Establishment of stable cell line for inducing KAP1 protein expression

Generation of the stable cell lines is a highly efficient tool in functional studies of certain genes or proteins, where the particular genes or proteins are inducibly expressed. The KRAB-associated protein-1 (KAP1) is an important transcription regulatory protein, which is investigated in several molecular biological studies. In this study, we have aimed to generate a stable cell line for inducing KAP1 expression. The recombinant plasmid pcDNA5/FRT/TO-KAP1 was constructed at first, which was then transfected into Flp-In™T-REx™-HEK293 cells to establish an inducible pcDNA5/FRT/TO-KAP1-HEK293 cell line. The Western blot analysis showed that the protein level of KAP1 is over-expressed in the established stable cell line by doxycycline induction, both dose and time dependently. Thus we have successfully established stable pcDNA5/FRT/TO-KAP1-HEK293 cell line, which can express KAP1 inducibly. This inducible cell line might be very useful for KAP1 functional studies

Development of Diagnostic SCAR Markers for Genomic DNA Amplifications in Breast Carcinoma by DNA Cloning of High-GC RAMP-PCR Fragments

Cancer is genetically heterogeneous regarding to molecular genetic characteristics and pathogenic pathways. A wide spectrum of biomarkers, including DNA markers, is used in determining genomic instability, molecular subtype determination and disease prognosis, and estimating sensitivity to different drugs in clinical practice. In a previous study, we developed highly effective DNA markers using improved random amplified polymorphic DNA (RAPD) with high-GC primers, which is a valuable approach for the genetic authentication of medicinal plants. In this study, we applied this effective DNA marker technique to generate genetic fingerprints that detect genomic alterations in human breast cancer tissues and then developed sequence-characterized amplified region (SCAR) markers. Three SCAR markers (BC10-1, BC13-4 and BC31-2) had high levels of genomic DNA amplification in breast cancer. The PHKG2 and RNF40 genes are either overlapping or close to the sequences of SCAR marker BC13-4, while SCAR marker BC10-1 is in the intron and overlap the DPEP1 gene, suggesting that alterations in the expression of these genes could contribute to cancer progression. Screening of breast cancer cell lines showed that the mRNA expression levels for the PHKG2 and DPEP1 were lower in non-tumorigenic mammary epithelial cell MCF10A, but elevated in other cell lines. The DPEP1 mRNA level in invasive ductal carcinoma specimens was significantly higher than that of the adjacent normal tissues in women. Taken together, high-GC RAMP-PCR provides greater efficacy in measuring genomic DNA amplifications, deletion or copy number variations. Furthermore, SCAR markers BC10-1 and BC13-4 might be useful diagnostic markers for breast cancer carcinomas

Evaluation and characterization of HSPA5 (GRP78) expression profiles in normal individuals and cancer patients with COVID-19

HSPA5 (BiP, GRP78) has been reported as a potential host-cell receptor for SARS-Cov-2, but its expression profiles on different tissues including tumors, its susceptibility to SARS-Cov-2 virus and severity of its adverse effects on malignant patients are unclear. In the current study, HSPA5 has been found to be expressed ubiquitously in normal tissues and significantly increased in 14 of 31 types of cancer tissues. In lung cancer, mRNA levels of HSPAS were 253-fold increase than that of ACE2. Meanwhile, in both malignant tumors and matched normal samples across almost all cancer types, mRNA levels of HSPAS were much higher than those of ACE2. Higher expression of HSPAS significantly decreased patient overall survival (OS) in 7 types of cancers. Moreover, systematic analyses found that 7.15% of 5,068 COVID-19 cases have malignant cancer coincidental situations, and the rate of severe events of COVID-19 patients with cancers present a higher trend than that for all COVID-19 patients, showing a significant difference (33.33% vs 16.09%, p<0.01). Collectively, these data imply that the tissues with high HSPA5 expression, not low ACE2 expression, are susceptible to be invaded by SARS-CoV-2. Taken together, this study not only indicates the clinical significance of HSPA5 in COVID-19 disease and cancers, but also provides potential clues for further medical treatments and managements of COVID-19 patients

Effects of a particular heptapeptide on the IFN-α-sensitive CML cells

Abstract: Using the phage display biopanning technique, we have previously identified a heptapeptide KLWVIPQ which specifically bind to the surface of the IFN-α sensitive but not the IFN-α-resistant CML cells. The effects of this heptapeptide to the IFN-α-sensitive CML cells were investigated in the present study. IFN-α-sensitive KT-1/A3 and IFN-α-resistant KT-1/A3R CML cells were transfected by pEGFP KLWVIPQ expression vector and/or induced by IFN-α. WST-1 cell proliferation assay, flow cytometry and western blotting were performed to determine the effects of this heptapeptide and/or IFN-α on CML cells. The viability of the KT-1/A3 cells w as inhibited and apoptosis was induced by either expression of the heptapeptide KLWVIPQ or IFN-α treatment with concurrent up-regulation of P53 and down-regulation of P210bcr/abl. However, these effects were not observed in the IFN-α-resistant KT-1/A3R cells. These results suggest that the heptapeptide KLWVIPQ shares a similar mechanism w ith IFN-α in the regulat ion of CML cell growth and apoptosis, implying that the heptapeptide KLWVIPQ could be a novel target to go further into mechanisms of IFN-α sensitivity and/or resistance in CML

Development and significance of RAPD-SCAR markers for the identification of Litchi chinensis Sonn. by improved RAPD amplification and molecular cloning

Background: Analysis of genetic diversity is important for the authentication of a species. Litchi ( Litchi chinensis Sonn.) is a subtropical evergreen tree. Recently, L. chinensis has been characterized by an improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis. The goal of this study was to develop sequence-characterized amplified region (SCAR) markers from the improved RAPD fragments for the genetic analysis of L. chinensis. Results: The improved RAPD fragments from L. chinensis were cloned, sequenced and converted into stable SCAR markers. Sequencing of three cloned RAPD fragments revealed that the clone L7-16 consisted of 222 nucleotides (GenBank accession number KM235222), clone L9-6 consisted of 648 nucleotides (GenBank accession number KM235223), and clone L11-26 consisted of 369 nucleotides (GenBank accession number KM235224). Then, specific primers for SCAR markers L7-16, L9-6, and L11-26 were designed and synthesized. PCR amplification was performed using DNA templates from 24 different samples, including 6 samples of L. chinensis and other plants. The SCAR marker L9-6 was specific for all of the L. chinensis samples, the SCAR marker L11-26 specific for five L. chinensis samples, and the SCAR marker L7-16 only specific for the samples from Luzhou. Conclusions: This study developed stable SCAR markers for the identification of L. chinensis by the cloning of the improved RAPD fragments. Combining RAPD and SCAR markers provides a simple and reliable tool for the genetic characterization of plant species

A Novel Variant of the FZD4 Gene in a Chinese Family Causes Autosomal Dominant Familial Exudative Vitreoretinopathy

Background/Aims: Familial exudative vitreoretinopathy (FEVR) is a complex hereditary eye disorder characterized by incomplete development of the retinal vasculature, thereby affecting retinal angiogenesis. Methods: In this study, a Chinese autosomal dominant FEVR pedigree was recruited. Ophthalmic examinations were performed, targeted next-generation sequencing was used to identify the causative gene, and Sanger sequencing was conducted to verify the candidate mutation. Co-segregation analysis was performed to evaluate pathogenicity. Semi-quantitative reverse transcription-PCR was applied to investigate the spatial and temporal expression patterns of the frizzled class receptor 4 (FZD4) gene in the mouse. Results: A novel heterozygous, deleterious variant of the FZD4 gene, c.A749G (p.Y250C), was identified in this FEVR pedigree, which co-segregated with the clinical phenotype. The amino acid tyrosine (Y) is highly conserved both orthologously and paralogously. The FZD4 gene was highly expressed in the retina, sclera of the eye, ovary, kidney, and liver; ubiquitously expressed in other tissues; and highly expressed in 6 different developmental stages/times of retinal tissue. Conclusion: Our study is the first to identify that the novel heterozygous variant c.A749G (p.Y250C) in the FZD4 gene may be the disease-causing mutation in this FEVR family, extending its mutation spectrum. These findings further our understanding of the molecular pathogenesis of FEVR and will facilitate the development of methods for the diagnosis, prevention, and genetic counseling of this disease

Resveratrol Enhances Polyubiquitination-Mediated ARV-7 Degradation in Prostate Cancer Cells

Although androgen deprivation therapy (ADT) serves as the primary treatment option for localized or metastatic prostate cancer, most cases eventually develop into castration-resistant prostate cancer (CRPC). However, androgen receptor (AR) continues to be functional in CRPC through various mechanisms, including the development of AR splicing variants, especially ARV7. Since it lacks the ligand binding domain but retains the intact DNA binding domain, ARV7 is constitutively active, which makes ARV7-positive prostate cancer responsive to neither abiraterone nor enzalutamide. In this study, we explored the effect of resveratrol on ARV7 transcriptional activity and the potential for development of resveratrol as a treatment for ARV7-positive prostate cancer. First, we ectopically expressed ARV7 in PC3 cells, an AR-negative prostate cancer cell line, and demonstrated that resveratrol is capable of inhibiting ARV7 transcriptional activity by downregulating ARV7 protein levels. Of note, resveratrol does not affect the mRNA levels of ARV7 nor its nuclear translocation. Next, we demonstrated that resveratrol is capable of downregulating the levels of the endogenously expressed ARV7 as well as AR target gene mRNAs in 22RV1 prostate cancer cells. Mechanistically, resveratrol downregulates ARV7 by enhancing ARV7 polyubiquitination and subsequent proteasome-mediated degradation. These findings suggest that resveratrol could be a potential treatment for ARV7-positive CPRC

TWIST Represses Estrogen Receptor-alpha Expression by Recruiting the NuRD Protein Complex in Breast Cancer Cells

Loss of estrogen receptor α (ERα) expression and gain of TWIST (TWIST1) expression in breast tumors correlate with increased disease recurrence and metastasis and poor disease-free survival. However, the molecular and functional regulatory relationship between TWIST and ERα are unclear. In this study, we found TWIST was associated with a chromatin region in intron 7 of the human ESR1 gene coding for ERα. This association of TWIST efficiently recruited the nucleosome remodeling and deacetylase (NuRD) repressor complex to this region, which subsequently decreased histone H3K9 acetylation, increased histone H3K9 methylation and repressed ESR1 expression in breast cancer cells. In agreement with these molecular events, TWIST expression was inversely correlated with ERα expression in both breast cancer cell lines and human breast ductal carcinomas. Forced expression of TWIST in TWIST-negative and ERα-positive breast cancer cells such as T47D and MCF-7 cells reduced ERα expression, while knockdown of TWIST in TWIST-positive and ERα-negative breast cancer cells such as MDA-MB-435 and 4T1 cells increased ERα expression. Furthermore, inhibition of histone deacetylase (HDAC) activity including the one in NuRD complex significantly increased ERα expression in MDA-MB-435 and 4T1 cells. HDAC inhibition together with TWIST knockdown did not further increase ERα expression in 4T1 and MDA-MB-435 cells. These results demonstrate that TWIST/NuRD represses ERα expression in breast cancer cells. Therefore, TWIST may serve as a potential molecular target for converting ERα-negative breast cancers to ERα-positive breast cancers, allowing these cancers to restore their sensitivity to endocrine therapy with selective ERα antagonists such as tamoxifen and raloxifene