Expression changes and regulation of AR and IGF-1 in PC3 prostate cancer cells treated with sexual hormones and flutamide.

The study aims to investigate the changes and regulation of androgen receptor and insulin-like growth factor-1 in the PC3 prostate cells treated with 5α-dihydrotestosterone, estrone, and flutamide. The PC3 cells were cultured and treated with 5α-dihydrotestosterone, estrone, and flutamide. Immunocytochemistry and Western blot were used to detect the expression of androgen receptor and insulin-like growth factor-1. The androgen receptor expression was analyzed by Western blot and optic density scan in the presence or absence of various kinase inhibitors. The statistical calculations were performed with the statistics-analyzing software package SPSS 13.0. A P <0.05 was considered statistically significant. The concentrations of 5α-dihydrotestosterone and flutamide could almost not change the expression of androgen receptor and insulin-like growth factor-1 in PC3. But, the concentrations of estrone could increase the expression of androgen receptor and insulin-like growth factor-1 when PC3 cells are exposed to the studied concentration at various times. The expression of androgen receptor was regulated by the inhibitor of signal pathways of PI3, MEK1/2, and JUK. The expressions of androgen receptor and insulin-like growth factor-1 were influenced by estrone and were not influenced by 5α-dihydrotestosterone and flutamide in PC3 cells. And, the expression of androgen receptor was regulated by multiple signal pathways.

Expressions of Osteopontin (OPN), ανß3 and Pim-1 Associated with Poor Prognosis in Non-small Cell Lung Cancer (NSCLC).

OBJECTIVE: To examine the expressions of osteopontin (OPN), (α) (ν) (ß) (3) and Pim-1 in non-small cell lung cancer (NSCLC), and investigate their potential pathogenic roles in the development of NSCLC. METHODS: Immunohistochemistry was used to examine the expressions of OPN, (α) (ν) (ß) (3) and Pim-1 in cohort (136 cases) of NSCLC samples and their adjacent normal lung tissue specimens. Statistical analysis was performed to evaluate the relationships among expressions of OPN, (α) (ν) (ß) (3) and Pim-1 and their associations with patients clinico- pathological parameters. RESULTS: The expressions of OPN and Pim-1 were predominantly observed in cytoplasm. The expression of (α) (ν) (ß) (3) was mostly detected in cytoplasm and/or membrane. In NSCLC samples, the positive rates of OPN, (α) (ν) (ß) (3) and Pim-1 expressions were 68.4% (93/136), 77.2% (105/136) and 57.4% (78/136), respectively. In normal lung tissues, in contrast, the positive rates of OPN, (α) (ν) (ß) (3) and Pim-1 were 24.0% (12/50), 26.0% (13/50) and 16.0% (8/50), respectively. There were significant differences of the positive expression rates of OPN, (α) (ν) (ß) (3) and Pim-1 between NSCLCs samples and normal lung tissues (P<0.01). In addition, the positive expression of OPN, (α) (ν) (ß) (3) and Pim-1 in NSCLCs samples was significantly associated with increased pathological grade, lymph node metastasis and advanced clinical stage (P<0.01), and they were independent of other clinicopathological parameters (P>0.05). Furthermore, a significantly positive correlation between the expression of OPN and (α) (ν) (ß) (3) (r=0.38, P<0.01), OPN and Pim-1 (r=0.37, P<0.01), or (α) (ν) (ß) (3) and Pim-1 (r=0.20, P<0.05) was evaluated in our NSCLC cohort. CONCLUSION: OPN, (α) (ν) (ß) (3) and Pim-1 proteins are frequently overexpressed in NSCLC, and they may play important roles in the development and/or progression of NSCLC.

[Genetic polymorphisms of Investigator Argus X-12 amplification system in Guangdong Han population].

OBJECTIVE: To investigate the genetic polymorphisms of 12 X chromosome short tandem repeat (X-STR) loci of Investigator Argus X-12 amplification kit in Guangdong Han population. METHODS: DNA samples from 200 unrelated individuals (100 males and 100 females) and 103 families (59 father-mother-daughter trios and 44 mother-son duos) were extracted and amplified with fluorescence labeled multiplex PCR system. PCR products were separated and genotyped with capillary array electrophoresis. RESULTS: One hundred and thirty-seven alleles,including 9 off ladder alleles (OL allele) were observed at the 12 X-STR loci in the population. Six mutations were observed in 162 meioses. The combined power of discrimination (DP) was 0.999 999 997 in males and 0.999 999 999 in females, and the combined mean exclusion chance (MEC) was 0.999 999 988 in the trio cases and 0.999 998 013 in the duo cases. CONCLUSION: Investigator-Argus X-12 amplification system is highly polymorphic in Guangdong Han population and it is powerful for personal identification and paternity testing.

Polymorphism analysis and evaluation of nine non-CODIS STR loci in the Han population of Southern China.

BACKGROUND: Knowledge of allele and genotype frequencies is an essential prerequisite to the use of any human polymorphism in forensic work. AIM: To study the genetic polymorphism and evaluate the application value of nine STR loci. SUBJECTS AND METHODS: Genotyping of nine STR loci, including D11S2368, D12S391, D13S325, D18S1364, D22-GATA198B05, D6S1043, D2S1772, D7S3048 and D8S1132, of 1050 unrelated individuals was performed with the STR_Typer_10_v1 kit and Genetic Analyzer 3100 and analyzed with PowerState V12.xls and Arlequin ver 3.11 analyzing software. RESULTS: Allele frequency distribution was statistically analyzed and Hardy-Weinberg equilibrium determined. Several common parameters used in forensic sciences were found: the heterozygosity (H) ranged from 0.827 to 0.892; the matching probability (MP) ranged from 0.029 to 0.074; the power of discrimination (PD) ranged from 0.926 to 0.971; the power of exclusion (PE) ranged from 0.649 to 0.779; the polymorphic information content (PIC) ranged from 0.77 to 0.86; and the typical paternity index (TPI) ranged from 2.88 to 4.62. CONCLUSION: The results indicate that nine STR loci are high polymorphic among the Han population in Southern China. This set of polymorphic STR loci is a useful tool in forensic paternity testing and anthropological study.

[Polymorphism of 7 Y-STR loci in Chinese populations by multiplex PCR genotyping using fluorescein-labeled primers].

We reported the multiplex-PCR-based genotyping method for 7 Y-STR loci, including DYS456, DYS464a/b/c/d, DYS527a/b labeled with FAM (blue) and DYS531, DYS709, DYS448, DYS522 labeled with JOE (green). We investigated the haplotype distribution of these 7 Y-STR loci among 151 unrelated Han males in the Guangdong Province and 106 unrelated males in the Henan Province, and evaluated this method for forensic practice. The results showed that this method could successfully determine the genotypes using as little as 0.02 ng genomic DNA, and the male's Y-STR genotypes could be detected in a DNA mixture in which the ratio of male/female components was 1:150 (160 ng in total amount of DNA template). There were 150 and 105 haplotypes found of these 7 Y-STR loci in these two Chinese populations, out of them 149 and 104 haplotypes appeared only once, respectively. The haplotype diversity in the two populations were 0.999912 and 0.999820, respectively. The distribution variation of the 7 Y-STR haplotypes between Guangdong and Henan Chinese populations was statistically significant (P<0.001). Thus, the fluorescein-labeled multiplex-PCR genotyping of 7 Y-STR loci is a valuable tool for forensic medicine practice and for human anthropology study.

[Genetics of heteroplasmy in the mtDNA control region among the Chinese Han population].

OBJECTIVE: To explore the distribution and genetic pattern of heteroplasmy of mtDNA control region among Chinese Han population. METHODS: The human mtDNA control region was amplified into 6 amplicons overlapped partially each other. Then these amplicons were analyzed by DHPLC which we developed to detect low heteroplasmic signals. RESULTS: There were 51 heteroplasmic cases (34%) found from different tissues of 150 unrelated individuals of the Chinese Han population. mtDNA heteroplasmy shows non-uniform distribution in various tissues. The highest occurrence of heteroplasmy was in brain tissues (50/150) and myocardium (48/150), the lowest was in bone tissues (22/150). 36 sites of heteroplasmy were identified in our samples. Three sites of mtDNA heteroplasmy rarely co-existed in one individual. No sex differences were detected in the frequency of mtDNA heteroplasmy. No change in the mtDNA heteroplasmy profile was detected of blood samples from the same individuals within 2 years. Individuals older than 41 years showed a heteroplasmy frequency significantly higher than their younger counterparts. Members from the same maternal pedigree in a family can share the same sites of mtDNA heteroplasmy but may have different heteroplasmy contents at those sites. CONCLUSION: DHPLC is a highly sensitive technique in detecting heteroplasmy. mtDNA heteroplasmy widely exists in the Chinese Han population. The results shown here could potentially have a guidable value in forensic individual identification and parentage testing.

Genetic polymorphisms and mutation rates of 27 Y-chromosomal STRs in a Han population from Guangdong Province, Southern China.

In this study, we collected blood samples from 1033 father-son pairs of a Han population from Guangdong Province, Southern China, of which 1007 fathers were unrelated male individuals. All together, 2040 male individuals were analyzed at 27 Y-chromosomal short tandem repeats (Y-STRs) with Yfiler(®) Plus system. A total of 1003 different haplotypes were observed among 1007 unrelated fathers, with the overall haplotype diversity (HD) 0.999992 and discrimination capacity (DC) 0.996. The gene diversity (GD) values for the 27 Y-STR loci ranged from 0.4400 at DYS438 to 0.9597 at DYS385a/b. 11 off-ladder alleles and 25 copy number variants were detected in 1007 males. Population relationships were analyzed by comparison with 19 other worldwide populations. With 27,920 allele transfers in 1033 father-son pairs, 124 mutation events occurred, of which 118 were one-step mutations and 6 were two-step mutations. Eleven father-son pairs were found to have mutations at two loci, while one pair at three loci. The estimated locus-specific mutation rates varied from 0 to 1.74×10(-2), with an average estimated mutation rate 4.4×10(-3) (95%CI: 3.7×10(-3) to 5.3×10(-3)). Mutations were most frequently observed at three rapidly mutating Y-STRs (RM Y-STRs), DYS576, DYS518 and DYS627. However, at DYS570, DYS449 and DYF387S1 loci, which were also described as RM Y-STRs, the mutation rates in Guangdong Han population were not as high as estimated in other populations.

Mutations of 15 short tandem repeat loci in Chinese population.

OBJECTIVE: To explore the mutations of 15 short tandem repeat (STR) loci in PlowerPlex16 System which are world-widely used in parentage testing. METHODS: Mutations of 15 STR loci in PlowerPlex16 System were investigated in 1921 parentage testing cases from Chinese population. RESULTS: In 1921 parentage cases, seventy cases (3.644%) were found to have mutations. Among these were one case with double mutations (D21S11 and PentaD) and another case with two different mutations (D7S820 and D16S539) in two children. The total number of mutated STR loci observed was 72 over 3764 meiosis with a mutation rate of 0.128% +/- 0.1104% x 10(-3). The highest mutation rate was 0.292% at vWA and D21S11. No mutation was observed at TH01 or at TPOX. The mutated alleles coming from father were five times more than those from mother. The majority (98.611%) of mutated alleles were the results of one-step mutation. The ratio of one-step gain versus loss was 1.826:1. There was only one multiple-step mutation with a double-repeat gain observed at PentaD locus. In the PlowerPlex16 System, nine loci, namely D8S1179, Penta D, D13S317, D16S539, D7S820, D5S818, D3S1358, TH01 and TPOX, have lower mutation rates and are more suitable for parentage testing. CONCLUSION: Mutation of STR is relatively common and often makes parentage testing more complicated. Selecting stable STR locus with low mutation rate is more important in parentage testing.

Closely linked polymorphic marker: successful application in preimplantation genetic diagnosis for beta-thalassemia.

OBJECTIVE: To evaluate the applicability of the polymorphic marker closely linked with beta-globin gene for the preimplantation genetic diagnosis (PGD) in couples at risk of having child with beta-thalassemia. METHODS: Single cell multiplex nested PCR which coamplifies the beta-globin gene and the closely linked polymorphic marker, HumTHO1 gene, was applied in six clinical PGD cycles for four couples with beta-thalassemia. RESULTS: In six clinical PGD cycles, a total of 44 embryos were biopsied and 44 blastomeres were obtained. Forty-one blastomeres were amplified and thirty-five embryos were given definite diagnoses. Fourteen embryos were transferred back to the uterus of the patients and one pregnancy went on well and ended with one live healthy birth, which confirmed the results of PGD. The average amplification efficiency of single blastomere was 89.7% and the average allele drop-out(ADO) rate was 14.4%. The coamplification of HumTHO1 could help to detect the existence of ADO and contamination. CONCLUSION: This is the first report on unaffected pregnancy resulting from PGD using multiplex nested PCR in China. The simultaneous amplification of polymorphic marker closely linked to beta-globin gene(HumTHO1) could help to resist the risk of misdiagnosis in PGD caused by ADO and contamination.

[Successful preimplantation genetic diagnosis for alpha-thalassemia using fluorescent polymerase chain reaction].

OBJECTIVE: To develop single-cell fluorescent gap polymerase chain reaction (PCR) assay for preimplantation genetic diagnosis (PGD) in couples at risk of having child with alpha-thalassemia. METHODS: Single cell fluorescent gap PCR which can detect the alpha-thalassemia Southeast Asia deletion (-SEA deletion), was applied to single lymphocytes and blastomeres which coming from four clinical PGD cycles. RESULTS: The Single cell fluorescent gap PCR can detect the alpha-thalassemia -SEA deletion, which account for 94% of hydrop fetalis in Chinese population with an amplification efficiency of 90.0% (72/80) and allele drop-out (ADO) rate of 8.3% (6/72) in lymphocytes. In four clinical PGD cycles, a total 38 embryos were detected and 38 blastomeres were obtained. Thirty-four blastomeres were amplified with the amplification efficiency of 89.5% (34/38) and ADO rate of 5.9% (2/34). Eleven embryos were shown to be normal homozygous, eight embryos were shown to be heterozygous and 15 embryos were shown to be affected homozygous. Eleven embryos were transferred back to the uterus of the patients. Two pregnancy achieved, resulting in two live healthy births, which confirmed the results of PGD. CONCLUSION: This first reported unaffected pregnancy resulting from PGD using fluorescent gap PCR for alpha-thalassemia demonstrates that this technique, as an alternative to prenatal diagnosis, is a reliable and effective way to help those carrier couples to get a healthy baby.

[Studying the polymorphism at DYF155S1 locus in Chinese Han population by MVR-PCR marked with fluorescence].

The simple and useful genotyping methods of DYF155S1 locus by silver staining and fluorescence detection have been established. The blood samples from 155 unrelated males in Chinese Han population were typed by these two methods respectively and the same results were obtained. Among the 155 samples 66 alleles were found, out of them 38 were observed once only. The most frequent alleles named 18 or 22, which frequency was 0.065, the size of their first DNA fragments was 180 bp and the number of fragments was 16 or 17. The gene diversity (h) was 0.9789. Out of the 155 samples, 25 samples had one or two bands lost (null repeat) among the successive bands. It was demonstrated by sequencing that the position of the lost bands corresponded to type 3 repeats. Our results indicated that the method, which revealed the 5' end diversity of DYF155S1 locus, was a technique that could obtain the most Y-specific polymorphic information only by one PCR reaction. The allele frequencies of DYF155S1 locus in Chinese Han population provided the basic data for the study of population genetics and forensic practices.

[The characteristics of polymorphism and gene structure of DYF155S1 locus in Y-specific minisatellite from Chinese Uygur population].

The study is to reveal the diversity and gene structure of 5' and 3' end of DYF155S1 locus in Y-chromosome minisatellite among Chinese Uygur population. Fluorescent MVR-PCR(minisatellite variant repeat by PCR), Amp-FLP(Amplified fragment length polymorphism) and DNA sequencing methods were used respectively to detect 106 unrelated males among Chinese Uygur population. The polymorphisms of DYF155S1 locus could be revealed in three aspects: (1) polymorphic length: the sizes of amplified fragments ranged from 1405 to 2505 bp. There are 37 types found among the 106 unrelated males. (2) polymorphism at 5' end of DYF155S1 locus, 68 types found among the 106 unrelated males. (3) polymorphism at 3' end of DYF155S1 locus, 23 types found among the 106 unrelated males. In combination of these three aspects of polymorphism, none of the 106 unrelated males tested had the same allele, and the gene diversity (h) was over 0.9999. Seven and two types of modular structure were founded in the 5' and 3' end of DYF155S1 locus, respectively, by DNA sequencing. The alleles at DYF155S2 locus showed yes/no dimorphism and the rate of deletion was 4.7%. The polymorphisms of DYF155S1 locus were fully revealed by using combination of MVR-PCR, Amp-FLP and DNA sequencing methods, and we suggested the nomenclature for alleles of MVR loci. These methods are useful tools and provide basic data for the study of human genetics and forensic medicine.

[DNA analysis of a 500 year mummy sample].

OBJECTIVE: To accumulate experience for dated forensic matter analysis, for example, Mummy. METHODS: DNA are extracted by methods of phenol-chloroform and are purified by Wizard DNA clean-up system. The STRs locus are ampolification with Promega Powerplus 16 system. The mtDNA hypervariable region 1 (HV1) is amplificated by '3 pair primers'. The products were sequenced with 377 DNA sequencer. RESULTS: The STRs locus very distinctness and mtDNA sequence is correct. CONCLUSION: It is a valuable method for special forensic matters.

Overexpression of osteopontin, αvß3 and Pim-1 associated with prognostically important clinicopathologic variables in non-small cell lung cancer.

In this study, we examined the expression of osteopontin (OPN), αvß3 and Pim-1 in non-small cell lung cancer (NSCLC) and investigated the potential clinical implications of their expression patterns in NSCLC. Immunohistochemical assays were used to examine the protein expression of OPN, αvß3 and Pim-1 in 208 NSCLC samples and their adjacent normal lung tissue specimens. Statistical analyses were performed to evaluate the relationships between OPN, αvß3 and Pim-1 expression patterns, and their association with the clinical-pathological parameters of NSCLC patients. In NSCLC tissues, the positive rates of OPN, αvß3 and Pim-1 expression were 67.8% (141/208), 76.0% (158/208) and 58.7% (122/208), respectively. However, in the adjacent normal lung tissues, the positive rates of OPN, αvß3 and Pim-1 were 20.2% (42/208), 24.0% (50/208) and 14.9% (31/208), respectively. The differences in the positive expression rates of OPN, αvß3 and Pim-1 between NSCLCs and the adjacent normal lung tissues were all significant (P<0.01). Additionally, the positive expression of OPN, αvß3 and Pim-1 in NSCLCs was associated with an increase in pathological grade, lymph node metastasis and advanced clinical stage (all P<0.01). Furthermore, associations between the expression of OPN and αvß3, OPN and Pim-1, and αvß3 and Pim-1 were also observed in our NSCLC cohort (all P<0.01). The OPN, αvß3 and Pim-1 proteins are frequently overexpressed in NSCLC and are associated with some clinicopathologic variables that are of known prognostic importance in NSCLC, suggesting that they may play an important role in the development and/or progression of NSCLC.

Changes of androgen receptor and insulin-like growth factor-1 in LNCaP prostate cancer cells treated with sex hormones and flutamide.

Changes of androgen receptor (AR) and insulin-like growth factor-1 (IGF-1) were investigated in LNCaP cells treated with 5α-dihydrotestosterone (DHT), estrone and flutamide. Real-time PCR, immunocytochemistry and western blotting were used to detect the expression of AR and IGF-1 in the presence or absence of various kinase inhibitors. Low concentrations of DHT, estrone and flutamide increased the expression of AR and IGF-1, especially estrone, with concentration and time dependence. With DHT and flutamide, there was a significant alteration in AR expression (p<0.001). The results indicated expression of AR and IGF-1 genes to be influenced by DHT, estrone and flutamide in LNCaP cells, regulated by multiple signal pathways.