Objective:To investigate the role of miR-144-3p in cisplatin resistance of bladder cancer.Methods:Bladder cancer T24 cells were cultured in vitro and divided into blank group (untreated), mimetic control group, miR-144-3p mimetic transfection group, inhibitor control group, and miR-144-3p inhibitor transfection group. Real time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to verify the transfection effect, methyl thiazolyl tetrazolium (MTT) method was used to detect the survival rate of cells treated with cisplatin in each group, and Western blot was used to detect the expression of the target protein. The targeting relationship between miR-144-3p and nuclear factor E2 related factor 2 (Nrf2) was validated using dual fluorescence reporter gene experiments. Furthermore, Nrf2 was knocked out in each group of cells, and the mRNA and protein expression levels of HO-1, Bcl-2, and Caspase-3 were detected by qRT-PCR and Western blot in each group of cells.Results:Compared with the control group, bladder cancer cells in the miR-144-3p mimetic transfection group were more sensitive to cisplatin, while the miR-144-3p inhibitor transfection group had the opposite effect; The miR-144-3p simulant transfection group can effectively inhibit the mRNA and protein expression level of Nrf2 in bladder cancer cells (all P<0.05), while the miR-144-3p inhibitor transfection group can up regulate the mRNA and protein level of Nrf2 (all P<0.05). The miR-144-3p mimetic transfection group showed significant downregulation of mRNA and protein expression of HO-1 and Bcl-2, while the expression of Caspase-3 was upregulated (all P<0.05), while the miR-144-3p inhibitor transfection group showed the opposite results. The luciferase results confirmed that miR 144 3p can directly bind to the 3′- UTR region of Nrf2, reducing the mRNA level of Nrf2. When Nrf2 was knocked out, whether miR-144-3p mimetic transfection group or miR-144-3p inhibitor transfection group, the mRNA and protein expression levels of HO-1, Bcl-2 and Caspase-3 did not change significantly, and miR-144-3p lost the ability to regulate the cisplatin sensitivity of bladder cancer cells. Conclusions:miR-144-3p targets to regulate the sensitivity of Nrf2 to cisplatin in bladder cancer, and miR-144-3p is expected to become a new target for the treatment of cisplatin resistant or refractory bladder cancer.

AIM:To investigate the effects of the γ-aminobutyric acid(GABAergic)neural pathway from the anterior cingulate cortex(ACC)to the nucleus accumbens(NAc)on the regulation of irritable bowel syndrome(IBS)and its underlying mechanisms in mice.METHODS:(1)A C57BL/6J mice model of IBS was established by using chronic acute combing stress(CACS).The mice were divided into a normal group and an IBS group(n=8).The presence of IBS-like symptoms was determined through behavioural tests,an intestinal motility test and abdominal withdrawal reflex scores.(2)Fluorescence gold(FG)retrograde tracing and immunohistochemistry were used to investigate the ACC-NAc GABAergic neural pathway and to examine the activation of GABA in the ACC in IBS mice(n=8).(3)A total of 1.5 μL of normal saline(NS),GABAA receptor antagonist bicuculline(BIC)or agonist isoguvacine hydrochloride(Isog)was ad-ministered via a preburied catheter into the NAc of mice in IBS and normal groups.The mice were randomly divided into three groups(n=8):NS group,BIC group and Isog group.IBS-like symptoms were assessed.(4)The mice were prein-jected with AAV2/9-mDlx-iCre-WPRE-pA in the ACC and AAV2/2Retro Plus-hSyn-DIO-hM3D(Gq)-eGFP-WPRE-pA in the NAc and subsequently divided into four groups(n=8):NS(intraperitoneal injection)+NS(NAc microinjection)group,NS+BIC group,clozapine N-oxide(CNO)+NS group and CNO+BIC group.The mice who received AAV2/2Retro-hSyn-DIO-hM4D(Gi)-EGFP-WPRE-pA in the NAc were randomly divided into three groups(n=8):NS+NS group,NS+BIC group and CNO+NS group.Enzyme-linked immunosorbent assay(ELISA)was employed to estimate the expression levels of histamine and 5-hydroxytryptamine(5-HT)in colon tissue,and the effects of GABAergic neural pathways from ACC to NAc on IBS were studied.RESULTS:CACS induced IBS-like symptoms in mice.The results of FG retrograde tracing combined with fluorescence immunohistochemistry showed that GABA neurons of ACC could project to NAc.The injection of BIC in the NAc was found to significantly reduce anxiety-like behaviours,diarrheal symptoms and visceral hy-persensitivity in the IBS mice(P<0.05).Chemogenetic inhibition of the ACC-NAc GABAergic neurons ameliorated IBS-like symptoms in mice(P<0.05).CONCLUSION:The GABAergic pathway of ACC-NAc might be involved in the regu-lation of IBS in mice,which may be related to the release of histamine and 5-HT in colon tissue.

Objective:To analyze the gene variants in patients with primary distal renal tubular acidosis (dRTA), and explore the correlation between the genotype and phenotype.Methods:The Sanger direct sequencing or whole-exome sequencing was used to identify causal variants and the variation pathogenicity was evaluated according to 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines in 44 dRTA patients (37 families) diagnosed in the Affiliated Qingdao Municipal Hospital of Qingdao University and the Affiliated Hospital of Qingdao University from April 2010 to September 2020. The clinical features of the patients were summarized, and the correlation between the genotype and phenotype was investigated.Results:Seven variants of SLC4A1 gene, 17 variants of ATP6V0A4 gene, and 15 variants of ATP6V1B1 gene were identified in 44 patients with dRTA, and of which 11 variants were new ones. According to ACMG guidelines, the pathogenic, likely pathogenic, benign variants among the 39 variants were 22, 16 and 1, respectively. Nine patients were autosomal dominant hereditary dRTA caused by SLC4A1 gene mutation, 4 patients with autosomal recessive hereditary dRTA complicated with Southeast Asian ovalocytosis and anemia were caused by SLC4A1 gene mutation, and 14 patients caused by ATP6V0A4 gene mutation and 8 patients caused by ATP6V1B1 gene mutation were autosomal recessive hereditary dRTA; Two children with dRTA were found to carry one monoallelic defect in ATP6V1B1, and no causal gene mutation was identified in 7 patients. One patient showed incomplete dRTA, and the other 43 patients showed complete dRTA. The prevalence of sensory neural hearing loss caused by ATP6V0A4 and ATP6V1B1 mutation were 2/14 and 6/10 respectively. The frequency of chronic kidney disease in adults, children and infants were 4/4, 2/4, and 1/36, separately. After the drug treatment based on potassium citrate and sodium citrate, the growth and development (28/40) and electrolyte disturbance (41/44) of most patients were significantly improved. Conclusions:The present study has identified 39 variants of SLC4A1, ATP6V0A4 and ATP6V1B1 genes in 44 patients with dRTA, including 11 novel ones. There is a close relationship between genotype and phenotype in dRTA patients and most patients' conditions were improved after proper treatment. This study enriches the human gene mutation database and provides valuable references for diagnosis, treatment and genetic counseling in patients with dRTA.

Objective: To identify and analyze the variants of the KCNJ1 gene in five Chinese patients with Bartter syndrome type 2 (BS2), and to describe their clinical features as well as treatment results. Methods: Data and blood samples of five BS2 patients and their relatives confirmed by Qingdao Municipal Hospital from June 2012 to January 2019 were collected. Whole-exome-sequencing (WES) based on the second generation high throughput sequencing was performed to detect variants. The 2015 American College of Medical Genetics and Genomics Standards and Guidelines were applied to analyze the pathogenicity of the variants. The clinical features and laboratory results were retrospectively studied. The response to treatment and follow-up data were reviewed. Results: Ten variants including six novel ones of KCNJ1 gene were identified through WES and verified by Sanger dideoxy sequencing. Missense variants accounted for the highest proportion. The common symptoms and signs of five BS2 patients from high to low incidence were polydipsia and polyuria (5/5), one of them (1/5) presented with diabetes insipidus; maternal polyhydramnios and premature delivery (4/5); growth retardation (3/5). Initially, two patients presented with hypochloremic metabolic alkalosis and hypokalemia, whereas the acid-base disturbance was absent in the others. One patient experienced hyperkalemia. In terms of calcium-phosphorus metabolism, one patient had evident parathyroid hormone (PTH) resistance (hypocalcemia, hyperphosphatemia and markedly elevated serum intact PTH levels), three presented with PTH overacting (hypercalcemia, hypophosphatemia and mild elevated serum intact PTH levels), and one showed normal blood calcium and phosphorus concentrations with high-normal serum intact PTH levels. All patients had nephrocalcinosis or hypercalciuria, and one of them complicated with nephrolithiasis. Indomethacin helped to correct the growth retardation, halt polydipsia polyuria, decrease the elevated urinary calcium excretion, and normalize electrolyte disturbance as well as PTH parameters in some patients. Conclusions This investigation identifies ten variants of KCNJ1 gene, including six ones that have not been previously reported, which will enrich the human gene mutation database (HGMD). These patients in our study have atypical BS phenotype, so that careful differentiation from other parathyroid diseases will be required for clinicians.