The H19 Long non-coding RNA in cancer initiation, progression and metastasis – a proposed unifying theory

Up-regulation of long non-coding RNA H19 ameliorates renal tubulointerstitial fibrosis by reducing lipid deposition and inflammatory response through regulation of the microRNA-130a-3p/long-chain acyl-CoA synthetase 1 axis

To explore the relationship between long non-coding RNA (lncRNA) H19 expression and prognosis of hepatitis B-related hepatocellular carcinoma (HBV-related HCC), and its underlying mechanism. Expression level of lncRNA H19 in 36 HBV-related HCC tissues and para-cancerous tissues was detected by quantitative Real-time polymerase chain reaction (qRT-PCR). The relationship between lncRNA H19 expression and prognosis of HBV-related HCC was analyzed by Kaplan-Meier method. Serum DNA levels of HBV were detected by fluorescence quantitative polymerase chain reaction (FQ-PCR). For in vitro experiments, lncRNA H19 expression in HCC cell line, HBV-related HCC cell line and normal liver cell line was detected by qRT-PCR. After plasmids construction, the effects of lncRNA H19 on cell viability, migration, and invasion were detected by cell counting kit-8 (CCK-8), colony formation and transwell assay, respectively. Finally, protein levels of epithelial-mesenchymal transition (EMT) pathway-related genes were detected by Western blot. LncRNA H19 was highly expressed in HBV-related HCC tissues. The expression of lncRNA H19 was positively correlated with lymph node metastasis and distant metastasis, whereas negatively correlated with the overall survival of HBV-related HCC patients. Results of in vitro experiments showed that lncRNA H19 knockdown significantly downregulated cell proliferation and invasion. However, lncRNA H19 knockdown significantly upregulated apoptosis of HBV-related HCC cells. Western blot results demonstrated that lncRNA H19 remarkably decreased the protein expressions of EMT pathway-related genes, including N-cadherin, Vimentin, β-catenin and MMP-9. In addition, rescue experiments demonstrated that lncRNA H19 remarkably promoted malignant development of HBV-related HCC via regulating microRNA-22. LncRNA H19 promotes malignant development of HBV-related HCC through regulating microRNA-22 via EMT pathway.

BackgroundEmerging evidence suggests that epithelial mesenchymal transition (EMT) and epigenetic mechanisms promote metastasis. Histone deacetylases (HDACs) and noncoding RNAs (ncRNAs) are important epigenetic regulators. Here, we elucidated a novel role of histone deacetylase 2 (HDAC2) in regulating EMT and CRC metastasis via ncRNA.MethodsThe expression of HDACs in CRC was analyzed using the public databases and matched primary and metastatic tissues, and CRC cells with different metastatic potentials (DLD1, HCT116, SW480 and SW620). Microarray analysis was used to identify differential genes in parental and HDAC2 knockout CRC cells. EMT and histone modifications were determined using western blot and immunofluorescence. Migration ability was assessed by transwell assay, and metastasis was assessed in vivo using a tail vain injection. Gene expression and regulation was assessed by RT-PCR, chromatin immunoprecipitation and reporter assays. Protein interaction was assessed by immunoprecipitation. Specific siRNAs targeting H19, SP1 and MMP14 were used to validate their role in HDAC2 loss induced EMT and metastasis.ResultsReduced HDAC2 expression was associated with poor prognosis in CRC patients and found in CRC metastasis. HDAC2 deletion or knockdown induced EMT and metastasis by upregulating the long noncoding RNA H19 (LncRNA H19). HDAC2 inhibited LncRNA H19 expression by histone H3K27 deacetylation in its promoter via binding with SP1. LncRNA H19 functioned as a miR-22-3P sponge to increase the expression of MMP14. HDAC2 loss strongly promoted CRC lung metastasis, which was suppressed LncRNA H19 knockdown.ConclusionOur study supports HDAC2 as a CRC metastasis suppressor through the inhibition of EMT and the expression of H19 and MMP14.

The integrity of lens epithelial cells (LECs) lays the foundation for lens function and transparency. By contrast, epithelial-mesenchymal transition (EMT) of LECs leads to lens fibrosis, such as anterior subcapsular cataracts (ASC) and fibrotic forms of posterior capsule opacification (PCO). However, the underlying mechanisms remain unclear. Here, we aimed to explore the role of long non-coding RNA (lncRNA) H19 in regulating TGF-β2-induced EMT during lens fibrosis, revealing a novel lncRNA-based regulatory mechanism. In this work, we identified that lncRNA H19 was highly expressed in LECs, but downregulated by exposure to TGF-β2. In both human lens epithelial explants and SRA01/04 cells, knockdown of H19 aggravated TGF-β2-induced EMT, while overexpressing H19 partially reversed EMT and restored lens epithelial phenotypes. Semi-in vivo whole lens culture and H19 knockout mice demonstrated the indispensable role of H19 in sustaining lens clarity through maintaining LEC features. Bioinformatic analyses further implied a potential H19-centered regulatory mechanism via Smad-dependent pathways, confirmed by in vitro experiments. In conclusion, we uncovered a novel role of H19 in inhibiting TGF-β2-induced EMT of the lens by suppressing Smad-dependent signaling, providing potential therapeutic targets for treating lens fibrosis.

Advanced gallbladder cancer (GBC) is one of the most malignant of all types of biliary tract cancers that is associated with poor prognosis and high mortality. Accumulating evidence suggest that the B7 family of proteins serve an essential role in various types of cancers, including GBC. However, the potential function and regulatory mechanism of human endogenous retrovirus-H long terminal repeat-associating protein 2 (HHLA2; also known as B7-H7 or B7H5) in GBC remain poorly understood. In the present study, immunohistochemistry was used to examine the expression pattern of HHLA2 in samples from 89 patients with GBC. The possible association between HHLA2 expression and the clinicopathological parameters, including prognosis, were then assessed. Using lentiviruses, overexpression of HHLA2 plasmid or short-hairpin RNA (shRNA) of HHLA2 were transfected into GBC-SD cells to overexpress or knock down HHLA2 expression, respectively. The effects of HHLA2 overexpression and knockdown on the epithelial-mesenchymal transition (EMT) process on GBC-SD cells were measured by the western blotting and immunofluorescence staining of collagen I, N-cadherin, E-cadherin, vimentin and α-SMA. By contrast, changes in cell proliferation were measured using EdU assay. Cell invasion and migration were assessed using Transwell and wound-healing assays, respectively. In addition, a xenograft mouse model was established to evaluate the tumorigenic ability of the GBC cell line in vivo after stable transfection with lentivirus for HHLA2 overexpression or shRNA for HHLA2 knockdown. The regulatory relationships among TGF-β1, long non-coding RNA (lncRNA) H19 (H19) and HHLA2 were then investigated. The mRNA expression of lncRNA H19 were assessed using reverse transcription-quantitative PCR, whereas the expression levels of HHLA2 were detected by western blotting and immunofluorescence staining. HHLA2 expression was found to gradually increase as the stages of the GBC samples become more advanced. In addition, the expression level of HHLA2 was calculated to be positively associated with the Nevin stage, American Joint Committee on Cancer stage, tumor invasion and regional lymph node metastasis but was negatively associated with the overall patient survival (OS). In vitro experiments demonstrated that overexpression of HHLA2 promoted GBC migration, invasion, proliferation and EMT, whereas in vivo experiments found a promoting role of HHLA2 overexpression on GBC tumor growth. After transfection with lentiviruses encoding the overexpression plasmid of lncRNA H19, GBC migration, invasion, proliferation and EMT were increased. By contrast, knocking down HHLA2 expression suppressed TGF-β1- or lncRNA H19 overexpression-induced GBC migration, invasion, proliferation and EMT. In addition, HHLA2 knockdown significantly reduced the sizes of the GBC tumors in vivo. These results suggest that HHLA2 overexpression can promote GBC progression. Conversely, ablation of HHLA2 expression inhibited both TGF-β1- and lncRNA H19-induced GBC progression, suggesting that HHLA2 is a potential therapeutic target for this disease.

The Functions of Long Non-Coding RNA (lncRNA) H19 in the Heart

The abnormally expressed long non-coding RNA (lncRNA) H19 has a crucial function in the development and progression of cardiovascular disease; however, its role in atherosclerosis is yet to be known. We aimed to examine the impacts of lncRNA H19 on atherogenesis as well as the involved mechanism. The outcomes from this research illustrated that the expression of lncRNA H19 was elevated in mouse blood and aorta with lipid-loaded macrophages and atherosclerosis. Adeno-associated virus (AAV)-mediated lncRNA H19 overexpression significantly increased the atherosclerotic plaque area in apoE−/− mice supplied with a Western diet. The upregulation of lncRNA H19 decreased the miR-146a-5p expression but increased the levels of ANGPTL4 in mouse blood and aorta and THP-1 cells. Furthermore, lncRNA H19 overexpression promoted lipid accumulation in oxidized low-density lipoprotein (ox-LDL)-induced THP-1 macrophages. However, the knockdown of lncRNA H19 served as a protection against atherosclerosis in apoE−/− mice and lowered the accumulation of lipids in ox-LDL-induced THP-1 macrophages. lncRNA H19 promoted the expression of ANGPTL4 via competitively binding to miR-146a-5p, thus promoting lipid accumulation in atherosclerosis. These findings altogether demonstrated that lncRNA H19 facilitated the accumulation of lipid in macrophages and aggravated the progression of atherosclerosis through the miR-146a-5p/ANGPTL4 pathway. Targeting lncRNA H19 might be an auspicious therapeutic approach for preventing and treating atherosclerotic disease.

NLRP3 inflammasomes have been reported to be an essential mediator in the inflammatory response during early brain injury (EBI) following subarachnoid hemorrhage (SAH). Recent studies have indicated that NLRP3 inflammasome-mediated pyroptosis and long non-coding RNA (lncRNA) H19 can participate in the inflammatory response. However, the roles and functions of lncRNA H19 in NLRP3 inflammasome-mediated pyroptosis during EBI after SAH are unknown and need to be further elucidated. NLRP3 inflammasome proteins were significantly elevated in CSF of human with SAH induced EBI and presented a positive correlation with severity. In ipsilateral hemisphere cortex of rats, these NLRP3 inflammasome proteins were also increased and accompanied with upregulation of H19, and both of NLRP3 and H19 were peaked at 24h after SAH. However, knockdown of H19 markedly decreased the expression of NLRP3 inflammasome proteins at 24h after SAH in rats and also ameliorated EBI, showing improved neurobehavioral deficits, cerebral edema, and neuronal injury. Moreover, knocking down of H19 downregulated the expression of Gasdermin D (GSDMD) in microglia in SAH rats. Similarly, knockdown of H19 also alleviated OxyHb-induced pyroptosis and NLRP3-mediated inflammasomes activation in primary microglia. Lastly, H19 competitively sponged with rno-miR-138-5p and then upregulated NLRP3 expression in the post-SAH inflammatory response. lncRNA H19 promotes NLRP3-mediated pyroptosis by functioning as rno-miR-138-5p sponge in rats during EBI after SAH, which might provide a potential therapeutic target for post-SAH inflammation regulation.

The evolving concept of cancer stem-like cells in thyroid cancer and other solid tumors

Cancer is one of the leading causes of death. A primary tumor forms when cells start to proliferate in an uncontrolled way and stop reacting to restraining signals. Tumors that reach a critical volume induce angiogenesis, a vascular remodeling process that provides nutrients and oxygen to the degenerated cell mass. Upon further tumor progression to a malignant cancer, cells acquire the ability to invade the surrounding tissue. In order to do so, formerly epithelial tumor cells undergo an epithelial to mesenchymal transition (EMT). This process of cell-cell detachment, breaching through the basement membrane and gaining migratory capabilities is the first step of the metastatic cascade. Metastasis is a process that allows tumor cells to leave the primary lesion and disseminate via the vascular system to secondary sites. Metastases, as the fatal feature of cancer, lead in most of the cases to patients’ death. Furthermore, metastasis, as the end stage of malignant disease, until now is incurable. This is because metastases are spread systemically throughout the body. Moreover, they even can establish from a disseminated tumor cell months after the primary tumor has already been surgically removed. Another barely controllable feature of cancer is tumor relapse. Even after a thorough surgery with the aim to completely remove the primary tumor and eventual draining lymph nodes combined with chemotherapy, patients relapse. Resistance to chemotherapy and establishment of metastases has both been accounted for to the abundance of cancer stem cells (CSC) within the tumor mass. These CSCs are endowed with the ability to evade chemotherapeutic drugs, they are mesenchymal in nature, migratory and invasive, and, most importantly, they are able to establish cancer and metastasis de novo. The work of my thesis has been dedicated to investigate the process of metastasis and the function of cancer initiation. Tumor initiation has recently been associated with EMT. To understand the functional circumstances how EMT cells gain the ability to form tumors, I used cellular murine breast cancer models. These model systems allowed me to study cells’ behavior before and after EMT in vitro and in vivo. In vitro experiments validated the observation of others that EMT cells indeed resemble cancer stem cells by being able to form hollow spheres and being susceptible to the CSC-specific drug salinomycin. In vivo studies revealed that EMT cells initiate tumors with a much earlier onset and with a higher efficiency when limited amounts of cells were injected orthotopically into mice compared to their epithelial counterpart. Moreover, EMT cell-generated cancers are highly vascularized already in the early phase of tumor establishment. Knockdown studies of the main pro-angiogenic factor VEGF-A revealed that it is required for early tumor onset. Thus, tumor angiogenesis is not only an effect of early and fast EMT-tumor progression. Further supporting this notion are limiting dilution experiments, which suggest that tumor initiation in EMT cells is a multifactorial event. This concept was validated by the observation that 10 EMT cells could initiate a tumor, whereas VEGF‑A knockdown cells could not. Hence, EMT-induced tumor initiation is achieved by the ability of promoting angiogenesis. EMT can be induced by the cytokine TGFβ. Normally, cells that experience TGFβ signaling become quiescent or die due to induction of apoptosis. Cancer cells can overcome these effects and react to TGFβ by undergoing EMT. As part of my thesis, I could show that the transcription factor Dlx2 is an important switch that allows cells to react to TGFβ by undergoing EMT without facing apoptosis induction. Dlx2 exerts its anti-apoptotic, pro-survival function by directly reducing TGFβRI expression and inducing the expression of the epidermal growth factor receptor ligand betacellulin. Another feature of EMT is the gain of cell motility. Cell migration is extremely important for cancer cells in order to leave their primary site and to disseminate. I have found ephrinB2 expression upregulated during EMT. EphrinB2 is a member of the Eph-ephrin signaling network that is known to be crucial for cell-cell communication. Thereby, cells that do not belong to the same entity repulse each other, restricting intermingling of tissue. Furthermore, ephrinB2 is required for neuronal axon guidance and angiogenesis. In my thesis, I showed that EMT cells need ephrinB2 to efficiently migrate. As an explanation for this phenotype I described that a knockdown for ephrinB2 led to an over-stabilization of focal adhesions. Cells normally use focal adhesions to hold on to an extracellular matrix (ECM) surface. Over-stabilization of focal adhesions attaches cells too firmly to the ECM and, hence, cells cannot retract their rear-end anymore which decreases cell motility. In summary, I succeeded in gathering further insights into tumor initiation, EMT, and with this, the metastatic process.

The Weathering Hypothesis states BIPOC face more stressors, by which over a lifetime they are subjected to the negative consequences of stress (e.g., poorer emotional health). Using ecological momentary assessments, we examined whether subtle discrimination moderated the within-person stressor slope on positive and negative affect. We predicted emotional wellbeing would be worse at stressor moments, and those with greater discrimination experience would be more impacted by stressors. Participants were 334 diverse adults (25-65 years, Mage = 47, 63% Female) from Bronx, New York. Positive affect decreased and negative affect increased significantly at stressor moments (p<.0001). Unexpectedly, subtle discrimination was not a significant moderator for the within-person stressor slope on positive affect and negative affect. Unlike the predictions of the Weathering Hypothesis, these results show that prior discrimination experiences may not exacerbate responses to stressors and entail additional risk in daily life.

The long non-coding RNA (lncRNA) H19 is a maternally imprinted gene transcript that, in conjunction with the neighboring Igf2 gene, is critical in controlling embryonic growth. Loss of H19 results in fetal overgrowth associated with Beckwith Weidemann syndrome, while elevated H19 occurs in human cancers. In the adult, H19 functions in cancer cells where it promotes migration and is correlated with poor prognosis, and in adult stem cells where it is a key regulator of cell fate decisions during differentiation. While the function of H19 in primary somatic cells has not been defined, a reduction in the abundance of H19 has been reported during senescence in endothelial cells. Given the critical importance of H19 in cell fate decisions, it is likely that understanding the precise function of H19 in somatic cells in general and why reduced levels occur with cellular senescence will provide novel insights into both somatic cell maintenance and the senescence program. Towards this end, we examined the role of H19 in somatic cell growth using cardiac interstitial fibroblasts. Our results indicate that H19 is not only vital for somatic cell proliferation and survival, but that depletion of H19 leads to cell cycle arrest and the formation of abnormal nuclei resulting in senescent cells. We are defining both the upstream regulators of H19 and the downstream mediators of senescence following H19 depletion. Overall, these results indicate an essential role for H19 in cell cycle progression, chromatin structure, and possibly proper mitotic division.

Career situation of first and presenting authorStudent for a master or a PhD.IntroductionOsteoarthritis (OA) and osteoporosis are destructive bone diseases causing chronic pain and leading to disability. Mesenchymal stromal cells...

The purpose of this study was to investigate the role of lncRNA H19 in epithelial-mesenchymal transition (EMT) and its molecular mechanism in fibrotic cataracts. TGF-β2-induced EMT was induced in human lens epithelial cell line (HLECs) and rat lens explants to mimic posterior capsular opacification (PCO) in vitro and in vivo. Anterior subcapsular cataract (ASC) was induced in C57BL/6J mice. The long noncoding RNA (lncRNA) H19 (H19) expression was detected by RT-qPCR. Whole-mount staining of lens anterior capsule was used to detect α-SMA and vimentin. Lentiviruses carrying shRNA or H19 vector were transfected in HLECs to knockdown or overexpress H19. Cell migration and proliferation were characterized by EdU, Transwell, and scratch assay. EMT level was detected by Western blotting and immunofluorescence. The rAAV2 carrying mouse H19 shRNA was injected into ASC model mouse anterior chambers as a gene therapy to determine its therapeutic potential. PCO and ASC models were built successfully. We found H19 upregulation in PCO and ASC models in vivo and in vitro. Overexpression of H19 by lentivirus transfection increased cell migration, proliferation, and EMT. In addition, H19 knockdown by lentivirus suppressed cell migration, proliferation, and EMT levels in HLECs. Moreover, transfection of rAAV2 H19 shRNA alleviated fibrotic area in ASC mouse lens anterior capsules. Excessive H19 participates in lens fibrosis. Overexpression of H19 increases, whereas knockdown of H19 ameliorates HLECs migration, proliferation, and EMT. These results demonstrate H19 might be a potential target for fibrotic cataracts.

Objective To investigate the expression of long non-coding RNA (lncRNA)H19 in gallbladder cancer and its relationship with clinicopathological parameters and prognosis. Methods 74 cases of allbladder cancer resected in our hospital were collected and studied. At the same time, 66 cases of benign gallbladder lesions were selected as benign lesion group. The expression of lncRNA H19 in tissues was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). The correlation between lncRNA H19 expression and clinicopathological parameters of gallbladder cancer patients was analyzed.The patients were followed up for 3 years and COX was used to analyze the prognostic factors affecting the survival rate.The survival was analyzed by Kaplan-Meier curve. The survival differences of lncRNA H19 expression groups were detected by Log-Rank method. Results The expression level of lncRNA H19 in gallbladder cancer was significantly higher than that in benign lesions (P 0.05). The survival rate of patients with differentiated tumors and non-metastatic lymph nodes were higher (P<0.05). The survival rate of patients with low expression of lncRNA H19 was significantly higher than those with high expression of lncRNA H19 (P<0.05). Tumor differentiation, lymph node metastasis and lncRNA H19 expression were independent risk factors affecting the prognosis of patients with gallbladder cancer. Conclusions LncRNA H19 is highly expressed in gallbladder cancer, which is closely related to the degree of differentiation, lymph node metastasis and survival rate of patients. Key words: RNA, long noncoding; Gallbladder neoplasms; Factor analysis, statistical