Objective:To investigate the effect of CIRc_0000267 on proliferation, migration, invasion and apoptosis of gastric cancer cells in vitro.Methods:Gastric cancer cell lines with circ_0000267 knockdown and miR-661 overexpression were constructed in vitro, and the expressions of circ_0000267, miR-661 and NGAL mRNA in gastric cancer tissues and cells were detected by qRT-PCR. Cell proliferation, migration, invasion and apoptosis were detected by clonogenesis, Transwell chamber and flow cytometry, and related protein expression was detected by Western blot. Online prediction combined with double luciferase assay and RNA pull down assay was used to verify the targeting relationship between circ_0000267, miR-661 and NGAL. Tumogenesis assay in nude mice was used to observe the effect of circ_0000267 knockout on the growth of gastric cancer cells in vivo. Results:Circ_0000267 was highly expressed in gastric cancer tissues and cells. Knockdown circ_0000267 inhibited the proliferation, migration and invasion of gastric cancer cells and promote apoptosis. Circ_0000267 targeted miR-661, and inhibition of miR-661 partially reversed the effect of circ_0000267 on gastric cancer cells. NGAL was the target gene of miR-661, and overexpression of miR-661 regulated the proliferation, migration, invasion and apoptosis of gastric cancer cells by inhibiting NGAL. Knockdown circ_0000267 targeting miR-661 inhibited NGAL expression in gastric cancer cells; Knockdown circ_0000267 inhibited the growth of gastric cancer cells in vivo.Conclusions:Circ_0000267 may regulate the proliferation, migration, invasion and apoptosis of gastric cancer cells and inhibit the growth of tumors in vivo by regulating the expression of miR-661/NGAL.

Objective:To investigate the effects of serum exosomes of patients with gastric cancer on cisplatin resistance, clonal formation, migration, invasion and apoptosis of the AGS gastric cancer cells, and the corresponding molecular mechanisms.Methods:The experimental study was conducted. The exosomes of patients with gastric cancer was separated from their serum, and the expression of lncRNA HOTTIP was analyzed using the quantitative real time polymerase chain reaction (qRT-PCR). Normal gastric epithelial cell line GES1, gastric cancer cell line AGS and human embryonic kidney cell 293T were cultured in vitro. AGS cells were incubated with exosomes (Exo),with phos-phate buffered saline (PBS) treatment as control, and transfected with si-NC or si-HOTTIP-3, named as Exo group, PBS group, si-NC+Exo group, and si-HOTTIP-3+Exo group. The AGS cells were trans-fected with si-NC, si-HOTTIP-1, si-HOTTIP-2, si-HOTTIP-3, oe-HOTTIP, vector, oe-HOTTIP+miR-138-5p mimic, oe-HOTTIP+mimic NC, miR-138-5p inhibitor, inhibitor NC, miR-138-5p inhibitor+si-TJP1 and miR-138-5p inhibitor+si-NC. They were recorded as si-NC group, si-HOTTIP-1 group, si-HOTTIP-2 group, si-HOTTIP-3 group, oe-HOTTIP group, vector group, oe-HOTTIP+miR-138-5p mimic group, oe-HOTTIP+mimic NC group, miR-138-5p inhibitor group, inhibitor NC group, miR-138-5p inhibitor+si-TJP1 group and miR-138-5p inhibitor+si-NC group. The 293T cells transfected with mimic NC+HOTTIP wt, miR-138-5p mimic+HOTTIP wt, mimic NC+HOTTIP mut, miR-138-5p mimic+HOTTIP mut, mimic NC+TJP1 3'UTR wt, miR-138-5p mimic+TJP1 3'UTR wt, mimic NC+TJP1 3'UTR mut, miR-138-5p mimic+TJP1 3'UTR mut were recorded as the mimic NC+HOTTIP wt group, miR-138-5p mimic+HOTTIP wt group, mimic NC+HOTTIP mut group, miR-138-5p mimic+HOTTIP mut group, mimic NC+TJP1 3'UTR wt group, miR-138-5p mimic+TJP1 3'UTR wt group, mimic NC+TJP1 3'UTR mut group, miR-138-5p mimic+TJP1 3'UTR mut group. The cell counting kit-8 (CCK8) was used to analyze the cisplatin sensitivity of gastric cancer cells. The colony formation experiment was used to analyze the colony formation of gastric cancer cells. The Transwell experiment was used to analyzed cell migration and invasion of gastric cancer cells. The flow cytometry experiment was used to analyze cell apoptosis of gastric cancer cells. The Western bolt assay was used to analyze the expression of exosome marker proteins, including the CD63 and CD81, and the protein of TJP1, the drug-resistance related proteins, including the P-gp and MCL-1. The dual-luciferase assay was used to analyze the targeted relationships among lncRNA HOTTIP, miR-138-5p and TJP1. Observation indicators: (1) expression of lncRNA HOTTIP; (2) resistance of gastric cancer cells to cisplatin regulated by exosome-mediated lncRNA HOTTIP; (3) regulation of cisplatin resistance in gastric cancer cells mediated by miR-138-5p through lncRNA HOTTIP overexpression; (4) targeting of TJP1 gene 3′-untranslated region (UTR) by miR-138-5p; (5) regulation of cisplatin resistance in gastric cancer cells by TJP1 through miR-138-5p inhibition. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was conducted using the t test. The one-way ANOVA was used for comparison for multiple groups and the Tukey′s test was used for further pairwise compari-son. Count data were described as absolute numbers, and the chi-square test was used for comparison. Correlation analysis was conducted using the Pearson′s test. Results:(1) Expression of lncRNA HOTTIP. The expression of lncRNA HOTTIP in the serum exosome of patients with gastric cancer was higher than that in healthy volunteers, showing a significant difference ( P<0.05). Results of transmi-ssion electron microscopy examination showed that the serum exosomes were circular or oval in shape. Results of Western bolt assay showed the expression of marker proteins of CD63 and CD81 in serum exosomes. (2) Resistance of gastric cancer cells to cisplatin regulated by exosome-mediated lncRNA HOTTIP. Compared with the PBS group, the biochemical half maximal inhibitory concentra-tion (IC50), the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P-gp protein, expression of MCL-1 protein in the Exo group increased, while the cell apoptosis rate decreased, showing significant differences between them ( P<0.05). Compared with the si-NC+Exo group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P-gp protein, expression of MCL-1 protein in the si-HOTTIP-3+Exo group decreased, while the cell apoptosis rate increased, showing significant differences between them ( P<0.05). (3) Regulation of cisplatin resistance in gastric cancer cells mediated by miR-138-5p through lncRNA HOTTIP overexpression. Compared with the vector group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P-gp protein, expression of MCL-1 protein in the oe-HOTTIP group increased, while the cell apoptosis rate decreased, showing significant differences between them ( P<0.05). Compared with the oe-HOTTIP+mimic NC group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P-gp protein, expression of MCL-1 protein in the oe-HOTTIP+miR-138-5p mimic group increased, while the cell apoptosis rate decreased, showing significant differences between them ( P<0.05). (4) Targeting of TJP1 gene 3′-UTR by miR-138-5p. Results of dual-luciferase assay showed that the luciferase activity in 293T cells treatment with mimics of control+vectors of wild type of TJP1 gene 3′-UTR and 293T cells treatment with mimics of miR-138-5p+vectors of wild type of TJP1 gene 3′-UTR was 1.00±0.09 and 0.21±0.03, respectively, showing a significant difference between them ( t=15.02, P<0.05). (5) Regulation of cisplatin resistance in gastric cancer cells by TJP1 through miR-138-5p inhibition. Compared with the inhibitor group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P-gp protein, expression of MCL-1 protein in the miR-138-5p inhibitor group increased, while the cell apoptosis rate decreased, showing significant differences between them ( P<0.05). Compared with the miR-138-5p inhibitor+si-NC group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P-gp protein, expression of MCL-1 protein in the miR-138-5p inhibitor+si-TJP1 group decreased, while the cell apoptosis rate increased, showing significant differences between them ( P<0.05). Conclusion:Serum exosomes-mediated lncRNA HOTTIP can promote cisplatin resistance, clonal formation, migration, invasion and apoptosis of gastric cancer cells and inhibit cell apoptosis of gastric cancer cells through regulating the expression of miR-138-5p/TJP1.

Objective: To examine genetic variants of familial hematuria (FH) associated genes in 3 families with hematuria with probands initially diagnosed with IgA nephropathy (IgAN). Methods: A retrospective analysis was performed on the clinical data, laboratory tests and genetic test results of three children with hematuria and the probands in three families with hematuria. The families were ascertained at the Department of Pediatrics, Fuzhou General Hospital of Nanjing Military Command from August 2014 to May 2018. Results: The proband of Family One, an 8-year-old boy, manifested gross hematuria. His renal biopsy pathology revealed IgAN. His father also manifested hematuria. Genetic testing showed that the proband and his father carried a heterozygous variant of the CFHR5 gene,533A>G (Asn178Ser). The child of Family Two, a 4-year-old girl, manifested hematuria. Her father, the proband of the family, was 36 years old, and manifested hematuria, proteinuria, high-frequency sensorineural deafness and renal insufficiency. He was diagnosed as IgAN according to clinical manifestations, renal pathology and routine immunohistochemistry without renal biopsy electron microscopy, renal tissue type Ⅳ collagen α3, α4, α5 chains immunofluorescence and skin type Ⅳ collagen α5 chain immunofluorescence. Genetic testing showed that the girl carried a heterozygous variant of the COL4A5 gene,566G>T (Gly189Val), and her father carried the hemizygous variant. The child of Family Three, a 7-year-old girl, manifested hematuria and proteinuria. Her mother, the proband of the family, was 34 years old, and manifested hematuria and proteinuria as well. The proband was diagnosed as IgAN by the same method used for Family Two. The girl′s grandfather died of uremia at the age of 44. Genetic testing showed that the girl and her mother carried a heterozygous variant 539G>A (Gly180Glu)in COL4A5 gene. Conclusions The variant of the CFHR5 gene identified in Family One is of uncertain signifance, and the two variants of the COL4A5 gene identified in Families Two and Three are pathogenic. The probands of Families Two and Three are diagnosed as Alport syndrome. The study suggests that clinicians should examine genetic variants of FH associated genes in families with hematuria when the probands were diagnosed as IgAN by their clinical manifestations, renal pathology and routine immunohistochemistry.