STAT3-activated lncRNA XIST accelerates the inflammatory response and apoptosis of LPS-induced acute lung injury.

Acute lung injury (ALI) is a severe lung respiratory failure characterized by high morbidity and mortality. Novel findings demonstrated the critical roles of long non-coding RNA (lncRNA) in ALI. Here, we tried to investigate the roles and potential mechanism of lncRNA X-inactive specific transcript (XIST) in ALI. Results illustrated that lncRNA XIST was up-regulated in the lipopolysaccharide (LPS)-induced ALI mice models and pulmonary endothelial cells. Biofunctional assays unveiled that knockdown of XIST repressed the inflammatory response and apoptosis in LPS-induced endothelial cells. Mechanistically, XIST acted as the miR-146a-5p sponge to positively regulate STAT3. Moreover, STAT3 combined the promoter region of XIST to accelerate the transcription, constituting the positive feedback loop of XIST/miR-146a-5p/STAT3 in ALI. Collectively, these findings suggested that XIST knockdown attenuates the LPS-induced ALI, providing a potential therapeutic target.

TQFL12, a novel synthetic derivative of TQ, inhibits triple-negative breast cancer metastasis and invasion through activating AMPK/ACC pathway.

Thymoquinone (TQ) has been reported as an anti-tumour drug widely studied in various tumours, and its mechanism and effect of which has become a focus of current research. However, previous studies from our laboratory and other groups found that TQ showed weak anti-tumour effects in many cancer cell lines and animal models. Therefore, it is necessary to modify and optimize the structure of TQ to obtain new chemical entities with high efficiency and low toxicity as candidates for development of new drugs in treating cancer. Therefore, we designed and synthesized several TQ derivatives. Systematic analysis, including in vitro and in vivo, was conducted on a panel of triple-negative breast cancer (TNBC) cells and mouse model to demonstrate whether TQFL12, a new TQ derivative, is more efficient than TQ. We found that the anti-proliferative effect of TQFL12 against TNBC cells is significantly stronger than TQ. We also demonstrated TQFL12 affects different aspects in breast cancer development including cell proliferation, migration, invasion and apoptosis. Moreover, TQFL12 inhibited tumour growth and metastasis in cancer cell-derived xenograft mouse model, with less toxicity compared with TQ. Finally, mechanism research indicated that TQFL12 increased AMPK/ACC activity by stabilizing AMPKα, while molecular docking supported the direct interaction between TQFL12 and AMPKα. Taken together, our findings suggest that TQFL12, as a novel chemical entity, possesses a better inhibitory effect on TNBC cells and less toxicity in both in vitro and in vivo studies. As such, TQFL12 could serve as a potential therapeutic agent for breast cancer.

m6 A transferase METTL3-induced lncRNA ABHD11-AS1 promotes the Warburg effect of non-small-cell lung cancer.

N6 -methyladenosine (m6 A) and long noncoding RNAs (lncRNAs) are both crucial regulators in non-small-cell lung cancer (NSCLC) tumorigenesis. However, the pathological roles of m6 A and lncRNAs in NSCLC progression are still limited and undefined. Here, lncRNA ABHD11-AS1 was upregulated in NSCLC tissue specimens and cells and the ectopic overexpression was closely correlated with unfavorable prognosis of NSCLC patients. Functionally, ABHD11-AS1 promoted the proliferation and Warburg effect of NSCLC. Mechanistically, m6 A profile was analyzed by methylated RNA immunoprecipitation sequencing (MeRIP-Seq). MeRIP-Seq presented that there was m6 A modification site in ABHD11-AS1. m6 A methyltransferase-like 3 (METTL3) installed the m6 A modification and enhanced ABHD11-AS1 transcript stability to increase its expression. In conclusion, our findings highlight the function and mechanism of METTL3-induced ABHD11-AS1 in NSCLC and inspire the understanding of m6 A and lncRNA in cancer biology.

Identification of a novel germline BRCA2 variant in a Chinese breast cancer family.

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related deaths in women worldwide. In this study, a large Chinese pedigree with breast cancer including a proband and two female patients was recruited and a familial history of breast cancer was collected by questionnaire. Clinicopathological assessments and neoadjuvant therapy-related information were obtained for the proband. Blood samples were taken, and gDNA was extracted. The BRCA1/2 and PALB2 genes were screened using next-generation sequencing by a targeted gene panel. We have successfully identified a novel, germline heterozygous, missense mutation of the gene BRCA2: c.7007G>T, p.R2336L, which is likely to be pathogenic in the proband and her elder sister who both had breast cancer. Furthermore, the risk factors for developing breast cancer in this family are discussed. Thus, genetic counselling and long-term follow-up should be provided for this family of breast cancer patients as well as carriers carrying a germline variant of BRCA2: c.7007G>T (p.R2336L).

Expressions and significances of the angiotensin-converting enzyme 2 gene, the receptor of SARS-CoV-2 for COVID-19.

The ACE2 gene is a receptor of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) for COVID-19 (coronavirus disease 2019). To analyze the expression profiles and clinical significances for this gene in humans, RNA-seq data representing 27 different tissues were analyzed using NCBI; total RNA was extracted from different tissues of mouse and semi-quantitative reverse transcriptional-polymerase chain reaction (Q-RT-PCR) was carried out. Immunohistochemistry expression profiles in normal tissues and cancer tissues and TCGA survival analysis in renal and liver cancer were conducted. ACE2 was highly conserved in different species. In normal tissues, ACE2 expression distributions were organ-specific, mainly in the kidney, male testis and female breast, and cardiovascular and gastrointestinal systems. High level of expression in testis, cardiovascular and gastrointestinal system indicated that SARS-CoV-2 might not only attack the lungs, but also affect other organs, particularly the testes, thus it may severely damage male sexual development for younger male and lead to infertility in an adult male, if he contracted COVID-19. On the other side, high expression of ACE2 was correlated with increased survival rate in renal and liver cancer, indicating that ACE2 is a prognostic marker in both renal cancer and liver cancers. Thus, the ACE2 is a functional receptor for SARS-CoV-2 and has a potential anti-tumor role in cancer. Taken together, this study may not only provide potential clues for further medical pathogenesis of COVID-19 and male fertility, but also indicate the clinical significance of the role of the ACE2 gene in cancer.

Anti-PD-L1 immune checkpoint inhibitors in combination with etoposide and platinum for extensive-stage small cell lung cancer: a case report.

The conventional etoposide-platinum (EP) regimen and adjuvant radiotherapy remain the gold-standard treatment for small cell lung cancer (SCLC). However, most patients already have multiple metastases when they are first diagnosed with SCLC. The objective response rate (ORR) and 1-year survival rate are low in these patients despite active radiotherapy and chemotherapy. SCLC is oncologically featured by the high tumor mutational burden (TMB) of multiple genes, which makes immunotherapy a possible new treatment strategy for SCLC. New data from the IMpower133 and CASPIAN trials will shed new light on the treatment of SCLC. In 2020, the results from the phase 3 CASPIAN trial have already suggested that programmed cell death-ligand 1 (PD-L1) inhibitors may represent breakthroughs in the management of SCLC. Here, we report a patient with extensive-stage SCLC (ES-SCLC) treated with first-line anti-PD-L1 immune checkpoint inhibitor (PD-L1 inhibitor) (i.e., durvalumab) combined with the EP regimen for 6 cycles. The patient consistently achieved partial response (PR) [nearly complete response (CR)], and no immune-related adverse events were noted during this period. The Karnofsky performance status (PS) score maintained at 1-2 points. We further review the history of SCLC treatment and elucidate the role of combination with immunotherapy in treating SCLC in the coming years.

Epigenetic modification and a role for the E3 ligase RNF40 in cancer development and metastasis.

RNF40 (OMIM: 607700) is a really interesting new gene (RING) finger E3 ubiquitin ligase containing multiple coiled-coil domains and a C-terminal RING finger motif, which engage in protein-DNA and protein-protein interactions. RNF40 encodes a polypeptide of 1001 amino acids with a predicted molecular mass of 113,678 Da. RNF40 and its paralog RNF20 form a stable heterodimer complex that can monoubiquitylate histone H2B at lysine 120 as well as other nonhistone proteins. Cancer is a major public health problem and the second leading cause of death. Through its protein ubiquitylation activity, RNF40 acts as a tumor suppressor or oncogene to play major epigenetic roles in cancer development, progression, and metastasis, highlighting the essential function of RNF40 and the importance of studying it. In this review, we summarize current knowledge about RNF40 gene structure and the role of RNF40 in histone H2B monoubiquitylation, DNA damage repair, apoptosis, cancer development, and metastasis. We also underscore challenges in applying this information to cancer prognosis and prevention and highlight the urgent need for additional investigations of RNF40 as a potential target for cancer therapeutics.

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 HSPA5 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 HSPA5 were much higher than those of ACE2. Higher expression of HSPA5 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.

Novel compound heterozygous nonsense variants, p.L150* and p.Y3565*, of the USH2A gene in a Chinese pedigree are associated with Usher syndrome type IIA.

Usher syndrome refers to a group of genetically and clinically heterogeneous autosomal recessive diseases with retinitis pigmentosa (RP) and hearing deficiencies. The association between Usher syndrome­causative genes and resultant Usher syndrome phenotypes in patients are highly variable. In the present study, a Chinese family with Usher syndrome was recruited, and targeted next­generation sequencing, Sanger sequencing and segregation analysis were performed. The expression profiles and functional effects of the pathogenic variants of USH2A identified were analyzed. Novel nonsense compound heterozygous variants, c.T449G (p.L150*) and c.T10695A (p.Y3565*), were identified in the USH2A gene, which showed co­segregation with the disease phenotype causing Usher syndrome type IIA in the recruited Chinese pedigree. The p.L150* variant was predicted to produce a truncated protein which lacked almost all the functional domains of USH2A, whereas the p.Y3565* variant is located in one of the fibronectin type 3 domains, resulting in the loss of several fibronectin type 3 domains at the C­terminus of USH2A by producing the truncated protein. It was shown that Ush2a mRNA expression levels were higher in the retina compared with those in the eye tissues (lens, sclera and cornea), uterus, ovary, breast, testis, spleen, kidney, liver, intestine, brain, skeletal muscle and blood. Additionally, the protein structure was shown to be highly conserved by comparing Homo sapiens USH2A to eight other species. To the best of our knowledge, the present study is the first to identify two novel pathogenic variants, c.T449G (p.L150*) and c.T10695A (p.Y3565*), in the USH2A gene in a patient with Usher syndrome type IIA, thereby expanding the known spectrums of USH2A causative mutations. The present discovery may assist in understanding the molecular pathogenesis underlying the development of RP and Usher syndrome type IIA, and in the development of diagnostic, therapeutic and genetic counseling strategies in patients with Usher syndrome type IIA disease.

SCAR marker for identification and discrimination of specific medicinal Lycium chinense Miller from Lycium species from ramp-PCR RAPD fragments.

In the current study, ramp-PCR fragments from improved RAPD (random amplified polymorphic DNA) amplification of Lycium (Goji) species or cultivars were cut and cloned into the vector of pGEM-T. A positive clone 10-5 was screened by PCR amplification, enzymatic digestion, and Sanger sequencing. A SCAR (sequence-characterized amplified region) marker, named Goji 10-5, with 949 nucleotides in length, was identified. Goji 10-5 is specific to Goji species Lycium chinense Miller from Jiangxi in China and Texas in the USA. A BLAST search of this nucleotide sequence in the GenBank database indicated that it shows no identity with any other species, including no any other Lycium species. As a new sequence, we have deposited it in the GenBank database with accession No. MN862323. PCR assays were developed and converted the nucleotide sequence to become a novel molecular marker for Lycium chinense Miller, named Goji 10-5. This marker may be used for the genetic identification of other samples. This study has successfully developed Goji 10-5, a specific SCAR marker to identify L. chinense and distinguish it from other species, including other Lycium species from different locations.

Macrophage migration inhibitory factor promotes Warburg effect via activation of the NF­κB/HIF­1α pathway in lung cancer.

Macrophage migration inhibitory factor (MIF) is upregulated in various solid tumors, a process that is associated with tumor progression and metastasis. The present study aimed to investigate the role and the underlying mechanism of MIF in human lung cancer. Human lung cancer H358, H460, H524, H1650, H838, H1975 and A549 cell lines were used to examine the expression of MIF by real time­quantitative polymerase chain reaction and western blotting. The lentivirus was used to overexpress MIF and the expression of MIF and hypoxia­inducible factor 1­α (HIF­1α) were knocked down by shRNA or siRNA. The proliferation of cell lines was detected by MTT assay. Glucose uptake, adenosine 5'­triphosphate (ATP) production, the glycolytic rate and lactate production were used to examine the Warburg effect in cells. BAY 11­7082 (BAY) was used to inhibit the nuclear translocation of nuclear factor­κB (NF­κB), which was detected using immunofluorescence. It was revealed that overexpression of MIF promoted cell proliferation and the Warburg effect in lung cancer, whereas knockdown of MIF inhibited cell proliferation and the Warburg effect. Mechanistically, MIF promoted the Warburg effect by upregulating HIF­1α. Knockdown of HIF­1α largely abolished the promotional effect of MIF on the Warburg effect. Additionally, the results in the current study provided evidence that MIF regulates HIF­1α through NF­κB. In conclusion, the findings of the present study demonstrated that MIF is a key component in lung cancer progression through promoting the Warburg effect, and that the novel MIF/NF­κB/HIF­1α axis may prove to be useful for the development of new strategies for treating patients with lung cancer.

Epithelial-mesenchymal transition and GALC expression of circulating tumor cells indicate metastasis and poor prognosis in non-small cell lung cancer.

BACKGROUND: Circulating tumor cells (CTCs) is a promising biomarker for cancer prognosis and monitoring. Molecular characterizing of CTCs could provide beneficial information on the basis of CTCs counting. OBJECTIVE: To investigate the epithelial-mesenchymal transition (EMT) phenotypes and GALC mRNA expression of CTCs in non-small cell lung cancer (NSCLC) patients. METHODS: We analyzed the baseline number, EMT classification, and GALC expression of CTCs in 47 NSCLC patients using CanPatrol platform and RNA in situ hybridization technique. RESULTS: CTCs were detected in 91.5% patients ranging 0-47/5 mL blood. Increased CTCs were associated with advanced tumor stages (6/5 mL) compared with early stages (3.5/5 mL). Patients with effective treatment response presented lower CTCs (3.5/5 mL) than patients with insufficient response (7/5 mL). Epithelial, hybrid and mesenchymal CTCs were detected in 55.4%, 78.7% and 61.7% patients, respectively. Patients with distant metastasis and poor curative outcomes presented higher level of EMT-CTCs. GALC expression was positive in CTCs of 80.6% patients and closely correlated with tumor number and distant metastasis and treatment outcomes. CONCLUSIONS: EMT phenotypes and GALC expression of CTCs are correlated with cancer metastasis and therapeutic outcomes, suggesting them to be potential markers for the prognosis of NSCLC.

The effect of foxp3-overexpressing Treg cells on non-small cell lung cancer cells.

The aim of the present study was to investigate the novel mechanisms of forkhead box protein P3 (foxp3) in T regulatory (Treg) cells in lung cancer behavior. Treg cells were isolated from the peripheral blood of healthy volunteers and then co­cultured with 95D cells. A plasmid overexpressing foxp3 was constructed and transfected into Treg cells and an MTS assay was performed to assess cell viability. Flow cytometry was performed to evaluate cell apoptosis and reverse transcription­quantitative polymerase chain reaction was used to measure mRNA expression. A Transwell assay was used to assess cell invasion. Treg cells were successfully isolated from peripheral blood with purity of 94.26%. Foxp3 expression in Treg cells was significantly increased following co­culture with 95D cells, while matrix metalloproteinase­9 expression was upregulated in 95D cells co­cultured with Treg cells. The apoptosis, invasion and migration abilities of 95D cells were suppressed by co­culture with Treg cells, whereas the adhesive ability was enhanced. Foxp3 overexpression in Treg cells enhanced the viability and invasiveness of 95D cells, whereas cell adhesion and migration were decreased. The results of the present study demonstrate that the viability and invasiveness of 95D cells are enhanced by foxp3 overexpression in Treg cells, indicating that increased levels of foxp3 in the tumor microenvironment may promote tumor cell growth.

DNA promoter hypermethylation contributes to down-regulation of galactocerebrosidase gene in lung and head and neck cancers.

Galactocerebrosidase (GALC) is a lysosomal enzyme responsible for glycosphingolipids degradation byproducts of which are important for synthesis of apoptosis mediator ceramide. Reduced expression of GALC has been identified in human malignancies; however, molecular mechanisms underlying down-regulation of GALC expression in cancer remain unknown. We performed methylation and expression analysis on GALC gene in a panel of head and neck cancer (HNC) and lung cancer cell lines, attempting to understand the regulation of GALC in human cancer. QRT-PCR and western blot analysis were performed to detect the expression of GALC in HNC. Bisulfite DNA sequencing and real-time qMSP were used to detect the methylation of GALC in HNC and lung cancer cell lines. 5aza-dC treatment assay was used to analysis the functional effect of GALC methylation on GALC expression in HNC. Reduction or complete absence of GALC expression was observed in more than a half of the tested HNC cell lines (8/14). 7 out of 8 cell lines with down-regulated expression harbored heavy CpG island methylation, while all cell lines with abundant expression of the gene contained no methylation. Hypermethylation was also found in primary HNC tumor tissues and lung cancer cell lines whereas absent in normal oral mucosa tissues. Demethylating treatment demonstrated that 5aza-dC significantly restored GALC expression in cell lines with methylated promoter while showed no effect on cell lines without promoter hypermethylation. Our findings for the first time demonstrated that promoter hypermethylation contributed to down-regulation of GALC Gene, implicating epigenetic inactivation of GALC may play a role in tumorigenesis of cancer.

[Clinical significance of tumor interstitial T lymphocyte subset activity in non-small-cell lung cancer].

OBJECTIVE: To study the relation of tumor interstitial T lymphocyte subset activity to the clinical staging of non-small-cell lung cancer (NSCLC) and the immune response. METHODS: Immunohistochemical staining for CD4(+), CD8(+) and CD4(+)CD25(+) Foxp3(+) (regulatory T cells, Treg) T cells was performed on paraffin-embedded tissues from 60 NSCLC cases. RESULTS: Compared to stage I/II NSCLC patients, patients in stage III/IV showed a significant decrease in the percentage of CD4(+) and CD4(+)/CD8(+) T cells (P<0.05) and an increase in CD8(+) and CD4(+)CD25(+) Foxp3(+) T cells (P<0.05). Treg cells were enriched in the tumor tissue as compared with those in the adjacent tissues. CONCLUSIONS: The proportion of CD4(+)CD25(+) Foxp3(+) Treg cells is positively correlated to the clinical staging of NSCLC, in which T cell-mediated immune response is suppressed.