Objective:To explore the effect and mechanism of c-Myc on regulating the expression of immune-related ligands in Y subtype small-cell lung cancer (SCLC) characterized by high expression of immune-related molecules.Methods:The Y subtype SCLC cell line H196 was randomly divided into the control group, c-Myc inhibitor 10058-F4 group, histone deacetylase 1 (HDAC1) inhibitor pyroxamide group, and 10058-F4 plus pyroxamide group. The co-culture system with NK-92MI cells was used to determine the effect of H196 cells on the function of natural killer (NK) cells. Western Blotting and co-immunoprecipitation assays were used to detect the effect of c-Myc on class Ⅰ HDAC, and flow cytometry was used to detect the regulatory effect of c-Mycon CD47, programmed cell death ligand 1 (PD-L1), and CD155, which are highly expressed immune checkpoints in Y subtype SCLC, and major histocompatibility complex classⅠ-related chains A and (MICA/B), which is a poorly expressed immune-activating ligand in SCLC, and the role of HDAC. Chromatin immunoprecipitation (ChIP) assay and real-time quantitative polymerase chain reaction (RT-qPCR) were used to determine the regulatory mechanism of c-Myc-HDAC1 on MICA/B expression.Results:Inhibition of c-Myc decreased the mortality of H196 cells in the co-culture system and down-regulated the expression of MICA/B. Compared with the NK+H196 group [(42.54±2.47)%], the proportion of cells killed by NK-92MI cells in the NK+H196+10058-F4 group was lower [(28.48±3.38)%, P＜0.001]. The mean fluorescence intensity (MFI) of MICA/B on the cells in the 10058-F4 group (36.40±0.82) was lower than that in the control group (91.23±8.60, P＜0.001). And c-Myc could bind to HDAC1, whose protein level was up-regulated by 10058-F4 while the mRNA level was not. Compared with the cells in the control group (90.10±4.91), the MFI of MICA/B on the cells in the pyroxamide group was significantly increased (145.70±5.86, P＜0.001), and the MFI of MICA/B on the cells in the 10058-F4+pyroxamide group (54.60±2.88) was significantly increased compared with the cells in the 10058-F4 group (35.97±1.60, P＜0.001). The percentage of MICA promoter gene fragments in the c-Myc antibody precipitation group (0.125±0.037) was significantly higher than that in the IgG group (0.000 8±0.000 3, P=0.004). MICB had a similar trend, suggesting that the c-Myc-HDAC1 complex could bind to the promoter region of MICA/B. The MFI of CD47 on the cells in the 10058-F4 group (60.07±0.21) was significantly lower than cells in the control group (70.27±1.37, P＜0.001), but the MFIs of PD-L1 (13.50±0.61) and CD155 (829.70±41.19) were significantly higher than those on the cells in the control group (9.23±0.94, P＜0.01; 496.00±4.36, P＜0.001, respectively). Conclusions:c-Myc may promote the expression of MICA/B and CD47 in Y subtype SCLC cells by binding and inhibiting HDAC1, while it may also be involved in inhibiting the expression of PD-L1 and CD155 in SCLC cells.

Objective:To explore the effect and mechanism of c-Myc on regulating the expression of immune-related ligands in Y subtype small-cell lung cancer (SCLC) characterized by high expression of immune-related molecules.Methods:The Y subtype SCLC cell line H196 was randomly divided into the control group, c-Myc inhibitor 10058-F4 group, histone deacetylase 1 (HDAC1) inhibitor pyroxamide group, and 10058-F4 plus pyroxamide group. The co-culture system with NK-92MI cells was used to determine the effect of H196 cells on the function of natural killer (NK) cells. Western Blotting and co-immunoprecipitation assays were used to detect the effect of c-Myc on class Ⅰ HDAC, and flow cytometry was used to detect the regulatory effect of c-Mycon CD47, programmed cell death ligand 1 (PD-L1), and CD155, which are highly expressed immune checkpoints in Y subtype SCLC, and major histocompatibility complex classⅠ-related chains A and (MICA/B), which is a poorly expressed immune-activating ligand in SCLC, and the role of HDAC. Chromatin immunoprecipitation (ChIP) assay and real-time quantitative polymerase chain reaction (RT-qPCR) were used to determine the regulatory mechanism of c-Myc-HDAC1 on MICA/B expression.Results:Inhibition of c-Myc decreased the mortality of H196 cells in the co-culture system and down-regulated the expression of MICA/B. Compared with the NK+H196 group [(42.54±2.47)%], the proportion of cells killed by NK-92MI cells in the NK+H196+10058-F4 group was lower [(28.48±3.38)%, P＜0.001]. The mean fluorescence intensity (MFI) of MICA/B on the cells in the 10058-F4 group (36.40±0.82) was lower than that in the control group (91.23±8.60, P＜0.001). And c-Myc could bind to HDAC1, whose protein level was up-regulated by 10058-F4 while the mRNA level was not. Compared with the cells in the control group (90.10±4.91), the MFI of MICA/B on the cells in the pyroxamide group was significantly increased (145.70±5.86, P＜0.001), and the MFI of MICA/B on the cells in the 10058-F4+pyroxamide group (54.60±2.88) was significantly increased compared with the cells in the 10058-F4 group (35.97±1.60, P＜0.001). The percentage of MICA promoter gene fragments in the c-Myc antibody precipitation group (0.125±0.037) was significantly higher than that in the IgG group (0.000 8±0.000 3, P=0.004). MICB had a similar trend, suggesting that the c-Myc-HDAC1 complex could bind to the promoter region of MICA/B. The MFI of CD47 on the cells in the 10058-F4 group (60.07±0.21) was significantly lower than cells in the control group (70.27±1.37, P＜0.001), but the MFIs of PD-L1 (13.50±0.61) and CD155 (829.70±41.19) were significantly higher than those on the cells in the control group (9.23±0.94, P＜0.01; 496.00±4.36, P＜0.001, respectively). Conclusions:c-Myc may promote the expression of MICA/B and CD47 in Y subtype SCLC cells by binding and inhibiting HDAC1, while it may also be involved in inhibiting the expression of PD-L1 and CD155 in SCLC cells.

Melanoma is a highly invasive and lethal skin malignant tumor derived from melanocytes. It has the characteristics of high early metastasis and high mortality. In recent years, with the in-depth study of the pathogenesis of melanoma, it has been found that epigenetic modification, especially DNA methylation, is considered to be a universal intrinsic feature of melanoma development and evolution. This article reviews the research progress of abnormal DNA methylation genes in melanoma in detail, and summarizes the biomarker effect of DNA methylation genes, suggesting that the detection of abnormal DNA methylation genes in melanoma patients is hopeful as an early screening index and diagnostic marker for melanoma patients.

Melanoma is a highly invasive and lethal skin malignant tumor derived from melanocytes. It has the characteristics of high early metastasis and high mortality. In recent years, with the in-depth study of the pathogenesis of melanoma, it has been found that epigenetic modification, especially DNA methylation, is considered to be a universal intrinsic feature of melanoma development and evolution. This article reviews the research progress of abnormal DNA methylation genes in melanoma in detail, and summarizes the biomarker effect of DNA methylation genes, suggesting that the detection of abnormal DNA methylation genes in melanoma patients is hopeful as an early screening index and diagnostic marker for melanoma patients.

Objective: To investigate the expression of succinate dehydrogenase complex subunit protein in succinate dehydrogenase-deficient gastrointestinal stromal tumors (SDH-deficient GISTs). Methods: Three hundred fifty-two cases of GISTs were collected from January 2003 to January 2017 at the Affiliated Hospital of Guizhou Medical University and West China Hospital of Sichuan University.The expression of succinate dehydrogenase subunit protein was detected by immunohistochemical EnVision technique in 352 cases of GISTs, and the negative cases were analyzed for clinicopathologic features and outcome. The gene segments of CKIT exons 9, 11, 13 and 17 and PDGFRA exons 12 and 18 were amplified and detected in SDH-deficient (negative) cases. Results: A total of 15 SDHB-deficient (negative) GISTs (4.3%, 15/352) were found among 352 cases of GISTs. Six patients were male and nine were female. The age of initial diagnosis ranged from 15 to 84 years (median=53 years, mean=47 years). The tumor involved stomach (14 cases) and mesentery (1 case). The tumor sizes varied from 0.5 cm to 15.0 cm (mean=6.9 cm). There were six, six and three cases of epithelioid, mixed and spindle cell types respectively. Eight cases showed multi-nodularity in the wall of stomach. Metastasis to lymph node was noted in four cases, one case showed intraperitoneal implantation metastasis. Metastases to liver, pancreas and lymph node were found in one case, and one case showed vascular invasion. Among SDHB-deficient GISTs, two SDHA-deficient (negative) cases were found (0.6%, 2/352), but there were no SDHC and SDHD deficient (negative) cases. Five of the fifteen SDH-deficient GISTs had follow-up data: one patient died 8 months after surgery from unknown cause, four had no recurrences or metastases, and there was no history of paraganglioma and pulmonary chondroma found in patients and their families. No mutation in CKIT and PDGFRA gene was identified in 15 cases of SDH-deficient GISTs. Conclusion SDH-deficient GISTs have unique clinicopathologic features and a favorable prognosis, and a small proportion of cases are SDHA-deficient.

Objective To investigate the expression of succinate dehydrogenase complex subunit protein in succinate dehydrogenase?deficient gastrointestinal stromal tumors(SDH?deficient GISTs). Methods Three hundred fifty?two cases of GISTs were collected from January 2003 to January 2017 at the Affiliated Hospital of Guizhou Medical University and West China Hospital of Sichuan University.The expression of succinate dehydrogenase subunit protein was detected by immunohistochemical EnVision technique in 352 cases of GISTs, and the negative cases were analyzed for clinicopathologic features and outcome. The gene segments of CKIT exons 9,11,13 and 17 and PDGFRA exons 12 and 18 were amplified and detected in SDH?deficient(negative)cases. Results A total of 15 SDHB?deficient(negative)GISTs (4.3%,15/352)were found among 352 cases of GISTs. Six patients were male and nine were female. The age of initial diagnosis ranged from 15 to 84 years(median=53 years,mean=47 years). The tumor involved stomach(14 cases)and mesentery(1 case). The tumor sizes varied from 0.5 cm to 15.0 cm(mean=6.9 cm). There were six, six and three cases of epithelioid, mixed and spindle cell types respectively.Eight cases showed multi?nodularity in the wall of stomach. Metastasis to lymph node was noted in four cases,one case showed intraperitoneal implantation metastasis.Metastases to liver,pancreas and lymph node were found in one case,and one case showed vascular invasion. Among SDHB?deficient GISTs,two SDHA?deficient(negative)cases were found(0.6%, 2/352), but there were no SDHC and SDHD deficient (negative)cases. Five of the fifteen SDH?deficient GISTs had follow?up data: one patient died 8 months after surgery from unknown cause, four had no recurrences or metastases, and there was no history of paraganglioma and pulmonary chondroma found in patients and their families. No mutation in CKIT and PDGFRA gene was identified in 15 cases of SDH?deficient GISTs. Conclusion SDH?deficient GISTs have unique clinicopathologic features and a favorable prognosis, and a small proportion of cases are SDHA?deficient.

Objective To investigate the expression of succinate dehydrogenase subunit A(SDHA)and subunit B (SDHB) in patients with renal cell carcinoma (RCC),and its relationship with the clinicopathologic characteristics. Methods The expression of SDHA and SDHB was detected in 179 cases of RCC by immunohisto-chemistry and the relationship between the SDHA ,SDHB expression and the clinicopathologic characteristics of RCC were analyzed. Results In 179 cases of RCC,18 cases(10.1%)were shown with negative or suspicious-negative expression of SDHA and SDHB,including 2 cases(1.1%)with suspicious-negative expression of SDHA and SDHB. 12 cases(66.7%)had tumor located in left kidney and 6 cases(33.3%)had tumor located in right kidney. The well-differentiation group contained 15 cases(83.3%),moderate-differentiation group contained 1 case (5.6%)and poor-differentiation group contained 2 cases(11.1%). Most of SDH negative and suspicious negative RCC had the following characteristics that the normal renal tubule could be seen within the tumor. The tumor was composed of polygonal cells arranged in nest ,and the polygonal cells were separated by a fine fibrovascular stroma. The nuclei were shown around with vesicular chromatin. The eosinophilic flocculet material ,vacuole and eosino-philic inclusion body could be seen in the cytoplasm of tumor cells. Conclusion The RCC cases with negative expression of SDHA and SDHB protein have unique histological features ,with significantly higher incidence in the left kidney than that in the right kidney.

OBJECTIVETo investigate clinicopathologic features of succinate dehydrogenase-deficient gastrointestinal stromal tumors (SDH-deficient GIST).

METHODSImmunohistochemical EnVision technique was used to assess the expression of succinate dehydrogenase subunit B (SDHB) in 192 cases of GIST. Cases of SDH-deficient GIST were further evaluated for the presence of CKIT exons 9, 11, 13 and 17 mutations and PDGFRA exons 12 and 18 mutations with clinical followed-up data.

RESULTSSeven of the 192 cases showed SDHB-deficiency (3.6%, 7/192). The patients ranged in age from 35 to 84 years (median=56 years; mean=60 years). Four were male and three were female. Six tumors involved stomach and one involved mesentery. Histopathologic features of SDHB-deficient GIST included four cases of mixed-cell type and three of epithelioid cell type. The tumors commonly involved muscularis propria of the stomach as multiple nodules, creating a plexiform pattern. The tumors had high cellularity with cytoplasmic vacuolization. Five cases developed lymph node metastases including one also metastasizing to liver and pancreas. Two cases showed no evidence of metastasis. None of the 7 cases of the SDHB-deficient GIST had CKIT exons 9, 11, 13 and 17 mutations and PDGFRA exons 12 and 18 mutations. Three of the seven SDHB-deficient GIST cases had followed-up data: two did not recur and one died after 24 months of surgery of unknown cause.

CONCLUSIONSDHB-deficient GIST has characteristic clinicopathologic features with wide-type CKIT gene and a favorable prognosis.

Adult ; Aged ; Aged, 80 and over ; Exons ; Female ; Gastrointestinal Stromal Tumors ; diagnosis ; genetics ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Mutation ; Prognosis ; Succinate Dehydrogenase ; genetics

Objective To detect the expressions of chemokine CXCL5 and its receptor CXCR2 in hepatocellular carcinoma (HCC), investigate their relationships between CXCL5, CXCR2 and clinicopathologic features and probe into the significance in the evaluation of prognosis. Methods Immunohistochemical Envision method was utilized to detect the expressions of CXCL5 and CXCR2 in HCC , to explore their relationship between CXCL5, CXCR2 and clinicopathologic features and MVD in HCC. Results (1) The high expression rates of CXCL5 and CXCR2 protein was 64.7% and 68.6%, respectively, significantly higher than those in adjacent tissues of HCC(17.6%, 15.7%, P < 0.05). The overexpression of CXCL5 protein had correlation with tumor size, differentiation, TNM stage and vascular invasion (P < 0.05) and the overexpression of CXCR2 protein did with tumor grade and vascular invasion (P < 0.05). (2) The value of MVD was higher in HCC than that in the adjacent tissues of HCC (P < 0.05), and had positive correlation with the expressions of CXCL5, and CXCR2 proteins. (3) The higher rates of recurrence and metastasis were in the groups of higher expression of CXCL5 and CXCR2 proteins (P < 0.05). Conclusions The overexpressions of CXCL5 and CXCR2 may promote the occurrence and development of HCC as well as the neovasculation in HCC. Therefore, they can be used as markers to evaluate the prognosis of HCC.

We expressed 17-hydroxysteroid dehydrogenase10 (17β-hsd10) recombinant protein, prepared anti-17β- hsd10 polyclonal antibodies and established sandwich enzyme linked immunosorbent assay (ELISA) test for detection of 17β-hsd10. RT-PCR was used to get the gene of 17β-hsd10 of mouse liver, and a prokaryotic protein expression system pET 15b-17β-hsd10/Escherichia coli BL21 (DE3) which induced with isopropyl-1-thio-β-galactopyranoside (IPTG) for recombinant protein expression was constructed subsequently. The target protein purified using His-Binding-resin column was used to immunize BALB/c mice and rabbits, serum total IgGs from immunized animals were purified by ammonium sulfate precipitation method. We established a Double-antibody Sandwich enzyme linked immunosorbent assay about 17β-hsd10 using the two antibodies we prepared. We got the concentration of 1.5 mg/mL of 17β-hsd10 protein with molecular weight of 29.5 kDa, and polyclonal antibodies from mouse and rabbit with the tite 1.25 x 10(4) and 2.5 x 10(4) respectively. The concentration of 0.1 g/mL of 17β-hsd10 can be detected by the Double-antibody Sandwich ELISA we established, and the assay was sensitive and specific. It can be widely used in clinical and experimental study.
3-Hydroxyacyl CoA Dehydrogenases ; genetics ; immunology ; Animals ; Antibodies ; immunology ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; Immunization ; Mice ; Mice, Inbred BALB C ; Rabbits ; Recombinant Proteins ; genetics ; immunology