[Long-term efficacy of low-dose rituximab treatment in patients with primary membranous nephropathy].

Objective: To explore the long-term efficacy of low-dose rituximab (RTX) treatment in patients with primary membranous nephropathy (PMN). Methods: Patients with biopsy-proven PMN who received low-dose RTX as initial or second-line regimen from August 2018 to May 2020 in the Department of Nephrology, Tianjin Medical University General Hospital were respectively enrolled. The clinical parameters of patients were urinary protein>3.5 g/24 h, serum albumin<30 g/L and estimated glomerular filtration rate (eGFR)>20 ml·min-1·(1.73 m2)-1. The treatment response of patients with PMN was observed during follow-up, and the remission rate of patients with urinary protein<8 g/24 h or ≥8 g/24 h, anti-PLA2R antibody<150 RU/ml or ≥150 RU/ml, eGFR≥ 60 ml·min-1·(1.73 m2)-1 or<60 ml·min-1·(1.73 m2)-1 were analyzed, respectively. Results: A total of 40 patients were enrolled, including 26 males and 14 females, aged (53±15) years. There were 14 patients received RTX as initial treatment and 26 patients as second-line therapy. The total median dose of RTX in the first course was 800 (425, 1 075) mg. The overall remission rate at the 1st, 3rd, 6th, 12th and 24th months were 12.5% (5/40), 17.5% (7/40), 47.5% (19/40), 57.5% (23/40), 60% (24/40), respectively. The median overall response time was 6.0 (3.0, 7.5) months. Two cases relapsed. Patients with remission (n=24) had a higher level of baseline eGFR [(93.9±28.0) vs (62.4±28.1) ml·min-1·(1.73 m2)-1, P=0.001), and a lower level of both urinary protein [5.9 (5.0, 6.5) vs 11.7 (8.6, 15.5) g/24 h, P<0.001] and anti-PLA2R antibody level [73 (29, 132) vs 453 (182, 950) RU/ml, P=0.004] than those without remission (n=16) 24 month after treatment. There was no statistically significant difference in the remission rate between initial and second-line treatment (P=0.101). Moreover, patients had a higher remission rate in urinary protein<8 g/24 h group (21/26 vs 3/14, P<0.001), anti-PLA2R antibody<150 RU/ml group (16/19 vs 5/16, P=0.002) and eGFR ≥ 60 ml·min-1·(1.73 m2)-1 group (22/29 vs 2/11, P=0.003). Conclusions: Low-dose RTX treatment in PMN is effective during long-term follow-up, and has a lower recurrence rate. The results also suggest that it is more suitable for patients with baseline urinary protein<8 g/24 h, anti-PLA2R antibody<150 RU/ml and eGFR≥ 60 ml·min-1·(1.73 m2)-1.

Sevoflurane induction alleviates the progression of gastric cancer by upregulating the miR-34a/TGIF2 axis.

OBJECTIVE: This study aims to elucidate how sevoflurane affects the malignant progression of gastric cancer (GC) and its pharmacological mechanism. MATERIALS AND METHODS: Dose-dependent and time-dependent regulations of sevoflurane on proliferation inhibition rate in AGS and BGC-823 cells were examined, and thus the optimal dose and treatment time of sevoflurane on GC cells were selected. Subsequently, proliferative and migratory abilities in sevoflurane-induced AGS and BGC-823 cells (3.4% sevoflurane induction for 6 h) were detected by CCK-8 and transwell assay, respectively. After sevoflurane induction, relative levels of miR-34a and TGIF2 in GC cells were determined by qRT-PCR and Western blot. Regulatory effects of miR-34a on GC cell phenotypes were also assessed. Furthermore, the in vivo function of miR-34a in GC growth was explored by generating xenografted GC in nude mice. RESULTS: Sevoflurane induction time-dependently and dose-dependently enhanced proliferation inhibition rate in AGS and BGC-823 cells. The proliferative and migratory abilities in GC cells induced with 3.4% sevoflurane for 6 h were markedly attenuated. sevoflurane induction upregulated miR-34a, but downregulated TGIF2 in GC cells. TGIF2 was negatively regulated by miR-34a. Notably, overexpression of miR-34a inhibited proliferative and migratory abilities in sevoflurane-induced GC cells, and knockdown of miR-34a yielded the opposite results. In nude mice with xenografted GC tissues, sevoflurane treatment markedly reduced tumorigenic ability, which was improved by knockdown of miR-34a. CONCLUSIONS: Sevoflurane weakens proliferative and migratory abilities in GC by upregulating miR-34a and downregulating TGIF2.

The clinical significance of MIA gene in tumorigenesis of lung cancer.

Lung cancer is a common malignant disease in humans. Both the incidence rate and death rate keep growing in recent years and the prognosis of lung cancer patients is disappointing. Melanoma inhibitory activity (MIA) is a secreted protein and a serum marker for metastasis of melanoma. MIA was reported as an oncogene in several cancers. But its role in lung cancer was unknown. In this study, MIA level was shown to be increased in peripheral blood of 216 patients with lung cancer. And it was expressed much higher in tumor tissues than the normal control. Moreover, MIA expression was associated with the clinical stage of lung cancer. When MIA was knocked down, the viability, migration and invasion of A549 cells were remarkably suppressed. But the cell apoptosis rate was enhanced reversely. In contrast, overexpression of MIA promoted cell proliferation, migration and invasion while cell apoptosis was inhibited. Mechanically, the anti-apoptosis marker Bcl-2 was increased and pro-apoptosis marker Bax was decreased after MIA was overexpressed in A549 cells, and vice versa. The level of PCNA and PI3K/mTOR signaling molecules was also increased when MIA was upregulated but declined after knockdown of MIA. In conclusion, MIA plays an oncogenic role in lung cancer and might be a potential marker for the diagnosis of lung cancer.

Snail1-dependent cancer-associated fibroblasts induce epithelial-mesenchymal transition in lung cancer cells via exosomes.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is an essential component of metastasis. Our previous study demonstrated that cancer-associated fibroblasts (CAFs) induce EMT in lung cancer cells. In recent years, many studies have demonstrated that CAFs induce metastasis and drug resistance in cancer cells via exosomes. AIM: We sought to discover the mechanism underlying how CAFs induce EMT in lung cancer cells, unveiling the role of exosomes in lung cancer progression. DESIGN: We cultured lung cancer cell (i) with control medium, normal fibroblasts (NFs) or CAFs; (ii) with SNAI1-transfected or NC (negative control)-transfected CAFs; (iii) with exosomes extracted from NF- or CAF-conditioned medium; (iv) with exosomes released by SNAI1 or NC-transfected CAFs; (v) with CAF-conditioned medium or exosome-depleted CAF-conditioned medium. METHODS: qRT-PCR was conducted to examine the expression of CDH1 (gene of E-cadherin) and VIM (gene of Vimentin), western blotting was conducted to examine E-cadherin and vimentin levels in lung cancer cells. RESULTS: Exosomes released by CAFs-promoted EMT in lung cancer cells. Interestingly, SNAI1 levels in exosomes secreted from CAFs were correlated with SNAI1 expression in CAFs. Furthermore, the level of SNAI1 in exosomes was crucial for inducing EMT in lung cancer cells. Finally, treatment of CAFs with GW4869, an inhibitor of exosome release, noticeably inhibited their EMT-inducing effect on recipient epithelial cells. CONCLUSIONS: The molecular mechanism underlying how CAFs induce EMT in cancer cells may be that CAFs deliver SNAI1 to recipient cancer cells via exosomes.

[Role of miR-155 in invasion and metastasis of lung adenocarcinoma A549 cells].

OBJECTIVE: To investigate the role and mechanism of miR-155 in invasion and metastasis of lung adenocarcinoma A549 cells. METHODS: Real-time PCR and fluorescence in situ hybridization were used to detect the miR-155 expression in patients' lung adenocarcinoma and adjacent tissue and lymph nodes. Scratch test and Transwell migration assay were used to assess the effect of miR-155 on the A549 cell migration and invasion capability. Bioinformatics software was used to predict the target genes of miR-155, and using luciferase to assay the target gene. Western blot and real-time PCR were performed to confirm the role of miR-155 expression in the regulation of target gene PTEN. RESULTS: The real-time quantitative PCR showed that the miR-155 expression levels in adjacent normal tissue, lung adenocarcinoma and metastatic lymph nodes were 4.1±0.5, 9.6±3.1 and 7.8±2.2, respectively. The in situ hybridization showed that the expression rates of miR-155 in the adjacent normal tissue, lung adenocarcinoma and metastatic lymph nodes were (23.2±15.3)%, (75.4±20.2)% and (60.4±25.1)%, respectively. The Scratch assay showed that the wound healing rates in the miR-155 mimics group, miR-155 mimics NC group, miR-155 inhibitor group and miR-155 inhibitor NC group at 24 h were (43.2±2.2)%, (21.3±4.2)%, (24.3±5.3)%, and (35.2±5.1)%, and that at 48 h were (75.2±4.5)%, (52.6±5.2)%, (39.4±4.2)%, and (51.5±4.3)%, respectively. Dual luciferase reporter gene assay showed that the value of the luciferase in the miR-155 mimics group co-transfected with PTEN 3'UTR-containing wild-type and mutant plasmids were 4.7±0.5 and 7.3±0.7, and the miR-155 mimics luciferase values of the control group co-transfected with PTEN 3'UTR-containing wild-type and mutant plasmids were 7.8±0.9 and 7.5±0.8, respectively. The real-time quantitative fluorescence PCR showed that the relative expression of PTEN protein in the miR-155 mimics group, miR-155 mimics control group, miR-155 mimics inhibitor group, and miR-155 inhibitor control group were 0.5±0.3, 1.0±0.1, 2.2±0.2 and 1.2±0.1, respectively. The Western blot assay detected that the relative expression of PTEN protein levels in the miR-155 mimics group, miR-155 mimics control group, miR-155 inhibitor group and miR-155 inhibitor control group were 0.4±0.1, 1.0±0.3, 2.8±0.2 and 1.4±0.1, respectively. The differences in PTEN mRNA and protein expressions of the four groups were statistically significant (P<0.05 for all). CONCLUSIONS: miR-155 may promote the invasion and metastasis of lung adenocarcinoma through reducing the target PTEN gene expression.

Novel polymorphic microsatellite markers for Bellamya and their application in population genetics of three species.

Bellamya is a widely distributed freshwater snail genus in China; however, its genetic diversity is completely unknown. Sixty-five novel microsatellite loci were isolated and characterized from a microsatellite-enriched library of Bellamya aeruginosa genomic DNA. Most of the 65 loci were successfully amplified. We found high polymorphic information content values for these loci, ranging from 0.235 to 0.892. There were 3 to 12 alleles per locus, and the HE and HO varied from 0.425 to 0.953 and 0.026 to 1.000, respectively. Fifteen loci deviated significantly from Hardy-Weinberg equilibrium after Bonferroni's correction. All 65 SSR markers were tested in an additional five Bellamya species, and 96.9% of the 325 locus/taxon combinations tested resulted in cross-species amplification. Seven polymorphic microsatellite markers were randomly selected for comparison among nine populations of three species. All populations had moderate to high genetic diversity. In genetic distance-based cluster analysis, the populations of B. aeruginosa and B. dispiralis formed species-based clusters, whereas populations of B. angularia did not. The three examined Bellamya species could be differentiated using SSR markers. These microsatellite loci should be useful for genetic diversity analysis, analysis of phylogenetic relationship, and species delimitation of Bellamya.

Development of 18 microsatellite loci for the freshwater snail Bellamya aeruginosa (Mollusca, Gastropoda).

Although it is a major freshwater gastropod species, genetic diversity of Bellamya aeruginosa was completely unknown. Eighteen microsatellite loci were isolated and characterized from (AC)(15)-enriched genomic libraries of the freshwater snail B. aeruginosa. Most of the 18 loci were successfully amplified and high polymorphic information content values were found, ranging from 0.244 to 0.792 (mean 0.541). The number of alleles per locus ranged from 5 to 13 (mean 8.8), the expected heterozygosity varied from 0.347 to 0.950 (mean 0.815) and the observed heterozygosity varied from 0.087 to 0.782 (mean 0.431). Eight loci showed significant deviation from Hardy-Weinberg equilibrium after Bonferroni's correction and no significant genotypic linkage disequilibrium was detected between most locus pairs, except for TXH79-TXH97 and TXH113-TXH121. These 18 polymorphic microsatellite loci should be useful for population genetics analysis and species identification of Bellamya.

[Modulation of bradykinin on ATP-activated currents in isolated rat DRG neurons].

Modulation of bradykinin (BK) on ATP-activated membrane currents in the isolated rat dorsal root ganglion (DRG) neurons was investigated using whole-cell patch-clamp technique. In 56 neurons examined, BK (10(-6)-10(-4) mol/L)-induced responses were as follows: (1) inward current (40/56); (2) outward current (7/56); (3) no responses (9/56). In the majority of the neurons examined, ATP (10(-6)-10(-3) mol/L) activated a concentration-dependent inward current with obvious desensitization. ATP-activated current was potentiated markedly by preapplication of BK. The enhancement of BK was dependent on the concentration of BK and ATP. It was found that BK potentiated the peak value of ATP-activated currents predominately while the steady state value was not affected obviously. The onset of BK potentiation of ATP-activated currents needed preincubation of BK at least for 30 s and the time of the enhancement lasted over 20 min. The results suggest that BK may play a role in the facilitation of the excitatory effect of ATP on primary sensory neurons.