Duan Ma

The cell growth suppressor, mir-126, targets IRS-1.

miRNAs are a family of approximately 22-nuleotide-long noncoding RNAs involved in the formation and progress of tumors. Since traditional methods for the detection of miRNAs expression have many disadvantages, we developed a simple method called polyA RT PCR. With this method, we detected a series of miRNAs and found that mir-126 is one of the miRNAs underexpressed in breast cancer cells. Flow cytometry analysis showed that mir-126 inhibited cell cycle progression from G1/G0 to S. Further studies revealed that mir-126 targeted IRS-1 at the translation level. Knocking down of IRS-1 suppresses cell growth in HEK293 and breast cancer cell MCF-7, which recapitulates the effects of mir-126. In conclusion, we developed a simple method for high-throughput screening of miRNAs and found that mir-126, a cell growth suppressor, targets IRS-1.

Maternal Mosaicism Is a Significant Contributor to Discordant Sex Chromosomal Aneuploidies Associated with Noninvasive Prenatal Testing

BACKGROUND In the human fetus, sex chromosome aneuploidies (SCAs) are as prevalent as the common autosomal trisomies 21, 18, and 13. Currently, most noninvasive prenatal tests (NIPTs) offer screening only for chromosomes 21, 18, and 13, because the sensitivity and specificity are markedly higher than for the sex chromosomes. Limited studies suggest that the reduced accuracy associated with detecting SCAs is due to confined placental, placental, or true fetal mosaicism. We hypothesized that an altered maternal karyotype may also be an important contributor to discordant SCA NIPT results. METHODS We developed a rapid karyotyping method that uses massively parallel sequencing to measure the degree of chromosome mosaicism. The method was validated with DNA models mimicking XXX and XO mosaicism and then applied to maternal white blood cell (WBC) DNA from patients with discordant SCA NIPT results. RESULTS Sequencing karyotyping detected chromosome X (ChrX) mosaicism as low as 5%, allowing an accurate assignment of the maternal X karyotype. In a prospective NIPT study, we showed that 16 (8.6%) of 181 positive SCAs were due to an abnormal maternal ChrX karyotype that masked the true contribution of the fetal ChrX DNA fraction. CONCLUSIONS The accuracy of NIPT for ChrX and ChrY can be improved substantially by integrating the results of maternal-plasma sequencing with those for maternal-WBC sequencing. The relatively high frequency of maternal mosaicism warrants mandatory WBC testing in both shotgun sequencing- and single-nucleotide polymorphism-based clinical NIPT after the finding of a potential fetal SCA.

The Induction of MicroRNA Targeting IRS-1 Is Involved in the Development of Insulin Resistance under Conditions of Mitochondrial Dysfunction in Hepatocytes

Background Mitochondrial dysfunction induces insulin resistance in myocytes via a reduction of insulin receptor substrate-1 (IRS-1) expression. However, the effect of mitochondrial dysfunction on insulin sensitivity is not understood well in hepatocytes. Although research has implicated the translational repression of target genes by endogenous non-coding microRNAs (miRNA) in the pathogenesis of various diseases, the identity and role of the miRNAs that are involved in the development of insulin resistance also remain largely unknown. Methodology To determine whether mitochondrial dysfunction induced by genetic or metabolic inhibition causes insulin resistance in hepatocytes, we analyzed the expression and insulin-stimulated phosphorylation of insulin signaling intermediates in SK-Hep1 hepatocytes. We used qRT-PCR to measure cellular levels of selected miRNAs that are thought to target IRS-1 3′ untranslated regions (3′UTR). Using overexpression of miR-126, we determined whether IRS-1-targeting miRNA causes insulin resistance in hepatocytes. Principal Findings Mitochondrial dysfunction resulting from genetic (mitochondrial DNA depletion) or metabolic inhibition (Rotenone or Antimycin A) induced insulin resistance in hepatocytes via a reduction in the expression of IRS-1 protein. In addition, we observed a significant up-regulation of several miRNAs presumed to target IRS-1 3′UTR in hepatocytes with mitochondrial dysfunction. Using reporter gene assay we confirmed that miR-126 directly targeted to IRS-1 3′UTR. Furthermore, the overexpression of miR-126 in hepatocytes caused a substantial reduction in IRS-1 protein expression, and a consequent impairment in insulin signaling. Conclusions/Significance We demonstrated that miR-126 was actively involved in the development of insulin resistance induced by mitochondrial dysfunction. These data provide novel insights into the molecular basis of insulin resistance, and implicate miRNA in the development of metabolic disease.

miRNA-135a promotes breast cancer cell migration and invasion by targeting HOXA10

BackgroundmiRNAs are a group of small RNA molecules regulating target genes by inducing mRNA degradation or translational repression. Aberrant expression of miRNAs correlates with various cancers. Although miR-135a has been implicated in several other cancers, its role in breast cancer is unknown. HOXA10 however, is associated with multiple cancer types and was recently shown to induce p53 expression in breast cancer cells and reduce their invasive ability. Because HOXA10 is a confirmed miR-135a target in more than one tissue, we examined miR-135a levels in relation to breast cancer phenotypes to determine if miR-135a plays role in this cancer type.MethodsExpression levels of miR-135a in tissues and cells were determined by poly (A)-RT PCR. The effect of miR-135a on proliferation was evaluated by CCK8 assay, cell migration and invasion were evaluated by transwell migration and invasion assays, and target protein expression was determined by western blotting. GFP and luciferase reporter plasmids were constructed to confirm the action of miR-135a on downstream target genes including HOXA10. Results are reported as means ± S.D. and differences were tested for significance using 2-sided Students t-test.ResultsHere we report that miR-135a was highly expressed in metastatic breast tumors. We found that the expression of miR-135a was required for the migration and invasion of breast cancer cells, but not their proliferation. HOXA10, which encodes a transcription factor required for embryonic development and is a metastasis suppressor in breast cancer, was shown to be a direct target of miR-135a in breast cancer cells. Our analysis showed that miR-135a suppressed the expression of HOXA10 both at the mRNA and protein level, and its ability to promote cellular migration and invasion was partially reversed by overexpression of HOXA10.ConclusionsIn summary, our results indicate that miR-135a is an onco-miRNA that can promote breast cancer cell migration and invasion. HOXA10 is a target gene for miR-135a in breast cancer cells and overexpression of HOXA10 can partially reverse the miR-135a invasive phenotype.

An inhibitory effect of miR-22 on cell migration and invasion in ovarian cancer

OBJECTIVES Aberrant expression of microRNAs (miRNAs) has been implicated in ovarian carcinoma. However, roles of miRNAs in ovarian caner metastasis have not been comprehensively addressed. This work is aimed to identify selected miRNAs involved in ovarian cancer metastasis. METHODS We examined the distinct miRNA expression profiles between paired high-metastatic human serous ovarian cancer cell SKOV-3ip and low-metastatic human serous ovarian cell SKOV-3 using miRNA microarray. Subsequently, a validation with Real-time RT-PCR was performed for miR-22 expression level, and a functional study was carried out for miR-22. RESULTS Through a screen with microarray, we found there were a variety of miRNAs differentially expressed between paired high and low metastatic serous ovarian cancer cells. Particularly, miR-22 was identified as a potential metastasis-inhibitor in ovarian cancer. There was a negative correlation between miR-22 expression and the metastatic potential in ovarian cancer cells. Furthermore, both gain-of-function and loss-of-function studies displayed an inhibitory effect of miR-22 on cell migration and invasion in vitro without significantly affecting cell viability and apoptosis. Subsequent bioinformatics analysis revealed that miR-22 might regulate multiple pro-metastatic genes, which could provide an explanation to the inhibitory effects of miR-22 on cell migration and invasion. CONCLUSIONS Taken together, our findings suggested that miR-22 might be involved in inhibiting ovarian cancer metastasis.

Comprehensive expression profiling of microRNAs in laryngeal squamous cell carcinoma

MicroRNAs (miRNAs) are noncoding RNAs involved in posttranscriptional regulation of gene expression in cancer and provide new perspectives on the development of laryngeal squamous cell carcinoma (SCC).

Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells

BackgroundTissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz inhibitor that inhibits plasmin and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor progression, invasion and metastasis. Here, we have investigated the mechanism of DNA methylation on the repression of TFPI-2 in breast cancer cell lines.ResultsWe found that both protein and mRNA of TFPI-2 could not be detected in highly invasive breast cancer cell line MDA-MB-435. To further investigate the mechanism of TFPI-2 repression in breast cancer cells, 1.5 Kb TFPI-2 promoter was cloned, and several genetic variations were detected, but the promoter luciferase activities were not affected by the point mutation in the promoter region and the phenomena was further supported by deleted mutation. Scan mutation and informatics analysis identified a potential KLF6 binding site in TFPI-2 promoter. It was revealed, by bisulfite modified sequence, that the CpG island in TFPI-2 promoter region was hypermethylated in MDA-MB-435. Finally, using EMSA and ChIP assay, we demonstrated that the CpG methylation in the binding site of KLF-6 diminished the binding of KLF6 to TFPI-2 promoter.ConclusionIn this study, we found that the CpG islands in TFPI-2 promoter was hypermethylated in highly invasive breast cancer cell line, and DNA methylation in the entire promoter region caused TFPI-2 repression by inducing inactive chromatin structure and decreasing KLF6 binding to its DNA binding sequence.

Two cases of placental T21 mosaicism: challenging the detection limits of non‐invasive prenatal testing

Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai Medical College, Fudan University, Shanghai, China Maternity and Child Health Hospital of Anhui Province, The Maternal and Child Health Clinical College, Anhui Medical University, Hefei, China Prenatal Diagnostic Center, International Peace Maternal and Children’s Hospital, Shanghai Jiaotong University, Shanghai, China The First Affiliated Hospital of Nanjing Medical University, Nanjing, China Xijing Hospital, The Fourth Military Medical College, Xian, China Baoji Maternity and Child Health Hospital, Baoji, China Berry Genomics C., Limited, Beijing, China *Correspondence to: Weiwei Cheng. E-mail: 18017316001@163.com; David Cram. E-mail: david.cram@berrygenomics.com †These authors contributed equally to this work.

Tiam1, negatively regulated by miR-22, miR-183 and miR-31, is involved in migration, invasion and viability of ovarian cancer cells

Tiam1 has been implicated in the invasive phenotype of various carcinomas. However, its role in ovarian cancer remains to be elucidated, including its upstream regulatory mechanisms. In the present study, we examined the differential expression of Tiam1 in 10 normal ovarian tissues and 17 paired primary and corresponding metastatic ovarian cancer tissues by semi-quantitative immunohistochemistry. It was found that Tiam1 expression was remarkably increased in both primary and metastatic ovarian cancer tissues relative to normal ovarian tissues. Loss-of-function study revealed that downregulation of Tiam1 in SKOV-3ip and HO-8910PM cells lead to reduced cell migration and invasion, and growth inhibition without significantly affecting cell apoptosis. Subsequent regulatory study further confirmed the negative regulatory effects of miR-22, miR-183 and miR-31 on Tiam1 expression. Taken together, our data suggested that Tiam1 may be involved in the aggressive behavior of ovarian cancer, and differential expression profiles of microRNA (miRNA) may contribute to the dysregulation of Tiam1 abundance, which contributes to the invasive, migratory and viability properties of ovarian cancer cells.

Ubiquitin C-terminal Hydrolase 37, a novel predictor for hepatocellular carcinoma recurrence, promotes cell migration and invasion via interacting and deubiquitinating PRP19

Ubiquitin C-terminal hydrolase 37 (UCH37) plays a crucial role in numerous biological processes and is also involved in oncogenesis. In this study, clinicopathologic data showed that UCH37 was over-expressed in hepatocellular carcinoma (HCC) cancerous tissues and was a significant predictor for time to recurrence (TTR). In vitro, we discovered that UCH37 could promote cell migration and invasion. Subsequently, we utilized Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) to identify differentially expressed proteins in UCH37 over-expressing cells compared with the control cells, and found that PRP19, an essential RNA splicing factor, was up-regulated. The relationship between UCH37, PRP19 and the capability of cell migration and invasion was further confirmed. Collectively, this study demonstrated that UCH37 could promote cell migration and invasion in HCC cell lines through interacting and deubiquitinating PRP19, and suggested that UCH37 could be a novel predictor for HCC recurrence after curative resection.