Genomic profiling of non-small cell lung cancer with the rare pulmonary lymphangitic carcinomatosis and clinical outcome of the exploratory anlotinib treatment.

Background: To evaluate the potential treatment for patients with non-small cell lung cancer (NSCLC) and rare malignant pulmonary lymphangitis carcinomatosis (PLC), our study provided a genomic profile and clinical outcome of this group of patients. Methods: We retrospectively reviewed patients with NSCLC who developed PLC. The genomic alterations, tumor mutation burden (TMB), and microsatellite instability (MSI) based on DNA-based next-generation sequencing were reviewed and compared in a Chinese population with lung adenocarcinomas (Chinese-LUAD cohort). Clinical outcomes after exploratory anlotinib treatment and factors influencing survival are summarized. Results: A total of 564 patients with stage IV NSCLC were reviewed, and 39 patients with PLC were included. Genomic profiling of 17 adenocarcinoma patients with PLC (PLC-LUAD cohort) revealed TP53, EGFR, and LRP1B as the three most frequently altered genes. EGFR was less mutated in PLC-LUAD than Chinese-LUAD cohort of 778 patients (35.3% vs. 60.9%, P = 0.043). BRIP1 was mutated more often in the PLC-LUAD cohort (11.8% vs. 1.8%, P= 0.043). Two patients presented with high tumor mutational burden (TMB-H, 10 mutations/MB). Combing alterations in the patient with squamous cell carcinoma, the most altered pathways of PLC included cell cycle/DNA damage, chromatin modification, the RTK/Ras/MAPK pathway and VEGF signaling changes. Fourteen of the participants received anlotinib treatment. The ORR and DCR were 57.1% and 92.9%, respectively. Patients achieved a median progression-free survival of 4.9 months and a median overall survival of 7 months. The adverse effects were manageable. In patients with adenocarcinoma, the mPFS (5.3 months vs. 2.6 months) and mOS (9.9 months vs. 4.5 months) were prolonged in patients receiving anlotinib treatment compared to those receiving other treatment strategies (P < 0.05). Conclusion: Patients with PLC in NSCLC demonstrated distinct genetic alterations. The results improve our understanding of the plausible genetic underpinnings of tumorigenesis in PLC and potential treatment strategies. Exploratory anlotinib treatment achieved considerable benefits and demonstrated manageable safety.

Echinacoside Induces Mitochondria-Mediated Pyroptosis through Raf/MEK/ERK Signaling in Non-Small Cell Lung Cancer Cells.

Background: Various natural compounds are effective in cancer prevention and treatment with fewer side effects than conventional radiotherapy and chemotherapy. Considering the uncertainty of the antitumor mechanism of Echinacoside (Ech) and the fact that no study on Ech against non-small cell lung cancer (NSCLC) has been explored previously, this study inquired into the anti-NSCLC effect of Ech and explored its potential mechanisms. Methods: The IC50 to Ech of the NSCLC cells was calculated based on a series of cell viability assays. Different concentrations of Ech were used to treat the cells; the proliferation activity of the cells was evaluated using EdU staining. Mitochondrial membrane potential was detected by JC-1 staining. Levels of cytokines IL-1ß and IL-18 were measured by ELISA. GSH and MDA levels were measured by microplate reader. Expression of cytochrome c, NLRP3, caspase-1, IL-1ß, c-Myc, c-Fos, and Raf/MEK/ERK pathway proteins was evaluated by western blot. Meanwhile, we used xenograft, immunohistochemical staining, and H&E staining to evaluate the pharmacological effects of Ech in mice in vivo. Results: ECH inhibited the proliferation of NSCLC cells. Ech increased the expression of pyroptosis-related proteins. Besides, Ech perturbed the mitochondrial membrane potential with the release of mitochondrial cytochrome c, accompanied by increased oxidative stress. Ech inhibited the phosphorylation levels of Raf/MEK/ERK signaling pathway and subsequently reduced c-myc and c-fos protein expression. In addition, Ech effectively restrained the growth of tumors in vivo. Conclusions: Ech inhibited the Raf/MEK/ERK signaling. Impaired mitochondria activated inflammasome, which in turn led to the pyroptosis of NSCLC cells. These findings can provide some ideas on how to use pyroptosis to treat NSCLC.

CHML targeted by miR-199a-3p promotes non-small cell lung cancer cell growth via binding to Rab5A.

Choroideremia-like (CHML) has been demonstrated to be related to the development of urothelial carcinoma, multiple myeloma, and hepatocellular carcinoma. Whereas, the association between CHML and lung cancer remains dimness. CHML expression was analyzed in NSCLC patients from TCGA dataset and evaluated in our collected NSCLC tissues and NSCLC cell lines. The effects of CHML on the proliferation and apoptosis of NSCLC were investigated in A549 and H1299 cells that downregulation of CHML as well as in H1299-induced xenograft mouse model. An upstream miRNA of CHML was further analyzed. Moreover, bioinformatics analysis and co-immunoprecipitation assay were carried out to explore the mechanism of CHML in NSCLC. We found CHML expression was upregulated in NSCLC patients and cell lines compared with their controls. Knockdown of CHML suppressed the viability and BrdU-positive cell number, and elevated the proportion of Tunel-positive cells and levels of Bax/Bcl-2 and cleaved-caspase-3 in NSCLC cells. In mouse models, downregulation of CHML decreased tumor volume and weight, attenuated Ki-67 staining, whereas elevated numbers of Tunel-positive cells, and upregulated levels of Bax/Bcl-2 and cleaved-caspase-3. CHML was demonstrated to be a target of miR-199a-3p. miR-199a-3p inhibitor significantly promoted the proliferation, and attenuated the apoptosis of H1299 cells, which were abrogated by CHML silencing. CHML promoted the proliferation of NSCLC cells via directly binding to Rab5A. Taken together, this study revealed that CHML was an oncogene in NSCLC and it could promote the proliferation and inhibit apoptosis of NSCLC cells through binding to Rab5A. CHML was targeted by miR-199a-3p in this cancer.

A novel pyroptosis-related lncRNA signature for prognostic prediction in patients with lung adenocarcinoma.

Lung adenocarcinoma (LUAD) has been the major cause of tumor-associated mortality in recent years and has a poor prognosis. Pyroptosis is regulated via the activation of inflammasomes and participates in tumorigenesis. However, the effects of pyroptosis-related lncRNAs (PRlncRNAs) on LUAD have not yet been completely elucidated. Therefore, we attempted to systematically explore patterns of cell pyroptosis to establish a novel signature for predicting LUAD survival. Based on TCGA database, we set up a prognostic model by incorporating PRlncRNAs with differential expression using Cox regression and LASSO regression. Kaplan-Meier analysis was conducted to compare the survival of LUAD patients. We further simplified the risk model and created a nomogram to enhance the prediction of LUAD prognosis. Altogether, 84 PRlncRNAs with differential expression were discovered. Subsequently, a new risk model was constructed based on five PRlncRNAs, GSEC, FAM83A-AS1, AL606489.1, AL034397.3 and AC010980.2. The proposed signature exhibited good performance in prognostic prediction and was related to immunocyte infiltration. The nomogram exactly forecasted the overall survival of patients and had excellent clinical utility. In the present study, the five-lncRNA prognostic risk signature and nomogram are trustworthy and effective indicators for predicting the prognosis of LUAD.

Serum lncRNA HAND2-AS1 is downregulated in diabetic patients with chronic renal failure and ameliorates cell apoptosis.

BACKGROUND: LncRNA HAND2-AS1 has been reported to be a tumor suppressor in several types of malignancy, while its involvement in other human diseases is unclear. Our preliminary RNA-seq analysis revealed the downregulation of lncRNA HAND2-AS1 in diabetic patients with chronic renal failure, indicating the involvement of lncRNA HAND2-AS1 in this disease. This study was therefore carried out to explore the role of lncRNA HAND2-AS1 in the development of chronic renal failure in diabetic patients. METHODS: Mouse podocyte cells and plasma samples of diabetic patients (46 diabetic patients with chronic renal failure, 38 diabetic patients without obvious complications and 42 healthy volunteers) were used in this study. Cell apoptosis assay and PCR were performed. RESULTS: LncRNA HAND2-AS1 was downregulated in diabetic patients with chronic renal failure but not in diabetic patients without obvious complications. Downregulation of lncRNA HAND2-AS1 distinguished diabetic patients with chronic renal failure from diabetic patients and healthy controls. High glucose environment did not affect the expression of lncRNA HAND2-AS1 in mouse podocyte cells. Overexpression of lncRNA HAND2-AS1 inhibited the apoptosis of mouse podocyte cells under high glucose treatment. CONCLUSIONS: We therefore conclude that lncRNA HAND2-AS1 may participate in the development of chronic renal failure in diabetic patients by regulating cell apoptosis.

Silencing stomatin-like protein 2 attenuates tumor progression and inflammatory response through repressing CD14 in liver cancer.

PURPOSE: Toll-like receptor 4 (TLR4) is involved in the inflammation in liver cancer. High-expressed stomatin-like protein 2 (SLP-2) is commonly reported in many cancer types. This study aims to investigate the functions of SLP-2 in TLR4-mediated inflammatory responses and tumor progression of liver cancer. PATIENTS AND METHODS: Plasmid transfection technique was applied to silence and overexpress genes. Changes in cell viability and apoptosis were determined by performing cell counting kit-8 assay and flow cytometry. The levels of pro-inflammatory cytokines were determined by ELISA. We further measured the several types of the malignant transformation of SK-Hep1 cells to assess the effects of SLP-2 silencing on the cell migration and invasion, proliferation and angiogenesis of liver cancer in vitro. Western blot and RT-qPCR were performed for expression analysis. RESULTS: Lipopolysaccharide (LPS) promoted the cell proliferation of SK-Hep1 and production of tumor necrosis factor-α (TNF-α) and IL-6. SLP-2 silencing could inhibit the protein and mRNA levels of CD14 and Cdc42 and subsequently inhibited the levels of TNF-α and IL-6. Overexpressed CD14 not only remarkably reversed the proapoptotic ability of SLP-2 silencing and promoted the expression of Cdc42 and production of TNF-α and IL-6, but also notably reversed the inhibitory effects on the malignant abilities of SK-Hep1 cells by SLP-2 silencing. CONCLUSION: SLP-2 silencing could significantly attenuate the inflammatory responses and tumor progression of liver cancer via inhibiting LPS/TLR4 signal transduction through the repression of CD14.

Pathological spectrum of glomerular disease in patients with renal insufficiency: a single-center study in Northeastern China.

Background: To investigate the pathological spectrum of glomerular disease in patients with renal insufficiency (RI) from 2008 to 2017. Methods and results: We calculated the estimated glomerular filtration rate (eGFR) with the Chronic Kidney Disease Epidemiology Collaboration creatinine (CKD-EPI) equation and defined RI as an eGFR <60 ml/min/1.73 m2. A total of 969 RI patients were included in our study. IgA nephropathy (IgAN) was the most common subtype of primary glomerulonephritis (37.2%). The frequencies of IgAN and non-IgA mesangioproliferative glomerulonephritis decreased from 27.3% and 9.5% during 2008-2012 to 20.7% and 2.6% during 2013-2017, respectively. However, the frequency of membranous nephropathy increased from 6.8% to 16.2%. Lupus nephritis was the most common subtype of secondary glomerulonephritis (32.1%). The frequencies of both ANCA-associated systemic vasculitis and diabetic nephropathy increased from 3.8% to 7.6% and from 4.3% to 7.6%, respectively. The number of elderly patients (≥60 years) in our study increased sharply, from 15.6% in 2008 to 35.0% in 2017. Membranous nephropathy, minimal change disease, membranoproliferative glomerulonephritis, lupus nephritis and renal amyloidosis are more frequently observed in the elderly patients than in nonelderly patients (<60 years) (p < .05). Excluding those with acute kidney injury, IgAN was the leading cause of RI (24.9%), followed by membranous nephropathy (13.3%) and lupus nephritis (12.0%). Conclusions: IgAN and lupus nephritis were the most prevalent primary glomerulonephritis and secondary glomerulonephritis in patients with RI, respectively. The frequencies of membranous nephropathy, ANCA-associated systemic vasculitis and diabetic nephropathy increased significantly. The number of elderly patients with RI increased sharply.

Clinical and Pathological Analysis of 4910 Patients Who Received Renal Biopsies at a Single Center in Northeast China.

PURPOSE: To identify the epidemiology and pathological types of kidney diseases and their changes during the past decade, in a population from Northeast China. METHODS: We retrospectively analysed clinical and renal pathological data from 4910 patients who received renal biopsies in the Second Hospital of Jilin University from 2008 to 2017. RESULTS: Males received more renal biopsies than females (p < 0.001). The average age (p < 0.001) and percentage of elderly patients (p < 0.001) increased over time. The pathological types were primary glomerulonephritis (PGN, 73.2%), secondary glomerulonephritis (SGN, 23.7%), tubular-interstitial nephropathy (TIN, 2.8%), and hereditary nephropathy (HN, 0.3%). The most common forms of PGN were membranous nephropathy (MN, 37.2%) and IgA nephropathy (IgAN, 29.9%). Over time, the prevalence of IgAN decreased, but the prevalence of MN increased. MN was more common in middle-aged and elderly patients, but IgAN was most common in young adults. Analysis of SGN data indicated that lupus nephritis (LN, 34.0%), Henoch-Schönlein purpura glomerulonephritis (HSPN, 17.9%), and diabetic nephropathy (DN, 11.7%) were the most common forms. Over time, the prevalence of DN (p = 0.003), hypertension-associated renal damage (p = 0.005), and systemic vasculitis-associated nephritis (SVARD, p < 0.001) increased, but the prevalence of HSPN (p < 0.001) and hepatitis B virus-associated glomerulonephritis (HBV-GN, p = 0.001) decreased. Nephrotic syndrome was the main clinical manifestation of PGN. CONCLUSION: From 2008 to 2017, renal biopsies were increasingly performed in the elderly. There were notable changes in the epidemiology and pathological types of kidney disease among renal biopsy patients at our centre.

IL18 rs360719 A>G, IL18R1 rs13015714 G>T, IL18RAP rs917997 C>T and IL28B rs8099917 T>G polymorphisms and risk of gastric cardiac adenocarcinoma.

The present study was conducted to investigate the association between gastric cardiac adenocarcinoma (GCA) and four functional single-nucleotide polymorphisms (SNPs), including interleukin 18 (IL18) rs360719 A>G, IL18 receptor 1 (IL18R1) rs13015714 G>T, IL18 receptor accessory protein (IL18RAP) rs917997 C>T and interleukin 28B (IL28B) rs8099917 T>G variants. A hospital-based case-control study was performed to evaluate the genetic effects of these SNPs. A total of 243 GCA cases and 476 controls were enrolled in this study. A custom-by-design 48-Plex SNPscan™ kit was used to determine the genotypes. When the IL18 rs360719 AA homozygote genotype was used as the reference group, the AG genotype was not associated with the risk for GCA; the GG genotype was also not associated with the risk for GCA. In the dominant model, the IL18 rs360719 AG/GG variants were not associated with the risk for GCA, compared with the IL18 rs360719 AA genotype. In the recessive model, when the IL18R1 rs13015714 AA/AG genotypes were used as the reference group, the GG homozygote genotype was not associated with risk for GCA. No association was observed between IL18R1 rs13015714 G>T, IL18RAP rs917997 C>T and IL28B rs8099917 T>G polymorphisms and the risk for GCA. These results demonstrated that the functional polymorphisms IL18 rs360719 A>G, IL18R1 rs13015714 G>T, IL18RAP rs917997 C>T and IL28B rs8099917 T>G do not contribute to GCA susceptibility. However, as the statistical power of our study was limited, large well-designed studies and further functional investigations are required to confirm our findings.

Tagging polymorphisms of methyl-CpG binding domain 4 and gastric cardiac adenocarcinoma risk in a Chinese population.

Potential effects of genetic factors on carcinogenesis of gastric cardiac adenocarcinoma (GCA) may exist. The present experiment specifically evaluated the genetic influence of single nucleotide in methyl-CpG binding domain 4 (MBD4) on GCA tumorigenesis. A case-control experiment based on hospital recruited 330 GCA patients and 608 non-cancer patients was carried out. We employed ligation detection reaction method to detect the genotypes. The results revealed that MBD4 rs3138373, rs2005618, and rs3138355 mutations had no significant association with the risk of GCA. However, a lower risk of GCA presented in male patients who carried the MBD4 rs3138355 G>A polymorphic loci by the stratified analyses. In general, The MBD4 gene polymorphism could not influence GCA hereditary predisposition. Nevertheless, whether the finding learned from our experiment could apply to other ethnic groups will remain vague until future multicenter studies further test and verify our conclusions.

Improving the refolding efficiency for proinsulin aspart inclusion body with optimized buffer compositions.

Successfully recovering proinsulin's native conformation from inclusion body is the crucial step to guarantee high efficiency for insulin's manufacture. Here, two by-products of disulfide-linked oligomers and disulfide-isomerized monomers were clearly identified during proinsulin aspart's refolding through multiple analytic methods. Arginine and urea are both used to assist in proinsulin refolding, however the efficacy and possible mechanism was found to be different. The oligomers formed with urea were of larger size than with arginine. With the urea concentrations increasing from 2 M to 4 M, the content of oligomers decreased greatly, but simultaneously the refolding yield at the protein concentration of 0.5 mg/mL decreased from 40% to 30% due to the increase of disulfide-isomerized monomers. In contrast, with arginine concentrations increasing up to 1 M, the refolding yield gradually increased to 50% although the content for oligomers also decreased. Moreover, it was demonstrated that not redox pairs but only oxidant was necessary to facilitate the native disulfide bonds formation for the reduced denatured proinsulin. An oxidative agent of selenocystamine could increase the yield up to 80% in the presence of 0.5 M arginine. Further study demonstrated that refolding with 2 M urea instead of 0.5 M arginine could achieve similar yield as protein concentration is slightly reduced to 0.3 mg/mL. In this case, refolded proinsulin was directly purified through one-step of anionic exchange chromatography, with a recovery of 32% and purity up to 95%. All the results could be easily adopted in insulin's industrial manufacture for improving the production efficiency.

Association between Cytotoxic T-lymphocyte antigen 4 (CTLA-4) +49 G>A (rs231775) polymorphism and esophageal cancer: from a case-control study to a meta-analysis.

The aim of this study was to evaluate the association between CTLA-4 +49 G>A polymorphism and esophageal cancer (EC) susceptibility in a hospital based case-control study and a subsequent meta-analysis. We implemented genotyping analyses for CTLA-4 +49 G>A polymorphism with 629 esophageal squamous cell carcinoma cases and 686 controls in a Chinese Han population. Polymerase chain reaction ligase detection reaction (PCR-LDR) method was used to identify genotypes of CTLA-4 +49 G>A polymorphism. We first assessed the association between CTLA-4 +49 G>A polymorphism and EC risk in a hospital based case-control study, and then performed a comprehensive meta-analysis to derive a more precise estimation. Our results demonstrated that CTLA-4 +49 G>A polymorphism was not associated with EC risk. This case-control study and further meta-analysis, failed to identify the association between CTLA-4 +49 G>A polymorphism and EC risk. And additional, further well designed studies with large sample sizes and detailed gene-environment data are required.

Vitamin D receptor gene polymorphisms and esophageal cancer risk in a Chinese population: a negative study.

Vitamin D receptor (VDR) gene polymorphisms have been reported to influence susceptibility to some malignant cancers. However, there were few published findings on the association between VDR polymorphisms and esophageal cancer susceptibility. Our investigation was aimed to obtain a precise estimation of the association between VDR polymorphisms and esophageal cancer susceptibility. We conducted a hospital-based case-control study to evaluate the genetic effects of functional single-nucleotide polymorphisms VDR rs2107301 T>C, rs2228570 C>T, rs1989969 C>T and rs11568820 G>A on the development of esophageal cancer. A total of 629 esophageal squamous cell carcinoma (ESCC) cases and 686 controls were enrolled for this study. The genotypes were determined using ligation detection reaction method. There were no significant associations between the four VDR variants and ESCC risk. Stratified analyses indicated a significantly increased risk of ESCC associated with VDR rs2107301 T>C polymorphism among patients who were drinking. These findings demonstrated that the risk of ESCC associated with VDR rs2107301 T>C polymorphism may be modified by lifestyle factors such as drinking. However, the results should be validated in larger well-designed studies in future.