MicroRNAs as Biomarkers for Glioblastoma and Therapeutic Opportunities

Abstract

Glioblastoma (GBM) is a highly malignant tumour with a low survival rate for patients that are diagnosed leaving early diagnosis as the most effective method for patient survival.  Researchers have found that MicroRNAs(miRNA), which are non-coding RNAs, have proven to be biomarkers for diseases such as GBM. Their abnormal expression levels can indicate the presence of a disease. Methods such as using AntagomiRs and miRNA sponges which can regulate the number of miRNAs expressed are already being researched and have resulted in the reduction of the size of GBM tumours. Researching miRNAs further could lead to the development of new therapeutic methods which is an important step towards advancement in the field and resulting in increase in survival rate.

Keywords: Glioblastoma, MicroRNAs, biomarkers, expression levels, AntagomiRs, miRNA sponges

Introduction

miRNAs are long, non-coding RNAs and are involved in processes such as differentiation, apoptosis, proliferation, development and gene regulation. Studies have shown that miRNA expression in tumour tissues differs from expression in normal tissues. Specific miRNAs are expressed differently in different types of cancer, either as tumour suppressors or as oncogenes. They are either over-expressed or under-expressed and therefore can be used as biomarkers for different types of cancers, especially GBM. Patients diagnosed with GBM have a very low survival rate and traditional therapeutic methods such as chemotherapy or radiation therapy has little effect. Understanding these miRNAs may help understand classify, diagnose and predict clinical course for patients suffering with GBM and other cancers and can bring vast advancements into the field helping with patient survival.  

This review discusses the role specific miRNAs play in GBM by looking at how differently they are expressed while understanding that therapeutics methods that have successfully regulated the expression of miRNAs and resulted in a decrease in size of GBM in patients.

Background on Glioblastoma

Glioblastoma multiforme, also known as GBM, is the most common malignant brain tumour (Fig 1)¹. Patients diagnosed usually do not have longer than 12 months to survive². Even after patients have undergone chemo and radiation therapy, the median survival time is 14.6 months². The five-year survival rate is only 0.05% to 4.7%¹. The tumour is more common in adults with a 10-15% chance in children³. Moreover, GBM is more common in men than in women, with women having a better survival rate³. The severity of the tumour makes the outcome for patients remain poor which is why early diagnosis is vital to achieve better outcomes. MiRNAs can be biomarkers for glioblastomas and other cancers which can help with better patient outcomes since miRNAs are known to be deregulated in cancers.

Figure 1. A comparative scan of two MRIs, one showcasing a GBM tumour and one of a healthy brain⁴

Background on miRNAs in cancer

MiRNAs are long, non-coding RNAs that contain complementary segments of one or more messenger RNAs (mRNA) as shown in Figure 2⁵. Through their interactions with mRNA, miRNAs primarily support post-transcriptional control by stifling the expression of certain genes. Put another way, these substances are post-transcriptional regulatory elements, which are present in a wide range of eukaryotic cells and primarily control gene expression⁶. MiRNAs are involved in several physiological processes, such as differentiation, apoptosis, proliferation, and development. Since many of these molecular structures serve as tumour suppressor genes and oncogenes in physiological and pathologic processes they have the potential to induce cancer⁶. Serum or plasma microRNAs related to cancer and tumours can therefore be measured noninvasively, with the exception of leukaemia, which is readily accessible to malignant cells. As shown in Table 1, there are different types of microRNAs that act as either tumour suppressors or oncogenes in certain types of cancers.

Figure 2. A figure explaining the process and pathway of a MiRNA⁷

Research has shown that the miRNAs in cancer cells have aberrant structure and function and that miRNAs are associated with functional variations in tumours and cancer stages⁶. Tumour features such as tissue of origin differentiation, invasiveness, and response to treatment are all correlated with microRNA expression. MiRNAs can be measured in human serum or plasma and used as diagnostic indicators⁶. By interfering with cell-cycle regulators, changes in miRNA expression that lead to oncogenesis have an impact on cell proliferation. The primary regulators of programmed cell death in carcinogenesis are microRNAs, and it is possible to manipulate these microRNAs to control the survival of cancer cells such as miR-34a⁶

A table listing the function of a particular MiRNA and the types of cancers it is present in.

MicroRNA	Function	Type Of Cancer
miR-145	Tumor Suppressor	Bladder, Cervical, Lung, Liver, Breast, Gastric and Prostate
miR-34a	Tumor Suppressor	Neuroblastoma, Glioblastoma, Liver, Prostate, Breast, Colon
miR-29b	Tumor Suppressor	Glioblastoma, Liver, lung, gastric, acute myelocytic leukaemia
miR155	Oncogene	Liver and Leukaemia
miR-21	Oncogene	Glioblastoma, Lung, Colon, Breast, Liver
miR-495	Tumor Suppressor	Glioblastoma, Acute Myelocytic Leukaemia, lung, breast, gastric, prostate

MiRNAs have been proven to be biomarkers for several types of cancer, they are either over-expressed or under-expressed in Glioblastomas which is a malignant brain tumour. Studying MiRNAs as biomarkers helps in early diagnosis of GBM which can help increase the survival rate.

Correlation between miRNAs and Glioblastoma

MiRNAs are thought to act as tumour suppressors by preventing proto-oncogene transcription or the growth, aggressiveness, and migration of tumour cells⁸. Many studies on GBM have discovered miRNAs with antitumorigenic effects and their modes of action. As mentioned above, miRNAs can be biomarkers for glioblastoma since they compare normal tissues to GMB tumours⁹. Therefore, studying the same can help with early diagnosis and improve survival rates. Table 2 lists specific miRNAs and how they are presented in Glioblastomas.

A study conducted at the Department of Neurosurgery at Tianjin Huanhu Hospital in the years 2011- 2012 takes the plasma samples of 10 patients with GBM and compares it with the plasma samples of 10 healthy donors to test the difference in the levels of nine miRNAs. The study concluded that expression of miRNAs was higher is patients with GBM.¹⁰

There are different methods that can be employed to study miRNAs in GMB. Characterizing the miRNA profiles in a patient’s peripheral bodily fluid is a more practical approach for non-invasively detecting GBM, even though miRNA profiles in the tumour tissues may aid in determining tumour grades, illness prognosis, and therapeutic targets⁹.

A table listing different miRNAs and how they present themselves in GBM 

Micro-RNA	Presented in GBM
miR-10b	Over expressed
miR-21	Over expressed
miR-25	Over expressed
miR-125b	Over expressed
miR-182	Over expressed
miR-7	Under expressed
miR-9	Under expressed
miR-29a	Under expressed
miR-128-3p	Under expressed
miR-93	Over expressed
miR-196	Over expressed
miR-15a	Over expressed
miR-140	Over expressed
miR-210	Over expressed
miR-16	Over expressed
miR-17	Over expressed
miR-335	Over expressed
miR-32	Under expressed
miR-34	Under expressed
miR-181	Under expressed

As mentioned in Table 2, miR- 21 is overexpressed in gliomas which results in low apoptosis and high proliferation⁹. Scientists are not completely certain but believe that miR-21 is a biomarker for GBM due to its specific sites where STAT3(a protein that promotes tumour growth and blood vessel formation) which boosts miR-21 production¹¹. This particular miRNA, miR-21, is expressed significantly higher in patients with GMB, even in patients whose disease had significantly progressed (Grade II or Grade III)¹². MiR-21 binds to PDCD4(a gene that helps trigger cell death and in tumour suppression) and suppresses it therefore reduces its activity and allows GBM cells to grow¹³. It has the ability to limit apoptosis by affecting the activity of P53(a tumour suppressor)¹⁴.

Moreover, miR-93 was associated with angiogenesis and the development of cells.  Higher miR-93 levels have been linked to early tumour invasion and poor survival, and its expression levels have been seen to be up-regulated in GBM tissues⁹. MiRNA- 93 helps reduce the levels of pro- inflammatory molecules. This is important because inflammation plays an important role in GBM growth and survival since the imbalance of inflammation molecules creates an environment that supports tumour growth¹⁴. In addition, miR-196 stimulates cell proliferation, suppresses cell death, and excessive expression of the miRNA is associated with a bad prognosis in GBM patients. Another example is miR-335 which has been discovered to interact with the cAMP/protein kinase A pathway to promote tumour invasion, differentiation, and proliferation. As a result, GBM tissues frequently showed elevated miR-335 expression, and miR-335 overexpression was linked to a worse prognosis in GBM⁹. 

Furthermore, by restricting epidermal growth factor which is a protein whose function is to proliferate different types of cells, miR-7 operates as a tumour suppressor². GBM tissues exhibited lower levels of miR-7 expression, and these levels were linked to more invasive GBM subtypes and worse treatment results. As miR-19 is thought to be involved in oncogenesis and has been found to be overexpressed in GMB tissues, greater grade GBM has been linked to a worse prognosis.

The expression of certain miRNA differed when patients were using TMZ, a chemotherapy drug, and when they were not. MiR-181 is usually overexpressed in GBM but with patients reacting positively to TMZ, it was under-expressed². Similarly, miR-210 and miR-195 are under-expressed in patients with GBM but were over-expressed with patients responding positively to the drug. This confirms that the expression of a certain miRNA depends on the size of the tumour therefore therapeutic methods to regulate miRNA can be effective.

GBM also interacts with the immune system in order to facilitate its growth by using immune checkpoints (mechanism that prevents immune system from attacking it) to avoid being destroyed. However, miRNA- 34a along with others blocks the expression of PD-L1( a protein on immune and GBM cells) restoring immune response and preventing further growth¹⁴. MiRNA-34a has also shown the ability to block the genes expressed for cell division and slow the growth of GBM tissues.¹³

MiRNAs are of several types and each is presented differently in GBM. The theory behind the use of miRNAs as therapeutic interventions is that GBM is characterized by dysregulated miRNA expression and that miRNA targeting can restore the malignant phenotype. OncomiRs (a class of miRNAs that are over-expressed in cancer cells) and tumour-suppressive miRNAs are potential therapeutic possibilities.⁸

Therapeutics

Finding ways to alter miRNA expression in GBM is an important one since it will help with early diagnosis and improve the life expectancy of patients. Specific types of miRNAs are expressed differently in GBM therefore regulating miRNAs could result in a positive therapeutic effect⁵. OncomiRs help in cell growth and survival and are usually under-expressed so over-expressing them would have a positive effect. Similarly, tumour suppressors are used for differentiation, cell death and many more and are overexpressed therefore under expressing them will have a positive effect.

There are two methods that are being investigated to aid in the process of regulating miRNAs. AntagomiRs are RNA molecules with modified nucleosides whose main function is to enhance stability and have a structure that is complementary to miRNA sequencing.⁵ The enhanced stability allows them to hide away miRNAs and prevent them from being expressed in GBM and other cancers. MiRNA sponges can be used to decrease miRNA activity⁵. MiRNA sponges are circular RNA with millions of miRNA binding sites therefore they can hide away miRNAs and allow the original amount to be expressed. MiRNA sponges can be used to target several miRNAs at the same time. Other methods include miRNA mimics which can restore the miRNAs that are silenced and under-expressed¹⁵. Single-stranded oligonucleotides(an acid) with miRNA complementary sequences can also be administered to stop the function of over-expressed miRNAs.

MiRNA treatments along with conventional chemotherapy and radiation therapy have proved to be helpful¹⁵. The GBM chemotherapy is called TMZ however many patients are resistant to the drug as well. MiR-21 causes an imbalance between proteins like Bax(promotes cell death) and Bcl2(prevents cell death) making GBM cells more resistant to TMZ¹¹.The presence of miR-221 when under expressed can make the tumour more sensitive to chemotherapy and radiation therapy therefore deregulating the expression of miR-221 can make traditional preventative methods more effective¹⁵. This can be done so using miRNA mimics.

However, these methods can become difficult to implement due to the presence of blood brain barrier (BBB). It blocks molecules from entering the cells in order to protect the brain. A process called pinocytosis allows some substances to move in via transporters. In cancers like GBM certain transporters are overactive therefore allow the cancer cells to take in nutrients and become more aggressive while actively removing drugs from the brain thus making the therapy ineffective. Scientists have developed technologies to overcome these challenges. Nanoparticles such as gold and silver are used to deliver drugs. Hybrid nanoparticles are used to deliver specific anti-cancer miRNAs to glioblastoma cells to suppress tumour growth. Some nanoparticles are combined with existing drugs to improve their effectiveness.¹⁶

Furthermore, these methods also face challenges because of concerns such as dosage. The methods are still being tested with many projects being abandoned in the middle. Animal studies do exist to guide dosing but are not completely accurate for the human body¹⁷. The process of creating miRNA based drugs is an expensive one due to its early stages therefore many countries, especially those that are less developed, do not employ these methods¹⁷. The lack of extensive research poses a challenge in implementing these therapeutic methods.

Methods are being researched to use miRNAs as therapeutics for GBM to increase life expectancy. The goal is to find ways to regulate miRNAs which will slow the growth of the tumour or make traditional methods such as chemotherapy and radiation therapy more effective.

Conclusion

GBM is an aggressive brain tumour and has an average survival period of fifteen months Discovering relevant biomarkers would be highly beneficial in diagnoses and developing targeted treatments. Numerous miRNAs are important for oncogenesis, development, and invasion are dysregulated in GBM tissues. Research has already proven that regulating miRNAs has shown improvement and resulted in the size of the tumour decreasing. MiRNAs have great potential to be used as therapeutics for several types of cancer, not only GBM, which can result in better patient recovery which is why it is vital to continue research and develop therapeutic methods that are accessible.

Methods

This literature review on mircoRNAs as biomarkers for Glioblastoma and its therapeutic opportunities was conducted by selecting and analysing primary research papers along with literature review papers that focus on microRNAs in cancer and then specifically in Glioblastoma along with therapeutic methods. The research was filtered to all papers published withing the last 5 years and excluding any papers that were not written in the English language. The search engine Google Scholar was used for the research while using a website called Sci-Hub to access papers that are blocked for students or can only be accessed through institutions. Papers were initially screened based on their abstracts and subheadings for relevant information that was focused on the topic, either miRNAs or application of miRNA on GBM.

This method is reliable because it quickly gives an overview of relevant papers and ensures recent studies are included to keep the literature review current. However, this method is biased because relying on titles, abstracts, and subheadings may overlook some details in the full text and potentially exclude valuable insights contained within studies that might seem less relevant at first.

Acknowledgments

This work would not have been possible without the Lumiere Foundation where I participated in the Individual Research Program. I am especially thankful to Veronika Mantziou, Ph.D. from University of Cambridge, who has been supportive of all my academic aspirations and who worked actively to provide me with resources and guidance through the process.

References

1. Huang S, Ali N, Zhong L, Shi J. MicroRNAs as biomarkers for human glioblastoma: progress and potential. Acta Pharmacologica Sinica [Internet]. 2018 Sep 1 [cited 2020 Nov 9];39(9):1405–13. [↩] [↩]
2. Huang S, Ali N, Zhong L, Shi J. MicroRNAs as biomarkers for human glioblastoma: progress and potential. Acta Pharmacologica Sinica [Internet]. 2018 Sep 1 [cited 2020 Nov 9];39(9):1405–13. [↩] [↩] [↩] [↩]
3. Colopi A, Fuda S, Santi S, Onorato A, Cesarini V, Salvati M, et al. Impact of age and gender on glioblastoma onset, progression, and management. Mechanisms of Ageing and Development [Internet]. 2023 Apr 1;211:111801. [↩] [↩]
4. File:Glioblastom (WHO Grad IV) im MRI preoperativ und postoperativ.png – Wikimedia Commons [Internet]. Wikimedia.org. 2020 [cited 2024 Sep 19]. [↩]
5. Ji W, Sun B, Su C. Targeting MicroRNAs in Cancer Gene Therapy. Genes. 2017 Jan 9;8(1):21. [↩] [↩] [↩] [↩] [↩]
6. Babaei K, Shams S, Keymoradzadeh A, Vahidi S, Hamami P, Khaksar R, et al. An insight of microRNAs performance in carcinogenesis and tumorigenesis; an overview of cancer therapy. Life Sci [Internet]. 2020;240(117077):117077. [↩] [↩] [↩] [↩] [↩] [↩]
7. File:MiRNA.svg – Wikimedia Commons [Internet]. Wikimedia.org. 2012 [cited 2024 Sep 19]. [↩]
8. Lei Q, Yang Y, Zhou W, Liu W, Li Y, Qi N, et al. MicroRNA-based therapy for glioblastoma: Opportunities and challenges. Eur J Pharmacol [Internet]. 2023;938(175388):175388 [↩] [↩]
9. Huang S, Ali N, Zhong L, Shi J. MicroRNAs as biomarkers for human glioblastoma: progress and potential. Acta Pharmacologica Sinica [Internet]. 2018 Sep 1 [cited 2020 Nov 9];39(9):1405–13 [↩] [↩] [↩] [↩] [↩] [↩]
10. Wang, Qiong, et al. “Plasma Specific MiRNAs as Predictive Biomarkers for Diagnosis and Prognosis of Glioma.” Journal of Experimental & Clinical Cancer Research, vol. 31, no. 1, 22 Nov. 2012 [↩]
11. Aloizou, Athina-Maria, et al. “The Role of MiRNA-21 in Gliomas: Hope for a Novel Therapeutic Intervention?” Toxicology Reports, vol. 7, 2020, pp. 1514–1530, https://doi.org/10.1016/j.toxrep.2020.11.001. [↩] [↩]
12. Wang, Qiong, et al. “Plasma Specific MiRNAs as Predictive Biomarkers for Diagnosis and Prognosis of Glioma.” Journal of Experimental & Clinical Cancer Research, vol. 31, no. 1, 22 Nov. 2012 [↩]
13. Makowska, Marianna, et al. “MicroRNAs (MiRNAs) in Glioblastoma Multiforme (GBM)—Recent Literature Review.” International Journal of Molecular Sciences, vol. 24, no. 4, 9 Feb. 2023, p. 3521, https://doi.org/10.3390/ijms24043521. [↩] [↩]
14. Sati, Isra Saif Eldin Eisa, and Ishwar Parhar. “MicroRNAs Regulate Cell Cycle and Cell Death Pathways in Glioblastoma.” International Journal of Molecular Sciences, vol. 22, no. 24, 17 Dec. 2021, p. 13550, https://doi.org/10.3390/ijms222413550. Accessed 30 July 2022. [↩] [↩] [↩]
15. Lei, Q., Yang, Y., Zhou, W., Liu, W., Li, Y., Qi, N., Li, Q., Wen, Z., Ding, L., Huang, X., Li, Y., & Wu, J. (2023). MicroRNA-based therapy for glioblastoma: Opportunities and challenges. European Journal of Pharmacology, 938(175388), 175388. [↩] [↩] [↩]
16. Pottoo, Faheem Hyder, et al. “Targeted Delivery of MiRNA Based Therapeuticals in the Clinical Management of Glioblastoma Multiforme.” Seminars in Cancer Biology, Apr. 2020, https://doi.org/10.1016/j.semcancer.2020.04.001. Accessed 25 Apr. 2020 [↩]
17. Reda El Sayed, Soha, et al. “MicroRNA Therapeutics in Cancer: Current Advances and Challenges.” Cancers, vol. 13, no. 11, 29 May 2021, p. 2680, https://doi.org/10.3390/cancers13112680. [↩] [↩]