Identification of an m6A-related lncRNA prognostic signature in colorectal cancer.

Data sets of colorectal cancer (CRC) were obtained from The Cancer Genome Atlas (TCGA), three N6-methyladenosine (m6A) subtypes were identified using 21 m6A-related long noncoding RNAs (lncRNAs) and differential m6A subtypes of different CRC tumors were determined in this study to evaluate the m6A expression and the prognosis of patients with CRC. Subsequently, eight key lncRNAs were identified based on co-expression with 21 m6A-related genes in CRC tumors using the single-factor Cox and least absolute shrinkage and selection operator. Finally, an m6A-related lncRNA risk score model of CRC tumor was established using multifactor Cox regression based on the eight important lncRNAs and found to have a better performance in evaluating the prognosis of patients in the TCGA-CRC data set. TCGA-CRC tumor samples were divided based on the risk scores: high and low. Then, the clinical characteristics, tumor mutation load, and tumor immune cell infiltration difference between the high- and low-risk-score groups were explored, and the predictive ability of the risk score was assessed for immunotherapeutic benefits. We found that the risk score model can determine the overall survival, be a relatively independent prognostic indicator, and better evaluate the immunotherapeutic benefits for patients with CRC. This study provides data support for accurate immunotherapy in CRC.

Reactivation of γ-globin expression using a minicircle DNA system to treat ß-thalassemia.

Reactivation of fetal hemoglobin by editing the B-cell lymphoma/leukemia 11A (BCL11A) erythroid enhancer is an effective gene therapy for ß-thalassemia. Using the CRISPR/Cas9 system, fetal γ-globin expression can be robustly reactivated to mitigate the clinical course of ß-thalassemia. In our study, we found that the transfection efficiencies of CD34+ hematopoietic stem/progenitor cells (HSPCs) were significantly and negatively correlated with the length of plasmids and greatly affected by the linearization of plasmids. Furthermore, the transgene expression of minicircles (MC) without plasmid backbone sequences was better both in vitro and in vivo compared with conventional plasmids. Thus, MC DNA was used to deliver the cassette of Staphylococcus aureus Cas9 (SaCas9) into HSPCs, and a single-guide RNA targeting the erythroid enhancer region of BCL11A was selected. After electroporation with MC DNA, an evident efficiency of gene editing and reactivation of γ-globin expression in erythroblasts derived from unsorted HSPCs was acquired. No significant off-target effects were found by deep sequencing. Furthermore, fragments derived from lentiviral vectors, but not MC DNA, were highly enriched in promoter, exon, intron, distal-intergenic, and cancer-associated genes, indicating that MC DNA provided a relatively safe and efficient vector for delivering transgenes. The developed MC DNA vector provided a potential approach for the delivery of SaCas9 cassette and the reactivation of γ-globin expression for ameliorating syndromes of ß-thalassemia.

Identification of a Prognostic Colorectal Cancer Model Including LncRNA FOXP4-AS1 and LncRNA BBOX1-AS1 Based on Bioinformatics Analysis.

Background: Knowledge about the prognostic role of long noncoding RNA (lncRNA) in colorectal cancer (CRC) is limited. Therefore, we constructed a lncRNA-related prognostic model based on data from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA). Materials and Methods: CRC transcriptome and clinical data were downloaded from the GSE20916 dataset and the TCGA database, respectively. R software was used for data processing and analysis. The differential lncRNA expression within the two datasets was first screened, and then intersections were measured. Cox regression and the Kaplan-Meier method were used to evaluate the effects of various factors on prognosis. The area under the curve (AUC) of the receiver operating characteristic curve and a nomogram based on multivariate Cox analysis were used to estimate the prognostic value of the lncRNA-related model. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were applied to elucidate the significantly involved biological functions and pathways. Results: A total of 11 lncRNAs were crossed. The univariate Cox analysis screened out two lncRNAs, which were analyzed in the multivariate Cox analysis. A nomogram based on the two lncRNAs and other clinicopathological risk factors was constructed. The AUC of the nomogram was 0.56 at 3 years and 0.71 at 5 years. The 3-year nomogram model was compared with the ideal model, which showed that some indices of the 3-year model were consistent with the ideal model, suggesting that our model was highly accurate. The GO and KEGG enrichment analyses showed that positive regulation of secretion by cells, positive regulation of secretion, positive regulation of exocytosis, endocytosis, and the calcium signaling pathway were differentially enriched in the two-lncRNA-associated phenotype. Conclusions: A two-lncRNA prognostic model of CRC was constructed by bioinformatics analysis. The model had moderate prediction accuracy. LncRNA BBOX1-AS1 and lncRNA FOXP4-AS1 were identified as prognostic biomarkers.

The Effect of Early vs. Deferred Antiretroviral Therapy Initiation in HIV-Infected Patients With Cryptococcal Meningitis: A Multicenter Prospective Randomized Controlled Analysis in China.

Background: The optimal timing for initiation of antiretroviral therapy (ART) in HIV-positive patients with cryptococcal meningitis (CM) has not, as yet, been compellingly elucidated, as research data concerning mortality risk and the occurrence of immune reconstitution inflammatory syndrome (IRIS) in this population remains inconsistent and controversial. Method: The present multicenter randomized clinical trial was conducted in China in patients who presented with confirmed HIV/CM, and who were ART-naïve. Subjects were randomized and stratified into either an early-ART group (ART initiated 2-5 weeks after initiation of antifungal therapy), or a deferred-ART group (ART initiated 5 weeks after initiation of antifungal therapy). Intention-to-treat, and per-protocol analyses of data for these groups were conducted for this study. Result: The probability of survival was found to not be statistically different between patients who started ART between 2-5 weeks of CM therapy initiation (14/47, 29.8%) vs. those initiating ART until 5 weeks after CM therapy initiation (10/55, 18.2%) (p = 0.144). However, initiating ART within 4 weeks after the diagnosis and antifungal treatment of CM resulted in a higher mortality compared with deferring ART initiation until 6 weeks (p = 0.042). The incidence of IRIS did not differ significantly between the early-ART group and the deferred-ART group (6.4 and 7.3%, respectively; p = 0.872). The percentage of patients with severe (grade 3 or 4) adverse events was high in both treatment arms (55.3% in the early-ART group and 41.8% in the deferred-ART group; p=0.183), and there were significantly more grade 4 adverse events in the early-ART group (20 vs. 13; p = 0.042). Conclusion: Although ART initiation from 2 to 5 weeks after initiation of antifungal therapy was not significantly associated with high cumulative mortality or IRIS event rates in HIV/CM patients compared with ART initiation 5 weeks after initiation of antifungal therapy, we found that initiating ART within 4 weeks after CM antifungal treatment resulted in a higher mortality compared with deferring ART initiation until 6 weeks. In addition, we observed that there were significantly more grade 4 adverse events in the early-ART group. Our results support the deferred initiation of ART in HIV-associated CM. Clinical Trials Registration: www.ClinicalTrials.gov, identifier: ChiCTR1900021195.

Genome variation in colorectal cancer patient with liver metastasis measured by whole-exome sequencing.

BACKGROUND: Liver metastasis of colorectal cancer (CRC) is an important cause of death from CRC, but its molecular mechanism is still unclear. In recent years, whole-exome sequencing has played an increasingly important role in the study of the occurrence and development of diseases, especially malignant tumors. Its high throughput and low cost advantages enable researchers to explore the pathogenic genes of diseases, and screen potential molecular markers and therapeutic targets from the level of genomics. METHODS: This study collected the primary tumor tissues, matched paracancerous, normal tissues, and liver metastases of 4 CRC patients admitted to the Department of General Surgery of the First Affiliated Hospital of Soochow University, and performed high-depth whole-exome sequencing, with the sequencing depth of each sample reaching 123× on average, then filtered the sequencing data, compared them, and analyzed the bioinformatics data. RESULTS: we found 8,565 single nucleotide variants (SNV) and 429 insertions/deletions (InDel) in the primary and hepatic lesion tissues, and the genes with the highest mutation frequency were titin (TTN), obscurin (OBSCN), and homeodomain-interacting protein kinase 2 (HIPK2). The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the mutant genes was conducted, and it was found that the mutant genes were mainly concentrated in the cells, cell parts, and cellular process of GO. The results of KEGG pathway analysis showed that mutations were mainly distributed in circadian entrainment, insulin secretion, and glutamatergic synapse. Further, we identified 723 SNV and Indel genes with high frequency mutations including TTN, OBSCN, and hydrocephalus-inducing protein homolog (HYDIN) across all tissues of liver metastases. The GO analysis showed that the mutated genes in liver metastatic tissues were mainly concentrated in cell, cell part, and cellular process. The KEGG pathway analysis showed that high frequency mutation genes were focused on gastric acid secretion, bile secretion, and melanogenesis. CONCLUSIONS: This study found some candidate genes related to the occurrence of CRC and liver metastasis through whole-exome sequencing of relevant tissues in CRC patients with liver metastasis, which is expected to provide new markers and therapeutic targets for such patients.

DRAM1 regulates the migration and invasion of hepatoblastoma cells via autophagy-EMT pathway.

DNA-damage regulated autophagy modulator 1 (DRAM1) is known as a target of TP53-mediated autophagy, and has been reported to promote the migration and invasion abilities of glioblastoma stem cells. However, the precise contribution of DRAM1 to cancer cell invasion and migration, and the underlying mechanisms remain unclear. In the present study, small interfering (si)RNA or short hairpin RNA mediated knockdown of DRAM1 was performed in hepatoblastoma cells and the migration and invasion abilities were detected in vitro and in vivo. To investigate the underlying mechanisms, western blotting and immunofluorescence were used to detect the expression of autophagy-associated proteins and epithelial-mesenchymal-transition (EMT)-associated markers. The results showed that DRAM1 knockdown by specific siRNA abrogated cell autophagy, as well as inhibited the migration and invasion of HepG2 cells in Transwell assays, which may be reversed by rapamycin treatment. In addition, DRAM1 knockdown increased the expression of E-Cadherin while decreased the expression of vimentin in HepG2 cells, which was also be reversed by rapamycin treatment. Taken together, these results suggest that DRAM1 is involved in the regulation of the migration and invasion of HepG2 cells via autophagy-EMT pathway.

MiR-26b regulates 5-FU-resistance in human colorectal cancer via down-regulation of Pgp.

Chemotherapy resistance frequently drives tumor progression. However, the underlying molecular mechanisms remain unclear. In this study, we found that the expression level of miR-26b was down-regulated in the human colorectal cancer tissues and the resistant cells strains: HT-29/5-FU and LOVO/5-FU cells. Meanwhile, we showed that miR-26b improved sensibility of colorectal cancer cells to 5-FU in vitro and enhanced the potency of 5-FU in the inhibition of tumor growth in vivo. We further demonstrated that the tumor suppressive role of miR-26b was mediated by negatively regulating P-glycoprotein (Pgp) protein expression. Furthermore, studies of colorectal cancer specimens indicated that the expression of miR-26b and Pgp had inverse correlation. Importantly, we found that CpG islands in the miR-26b promoter region were hypermethylated in 5-FU resistant cells. Our study is the first to identify the tumor suppressive role of over-expressed miR-26b in chemo-sensitivity. Identification of a novel miRNA-mediated pathway that regulates chemo-sensitivity in colorectal cancer will facilitate the development of novel therapeutic strategies in the future.

miR-133a-3p Targets SUMO-Specific Protease 1 to Inhibit Cell Proliferation and Cell Cycle Progress in Colorectal Cancer.

Dysregulation of SUMO-specific protease 1 (SENP1) expression has been reported in several kinds of cancer, including human colorectal and prostate cancers, proposing SENP1 as an oncogene with a critical role in cancer progression. miR-133a-3p has been reported as a tumor suppressor in several malignant neoplasias. However, the precise molecular mechanisms underlying its role in colorectal cancer remain largely unknown. The aim of this work was to investigate the relationship between miR-133a-3p and SENP1 in colorectal cancer cells. We found that miR-133a-3p expression was downregulated in colorectal cancer tissues. In silico analyses indicated that SENP1 is one of the target genes of miR-133a-3p. Overexpression of miR-133a-3p mimics was able to inhibit cell growth with G1 arrest of colorectal cancer cells. Overexpression of miR-133a-3p antisense promoted cell growth of colorectal cancer cells. The luciferase reporter experiments showed that miR-133a-3p regulated the expression of SENP1 by combining with its 3'-UTR and resulted in downregulation of SENP1 and upregulation of CDK inhibitors such as p16, p19, p21, and p27. These results suggest that the miR-133a-3p-SENP1 axis might play a role in cell proliferation and cell cycle regulation of colorectal cancer cells.

Inhibition of EZH2 expression is associated with the proliferation, apoptosis, and migration of SW620 colorectal cancer cells in vitro.

Epigenetic changes have been recently recognized as important in many human cancers. Enhancer of zeste homologue 2 (EZH2)gene has shown overexpression in various human cancers, consistent with a straightforward role of EZH2 as an oncogene, but its function in carcinogenesis is partly contradictory. The role of EZH2 in development of human colorectal cancer (CRC) has not yet been clarified. In present study, we observed up-regulation of EZH2 expression in tumor tissues from CRC patients [corrected]. The expression of EZH2 in CRC cell lines is consistent with the trend in cancer tissues using RT-PCR. We showed that TNM stage and lymph node metastasis in CRC patients are significantly correlated with EZH2 expression levels [corrected]. EZH2 level of transcription and protein was inhibited by small interfering RNA (siRNA). More importantly, EZH2-siRNA inhibited the proliferation and migration of SW620 cells while promoting their apoptosis, and inducing G0/G1 cell cycle arrest of CRC cells. Collectively, our results suggest that upregulated EZH2 expression may contribute to the progression of the patients with CRC. A comprehensive study of epigenetic mechanisms and the relevance of EZH2 in CRC is important for fully understanding this disease and as a basis for developing new treatment options in patients with CRC [corrected].

[Clinical characteristics of highly active antiretroviral therapy-associated immune reconstruction inflammation in acquired immunodeficiency syndrome].

OBJECTIVE: To summarize the morbidity, mortality, clinical manifestations and risk factors for IRIS (immune reconstruction inflammatory syndrome) during HAART (highly active antiretroviral therapy) in China. METHODS: From October 2007 to September 2009, a prospective cohort of 238 AIDS (acquired immunodeficiency syndrome) patients on HAART from Hunan and Jianxi provinces was recruited for a follow-up of 24 weeks. And 47 and 191 patients were assigned into the IRIS and non-IRIS groups respectively. The data of general information, clinical manifestations and treatment of two groups were collected and compared. Blood samples were collected in both groups at pre-and post-HAART 12 weeks, 24 weeks for HIV viral load and CD4(+) cell count examinations. A statistical analysis was performed. RESULTS: A total of 47 (19.7%) IRIS cases was analyzed. The median onset of IRIS was 28 (9 - 36) days. And 29 (61.7%) cases of tuberculosis IRIS were found. There was no significant difference in age, gender, route of transmission and antiretroviral regimens between the IRIS and non-IRIS groups. At baseline, Weeks 12 and 24, both groups showed a significant decline of viral load. And there was no significant difference between them. Both groups showed a significant increase of CD4(+) cell count. But there was no significant difference between two groups. However, the baseline CD4(+) cell count was markedly lower in the IRIS group than that in the non-IRIS group. In 85.1% (40/47) of cases, the CD4(+) cell count was < 100 × 10(6)/L in the IRIS group at the baseline of HAART. CONCLUSION: IRIS mostly occurs during 3 months of HAART initiation. The age, gender, route of transmission and antiretroviral treatment regimens of patients on HAART are not risk factors for the development of IRIS. The HIV RNA viral load decreases in both IRIS and non-IRIS groups without any significant difference. The patients with a CD4(+) cell count < 100/µl are more vulnerable to develop IRIS.

[Effects of different mutated sites in vpr gene of HIV on apoptosis of host cells: experiment with HeLa cells].

OBJECTIVE: To investigate the effects of different mutated sites in the vpr gene of HIV on the apoptosis of host cells, and the possible mechanism thereof. METHODS: Fourteen HIV-1 vpr fragments were obtained from HIV-infected persons. Eukaryotic expression vector pcDNA3.1 (+) plasmid was extracted, the PCR purified product was double-cut by HindIII and BamH, and the cut products were ligated to vectors, thus establishing the JM109 competent cells. Sequencing was used to confirm the reconstruction of pcDNA-vpr eukaryotic expression vectors that were then transfected into HeLa cells. Blank vectors were transfected as control group. Cells were harvested after 24 hours and underwent Hoechst 33258 staining and observed under fluorescence microscope. Annexin-FITC-PI staining and flow cytometry were used to observe the percentage of apoptosis. The caspase-3 activity was detected by enzyme labeling instrument. RESULTS: The apoptotic rates shown by Hoechst and annexin--FITC-PI staining methods, and caspase-3 activity levels of the HeLa cells transfected with the gene fragments with mutated sites 70, 85, 86, and 94 cells were all lower than the cells transfected with the gene fragments without these mutated sites. The apoptosis causing ability levels of the No 1-7 recombinant plasmids (all of the Vpr AE subtype) were all lower than those of the No 8-14 plasmids (of Vpr B, AB, C, and C/BC subtypes). CONCLUSION: The apoptosis causing ability of the HIV with the vpr sequence with mutated sites 70, 85, 86, 94 is significantly lower than those without these sites. AE subtype induces lower apoptotic behavior in the hoist cells, and decreased activation of the caspase-3 pathway may be one of the mechanisms.

[Influence of intranasal instilled titanium dioxide nanoparticles on monoaminergic neurotransmitters of female mice at different exposure time].

OBJECTIVE: To investigate the influence of intranasal instilling titanium dioxide (TiO2) nanoparticles on monoaminergic neurotransmitters at different-time exposure. METHODS: CD female mice were intranasally instilled three different-sized (25 nm, 80 nm and 155 nm) TiO, suspension every other day in a dose of 50 mg/kg body weight. The control group was instilled the same volume of Milli-Q water. Inductively coupled plasma-mass spectrometry (ICP-MS) was used to analyze the titanium contents in murine brain after exposure to TiO2 particles 2 days, 10 days, 20 days and 30 days. The monoaminergic neurotransmitters such as norepinephrine (NE), dopamine (DA), 5-hydroxytryptamine (5-HT), 5-hydroxyindole acetic acid (5-HIAA), 3, 4-dihydroxyphenylacetic acid (DOPAC), and homovanillic (HVA), were determined by reversed-phase high performance liquid chromatography (RP-HPLC) with electrochemical detector. RESULTS: After exposure to TiO, nanoparticles 10 days, the titanium contents in murine brain were increased, the titanium content in the 25 nm group was up to (1059.3 +/- 293.5) ng/g. In 20 days, the titanium content decreased slowly with the metabolism of titanium in vivo, but it kept at a high level, the content decreased to (654.7 +/- 269.2) ng/g in the 25 nm group. After exposure to TiO2 nanoparticles 30 days, the titanium contents had no obviously change. Because of the accumulation of TiO, in the brain, the contents of NE and 5-HT increased significantly after exposure to 80 nm and 155 nm TiO, nanoparticles 20 days, while the decreased contents of DA, DOPAC, HVA and 5-HIAA were observed. CONCLUSION: The inhaled TiO2 nanoparticles could be translocated to and deposited in murine brain after absorbing by nasal mucosa, and further influence the releases and metabolisms of monoaminergic neurotransmitters in brain.