Role of the lncRNA complex HCP5 in glioma treatment

Harini Balasubramanian, Anna University, Chennai

The investigation of non-coding regions of the DNA, especially miRNA and long non-coding RNA (lncRNA), and their role in various disease phenotypes, has drawn the attention of scientists in recent years. Gliomas are neoplasias usually found in the brain, a stage IV progression of which leads to glioblastoma (GBM). GBM is highly fatal with a median survival rate of merely 15 months. It is therefore essential that for a better understanding of the disease progression, more studies need to be conducted.

LncRNAs have been found to interact with miRNAs — short non-coding RNAs with up to 22 nucleotides. miRNAs regulate most of the post-transcriptional gene expression in a cell – to either stimulate malignancy or tumor suppression. lncRNA HLA complex P5 (HCP5) has been discovered to have carcinogenic properties by promoting cell proliferation, migration, and angiogenesis in follicular thyroid carcinoma cells. miR-205 is a miRNA of interest in this study, given its tumor-suppressive properties in gliomas, as established in previous studies.

So, this study investigated the previously unexplored role of HCP5 in glioma progression. Also, it further investigated the interaction of HCP5 with miR-205 and their combined molecular mechanisms along the VEGF-A axis (an essential factor in vascularizing tumors) in affecting glioma progression. 

Methodology:

1. RNA Sequencing, Data processing, Analysis
2. The culture of a patient’s primary GBM cell line (U251), Normal Human Astrocyte (NHA), and tumor tissue were obtained from patient.
3. Cell transfection: siRNA against HCP5, miR-205 mimics, and negative control mimics were synthesized. Vectors for shRNA targeting HCP5 and the human VEGF-A gene were transfected into primary GBM cells.
4. Cell viability assay, scratch wound healing assay for cell migration and invasiveness, and cell proliferation assays were conducted.
5. In vivo xenograft study in mice was performed by injecting them subcutaneously with U251 cells post-transfection.
6. qRT-PCR was performed to quantify expression levels of HCP5, miR-205, and VEGF-A.
7. Western Blot analysis was done to primarily quantify VEGF-A protein concentration.

Outcome & Inference:

In different types of cancers, HCP5 was found to be significantly more upregulated compared to healthy control samples. The RNA sequencing data derived from TCGA projects also revealed the prognosis and thereby, that the survival rate had a negative correlation with expression levels 0f HCP5. For gliomas, in particular, the results showed that overexpression of HCP5 may be implicated in glioma progression.

Conversely, it also proved that in cells with siRNA transfection and subsequent HCP5 knockdown, its downregulation prominently mitigated cell viability and migration. In vivo studies also showed slowed tumor growth in mice injected with HCP5-silenced GBM cells.

In addition, HCP5’s interaction with miR-205 downregulated (relative to NHA cell line) the former’s expression in GBM cell lines. It was also found that miR-205 expression has an inverse relationship with HCP5, as the latter-silenced GBM cells had elevated levels of miR-205.

Regarding the feedback loop modulating GBM progression, the researchers determined that HCP5 downregulation was strongly associated with the downregulation of VEGF-A, both of which strikingly decreased upon upregulation of miR-205. So, they ascertained that by targeting miR-205, HCP5 modulated the expression of VEGF-A.

The results of this study, therefore, provide potential theragnostic targets for glioma treatment and specific drug development.

Reference:

Cheng, R., Ji, L., Su, H., Wang, L., Jia, D., Yao, X. and Ji, H. Silencing of Long Noncoding RNA HLA Complex P5 (HCP5) Suppresses Glioma Progression through the HCP5-miR-205-Vascular Endothelial Growth Factor A Feedback Loop. BioMed Research International, 2022. https://pubmed.ncbi.nlm.nih.gov/35769676/

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