness and survival prognosis of patients. METHODS: By exploring the half maximum inhibitory concentration (IC₅₀) of axitinib on ccRCC cell lines 786-O and Caki-1, cell lines resistant to axitinib were constructed by repeatedly stimulated with axitinib at this concentration for 30 cycles in vitro. Cell lines that were not treated by axitinib were sensitive cell lines. The phenotypic differences of cell proliferation and apoptosis levels between drug resistant and sensitive lines were tested. Genes that might be involved in the drug resistance process were screened from the differentially expressed genes that were co-upregulated in the two drug resistant lines by transcriptome sequencing. The expression level of the target gene in the drug resistant lines was verified by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot (WB). The expression differences of the target gene in ccRCC tumor tissues and adjacent tissues were analyzed in the Gene Expression Profiling Interactive Analysis (GEPIA) public database, and the impact of the target gene on the prognosis of ccRCC patients was analyzed in the Kaplan-Meier Plotter (K-M Plotter) database. After knocking down the target gene in the drug resistant lines using RNA interference by lentivirus vector, the phenotypic differences of the cell lines were tested again. WB was used to detect the levels of apoptosis-related proteins in the different treated cell lines to find molecular pathways that might lead to drug resistance. RESULTS: Cell lines 786-O-R and Caki-1-R resistant to axitinib were successfully constructed in vitro, and their IC₅₀ were significantly higher than those of the sensitive cell lines (10.99 μmol/L, P < 0.01; 11.96 μmol/L, P < 0.01, respectively). Cell counting kit-8 (CCK-8) assay, colony formation, and 5-ethynyl-2 '-deoxyuridine (EdU) assay showed that compared with the sensitive lines, the proliferative ability of the resistant lines decreased, but apoptosis staining showed a significant decrease in the level of cell apoptosis of the resistant lines (P < 0.01). Although resistant to axitinib, the resistant lines had no obvious new replicated cells in the environment of 20 μmol/L axitinib. Nuclear protein 1 (NUPR1) gene was screened by transcriptome sequencing, and its RNA (P < 0.0001) and protein expression levels significantly increased in the resistant lines. Database analysis showed that NUPR1 was significantly overexpressed in ccRCC tumor tissue (P < 0.05); the ccRCC patients with higher expression ofNUPR1had a worse survival prognosis (P < 0.001). Apoptosis staining results showed that knockdown ofNUPR1inhibited the anti-apoptotic ability of the resistant lines to axitinib (786-O, P < 0.01; Caki-1, P < 0.05). WB results showed that knocking downNUPR1decreased the protein level of B-cell lymphoma-2 (BCL2), increased the protein level of BCL2-associated X protein (BAX), decreased the protein level of pro-caspase3, and increased the level of cleaved-caspase3 in the resistant lines after being treated with axitinib. CONCLUSION ccRCC cell lines reduce apoptosis through theNUPR1 -BAX/ BCL2 -caspase3 pathway, which is involved in the process of resistance to axitinib.
Humans ; Carcinoma, Renal Cell/metabolism* ; Axitinib/pharmacology* ; Kidney Neoplasms/metabolism* ; bcl-2-Associated X Protein ; Nuclear Proteins ; Cell Line, Tumor ; Apoptosis ; Cell Proliferation

Humans ; COVID-19 ; Consensus ; SARS-CoV-2 ; China

Objective: To investigate the clinicopathological features and differential diagnosis of NTRK3 gene rearrangement thyroid papillary carcinoma (PTC). Methods: The PTC cases without BRAF V600E mutation were collected at Fujian Provincial Hospital South Branch from January 2015 to January 2020. The cases of NTRK3 gene rearrangement PTC were examined using immunohistochemistry and fluorescence in situ hybridization (FISH). The clinical data, histopathological characteristics, immunohistochemical features and molecular pathological changes were retrospectively analyzed. Data from the TCGA PTC dataset and the literature were also studied. Results: A total of 3 PTC cases harboring NTRK3 gene rearrangement were confirmed. All the patients were female, aged from 26,49,34 years. Histologically, two of them demonstrated a multinodular growth pattern. Only one case showed prominent follicular growth pattern; the other two tumors showed a mixture of follicular, papillary and solid growth patterns. All tumors showed a typical PTC nuclear manifestation, with some nuclear pleomorphism, vacuolated foci and oncocytic features. The characteristic formation of glomeruloid follicular foci was present in two cases which also showed psammoma bodies, and tumoral capsular or angiolymphatic invasion. The background thyroid parenchyma showed chronic lymphocytic thyroiditis. Mitotic rates were low, and no cases had any tumor necrosis. The pan-TRK and TTF1 testing was both positive in 3 cases, while S-100 and mammaglobin were both negative in them. FISH studies confirmed the NTRK3 gene rearrangement in all 3 cases. Studies on the TCGA datasets and literature revealed similar findings. Conclusions: NTRK3 gene rearrangement PTC is rare. It may be easily misdiagnosed due to the lack of histological and clinicopathological characteristics. Molecular studies such as pan-TRK immunostaining, FISH and even next-generation sequencing are needed to confirm the diagnosis. Immunohistochemistry of pan-TRK performed in the PTC cases without BRAF V600E mutation can be used as a good rapid-screening tool. With the emergence of pan-cancer tyrosine receptor kinase inhibitors, proper diagnosis of these tumors can help determine appropriate treatments and improve their outcomes.
Biomarkers, Tumor ; Female ; Gene Rearrangement ; Humans ; In Situ Hybridization, Fluorescence ; Mutation ; Proto-Oncogene Proteins B-raf/genetics* ; Receptor, trkC ; Retrospective Studies ; Thyroid Cancer, Papillary/genetics* ; Thyroid Neoplasms/genetics*

Humans ; Encephalitis

Objective:To explore the effects and mechanism of electroacupuncture (EA) on expression of myostatin (MSTN), muscle-specific ring finger protein 1 (MuRF1/Trim63), F-box only protein 32 (Atrogin-1/ Fbxo32), myogenic differentiation antigen (Myod) and myogenin (Myog) in traumatic spinal cord injury (TSCI) rats. Methods:A total of 45 adult female Sprague-Dawley rats were randomly divided into sham operation group (n = 12) and operation group (n = 33). The TSCI model was established with the modified Allen's method. After modeling, there were 24 survival rats and they were randomly divided into model group (n = 12) and EA group (n = 12). EA group was electroacupunctured at Dazhui (DU 14), Mingmen (DU 4) and bilateral Zusanli (ST 36) for 10 minutes, once a day, six times a week for 28 days. Basso-Beattie-Bresnahan (BBB) score was tested before modeling, and three days, seven days, 14 days, 21 days and 28 days after modeling. The rats were measured their body mass before and 28 days after modeling. The ratio of gastrocnemius wet mass was calculated; the cross-sectional area (CSA) and fiber diameter were measured by HE staining; the expression of MSTN, Trim63, Fbxo32, Myod and Myog mRNA were tested with real-time quantitative polymerase chain reaction (qPCR). Results:Three days, seven days, 14 days, 21 days, and 28 days after modeling, the score of BBB was lower in the model group than in the sham operation group (P < 0.01); seven days, 14 days, 21 days, and 28 days after modeling, the score of BBB was higher in EA group than in the model group (P < 0.01). Compared with the sham operation group, the mass of rats, the gastrocnemius wet mass, the CSA and the diameter of the muscle fiber were smaller in the model group (P < 0.05), while the expression of MSTN, Trim63, Fbxo32, Myod and Myog mRNA were higher (P < 0.05). Compared with the model group, the mass of rats, the gastrocnemius wet mass, the CSA, the expression of Myod and Myog mRNA were higher (P < 0.05) in EA group, while the expression of MSTN, Trim63 and Fbxo32 mRNA were lower (P < 0.05). Conclusion:EA might delay the gastrocnemius atrophy in TSCI rats by down-regulating the expression of MSTN, Trim63, Fbxo32 mRNA and up-regulating the expression of Myod and Myog mRNA via controlling the differentiation of the muscle satellite cells and the degradation of protein in skeletal muscle cells.

OBJECTIVE: To evaluate the clinical efficacy of decitabine combined with arsenious acid in the treatment of patients with higher-risk myelodysplastic syndromes(MDS) and chronic myelomonocytic leukemia(CMML). METHODS: Totally 39 patients with MDS and 8 patients with CMML received the treatment of decitabine and arsenious acid from April 2016 to December 2018. Decitabine [20 mg/(m~2·d)] and arsenious acid [0.15 mg/(m~2·d)] were administered intravenously for 5 consecutive days every 4-6 weeks. Patients who achieved complete or partial remission entered into the consolidation cycle. Efficacy and influencing factor were analyzed. RESULTS: Clinical response were observed in 31 patients after a median of 2 courses(ranging 1-12) of treatment. The overall response rate(ORR) was 66.0%. The median duration of response was 16 weeks(ranging 2-52 weeks). There were 8 cases(17.0%) of complete remission(CR), 10 cases(21.3%) of partial remission(PR),12 cases(25.5%) of hematological improvement(HI), 1 case(2.1%) of marrow complete remission(mCR), 8 cases(17.0%) of stable disease(SD), and 1 case(2.1%) of progressive disease(PD). By next generation sequencing, 25 genes mutated with 70 times in 33 cases. The mutation frequency of epigenetic regulators(57.6%) was higher than splicing factors(33.5%), transcription factors and kinase signaling(54.5%),and TP53(21.2%)(P<0.01). There was no significant difference in response rates among these patients(47.4%, 54.5%, 50.0% and85.7%, P=0.977). Gene mutation frequency(VAF) of patients who responded to the regimen declined significantly(16.67% vs. 10.26%,P=0.014). CONCLUSION: Decitabine combined with arsenious acid has significant effect in the treatment of patients with higher-risk MDS and CMML and is well-tolerated. Gene mutation test results by next generation sequencing might be related to clinical response.

BACKGROUNDMitofusin-2 (MFN2), a well-known mitochondrial fusion protein, has been shown to participate in innate immunity, but its role in mediating adaptive immunity remains poorly characterized. In this study, we explored the potential role of MFN2 in mediating the immune function of T lymphocytes.

METHODSWe manipulated MFN2 gene expression in Jurkat cells via lentiviral transduction of MFN2 small interfering RNA (siRNA) or full-length MFN2. After transduction, the immune response and its underlying mechanism were determined in Jurkat cells. One-way analysis of variance and Student's t-test were performed to determine the statistical significance between the groups.

RESULTSOverexpression of MFN2 enhanced the immune response of T lymphocytes by upregulating Ca2+ (359.280 ± 10.130 vs. 266.940 ± 10.170, P = 0.000), calcineurin (0.513 ± 0.014 vs. 0.403 ± 0.020 nmol/L, P = 0.024), and nuclear factor of activated T cells (NFATs) activation (1.040 ± 0.086 vs. 0.700 ± 0.115, P = 0.005), whereas depletion of MFN2 impaired the immune function of T lymphocytes by downregulating Ca2+ (141.140 ± 14.670 vs. 267.060 ± 9.230, P = 0.000), calcineurin (0.054 ± 0.030 nmol/L vs. 0.404 ± 0.063 nmol/L, P = 0.000), and NFAT activation (0.500 ± 0.025 vs. 0.720 ± 0.061, P = 0.012). Furthermore, upregulated calcineurin partially reversed the negative effects of MFN2 siRNA on T cell-mediated immunity evidenced by elevations in T cell proliferation (1.120 ± 0.048 vs. 0.580 ± 0.078, P = 0.040), interleukin-2 (IL-2) production (473.300 ± 24.100 vs. 175.330 ± 12.900 pg/ml, P = 0.000), and the interferon-γ/IL-4 ratio (3.080 ± 0.156 vs. 0.953 ± 0.093, P = 0.000). Meanwhile, calcineurin activity inhibitor depleted the positive effects of overexpressed MFN2 on T cells function.

CONCLUSIONSOur findings suggest that MFN2 may regulate T cell immune functions primarily through the Ca2+-calcineurin-NFAT pathway. MFN2 may represent a potential therapeutic target for T cell immune dysfunction-related diseases.

OBJECTIVETo evaluate the effect of Fuzheng Kang'ai Formula (, FZKA) plus gefitinib in patients with advanced non-small cell lung cancer with epidermal growth factor receptor (EGFR) mutations.

METHODSA randomized controlled trial was conducted from 2009 to 2012 in South China. Seventy chemotherapynaive patients diagnosed with stage IIIB/IV non-small cell lung cancer with EGFR mutations were randomly assigned to GF group [gefitinib (250 mg/day orally) plus FZKA (250 mL, twice per day, orally); 35 cases] or G group (gefitinib 250 mg/day orally; 35 cases) according to the random number table and received treatment until progression of the disease, or development of unacceptable toxicities. The primary endpoint [progression-free survival (PFS)] and secondary endpoints [median survival time (MST), objective response rate (ORR), disease control rate (DCR) and safety] were observed.

RESULTSNo patient was excluded after randomization. GF group had significantly longer PFS and MST compared with the G group, with median PFS of 12.5 months (95% CI 3.30-21.69) vs. 8.4 months (95% CI 6.30-10.50; log-rank P<0.01), MST of 21.5 months (95% CI 17.28-25.73) vs. 18.3 months (95% CI 17.97-18.63; log-rank P<0.01). ORR and DCR in GF group and G group were 65.7% vs. 57.1%, 94.3% vs. 80.0%, respectively (P>0.05). The most common toxic effects in the GF group and G group were rash or acne (42.8% vs. 57.1%, P>0.05), diarrhea (11.5% vs. 31.4%, P<0.05), and stomatitis (2.9% vs. 8.7%, P>0.05).

CONCLUSIONPatients with advanced non-small cell lung cancer selected by EGFR mutations have longer PFS, MST with less toxicity treated with gefitinib plus FZKA than gefitinib alone.

Objective To compare the clinical efficacy and safety of insulin glulisine (GLU) and insulin aspam(ASP) combined with insulin glargine (GLA) in type 2 diabetes patients (T2DM) with secondary failure to oral hypoglycemic agents.Methods One hundred and twenty cases of T2DM with secondary failure to oral hypoglycemia agents were randomly divided into control group (n =60 cases) and treatment group (n =60 cases).The control group received subcutaneous injection of ASP before 0-10 min three meals daily,and the treatment group received subcutaneous injection of GLU before 0-10min three meals daily.The two groups were treated with subcutaneous injection of GLA,once daily.The initial dose of ASP and GLU were 0.8 U · kg-1 · d-1,and the initial dose of GLA was 0.2 U · kg-1 · d-1.Two groups of patients were hospitalized for 2 weeks.The changes of blood glucose,cases and days of blood glucose of achieving target were compared after treatment of hospitalization.The improvement of HbAlc and the frequency of hypoglycemia were observed after treatment of 12 weeks.Results The days of 2-hour postprandial blood glucose(2 h PG) of achieving target in the treatment group and the control group were (10.01 ± 1.99)d and (10.93 ± 1.52)d with 57 cases and 47 cases,and the days of fasting plasma glucose,postprandial blood glucose and 2 h PG of achieving target were (10.31 ± 1.04) d and (11.03 ± 1.38) d with 48 cases and 40 cases(all P ＜ 0.05).After treatment 12 weeks,the HbAlc in treatment and control groups were (6.78 ±0.59)％ and (7.07 ±0.49)％ without significant difference (P ＞ 0.05).During treatment of 12 weeks,the adverse drug reactions in the two groups were mainly hypoglycemic events,the incidence of hypoglycemia in the treatment group was 30.00％,and 25.00％ in the control group,the difference was not statistically significant (P ＞ 0.05).Conclusion Both GLU combined with GLA or ASP combined with GLA have similar efficacy and safety for T2DM with secondary failure to oral hypoglycemic agents.GLU combined with GLA is more rapid in controlling 2 h PG of these patients.

To research the intestinal toxicity of n-BuOH fraction in Phytolacca Radix before and after being processed with vinegar. Toxic n-BuOH fractions were separated from Phytolacca Radix. In the animal model, the level of intestinal edema, water content of intestine and stool, IC₅₀ values of HT-29 and IEC-6 were detected with MTT method to compare the changes in toxicity of n-BuOH fractions from Phytolacca Radix before and after being processed with vinegar. n-BuOH fractions of Phytolacca Radix could cause intestinal edema in mice, increase the edema of duodenum, jejunum and the water content in stool, inhibit the proliferation of HT-29 cells and IEC-6 cells, indicating its intestinal toxicity, with HT-29 IC₅₀ at 14.59 mg•L⁻¹ and IEC-6 IC₅₀ at 43.77 mg•L⁻¹. After being processed with vinegar, the level of intestinal edema, edema of duodenum and jejunum and the water content in stool and inhibition ratio of cells line were reduced, with HT-29 IC₅₀ at 58.51 mg•L⁻¹ and IEC-6 IC₅₀ at 84.37 mg•L⁻¹. After being processed with vinegar, the toxicity of n-BuOH fractions from Phytolacca Radix decreased obviously.