The lncRNA MIR4435-2HG is upregulated in hepatocellular carcinoma and promotes cancer cell proliferation by upregulating miRNA-487a.

BACKGROUND: Given the high mortality rate and unclear pathogenesis for liver cancer, investigation of its molecular mechanisms is essential. We focused on the long non-coding RNA (lncRNA) MIR4435-2HG, which was recently reported to be oncogenic in lung cancer and the microRNA miRNA-487a, which has been reported to be oncogenic in hepatocellular carcinoma (HCC). Our aim was to determine if the former has a role in HCC, and to further validate the role of the latter. METHODS: Samples from 64 patients with HCC were taken at The Third Affiliated Hospital of Sun Yat-Sen University. Cell transfection and PCR were applied. RESULTS: We found that MIR4435-2HG and miRNA-487a were upregulated in tumor tissues compared to adjacent healthy tissues from HCC patients. The expression of MIR4435-2HG was significantly affected by tumor size but not by tumor metastasis. Correlation analysis showed that MIR4435-2HG and miRNA-487a were positively correlated in both the tumor tissues and adjacent healthy tissues from HCC patients. Overexpression of MIR4435-2HG led to upregulation of miRNA-487a in the cells of HCC cell lines, while overexpression of miRNA-487a did not significantly affect MIR4435-2HG. Overexpression of MIR4435-2HG and miRNA-487a promoted the proliferation of cells of HCC cell lines, and miRNA-487a knockdown partially attenuated the enhancing effects of MIR4435-2HG overexpression on cancer cell proliferation. CONCLUSION: MIR4435-2HG is upregulated in HCC and promotes cancer cell proliferation possibly by upregulating miRNA-487a.

Genome-wide screen for universal individual identification SNPs based on the HapMap and 1000 Genomes databases.

Differences among SNP panels for individual identification in SNP-selecting and populations led to few common SNPs, compromising their universal applicability. To screen all universal SNPs, we performed a genome-wide SNP mining in multiple populations based on HapMap and 1000Genomes databases. SNPs with high minor allele frequencies (MAF) in 37 populations were selected. With MAF from ≥0.35 to ≥0.43, the number of selected SNPs decreased from 2769 to 0. A total of 117 SNPs with MAF ≥0.39 have no linkage disequilibrium with each other in every population. For 116 of the 117 SNPs, cumulative match probability (CMP) ranged from 2.01 × 10-48 to 1.93 × 10-50 and cumulative exclusion probability (CEP) ranged from 0.9999999996653 to 0.9999999999945. In 134 tested Han samples, 110 of the 117 SNPs remained within high MAF and conformed to Hardy-Weinberg equilibrium, with CMP = 4.70 × 10-47 and CEP = 0.999999999862. By analyzing the same number of autosomal SNPs as in the HID-Ion AmpliSeq Identity Panel, i.e. 90 randomized out of the 110 SNPs, our panel yielded preferable CMP and CEP. Taken together, the 110-SNPs panel is advantageous for forensic test, and this study provided plenty of highly informative SNPs for compiling final universal panels.

Development of New mRNA Markers for the Identification of Menstrual Blood.

BACKGROUND: The aim of this study was to screen 3 mRNA markers (i.e., PAEP, LAPR3, and HOXA10) with diverse expression in different body fluids and to develop a method for the identification of menstrual blood using these mRNA markers. METHODS: Body fluid (i.e., venous blood, menstrual blood, semen, and saliva) samples were collected and prepared under differing environmental conditions (temperature, humidity and time), and RNA was extracted and reverse transcribed. The expression specificity of these markers was assessed using TaqMan probe qPCR. RESULTS: A high mean cycle threshold value corresponds to a lower expression level. The mean cycle threshold value of the LAPR3, HOXA10, and PAEP genes are 8.37, 8.73, 4.67 in menstrual blood respectively. LAPR3 and PAEP were only expressed in menstrual blood. HOXA10 were expressed in blood, menstrual blood, and semen. No significant differences were found while the mean cycle threshold of MMP11 and PAEP were compared in the menstrual blood under common environmental conditions. There were no observed differences in the expression of the target genes in women of different ages and at different menstrual phases. The sensitivity of the expression of the 3 target genes could be examined in fluid amount range from 1 to 32 µl of body fluid. The expression of PAEP differed markedly from the expression of LAPR3 and HOXA10 in menstrual blood stains tested using mRNA-based assays (p<0.001). CONCLUSIONS: These markers, particularly PAEP, can likely be used for the identification of menstrual blood in certain forensic cases.

The overexpression of Rabl3 is associated with pathogenesis and clinicopathologic variables in hepatocellular carcinoma.

Overexpression of Rabl3 is associated with some malignancies. However, their relationship with hepatocellular carcinoma remains unclear. In this study, the expression of Rabl3 in hepatocellular carcinoma cell lines, and four pairs of matched hepatocellular carcinoma tissues and their adjacent normal hepatic tissues were detected by quantitative reverse transcription polymerase chain reaction and western blot. In addition, the protein expression of Rabl3 was examined in 162 cases of hepatocellular carcinoma by immunohistochemistry. Rabl3 in hepatocellular carcinoma cell lines was elevated at both messenger RNA and protein levels, and the Rabl3 protein was significantly upregulated by upto 3.3-fold in hepatocellular carcinoma compared with the paired normal hepatic tissues. Immunohistochemical analysis revealed that overexpressions of Rabl3 were 80.2% in hepatocellular carcinoma. Rabl3 is expressed at significantly higher rates in hepatocellular carcinoma compared with adjacent normal hepatic tissue (p < 0.01). Statistical analysis suggested the upregulation of Rabl3 was significantly associated with lymph node metastasis, tumor thrombus of the portal vein, and advanced clinical stage (p < 0.05). Furthermore, we found that overexpression of Rabl3 in hepatocellular carcinoma cells could significantly enhance cell proliferation and growth ability. Conversely, silencing Rabl3 by small hairpin RNA interference caused an inhibition of cell proliferation and growth. Our studies suggest that the Rabl3 is a valuable marker of hepatocellular carcinoma progression and that the overexpression of Rabl3 plays an important role in the development and pathogenesis of hepatocellular carcinoma.

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.

Investigation of the Application of miR10b and miR135b in the Identification of Semen Stains.

To evaluate the identification method using the microRNA markers miR10b and miR135b to distinguish semen stains from menstrual blood, peripheral blood, vaginal fluid and so on body fluid stains. The expression levels of miR10b and miR35b in semen stains and menstrual blood and so on were detected utilizing a real-time quantitative PCR technique with a specific fluorescence-labeled TaqMan probe. RNU6b was used as the internal reference gene; the difference in their expression was analyzed, and the specificity, sensitivity, and detection capability of the techniques were evaluated. The expression of miR10b and miR135b in semen stains was significantly higher than that of other body fluid stains, with a mean value of ΔCт from-6 to-7. However, it ranged from-2 to-4 for other body fluid stains. The initial criteria for judging which semen stains can be identified were determined by analyzing the research results. When the threshold value was set to 0.04, the CT value could be detected in the target genes miR10b, miR135b and in the internal reference gene RNU6b, and CT values are<40, ΔCT[10b-U6]<-5.5, and ΔCT[135b-U6]<-6, respectively, and the semen stain could be identified. The expression levels of miR10b and miR135b are higher in semen with strong tissue specificity; thus, they can be used to differentiate semen stains from other body fluid stains in forensic science.

The expression of Survivin and NF-κB associated with prognostically worse clinicopathologic variables in hepatocellular carcinoma.

Expressions of Survivin and nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) are associated with a poor prognosis in many malignancies. However, their relationship in hepatocellular carcinoma remains unclear. To investigate the protein expression of Survivin and NF-κB, determine their role in the pathogenesis of hepatocellular carcinoma, and correlate expression with patient survival outcome, immunohistochemistry was used to detect the protein expression of Survivin and NF-κB in 305 cases of hepatocellular carcinoma. Statistical analysis was performed to determine the relationship between the protein expression of Survivin and NF-κB and clinicopathological parameters, survival time, and prognosis. Survivin was expressed predominantly in the cytoplasm, and NF-κB was expressed mostly in the nucleolus. Survivin and NF-κB are expressed at significantly higher rates in hepatocellular carcinoma compared with benign tissue (75.7 vs 13.4 %, P < 0.01 and 79.0 vs 17.1 %, P < 0.01, respectively). Both Survivin and NF-κB expression levels are associated with poor prognostic factors, including tumor size, capsular invasion, tumor thrombus of the portal vein, metastasis of the lymph node, and clinical staging. There was an obvious positive correlation between the expression of Survivin and NF-κB in hepatocellular carcinoma (r = 0.23, P < 0.01). Patients expressing Survivin and NF-κB had significantly shorter survival compared with patients negative for protein expression (P < 0.01). The overexpressions of both Survivin and NF-κB are associated with worse survival outcome in patients with hepatocellular carcinoma. Thus, these proteins could be used as negative prognostic indicators.

Establishment of experimental implantation tumor models of hepatocellular carcinoma in Wistar rats.

Our aims were to investigate and establish simple and reliable implanted hepatocellular carcinoma (HCC) models in Wistar rats. Concentrated suspensions of CBRH-7919 cancer cell lines were injected subcutaneously into the scapular regions of nude mice. The developing tumor tissues were then implanted into the livers of 45 adult Wistar rats. Dexamethasone (2.5 mg/day) was injected intramuscularly daily for 1 week preoperatively and 2 weeks postoperatively. After 4 weeks of implantation, ultrasonography and nuclear magnetic resonance imaging (MRI) were performed to identify model rats with liver tumor growth and to analyze the growth and characteristics of the tumors. Five of these model rats were then sacrificed, and the tumors were removed from the liver for pathological examination. Three rats died during the operation; among the remaining 42 rats, 36 possessed a total of 43 liver tumors. The success rate of tumor implantation was 85.7 % (36/42), and the diameters of the tumors ranged from 5 to 10 mm. All tumor specimens were confirmed to be HCC by pathological examination. This study provides a new approach for establishing implanted HCC models in Wistar rats, which can be used for studying numerous biological features of HCC.

[Analysis of rare alleles of D13S325 falling in the range of adjacent locus].

OBJECTIVE: To analyze the rare alleles of D13S325 locus which fell in the size range of D12S391 locus with the STRtyper-10G kit. METHODS: Genotyping results of cases with suspected rare alleles of D13S325 were verified with Sinofiler(TM) kit and a singleplex amplification system. The rare alleles were separated and sequenced. RESULTS: Five families were detected with rare alleles of the D13S325 locus, which were misread as allele 20 of D12S391 locus. The alleles were named as 5.1 based on DNA sequences and have a frequency of 0.156 × 10(-2). CONCLUSION: As the rare allele 5.1 of D13S325 locus with the STRtyper-10G kit is prone to be mistyped, attention should be paid in the paternity testing, personal identification and DNA database search.

Identification of the sequence variations of 15 autosomal STR loci in a Chinese population.

BACKGROUND: DNA sequence variation including base(s) changes and insertion or deletion in the primer binding region may cause a null allele and, if this changes the length of the amplified fragment out of the allelic ladder, off-ladder (OL) alleles may be detected. AIM: In order to provide accurate and reliable DNA evidence for forensic DNA analysis, it is essential to clarify sequence variations in prevalently used STR loci. SUBJECTS AND METHODS: Suspected null alleles and OL alleles of PlowerPlex16® System from 21,934 unrelated Chinese individuals were verified by alternative systems and sequenced. RESULTS: A total of 17 cases with null alleles were identified, including 12 kinds of point mutations in 16 cases and a 19-base deletion in one case. The total frequency of null alleles was 7.751 × 10(-4). Eight hundred and forty-four OL alleles classified as being of 97 different kinds were observed at 15 STR loci of the PowerPlex®16 system except vWA. All the frequencies of OL alleles were under 0.01. CONCLUSION: Null alleles should be confirmed by alternative primers and OL alleles should be named appropriately. Particular attention should be paid to sequence variation, since incorrect designation could lead to false conclusions.

Identification of a rare off-ladder allele of the D13S325 locus during paternity testing.

This study demonstrates an unusual rare allele of D13S325 that was falsely categorized as an allele of D12S391 under the STRtyper™-10F/G system. The parentage cases with these rare alleles were analyzed using the Sinofiler™ system and singleplex amplification system, and the alleles of D13S325 extracted from the electrophoresis gel were sequenced. 5 Cases with the rare alleles misread as allele 20 of D12S391 were identified in total 2618 cases (including 3200 unrelated parents). This rare allele was designated as allele 5.1 of D13S325 based on its DNA sequence. Its frequency in the Chinese population was 1.6×10(-3). Because the rare allele 5.1 of D13S325 locus tends to be incorrectly labeled in the STRtyper™-10F/G system, particular attention should be paid when the system is used in paternity testing, personal identification, and DNA database comparisons.

Allele frequencies of 12 Y-chromosomal STRs in Chinese Tuvans in the Altay region.

Allele and haplotype frequencies of 12 Y-chromosomal short tandem repeats (STRs) included in the PowerPlex(®) Y Systems (Promega) were determined in a sample of 150 unrelated healthy male individuals of Chinese Tuvans living in the Altay region of Xinjiang Uygur Autonomous Region. Allele frequencies and gene diversity for each Y-STR locus were determined. The observed haplotype diversity value was 0.9708. The present results can be used as Chinese Tuvan genetic information resources in routine population study and forensic analysis.

Polymorphism analysis and evaluation of 19 STR loci in the Han population of Southern China.

BACKGROUND: The knowledge of allele and genotype frequencies is an essential prerequisite to the use of any human polymorphism in forensic medicine. AIM: To study the genetic polymorphism and evaluate the 19 STR loci using forensic medicine. SUBJECTS AND METHODS: Nineteen STR loci, which include D19S433, D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX, Penta E, TH01, D12S391, D2S1338 and FGA, were amplified simultaneously with Goldeneye(TM) DNA ID system 20A kit for 1161 unrelated Han individuals in Southern China. The PCR products were separated with the arrayed capillary electrophoresis. The allele frequency distribution and several parameters commonly used in forensic science were statistically analysed. RESULTS: The observed heterozygosity (Hobs) of these 19 STR loci ranged from 0.6072-0.9070; the expected heterozygosity (Hexp) ranged from 0.6052-0.9117; the power of discrimination (PD) ranged from 0.7836-0.9662; the power of exclusion (PE) ranged from 0.3479-0.8227 for trio paternity cases and 0.1959-0.6988 for duo paternity cases; the polymorphic information content (PIC) ranged from 0.5443-0.9050. CONCLUSION: The results indicate that 19 STR loci are polymorphic among the Han population in Southern China. This set of polymorphic STR loci is a useful tool in forensic paternity test and anthropological study.

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.

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.

Null alleles of the X and Y chromosomal amelogenin gene in a Chinese population.

The use of amelogenin locus typing as a gender marker incorporated in short tandem repeat (STR) multiplexes is a common practice in sex typing. Mutations in the X or Y homologue of the amelogenin gene can be misleading and result in serious mistakes in forensic applications and prenatal diagnosis. In these present studies, the amelogenin gene of 8,087 unrelated male individuals from Chinese Han population was genotyped with Powerplex(®)16 system. The samples that showed discordant results were taken for frequency calculation and further validated by re-amplification with different primer sets, Y-STR typing, and sequencing. Our results describe six amelogenin X-allele (AMELX) or amelogenin Y-allele (AMELY) null cases in these studied subjects with an overall prevalence of 0.074%. Further validation revealed point mutations in the amelogenin-priming sites associated with AMELX nulls (three cases, 0.037%) and deletions on the Y chromosome encompassing the AMELY and other Y-STR loci with three AMELY nulls (0.037%). These mutations and failure of the amplification of the AMELX and AMELY alleles have not been reported for the Chinese population. These and previous findings suggest that mutations in the amelogenin gene may result in amplification failure of the AMELX or AMELY allele, and an additional gender test for unambiguous sex determination may be needed.

[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 of 15 STR loci in a large sample of the Han population in southern China.

We have conducted genotyping experiments on 15 STR loci in over 5000 unrelated individuals of the Han population in Southern China. The loci are D31358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX and FGA. Our statistic analysis indicates that the 15 STR loci conform to the Hardy-Weinberg's equilibrium (p>0.05). We also report here the heterozygosity, matching probability, power of discrimination, probability of exclusion, polymorphism information content and typical paternity index for each locus. The results indicate that these loci are highly polymorphic in the Han population in Southern China.

[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.