Unveiling inflammatory and prehypertrophic cell populations as key contributors to knee cartilage degeneration in osteoarthritis using multi-omics data integration.

OBJECTIVES: Single-cell and spatial transcriptomics analysis of human knee articular cartilage tissue to present a comprehensive transcriptome landscape and osteoarthritis (OA)-critical cell populations. METHODS: Single-cell RNA sequencing and spatially resolved transcriptomic technology have been applied to characterise the cellular heterogeneity of human knee articular cartilage which were collected from 8 OA donors, and 3 non-OA control donors, and a total of 19 samples. The novel chondrocyte population and marker genes of interest were validated by immunohistochemistry staining, quantitative real-time PCR, etc. The OA-critical cell populations were validated through integrative analyses of publicly available bulk RNA sequencing data and large-scale genome-wide association studies. RESULTS: We identified 33 cell population-specific marker genes that define 11 chondrocyte populations, including 9 known populations and 2 new populations, that is, pre-inflammatory chondrocyte population (preInfC) and inflammatory chondrocyte population (InfC). The novel findings that make this an important addition to the literature include: (1) the novel InfC activates the mediator MIF-CD74; (2) the prehypertrophic chondrocyte (preHTC) and hypertrophic chondrocyte (HTC) are potentially OA-critical cell populations; (3) most OA-associated differentially expressed genes reside in the articular surface and superficial zone; (4) the prefibrocartilage chondrocyte (preFC) population is a major contributor to the stratification of patients with OA, resulting in both an inflammatory-related subtype and a non-inflammatory-related subtype. CONCLUSIONS: Our results highlight InfC, preHTC, preFC and HTC as potential cell populations to target for therapy. Also, we conclude that profiling of those cell populations in patients might be used to stratify patient populations for defining cohorts for clinical trials and precision medicine.

Intracellular expression of arginine deiminase activates the mitochondrial apoptosis pathway by inhibiting cytosolic ferritin and inducing chromatin autophagy.

BACKGROUND: Based on its low toxicity, arginine starvation therapy has the potential to cure malignant tumors that cannot be treated surgically. The Arginine deiminase (ADI) gene has been identified to be an ideal cancer-suppressor gene. ADI expressed in the cytosol displays higher oncolytic efficiency than ADI-PEG20 (Pegylated Arginine Deiminase by PEG 20,000). However, it is still unknown whether cytosolic ADI has the same mechanism of action as ADI-PEG20 or other underlying cellular mechanisms. METHODS: The interactions of ADI with other protein factors were screened by yeast hybrids, and verified by co-immunoprecipitation and immunofluorescent staining. The effect of ADI inhibiting the ferritin light-chain domain (FTL) in mitochondrial damage was evaluated by site-directed mutation and flow cytometry. Control of the mitochondrial apoptosis pathway was analyzed by Western Blotting and real-time PCR experiments. The effect of p53 expression on cancer cells death was assessed by siTP53 transfection. Chromatin autophagy was explored by immunofluorescent staining and Western Blotting. RESULTS: ADI expressed in the cytosol inhibited the activity of cytosolic ferritin by interacting with FTL. The inactive mutant of ADI still induced apoptosis in certain cell lines of ASS- through mitochondrial damage. Arginine starvation also generated an increase in the expression of p53 and p53AIP1, which aggravated the cellular mitochondrial damage. Chromatin autophagy appeared at a later stage of arginine starvation. DNA damage occurred along with the entire arginine starvation process. Histone 3 (H3) was found in autophagosomes, which implies that cancer cells attempted to utilize the arginine present in histones to survive during arginine starvation. CONCLUSIONS: Mitochondrial damage is the major mechanism of cell death induced by cytosolic ADI. The process of chromatophagy does not only stimulate cancer cells to utilize histone arginine but also speeds up cancer cell death at a later stage of arginine starvation.

Retraction Note: Long noncoding RNA LINC00968 inhibits proliferation, migration and invasion of lung adenocarcinoma through targeting miR-22-5p/CDC14A axis.

[This retracts the article DOI: 10.1007/s13205-021-02981-8.].

Genome-wide analysis of circular RNAs and validation of hsa_circ_0086354 as a promising biomarker for early diagnosis of cerebral palsy.

BACKGROUND: Cerebral palsy (CP) is a spectrum of non-progressive motor disorders caused by brain injury during fetal or postnatal periods. Current diagnosis of CP mainly relies on neuroimaging and motor assessment. Here, we aimed to explore novel biomarkers for early diagnosis of CP. METHODS: Blood plasma from five children with CP and their healthy twin brothers/sisters was analyzed by gene microarray to screen out differentially expressed RNAs. Selected differentially expressed circular RNAs (circRNAs) were further validated using quantitative real-time PCR. Receiver operating characteristic (ROC) curve analysis was used to assess the specificity and sensitivity of hsa_circ_0086354 in discriminating children with CP and healthy controls. RESULTS: 43 up-regulated circRNAs and 2 down-regulated circRNAs were obtained by difference analysis (fold change > 2, p < 0.05), among which five circRNAs related to neuron differentiation and neurogenesis were chosen for further validation. Additional 30 pairs of children with CP and healthy controls were recruited and five selected circRNAs were further detected, showing that hsa_circ_0086354 was significantly down-regulated in CP plasma compared with control, which was highly in accord with microarray analysis. ROC curve analysis showed that the area under curve (AUC) to discriminate children with CP and healthy controls using hsa_circ_0086354 was 0.967, the sensitivity was 0.833 and the specificity was 0.966. Moreover, hsa_circ_0086354 was predicted as a competitive endogenous RNA for miR-181a, and hsa_circ_0086354 expression was negatively correlated to miR-181a expression in children with CP. CONCLUSION: Hsa_circ_0086354 was significantly down-regulated in blood plasma of children with CP, which may be a novel competent biomarker for early diagnosis of CP.

Long noncoding RNA LINC00968 inhibits proliferation, migration and invasion of lung adenocarcinoma through targeting miR-22-5p/CDC14A axis.

Lung adenocarcinoma (LUAD) is a high aggressive human cancer which usually diagnosed at advanced stages. Accumulating evidences indicate that long noncoding RNAs (lncRNAs) are crucial participants in LUAD progression. In the present study, we found that lncRNA LINC00968 was significantly down-regulated in LUAD tissues and cell lines. LINC00968 level was positively correlated to survival rate, and negatively correlated to tumor node metastasis (TNM) stage, tumor size and lymph node metastasis of LUAD patients. We over-expressed LINC00968 in LUAD cells using lentivirus, inhibited proliferation and cell cycle arrest at G1 phase were detected. LINC00968 over-expression also suppressed migration, invasion and epithelial mesenchymal transition. We further validated that LINC00968 localized in cytoplasm and acted as an upstream regulator of microRNA miR-22-5p, which was up-regulated in LUAD tissues and cell lines. Besides, elevated miR-22-5p expression abolished the effect of LINC00968 over-expression on LUAD progression including in vivo tumor growth. In addition, we first validated that cell division cycle 14A (CDC14A), which was down-regulated in LUAD tissues, was a downstream target of miR-22-5p. We over-expressed CDC14A in LUAD cells and miR-22-5p induced LUAD progression was partially reversed. In conclusion, our study demonstrated that LINC00968 inhibited proliferation, migration and invasion of LUAD by sponging miR-22-5p and further restoring CDC14A. This novel regulatory axis might provide us with promising diagnostic and therapeutic target in LUAD treatment.

Arginine deiminase expressed in vivo, driven by human telomerase reverse transcriptase promoter, displays high hepatoma targeting and oncolytic efficiency.

Arginine starvation has the potential to selectively treat both primary tumor and (micro) metastatic tissue with very low side effects. Arginine deiminase (ADI; EC 3.5.3.6), an arginine-degrading enzyme, has been studied as a potential anti-tumor drug for the treatment of arginine-auxotrophic tumors. Though ADI-PEG20 (pegylated ADI by PEG 20,000) already passed the phase I/II clinical trials [1], it is just used as adjuvant therapy because of its low efficiency and less targeting. Then, this paper discussed the efficiency of arginine starvation mediated by ADI expressed in cytoplasm for liver cancers. In order to guarantee the tumor targeting, human telomerase reverse transcriptase (hTERT) promoter was used to drive the expression of ADI in vivo. To access the anti-tumor efficiency of ADI, p53 gene was used as the positive control. Thus, ADI displayed obvious cytotoxicity to BEL7402 and HUH7 cell lines in cytoplasm. The apoptosis rates rose from 15% to nearly 60% after changing the expression vectors from pcDNA4 plasmid to adenovirus. Compared with p53-adenovirus, ADI-adenovirus showed the higher oncolytic activity in the intratumoral injection model of mice. Tumor disappeared after the treatment of ADI-adenovirus for two weeks, and the mice pulled through all. Therefore, ADI is an ideal anti-tumor gene for caner targeting therapy with the help of hTERT promoter.