Blood always shows some immune changes after spinal cord injury (SCI), and detection of such changes in blood may be helpful for diagnosis and treatment of SCI. However, studies to date on blood immune changes after SCI in humans are not comprehensive. Therefore, to obtain the characteristics of blood immune changes and immunodiagnostic blood biomarkers of SCI and its different grades, a human blood transcriptome sequencing dataset was downloaded and analyzed to obtain differentially expressed immune-related genes (DEIGs), related functions and signaling pathways related to SCI and its various grades. Characteristic biomarkers of SCI and its different grades were identified by using weighted gene coexpression network analysis (WGCNA) and least absolute shrinkage and selection operator (LASSO) logistic regression. Expression of biomarkers was verified through experiments. The area under the curve (AUC) of biomarkers was calculated to evaluate their diagnostic value, and differences in immune cell content were examined. In this study, 17 kinds of immune cells with different contents between the SCI group and healthy control (HC) group were identified, with 7 immune cell types being significantly increased. Differences in the content of immune cells between different grades of SCI and the HC group were also discovered. DEIGs were identified, with alteration in some immune-related signaling pathways, vascular endothelial growth factor signaling pathways, and axon guidance signaling pathways. The SCI biomarkers identified and those of American Spinal Injury Society Impairment Scale (AIS) A and AIS D of SCI have certain diagnostic sensitivity. Analysis of the correlation of immune cells and biomarkers showed that biomarkers of SCI, AIS A grade and AIS D grade correlated positively or negatively with some immune cells. CKLF, EDNRB, FCER1G, SORT1, and TNFSF13B can be used as immune biomarkers for SCI. Additionally, GDF11and HSPA1L can be used as biomarkers of SCI AIS A grade; PRKCA and CMTM2 can be used as biomarkers of the SCI AIS D grade. Detecting expression of these putative biomarkers and changes in related immune cells may be helpful for predicting the severity of SCI.

e16523 Background: Non-clear cell renal carcinoma (nccRCC) usually had a poorer response to tyrosine kinase inhibitors (TKIs) due to its heterogeneity and rarity. The combination of immunotherapy with TKIs has shown promising efficacy in ccRCC. However, for nccRCC failed standard first-line TKI therapy, the second-line option is still limited. This real-world study aimed to evaluate the efficacy of immune-targeted combination therapy versus TKI monotherapy as the second-line regimen in patients with metastatic nccRCC who failed first-line TKI therapy. Methods: Demographic and clinicopathological data of patients with metastatic nccRCC who were admitted into the Sun Yat-sen University Cancer Center between October, 2011 and September, 2020 were retrospectively collected. All patients failed in first-line TKI therapy and received second-line TKI alone (TKI group) or TKI plus PD-1 therapy (combination group). Efficacy including the objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS) was calculated. The differences on baseline characteristics and efficacy between the two groups were compared. Results: Totally 67 patients were included, with a median age of 50 (interquartile 37-60) years. The median follow-up time was 32.7 (interquartile 21.6-53.0) months. The overall ORR, DCR was 37.3% and 56.7%, respectively. The overall second-line PFS was 7.2 (95% CI: 5.8-8.5) months and OS was 53.2 (18.4-88.0) months. Baseline characteristics between the combination group (n = 45) and the TKI group (n = 22) did not differ significantly except that the combination group had more proportion of liver metastasis (20.9% vs 4.5%, P = 0.025). The combination group had a significantly longer PFS compared with the TKI group [median PFS (95% CI): 9.2 (6.4-12.0) vs 5.2 (2.9-7.5) months, P = 0.001]. Similarly, ORR (48.9% vs 13.6, P = 0.005) and DCR (71.1% vs 27.3%, P = 0.001) was remarkably improved in the combination group. The superior efficacy over TKI monotherapy was more prominent in the younger (< 60 years), male, and patients with lower KPS, IMDC intermediate/poor-risk, non-hereditary RCC and multiple metastatic sites. Conclusions: Immune-targeted combination therapy was effective in the treatment of metastatic nccRCC who failed first-line TKIs.[Table: see text]

Abstract Radioresistance was the main reason for local recurrence and metastasis of nasopharyngeal carcinoma. Tetrandrine is reported as an antitumor drug via inducing cell cycle arrest and apoptosis. In this study, the radiosensitization effects of maximum noncytotoxic doses of tetrandrine in nasopharyngeal carcinoma were analyzed both in vitro and in vivo, using MTT assay, western blot, TUNEL, and HE staining. It was found that the maximum dose of tetrandrine inhibited the phosphorylation of ERK and MEK induced by irradiation, and significantly enhanced irradiation‐induced cell growth inhibition in nasopharyngeal carcinoma cells CNE1, CNE2, and C666‐1. The ERK activator and overexpression of ERK reversed the radiosensitization effect of tetrandrine. About 50 mg/kg of tetrandrine which was used as the maximum noncytotoxic dose of tetrandrine in vivo, enhanced the radiosensitivity of the xenograft tumor and increased the apoptosis rate of the xenograft tumor cells caused by irradiation, while did not raise the side effect of the treatment. Moreover, tetrandrine increased autophagy in nasopharyngeal carcinoma cells. These results suggested that the maximum noncytotoxic dose of tetrandrine sensitized nasopharyngeal carcinoma to irradiation by inhibiting MEK/ERK pathway and inducing autophagy.

Prostate cancer (PCa) remains the second leading cause of cancer‐related death among men in the United States, and its molecular mechanism remains to be elucidated. Recent studies have suggested that microRNAs may play an important role in cancer development and progression. By analyzing the Gene Expression Omnibus dataset, we found lower expression for miR‐488 in PCa than in normal tissues. Moreover, CCK‐8, EdU, glucose uptake, and lactate secrete assays revealed that overexpression of miR‐488 in PCa cell lines PC3 and DU145 resulted in inhibition of proliferation and glycolysis. In contrast, downregulation of miR‐488 expression promoted proliferation and glycolysis in PCa cells. Using a bioinformatic approach and dual‐luciferase reporter assays, we identified 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase, isoform3 (PFKFB3), as a direct target of miR‐488. Inhibition of PFKFB3 also suppressed PCa cell glycolysis and proliferation. Our study suggests that miR‐488 inhibits PCa cell proliferation and glycolysis by targeting PFKFB3, and thus, miR‐488 may be a novel therapeutic candidate for PCa.

Hyperglycaemia promotes the development of Prostate cancer (PCa). However, the roles of miRNAs in this disease process and the underlying mechanisms are largely unknown. In this study, we recruited 391 PCa patients in China and found that PCa patients with high level blood glucose (≥100 mg/dL) trended to have high Gleason score (GS ≥ 7). miRNA-301a levels were significantly higher in prostate cancer than that in normal prostate tissues. Hyperglycaemia or high glucose treatment induced miR-301a expression in prostate tissues or PCa cell lines. miR-301a suppressed the expression of p21 and Smad4, and subsequently promoted G1/S cell cycle transition and cell proliferation in vitro and xenograft growth in nude mice in vivo. Furthermore, knockdown of p21 and Smad4 mimicked the effects of miR-301a overexpression. Restoration of p21 and smad4 could interrupt the effects of miR-301a overexpression. Importantly, inhibition of miR-301a severely blocked high glucose-induced PCa cell growth both in vitro and in vivo. These results revealed a novel molecular link between hyperglycaemia and PCa. The miR-301a plays an important role in the hyperglycaemia-associated cancer growth, and represents a novel therapeutic target for PCa.

OBJECTIVE:Recent studies have shown that understanding the differences between Gleason 3+4 and Gleason 4+3 in PCa patients may improve their treatment. This study aimed to evaluate the different expression levels of glycolytic proteins for Gleason score of 4+3 and 3+4.METHODS:A total of 90 PCa patients, including 38 cases with a Gleason score of 7, were included in this study. The expression of glycolytic proteins in both prostate cancer and normal prostate tissues, in GGG2 and GGG3 as well were assessed by immunohistochemical staining.RESULTS:Compared with GGG3, the GGG2 cases displayed significantly lower expression of all proteins (P < 0.05). The correlation among all enzymes showed that the key glycolytic enzyme, HK2, was significantly positively related to another key enzyme, PKM2 (r = 0.550, P < 0.01), and the expression of PFKFB4 was correlated with the expression of HK2 (r = 0.236, P < 0.05) and PKM2 (r = 0.392, P < 0.01). Additionally, neither GLUT1 nor PFKFB3 was correlated with PFKFB4, HK2 or PKM2. Further analysis showed that HK2 (r = 0.297, P < 0.01) and PKM2 (r = 0.431, P < 0.01) were significantly positively related to the Gleason score in PCa tissues.CONCLUSIONS:Glycolytic proteins expression levels were upregulated in PCa tissues. Furthermore, GGG3 exhibits a higher level of glycolysis compared with GGG2 in PCa tissues. Additionally, the key glycolytic enzymes, HK2 and PKM2, are overexpressed simultaneously in PCa and significantly correlate with PCa progression as represented by the GS.

The increasing resistance of nasopharyngeal carcinoma to irradiation makes the exploration of effective radiosensitizers necessary. Tetrandrine is known to be an antitumor drug, but little is known regarding its radiosensitization effect on nasopharyngeal carcinoma. We investigated the effect of combined treatment of irradiation and maximum non-cytotoxic doses of tetrandrine on the nasopharyngeal carcinoma cell lines CNE1 and CNE2. The maximum non-cytotoxic doses of tetrandrine in CNE1 and CNE2 cells were assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The radiosensitization of cells receiving the maximum non-cytotoxic doses of tetrandrine was assessed by evaluating cell proliferation and DNA damage repair using MTT, clonogenic, comet assays and detection of caspase-3 and phosphorylated histone H2AX (γ-H2AX). The cell cycle was assessed by flow cytometry, and protein expression was detected by western blot analysis. The maximum non-cytotoxic doses of tetrandrine in CNE1 and CNE2 cells were 1.5 μmol/L and 1.8 μmol/L, respectively. When cells were exposed to irradiation and the maximum non-cytotoxic doses of tetrandrine, the survival fraction was decreased. DNA damage and γ-H2AX levels markedly increased. Moreover, tetrandrine abrogated the G2/M phase arrest caused by irradiation. Combined treatment with the maximum non-cytotoxic dose of tetrandrine and irradiation caused suppression of the phosphorylation of CDK1 and CDC25C and increase in the expression of cyclin B1. The study in vivo also showed that the maximum non-cytotoxic dose of tetrandrine could reduce tumor growth in xenograft tumor model. Our results suggest that the maximum non-cytotoxic dose of tetrandrine can enhance the radiosensitivity of CNE1 and CNE2 cells and that the underlying mechanism could be associated with abrogation of radiation-induced G2/M arrest via activation of the CDC25C/CDK1/Cyclin B1 pathway.

Objective To investigate the effect of micrioRNA (miRNA,miR)-488 on glycolysis and proliferation of prostate cancer cells and the underlying mechanism.Methods Real-time quantitative polymerase chain reaction (Real-time PCR) was used to examine the expression of miR-488 in prostate cancer cells.Bioinformatics prediction and luciferase reporter gene assay were utilized for the identification of the target genes of miR-488.The expression levels of 6-phosphofructo-2-kinase 3 (PFKFB3) mRNA and protein were detected by Real-time PCR and Western blotting after the miR-488 mimic was trausfected 24 h later.After miR-488 mimics was transfected,or co-transfected with PFKFB3 over-expression plasmid for 24 h,the capacity of proliferation and the glycolysis of prostate cancer cells (PC-3 and DU145) were determined by cell counting kit-8 (CCK-8),glucose uptake and lactate assay,respectively.Results Compared to the normal prostate epithelium cell RWPE-1,the expression of miR-488 in PC-3 and DU145 cell lines was 0.26 ± 0.03 (P =0.031) and 0.19 ± 0.04 (P =0.021),but there was no statistically significant difference in LNCaP and 22RV1 cells.The glucose uptake rate and the lactate secretion rate were 0.54 ±0.03 (P =0.042) and 0.52 ±0.01 (P=0.032),and 0.55 ± 0.02 (P =0.037) and 0.61 ± 0.17 (P =0.048) after up-regulating the expression of miR-488,which indicating the decline of glycolysis ability and the proliferation ability.Bioinformatics prediction and luciferase reporter gene assay showed that PFKFB3 was the target gene of miR-488.After the expression of miR-488 was up-regulated,the expression levels of PFKFB3 mRNA and protein were down-regulated.When miR-488 and PFKFB3 over-expression plasmids were co-transfected into the cells,the proliferation was elevated and the glucose uptake rate and lactate secretion rate were 0.79 ± 0.12 and 0.82 ± 0.11,and 0.77 ± 0.07 (P =0.015) and 0.83 ± 0.04 (P =0.026) respectively,indicating a higher glycolysis rate than the control.Conclusion MiR-488 can regulate the ability of proliferation and glycolsis of prostate cancer cells by targeting PFKFB3 gene.

Objective To investigate the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphalase-3 (PFKFB3) gene in the prostate cancer tissues and its impact on the proliferation of prostate cancer cells PC3 and DU145.Methods Immunohistochemical staining was used to detect the expression of PFKFB3 in the prostate cancer tissues and the adjacent tissues (antibody concentration 1∶50).Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR,40 cycles,188 bp) and Western blotting [30 μl,10％ sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE)] were applied to detect the mRNA and protein expression levels of PFKFB3 expression in the prostate cancer cells and prostate epithelial cells.After knocking out PFKFB3 gene,plate cloning (40 μmol/L H2O2) formation assay and cell growth assay [20 μl methyl thiazol tetrazolium (MTT)] were applied to measure the cell proliferation.Results The expression of PFKFB3 in the prostate cancer tissues (2.42 ±0.16) was significantly higher than that in the adjacent tissues (1.38 ±0.10,P =0.031).The mRNA and protein expression levels of PFKFB3 in prostate cancer cells were significantly higher than those in the prostate epithelial cells (P =O.015),2.0-2.6 times and 2.5-4.5 times respectively.When knocking out the PFKFB3 gene,cloning formation assay showed that the cloning formation rate decreased as 22％-35％ compared to the control (P =0.018).Cell grouth assay showed that the proliferation of prostate cancer cells was inhibited as 28％-30％ at day 4 (P =0.026).Conclusion PFKFB3 gene is highly expressed in prostate cancer cells and prostate cancer tissues.PFKFB3 plays a promotional role in the prostate cancer cells.

Pancreatic adenocarcinoma is one of the most highly malignant digestive system cancers,and current therapeutic strategies are often unsatisfactory.It is associated with a high mortality rate because of low resection rate,early metastasis,and poor chemoradiotherapy response.Identification and development of more efficacious therapies is urgently needed.The basic research promoted the development of immunotherapy for pancreatic cancer,and immunotherapy offered encouraging results in some preclinical trials during the last decade.The aim of this review is to summarize the recent advances in immunotherapy and to evaluate the future perspectives of immunotherapy in the treatment of pancreatic adenocarcinoma.

Cancer arises as the consequence of mutations and epigenetic alterations that activate oncogenes and inactivate tumor suppressor genes. Through a genome-wide screen for methylated genes in colon neoplasms, we identified aberrantly methylated RET in colorectal cancer. RET, a transmembrane receptor tyrosine kinase and a receptor for the glial cell-derived neurotrophic factor family ligands, was one of the first oncogenes to be identified, and has been shown to be an oncogene in thyroid cancer and pheochromocytoma. However, unexpectedly, we found RET is methylated in 27% of colon adenomas and in 63% of colorectal cancers, and now provide evidence that RET has tumor suppressor activity in colon cancer. The aberrant methylation of RET correlates with decreased RET expression, whereas the restoration of RET in colorectal cancer cell lines results in apoptosis. Furthermore, in support of a tumor suppressor function of RET, mutant RET has also been found in primary colorectal cancer. We now show that these mutations inactivate RET, which is consistent with RET being a tumor suppressor gene in the colon. These findings suggest that the aberrant methylation of RET and the mutational inactivation of RET promote colorectal cancer formation, and that RET can serve as a tumor suppressor gene in the colon. Moreover, the increased frequency of methylated RET in colon cancers compared with adenomas suggests RET inactivation is involved in the progression of colon adenomas to cancer.