Construction and identification of a eukaryotic expression vector for the small interfering RNA targeting nucleostemin gene / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a eukaryotic expression vector for the small interfering RNA (siRNA) targeting nucleostemin (NS) gene.</p><p><b>METHODS</b>The siRNA targeting NS gene was designed according to the sequence of NS mRNA available in GenBank. Three siRNA sequences were obtained, and the corresponding cDNAs were synthesized and inserted into plasmid pRNAT-U6.1 for constructing the recombinant plasmids, which were transformed into E.coli DH5alpha strain. The plasmids, after identification by PCR and DNA sequencing, were transfected into EC9706 cell line via liposome, and the mRNA and protein expressions of NS gene in the cells were determined by RT-PCR and Western blotting, respectively.</p><p><b>RESULTS</b>Three recombinant plasmids were identified by PCR and sequence analysis, the results of which showed correct insertion of the designed sequences in the plasmids. RT-PCR and Western blotting showed substantially decreased mRNA and protein expressions of NS gene in the transfected cells.</p><p><b>CONCLUSION</b>The recombinant plasmid expressing the siRNA targeting NS gene has been successfully constructed, which provides the basis for studying RNA interference of the NS gene.</p>

Expression of nucleostemin mRNA and protein in the esophageal squamous cell carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the mRNA and protein expression of nucleostemin (NS) in human esophageal squamous cell carcinoma.</p><p><b>METHODS</b>The mRNA and protein expression of NS were detected in 31 mucosal atypical hyperplasia specimens, 62 esophageal squamous cell carcinoma specimens and the matched normal esophageal mucosa samples by RT-PCR and immunohistochemistry method, respectively.</p><p><b>RESULTS</b>The positive expression rate of NS protein in normal esophageal mucosa, atypical hyperplasia and esophageal squamous cell carcinoma was 17.7% (11/62), 41.9% (13/31) and 69.4% (43/62), respectively. There was a significant difference among the above three groups (chi2 = 33.676, P < 0.01). The expression levels of NS mRNA in esophageal squamous cell carcinoma (0.971 +/- 0.121) was significantly higher than that in the atypical hyperplasia (0.913 +/- 0.085) and also in the normal esophageal mucosa (0.866 +/- 0.103; F = 14.829, P < 0.01). The expression level of both NS protein and mRNA was positively correlated with histological grade, infiltration depth, and lymph node metastasis (P < 0.05), but not with age, gender or pathological type (P > 0.05).</p><p><b>CONCLUSION</b>Our results indicate that nucleostemin mRNA and protein are over-expressed in human esophageal squamous cell carcinoma, and it may be related with its oncogenesis.</p>

Expression and significance of B7-H1 and its receptor PD-1 in human gastric carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>The B7-H1/PD-1 co-signaling pathway has recently been found to play a pivotal role in the immune evasion of tumor cells from host immune system. The aim of this study was to examine the B7-H1 and PD-1 expression and TILs status in gastric cancer and to elucidate the clinical relevance of B7-H1 and PD-1 to the pathogenesis of gastric carcinoma.</p><p><b>METHODS</b>Immunohistochemistry and ANAE histochemical staining were used to investigate the in situ expression of B7-H1 and PD-1 and TILs status in the gastric tissues. RT-PCR was used to explore B7-H1 and PD-1 expression at the transcriptional level. The B7-H1 expression at protein level was detected by Western blot.</p><p><b>RESULTS</b>Expression of B7-H1 and PD-1 was found to be increased in gastric carcinoma, but absent in normal gastric tissue. B7-H1 expression in gastric carcinoma was inversely correlated with TILs infiltration. B7-H1 but not PD-1 expression in tumor tissue was significantly correlated with some clinicopathhological variables including depth of invasion, lymph node metastasis and distant metastasis.</p><p><b>CONCLUSION</b>B7-H1 and PD-1 expressions are increased in gastric carcinoma. This signaling pathway may inhibit antitumor immune responses in gastric carcinoma. B7-H1 expression plays a critical role in the pathogenesis of human gastric carcinoma,and might be a promising prognostic marker and therapeutic target in the treatment of this disease.</p>

Screening effective sequences of small interfering RNAs targeting MDR1 gene in human gastric cancer SGC7901/VCR cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To screen effective sequences of small interfering RNA targeting MDR1 gene in human gastric cancer SGC7901/VCR cells.</p><p><b>METHODS</b>Four siRNAs (MDR1si326, MDR1si1513, MDR1si2631 and MDR1si3071) targeting MDR1 gene were designed and synthesized by in vitro transcription. The siRNA duplexes were used to transfect into the human gastric cancer SGC7901/VCR cells. The expression level of MDR1 mRNA and P-gp were detected by RT-PCR and Western blotting, respectively. The accumulation of intracellular adriamycin (ADR) was examined by flow cytometry and the cell sensitivity to ADR was demonstrated by MTT.</p><p><b>RESULTS</b>The SGC7901/VCR cells treated with 4 siRNAs led to reversal effect on multidrug resistance to different extents. Among the SGC7901/VCR cells treated by siRNAs for 48 h, the expression level of MDR1 mRNA in cells of MDR1si326 or MDR1si2631 group (0.42 +/- 0.07 or 0.49 +/- 0.02) was more decreased than that in cells of MDR1si1513 or MDR1si3071 group (P < 0.05). The accumulation of ADR in cells of MDR1si326 group was the most; in cells of MDR1si2631 group, more; in cells of MDR1si3071 group, lower and in cells of MDR1si1513 group, the lowest (P < 0.05). The relative reversal efficiency of cells of MDR1si2631 group to ADR was the highest and in cells of MDR1si326 group, higher (P < 0.05). There was no significant difference in the relative reversal efficiency between the cells of MDR1si1513 and MDR1si3071 groups (P > 0.05). The expression level of P-gp in cells of MDR1si326 group was the lowest among the SGC7901/VCR cells treated by siRNAs for 72 h.</p><p><b>CONCLUSION</b>The MDR1si326 with most, MDR1si2631 with more, MDR1si3071 with less and MDR1si1513 with least reversal effects on MDR1 gene mediated multidrug resistance were found in the human gastric cancer SGC7901/VCR cells.</p>

Effect of hTERT ASODN on the oncogenicity and the inductive apoptosis of HL-60 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of hTERT antisense oligodeoxynucleotide (ASODN) on the oncogenicity and the inductive apoptosis of HL-60 cells.</p><p><b>METHODS</b>Apoptosis of HL-60 cells was detected by flow cytometry (FCM) and agarose gel electrophoresis. Both treated and untreated HL-60 cells were collected and transplanted into 5 BALB/c nude mice respectively, the formation of transplanted neoplasm and its morphologic change were observed. After the transplanted neoplasms were uniform with the ameliorated method in another 10 BALB/c nude mice, they were divided into 2 groups and injected ASODN and PBS into the neoplasm respectively. Seven days later, the tumor were measured, its morphology were observed, and the apoptotic cells were detected with a TUNEL kit.</p><p><b>RESULTS</b>After 72 h treatment there were DNA ladders and early apoptosis peak in hTERT ASODN treated HL-60 cells but was none in SODN treated and blank control cells. In tumor formation experiment, neoplasms were formed in ASODN treated group at 16-17 d and untreated group at 12-13 d. Neoplasm was formed in 2 of 5 ASODN treated mice and 4 of 5 untreated mice respectively. In untreated mice tumor tissues were rich in blood vasa and stromal tissue compared with that in ASODN treated mice. In tumor therapy experiment, before treatment, there was no difference in the average neoplasm physical volume between ASODN treated group [(100.9 +/- 24.6) mm3] and PBS treated group [(98.4 +/- 23.1) mm3] (P > 0.05). After treatment, the neoplasm volume in ASODN treated group [(422.7 +/- 326.4) mm3] was smaller than that in PBS treated group [(786.4 +/- 357.6) mm3] (P < 0.05). Histologically, there were many apoptosis cells in ASODN treated group, but was seldom seen in PBS treated group. The TUNEL positive cells in ASODN treated group were much more than that in PBS treated group (P < 0.05).</p><p><b>CONCLUSION</b>The hTERT ASODN induces apoptosis of HL-60 cells in vitro, reduces the tumor formation in BALB/c nude mice and inhibits the growth of the transplanted neoplasm.</p>

Rapid Detection of Down Syndrome by Gene Diagnosis Using Short Tandem Repeat Polymorphisms / 实用儿科临床杂志

Objective To set a rapid,simple gene diagnosis method for Down syndrome.Methods Three short tandem repeats(D21S11,D21S1270,D21S1437)loci in or near Down syndrome critical region(DSCR) were analyzed and detected by polymerase chain reaction and DNA quantitative analysis in 11 core ancestry.Results There were four types by DNA quantitative analysis to different individuals at a short tandem repeats(STR) locus.In type one,a homozygote of one allelic gene was detected.In type two,a normal heterozygote of two allelic genes was found,the content or two DNA electrophoresis bands was approximately 1∶1.In type three,a Down syndrome patient of two allelic genes was discovered.The quantity of two electrophoresis bands was nearly 2∶1.In type four,the patient showed three DNA electrophoresis bands which the content was approximately 1∶1∶1.Conclusion A rapid gene diagnosis and prenatal diagnosis method for Down syndrome can be used for quantitative analysis of STR polymorphism loci.

Inhibition of hTERT antisense oligodeoxynucleotide on proliferation and telomerase activity in HL-60 cells / 中国实验血液学杂志

This study was purposed to investigate the inhibition of hTERT antisense oligodeoxynucleotide (ASODN) on the proliferation and telomerase activity in HL-60 cells and to explore the relativity between the telomerase activity and the expression of hTERT gene in HL-60 cells. After treated by hTERT ASODN the expression of hTERT was detected by RT-PCR, the morphological changes of HL-60 cells was observed with inverted microscopy, the cell proliferation was measured by MTT method, and the telomerase activity was determined with TRAP-ELISA and TRAP-PAGE. The results showed that after sealing hTERT gene with ASODN for 72 hours, the expression of hTERT gene was significantly inhibited, the cell growth was repressed and the ability of proliferation decreased, and the effect was specific in sequence and dependent in dose and time. OD(450-690) values were 2.648 +/- 0.42, 1.504 +/- 0.47, 1.223 +/- 0.39, 0.944 +/- 0.16 respectively, as the cells were treated with 0, 10, 20, 30 micromol/L ASODN for 72 hours. The difference was significant as compared 10, 20, 30 micromol/L groups with 0 micromol/L ASODN group respectively (P < 0.05), but the difference was no significant when compared 20 micromol/L SODN group (2.376 +/- 0.65) with untreated group (2.648 +/- 0.42) (P > 0.05). TRAP-PAGE detection revealed that comparing ASODN groups with SODN groups the telomerase image bands were decreased and least was found in groups of 30 +/- mol/L. It is concluded that the hTERT ASODN may inhibit the proliferation and down-regulate the telomerase activity in HL-60 cells by sealing the expression of hTERT gene.

Effects of c-myc antisense oligodeoxynucleotide on the telomerase activity and the induction of apoptosis in HL-60 cells / 中国实验血液学杂志

To investigate the effects of c-myc antisense oligodeoxynucleotide (ASODN) on the telomerase activity and the induction of apoptosis in HL-60 cells, and to explore the relationship between the telomerase activity and the expression of c-myc gene in HL-60 cells, after treatment by c-myc ASODN, the expression of c-myc was detected by RT-PCR, the apoptosis, cell cycle were detected with agarose gel electrophoresis and flow cytomety, and the telomerase activity was determined with TRAP-ELISA. The results showed that after blocking c-myc gene with ASODN for 72 hours, it is obvious that the expression of c-myc gene was inhibited. The percentage of S phase HL-60 cells decreased from 55.6% to 30%, the early apoptosis peak appeared (the percentage of apoptosis cells were 25.2%) and the DNA ladders were shown. OD(450 - 690) were 2.648 +/- 0.42, 2.324 +/- 0.36, 2.162 +/- 0.38, 1.952 +/- 0.14, 1.805 +/- 0.40, 1.616 +/- 0.41 and 2.466 +/- 0.29, respectively, as the cells were treated with 0, 1, 2, 3, 4, 5 micromol/L ASODN and 5 micromol/L SODN for 72 hours. The difference was significant when compared 3, 4, 5 micromol/L groups with 0 micromol/L ASODN group respectively (P < 0.05), but the difference was no significant when compared 1, 2 micromol/L ASODN and 5 micromol/L SODN groups with 0 micromol/L ASODN group (P > 0.05). It is concluded that the c-myc gene ASODN may induce the apoptosis of cells, inhibit cells from G(1) phase into S phase and regulate the telomerase activity down in HL-60 cells by blocking the expression of c-myc gene.

Detection of methylation of hMSH2 gene promoter region of esophageal cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To detect methylation in promoter region of hMSH2 gene in esophageal cancer.</p><p><b>METHODS</b>Specimens of cancer and normal tissues freshly removed from 32 cases of esophageal cancer patients without previous radiotherapy, chemotherapy or other treatment were preserved at -80 degrees C within 30 min. Methylation specific PCR (MSP) was used to detect methylation of mismatch repair gene (MMR) hMSH2 in promoter region in esophageal cancer and normal esophageal tissues.</p><p><b>RESULTS</b>The frequencies of methylation of hMSH2 gene in promoter region of cancer and normal esophageal tissues were 32.4% (11/32) and 0/30 (0%), respectively, and significant difference was found between the two groups (P < 0.01). The frequency of methylation in elder patients (> or = 70 years old) was significantly higher than that in younger patients (< 70 years old) (P < 0.05). Methylation was less frequently found in grade I-II (18.2%) than in grade III-IV (70.0%) (P < 0.05).</p><p><b>CONCLUSION</b>Methylation of hMSH2 gene in promoter region is related to patients' age and histopathological grade of the esophageal cancer.</p>