Objective:To investigate the molecular mechanism of miR-886-5p targeting BCL-2-associated X protein (BAX) to inhibit the proliferation, migration, and invasion of liver cancer cells.Methods:mRNA expression data of HCC patients were obtained from the Starbase database, including 370 liver cancer samples and 50 normal liver tissue samples adjacent to the cancer. Analyze the expression of miR-886-5p in the previously obtained data and investigate the relationship between miR-886-5p and BAX in liver cancer samples. After transfection of the corresponding plasmids into Huh7 and HepG2 cells, the following groups were established. Analyze the interaction between miR-886-5p and BAX in vitro, detect the protein expression by Western blotting, and verify the targeting relationship between the two by dual luciferase reporter gene assay.Results:Starbase database analysis found that the standardized expression level of miR-886-5p in 370 liver cancer samples was lower than that in normal liver tissue samples (0.12±0.07 vs. 0.73±0.27, t=-15.71, P<0.001), and the expression level of miR-886-5p was positively correlated with the expression level of BAX ( r=0.152, P=0.003). qRT-PCR analysis showed that the expression level of miR-886-5p in HL-7702 cells was higher than that in Huh7 (4.57±0.06 vs. 1.61±0.40, t=32.48) and HepG2 (4.57±0.06 vs. 1.03±0.13, t=143.9), and the expression level of BAX in HL-7702 cells was higher than that in Huh7 (4.01±0.12 vs. 1.28±0.09, t=82.20) and HepG2 (4.01±0.12 vs. 1.30±0.11, t=80.76), the differences were statistically significant (all P<0.001). The proliferation, migration, and invasion abilities of Huh7 and HepG2 cells decreased after transfection with miR-886-5p mimics, while the expression levels of BAX at the mRNA and protein levels increased. However, after inhibiting the expression of miR-886-5p, the above indicators of cells were the opposite, and the dif-ferences were statistically significant (all P<0.05). The viability, EdU positivity rate, cell migration rate, and number of transmembrane cells in the miR-886-5p+ BAX group were lower than those in the BAX group, and the relative expression levels of miR-886-5p, BAX mRNA, and BAX protein were higher than those in the BAX group. However, the above indicators in the Sponge+ BAX group showed opposite trends, and all differences were statistically significant (all P<0.05). There was a targeted binding site between miR-886-5p and BAX. Conclusion:Both miR-886-5p and BAX are downregulated in liver cancer, and miR-886-5p inhibits the proliferation, migration, and invasion of liver cancer cells by targeting BAX.

Objective:To investigate the molecular mechanism of miR-886-5p targeting BCL-2-associated X protein (BAX) to inhibit the proliferation, migration, and invasion of liver cancer cells.Methods:mRNA expression data of HCC patients were obtained from the Starbase database, including 370 liver cancer samples and 50 normal liver tissue samples adjacent to the cancer. Analyze the expression of miR-886-5p in the previously obtained data and investigate the relationship between miR-886-5p and BAX in liver cancer samples. After transfection of the corresponding plasmids into Huh7 and HepG2 cells, the following groups were established. Analyze the interaction between miR-886-5p and BAX in vitro, detect the protein expression by Western blotting, and verify the targeting relationship between the two by dual luciferase reporter gene assay.Results:Starbase database analysis found that the standardized expression level of miR-886-5p in 370 liver cancer samples was lower than that in normal liver tissue samples (0.12±0.07 vs. 0.73±0.27, t=-15.71, P<0.001), and the expression level of miR-886-5p was positively correlated with the expression level of BAX ( r=0.152, P=0.003). qRT-PCR analysis showed that the expression level of miR-886-5p in HL-7702 cells was higher than that in Huh7 (4.57±0.06 vs. 1.61±0.40, t=32.48) and HepG2 (4.57±0.06 vs. 1.03±0.13, t=143.9), and the expression level of BAX in HL-7702 cells was higher than that in Huh7 (4.01±0.12 vs. 1.28±0.09, t=82.20) and HepG2 (4.01±0.12 vs. 1.30±0.11, t=80.76), the differences were statistically significant (all P<0.001). The proliferation, migration, and invasion abilities of Huh7 and HepG2 cells decreased after transfection with miR-886-5p mimics, while the expression levels of BAX at the mRNA and protein levels increased. However, after inhibiting the expression of miR-886-5p, the above indicators of cells were the opposite, and the dif-ferences were statistically significant (all P<0.05). The viability, EdU positivity rate, cell migration rate, and number of transmembrane cells in the miR-886-5p+ BAX group were lower than those in the BAX group, and the relative expression levels of miR-886-5p, BAX mRNA, and BAX protein were higher than those in the BAX group. However, the above indicators in the Sponge+ BAX group showed opposite trends, and all differences were statistically significant (all P<0.05). There was a targeted binding site between miR-886-5p and BAX. Conclusion:Both miR-886-5p and BAX are downregulated in liver cancer, and miR-886-5p inhibits the proliferation, migration, and invasion of liver cancer cells by targeting BAX.

Objective:To learn about the single nucleotide polymorphism (SNP) population structure and regional distribution characteristics of Yersinia pestis in the natural focus of plague in Qinghai-Tibet Plateau. Methods:A total of 319 representative strains of Yersinia pestis isolated from natural focus of plague in Qinghai-Tibet Plateau from 1954 to 2020 were selected, and 2 298 SNP loci included in the global Yersinia pestis phylogenetic tree were compared by whole genome sequencing technology. MEGA 6.0 software was used to construct phylogenetic trees of 319 strains of Yersinia pestis from Qinghai-Tibet Plateau, determine the SNP population structure of Yersinia pestis in the focus, and describe its regional distribution characteristics. Results:The 319 strains of Yersinia pestis isolated from Qinghai-Tibet Plateau natural plague foci were distributed in 5 clades, namely 1.IN, 2.ANT, 3.ANT, 0.PE and 2.MED. The 1.IN clade contained 209 strains (65.52%, 209/319), which was the dominant population of strains in Qinghai Province, accounting for 90.51% (143/158). The 2.ANT clade contained 83 strains (26.02%, 83/319), which was the dominant population in Tibet Autonomous Region, accounting for 67.24% (78/116). The 3.ANT, 0.PE, and 2.MED clades contained 12 (3.76%, 12/319), 9 (2.82%, 9/319) and 6 strains (1.88%, 6/319), respectively, which were scattered in Qinghai Province, Gansu Province, Sichuan Province, Tibet Autonomous Region, and Xinjiang Uygur Autonomous Region under the jurisdiction of Qinghai-Tibet Plateau. Conclusion:The SNP population structure of Yersinia pestis in natural focus of plague in Qinghai-Tibet Plateau is relatively rich, and the strains are distributed in 5 clades: 1.IN, 2.ANT, 3.ANT, 0.PE and 2.MED, showing the distribution characteristics of specific regions.

Objective:To study the clustered regularly interspaced short palindromic repeats (CRISPR) genotype of Yersinia pestis and its regional distribution characteristics in natural plague focus of Tibet Autonomous Region. Methods:A total of 125 representative Yersinia pestis strains isolated from natural plague focus in Tibet Autonomous Region at different times, regions, hosts and vectors were selected as experimental strains, and the phenol chloroform mixed extraction method was used to extract Yersinia pestis DNA. Three pairs of CRISPR primers (for YPa, YPb, YPc locus) were used to amplify the DNA of the experimental strains, and the CRISPR genotype of Yersinia pestis was determined by sequencing. Results:All 125 strains of Yersinia pestis had three CRISPR locus: YPa, YPb, and YPc. A total of 18 spacer were found, including 8 in YPa loci, 6 in YPb loci, and 4 in YPc loci. Two new types of spacers had been discovered, namely b52 and c14. CRISPR typing revealed 10 genotypes, including G1, G7, G7-b4''', G7-b52, G7-c2 -, G8, G22, G22-a4 -, G22-b4''', and G22-c14, of which 6 were newly discovered genotypes. Among the 125 experimental strains, G7 was the main genotype, accounting for 65.6% (82/125), which was distributed in 6 prefecture level citys and 1 region of Tibet Autonomous Region. Next were G22 and G7-c2 - genetypes, accounting for 14.4% (18/125) and 11.2% (14/125), respectively. G22 gene type was distributed in Nagqu, Changdu, Lhasa citys, and Ngari Prefecture, while G7-c2 - genetype was distributed in Shigatse and Shannan cities. Conclusion:The CRISPR locus of Yersinia pestis in natural plague focus of Tibet Autonomous Region is highly polymorphic, and the Yersinia pestis strains with different genotypes have obvious regional distribution characteristics.

Objective:To investigate the effect of circular RNA-Hsa-0101216 (Hsa-circ-0101216) targeting microRNA-142-3p (miR-142-3p) on the proliferation,cloning,migration and invasion of pancreatic cancer cells, and explore its molecular mechanism.Methods:The differentially expressed miRNAs in pancreatic cancer were analyzed and screened with GEO database and the circRNA-miRNA network was constructed. The expression of Hsa-circ-0101216 miRNA and miR-142-3p in 5 strains of pancreatic cancer cells (BxPC-3, PANC1, MIA PaCa-2, Capan-2, CFPAC-1) and normal pancreatic duct epithelial cells (HPNE) was detected by quantitative real time PCR. The PANC1 and Capan-2 pancreatic cancer cells were divided into the Hsa-circ-0101216 small interference RNA transfection group (si-circRNA group), the senseless negative sequence siRNA transfection group (si-NC group), and the normal blank control (NC group); the miR-142-3p overexpression lentiviral vector transfection group (miR-142-3p mimic group), the empty vector transfection group (miR-CON group), the co transfection of Hsa-circ-0101216 siRNA and miR-142-3p mimic group (si-Hsa circ0101216+mimic miR-142-3p group), co transfection of Hsa-circ-0101216 siRNA and miR-142-3p mimic negative control sequence group (si-Hsa-circ-010126+mimic miR-142-3p-NC group). The changes of cell proliferation, cloning, migration and invasion were detected by CCK-8, EdU proliferative staining, plate cloning, cell scratch and Transwell assay. Detect the expression of ERK1 and ERK1 and ERK2 protein expression was measured by Western blotting, and the targeting relationship between Hsa-cic-0101216 and miR-142-3p was verified by dual luciferase reporter gene method.Results:The expression of miR-142-3p was significantly down-regulated by GEO analysis, and the Hsa-circ-0101216-miR-142-3p regulatory network was successfully constructed. The mRNA expression levels of Hsa circ0101216 in BxPC-3, PANC1, MIA PaCa-2, Capan-2, and CFPAC-1 cells were 5.64±0.34, 5.93±0.40, 5.66±0.14, 5.63±0.33, and 5.70±0.50, respectively, which were significantly higher than those in HPNE cells (1.27±0.06); the expression levels of miR-142-3p were 1.43±0.12, 1.20±0.09, 1.60±0.04, 1.16±0.25, and 1.42±0.11, respectively, which were significantly lower than those of HPNE cells (4.69±0.22), and all the differences were statistically significant (all P value <0.001). Among them, the expression level of Hsa-circ-0101216 mRNA in PANC1 cells was the highest, and the expression level in Capan 2 cells was the lowest. Compared with the NC group and si-NC group, the si-circRNA group showed a significant decrease in the absorbance value at 450 nm at 24, 48, and 72 hours ( A450 value), EdU positivity rate, cell clone number, migration rate, and transmembrane cell number of PANC1 and Capan-2 cells; the A450 value at the above time points, EdU positivity rate, cell clone number, migration rate, and transmembrane cell number of PANC1 and Capan-2 cells in the miR-142-3p mimic group were significantly lower than those in the NC group and miR-CON group. The A450 value, EdU positivity rate, cell clone number, migration rate, transmembrane cell number, ERK1 and ERK2 protein expression levels of PANC1 and Capan-2 cells in the si-Hsa-circ-0101216+mimic-miR-142-3p group were significantly reduced compared to the si-Hsa-circ-0101216+mimic miR-142-3p-NC group, and the differences were statistically significant (all P<0.001).The luciferase activity of PANC1 and Capan-2 cells in co transfected with wild-type (WT) Hsa-circ-0101216 and miR-142-3p mimic groups was significantly lower than that of the miR-CON+WT group (0.92±0.11 vs 2.33±0.21, 0.89±0.08 vs 2.30±0.17), and the differences were statistically significant (all P value <0.001), but there was no statistically significant difference on luciferase activity of PANC1 and Capan-2 cells between the co transfected mutant (MUT) Hsa-circ-0101216 and miR-142-3p mimic groups and the miR-CON+MUT group. Conclusions:Hsa-circ-0101216 is overexpressed in pancreatic cancer cells, while miR-142-3p is poorly expressed; Hsa-circ-0101216 can promote the proliferation, cloning, migration and invasion of pancreatic cancer cells by targeting miR-142-3p.

Objective:To investigate the effect of circular RNA-Hsa-0101216 (Hsa-circ-0101216) targeting microRNA-142-3p (miR-142-3p) on the proliferation,cloning,migration and invasion of pancreatic cancer cells, and explore its molecular mechanism.Methods:The differentially expressed miRNAs in pancreatic cancer were analyzed and screened with GEO database and the circRNA-miRNA network was constructed. The expression of Hsa-circ-0101216 miRNA and miR-142-3p in 5 strains of pancreatic cancer cells (BxPC-3, PANC1, MIA PaCa-2, Capan-2, CFPAC-1) and normal pancreatic duct epithelial cells (HPNE) was detected by quantitative real time PCR. The PANC1 and Capan-2 pancreatic cancer cells were divided into the Hsa-circ-0101216 small interference RNA transfection group (si-circRNA group), the senseless negative sequence siRNA transfection group (si-NC group), and the normal blank control (NC group); the miR-142-3p overexpression lentiviral vector transfection group (miR-142-3p mimic group), the empty vector transfection group (miR-CON group), the co transfection of Hsa-circ-0101216 siRNA and miR-142-3p mimic group (si-Hsa circ0101216+mimic miR-142-3p group), co transfection of Hsa-circ-0101216 siRNA and miR-142-3p mimic negative control sequence group (si-Hsa-circ-010126+mimic miR-142-3p-NC group). The changes of cell proliferation, cloning, migration and invasion were detected by CCK-8, EdU proliferative staining, plate cloning, cell scratch and Transwell assay. Detect the expression of ERK1 and ERK1 and ERK2 protein expression was measured by Western blotting, and the targeting relationship between Hsa-cic-0101216 and miR-142-3p was verified by dual luciferase reporter gene method.Results:The expression of miR-142-3p was significantly down-regulated by GEO analysis, and the Hsa-circ-0101216-miR-142-3p regulatory network was successfully constructed. The mRNA expression levels of Hsa circ0101216 in BxPC-3, PANC1, MIA PaCa-2, Capan-2, and CFPAC-1 cells were 5.64±0.34, 5.93±0.40, 5.66±0.14, 5.63±0.33, and 5.70±0.50, respectively, which were significantly higher than those in HPNE cells (1.27±0.06); the expression levels of miR-142-3p were 1.43±0.12, 1.20±0.09, 1.60±0.04, 1.16±0.25, and 1.42±0.11, respectively, which were significantly lower than those of HPNE cells (4.69±0.22), and all the differences were statistically significant (all P value <0.001). Among them, the expression level of Hsa-circ-0101216 mRNA in PANC1 cells was the highest, and the expression level in Capan 2 cells was the lowest. Compared with the NC group and si-NC group, the si-circRNA group showed a significant decrease in the absorbance value at 450 nm at 24, 48, and 72 hours ( A450 value), EdU positivity rate, cell clone number, migration rate, and transmembrane cell number of PANC1 and Capan-2 cells; the A450 value at the above time points, EdU positivity rate, cell clone number, migration rate, and transmembrane cell number of PANC1 and Capan-2 cells in the miR-142-3p mimic group were significantly lower than those in the NC group and miR-CON group. The A450 value, EdU positivity rate, cell clone number, migration rate, transmembrane cell number, ERK1 and ERK2 protein expression levels of PANC1 and Capan-2 cells in the si-Hsa-circ-0101216+mimic-miR-142-3p group were significantly reduced compared to the si-Hsa-circ-0101216+mimic miR-142-3p-NC group, and the differences were statistically significant (all P<0.001).The luciferase activity of PANC1 and Capan-2 cells in co transfected with wild-type (WT) Hsa-circ-0101216 and miR-142-3p mimic groups was significantly lower than that of the miR-CON+WT group (0.92±0.11 vs 2.33±0.21, 0.89±0.08 vs 2.30±0.17), and the differences were statistically significant (all P value <0.001), but there was no statistically significant difference on luciferase activity of PANC1 and Capan-2 cells between the co transfected mutant (MUT) Hsa-circ-0101216 and miR-142-3p mimic groups and the miR-CON+MUT group. Conclusions:Hsa-circ-0101216 is overexpressed in pancreatic cancer cells, while miR-142-3p is poorly expressed; Hsa-circ-0101216 can promote the proliferation, cloning, migration and invasion of pancreatic cancer cells by targeting miR-142-3p.

Abstract: Objective To understand the phenotypic and genetic characteristics of Yersinia pestis strains isolated from Himalayan marmot natural focus area and domestic rat plague focus area in southern China, and provide reference for mastering the pathogenic characteristics of Yersinia pestis of two plague foci. Methods A total of 412 of Yersinia pestis strains isolated from Himalayan marmot plague focus and domestic rat plague focus of southern China were subjected to to sorbitol fermentation assays, virulence factor, different region (DFR) typing, and clustered regularly interspaced palindromic repeats (CRISPR) typing. Results The biochemical types of Y. pestis from the two plague foci showed distinct regional distribution features. Five biochemical phenotypes were identified in Yersinia pestis isolated from Himalayan marmot natural focus area, while only one biochemical phenotype was identified in strains isolated from the domestic rat plague focus of Southern China. Most of the Yersinia pestis isolated from the two plague foci were capable of producing the virulence factors of Fl and PstI. Among the strains from Himalayan marmot focus, 70.53% (201/285) were VW-positive, 75.09% (214/285) were Pgm-positive, 20.00% (57/285) of the strains were Pgm-negative, and 5.26% (15/285) were Pgm mixed-type strains. Among strains from domestic rat plague focus of southern China, 37.80% (48/127) were VW-positive, 29.13% (37/127) were Pgm-positive, 58.27% (74/127) were Pgm-negative, and 12.60% (16/127) were Pgm mixed-type strains. DFR typing revealed 22 genotypes of Y. pestis from the Himalayan marmot plague focus, with the main genotypes being type 5, 7, 8, 10, 19, 32 and 49. All strains from domestic rat plague focus area in southern China belonged to type 9. CRISPR typing revealed that all strains from the Himalayan marmot natural focus were classified into 7 CRISPR gene clusters and 14 CRISPR genotypes, with the main genotypes being G7, G22, G26-a1'and G22-A1'. All strains from domestic rat plague focus area in southern China belonged to CRISPR genotype G30, with the gene cluster being Ca8. Conclusions The phenotypes and genotypes of the Yersinia pestis of Himalayan marmot plague focus are diverse, with an obvious characteristics of geographical distribution. The phenotype and genotype of the Yersinia pestis of domestic rat plague focus of Southern China are single. DFR and CRISPR genotyping methods with phenotypic characteristics can effectively identify the Yersinia pestis isolated from the two plague foci, thereby meeting the needs of identification and traceability research.

Objective:To investigate the genotypes of clustered regularly interspaced short palindromic repeats (CRISPR) of Yersinia pestis ( Y. pestis) and it's regional distribution in the plague natural foci of Qinghai-Tibet Plateau. Methods:In this study, 1 004 strains of Y. pestis isolated from different hosts and vectors in different regions from 1954 to 2011 were selected as experimental objects, which preserved in Qinghai Institute for Endemic Disease Prevention and Control, and DNA of Y. pestis was extracted by traditional phenol-chloroform method. Three CRISPR loci (YPa, YPb and YPc) were amplified by PCR and sequenced, respectively, and the CRISPR sequences were compared with the CRISPRDictionary database recently reported in the literature to identify CRISPR spacer. For the spacer discovered for the first time at each CRISPR locus, Blast sequence alignment was performed in the National Center for Biotechnology Information (NCBI) database to speculate the source of gene sequence. Genotyping of Y. pestis from Qinghai-Tibet Plateau was performed based on the polymorphism of CRISPR spacer array. Results:A total of 53 spacers were found in 1 004 strains of Y. pestis, of which 6 spacers were newly discovered, namely a105, a106, a107, b51, b52 and c14. The 1 004 strains of Y. pestis were divided into 10 groups with 44 different CRISPR genotypes, and 15 new genotypes were found. The dominant genotypes of Y. pestis were G26-a1', G7, G22, G24-a1', G22-a1', G9 and G26-a1'a60 in Himalayan Marmot plague natural foci, and G37-a6' in Qinghai Microtinae plague natural foci. Conclusion:The CRISPR genotypes of Y. pestis in the plague natural foci of Qinghai-Tibet Plateau are high diverse and have significant regional distribution characteristics.

Objective:To understand whether there are drug resistant and disinfectant resistant Yersinia pestis strains in China, and to provide accurate information for clinical treatment of plague. Methods:A total of 2 753 Yersinia pestis strains isolated from 10 natural plague foci in China from 1943 to 2016 were collected. According to National Center for Biotechnology Information (NCBI) released sequences of aminoglycoside streptomycin resistant genes strA, strB, β-lactam antibiotics resistant genes TEM, SHV and CTX-M, sulfamilamide resistant genes sul1, sul2 and sul3, and disinfectant resistant gene qacE△1-sul1, a pair of primers of each gene was designed for above-mentioned genes. Genomic DNA of 2 753 strains of Yersinia pestis was extracted, and the 9 target genes of all DNA samples were amplified by PCR. Results:Negative and positive controls of PCR detection were established. No corresponding target bands of aminoglycoside streptomycin resistant genes strA, strB, β-lactam antibiotics resistant genes TEM, SHV and CTX-M, sulfamilamide resistant genes sul1, sul2 and sul3, and disinfectant resistant gene qacE△1-sul1 were found in the DNA samples of 2 753 strains of Yersinia pestis.Conclusion:The above-mentioned genes of drug resistance and disinfectant resistance have not been detected in Yersinia pestis of China, but the monitoring of drug resistance of Yersinia pestis still needs to be carried out continuously.

Objective:To investigate the drug resistance of Yersinia pestis to 11 kinds of antibiotics in the natural foci of plague in Inner Mongolia Autonomous Region, and to provide a theoretical basis for scientifically and effectively selecting antibiotics for treatment of the plague. Methods:A total of 137 strains of Yersinia pestis isolated from the natural foci of plague in Inner Mongolia Autonomous Region at different times, regions, hosts and vectors were collected. According to Clinical and Laboratory Standard Institute (CLSI), the agar plate dilution method was used to determine the minimum inhibitory concentration (MIC) of the 11 kinds of antibiotics against 137 strains of Yersinia pestis, including ofloxacin, ciprofloxacin, kanamycin, streptomycin, ceftriaxone, ampicillin, chloramphenicol, spectinomycin, cefuroxime, tetracycline and sulfamethoxazole-trimethoprim. The MIC 50 and MIC 90 (the minimum concentration of drug which could inhibit 50% and 90% of bacterial growth) were calculated, and their sensitivity was determined according to CLSI standards. Results:Among 137 strains of Yersinia pestis tested, no strains of Yersinia pestis had single or multiple resistance to ofloxacin, ciprofloxacin, kanamycin, streptomycin, ceftriaxone, ampicillin, chloramphenicol, spectinomycin, cefuroxime, tetracycline and sulfamethoxazole-trimethoprim. According to CLSI standards, 137 strains of Yersinia pestis were all sensitive to the 11 kinds of antibiotics; among them, ofloxacin, ciprofloxacin, ceftriaxone and sulfamethoxazole-trimethoprim had higher antibacterial activity, with MIC 90 < 0.250 μg/ ml; the antibacterial activity of spectinomycin was the lowest, with MIC 90 of 16.000 μg/ml. Conclusions:The Yersinia pestis isolated from the natural foci of plague in Inner Mongolia Autonomous Region is not found to have single or multiple resistance to the 11 kinds of antibiotics. Continuous drug resistance monitoring of Yersinia pestis should be carried out to provide a basis for clinical medication.