Method of Inhibiting Metastasis in Hepatocellular Carcinoma
According to 2018 data, an estimated 609,640 people in the United States will die from cancer this year, while approximately 1.7 million new cases of cancer will be diagnosed1. Cancer is the second leading cause of death in the US and constitutes approximately 22 percent of deaths1. In addition, cancer creates huge financial burdens on both individuals and society. It is estimated that medical costs associated with cancer reached an astonishing $80.2 billion in 20151.
Research surrounding miRNAs has found that manipulation of miRNAs has led to inhibition of metastasis, and miRNAs can be used as biomarkers in the diagnosis and prognosis of cancers2. Due to these two potential uses of miRNAs, further research could lead to more effective treatment and diagnosis of cancer, thereby increasing patients’ likelihood of survival. miRNAs are of increased interest due to their broad range of molecular targets and cellular signaling pathways and further research should be conducted to understand the mechanism and function of miRNAs as potential cancer therapeutics3-6.
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How it works
Micro-RNAs are small non-coding RNA molecules that are involved in post-transcriptional regulation of gene expression3. Researchers have studied the effects of miRNAs on multiple cancers, Parkinson’s disease, and diabetes. Much research has specifically focused on miRNA-7 and miR-106 and their roles in cancer metastasis, as well as their functions as oncogenes or tumor suppressors in specific cancers4. Elevated expression of epidermal growth factor receptor (EGFR) is associated with the progression of and treatment resistance of head and neck cancer (HNC). Data suggests that miRNA-7 inhibits EGFR expression and HNC growth5. Studies on epithelial ovarian cancer showed that miRNA-7 inhibited tumor metastasis by targeting EGFR and downstream Akt activity6. Research on the role of miRNA-7 as a regulator of proteins other than EGFR have been conducted. One study focused on the interaction of miRNA-7 with mRNA that encodes for focal adhesion kinase (FAK), a protein which promotes cell migration and is overexpressed in a variety of solid tumors. Upregulated miRNA-7 was shown to significantly suppress FAK at transcriptional and translational levels, and inhibited metastasis7. Further research found that overexpression of miRNA-7 prevented gastric carcinogenesis and tumorigenesis8. A study on hepatocellular carcinoma (HCC) found that miRNA-7 was downregulated in HCC and a low expression of miRNA-7 was significantly associated with tumor size9.
While studies showed that overexpression of miRNA-7 resulted in tumor suppression, research on miR-106 suggests an oncogenic effect. A study on endometrioid endometrial cancer (ECC) concluded that downregulation of miRNA-106b led to apoptosis of cancerous cells and reduced cell proliferation and therefore supports the theory that miR-106b acts as an oncogene10. Additional research concluded that miR-106b-5p was upregulated in cervical cancer and its expression was highly correlated with the number of metastatic lymph nodes11. A study on hepatocellular carcinoma (HCC) found that miR-106b-25 was upregulated in hepatitis virus-associated HCC, and corresponded to a decreased survival time and increased recurrence rate12. miR-106 was found inhibit apoptosis and increase cell proliferation by inhibiting PTEN expression10. PTEN acts as a negative regulator of PI3K whose activation leads to the phosphorylation and activation of Akt. Akt mediates cellular responses such as cell migration, proliferation, and growth13, and regulates apoptosis related proteins, such as Caspase-9, and Bax12. Taken together, these finding suggest that miR-106b aids tumor progression12.
Protein kinase B, also referred to as Akt, is a protein that is involved in many cellular processes. More specifically, Akt1 inhibits apoptosis17 and is a downstream effector of PIP313. PI3K catalyzes the reaction of PIP2 to PIP3, thereby activating Akt, whereas PTEN opposes the activity of PI3K by dephosphorylating PIP3 and generating PIP213. PTEN is inhibited by miRNA-106 and miRNA-106 is upregulated in cancerous cells. In addition, overexpression of miRNA-7 inhibits EFGR expression and downstream Akt activity6. Caspase-9 is an enzyme that is involved in the apoptotic pathway. It functions as an initiating caspase by cleaving and activating downstream caspases that will initiate apoptosis15. B-cell lymphoma 2 (Bcl-2) is a regulatory protein that regulates apoptosis by either inducing or inhibiting cell death. Bax is a member of the Bcl-2 family and is a pro-apoptotic protein16. Overall, both miRNA-7 and miRNA-106 have been shown to effect the Akt pathway and the expression of downstream proteins needed for apoptosis. Therefore, miRNA engineering could be used to increase the activity of pro-apoptotic proteins, thereby potentially decreasing the proliferation and metastasis of cancerous HCC cells. Hepatocellular carcinoma (HCC) cell lines will be harvested from multiple patient’s livers. Synthetic precursor molecules that correspond to miRNA-7 and miRNA-106 will be sourced. These miRNA precursors will be transfected into HCC cell lines. Using primers specific to miRNA-7 and miRNA-106, the RNA will be extracted from HCC cell cultures and transcribed to produce DNA5,12. Using a microRNA Assay Kit, the levels of miRNA-7 and miRNA-106 will be measured to study the amount of expression in HCC cells relative to non-cancerous cells12.
Shikonin, a natural naphthoquinone, has been found to suppress proliferation, promote apoptosis and decrease metastasis as it effects the miRNA-106 signaling pathway10. As such, it will be used to downregulate miRNA-106 in HCC cell lines. miRNA-7 will be overexpressed using lentiviral (LV) miRNA expression vectors14. In order to study the effect of overexpressing miRNA-7 and downregulating miRNA-106, the amount of Caspase-9 and Bax must be measured in HCC cell lines that have not been altered and those that have. Quantitation of each protein will be performed using three experimental methods: Western Blot analysis, protein microarrays, and high performance liquid chromatography (HPLC).
To prepare samples for Western Blot analysis, cells will be lysed by centrifugation. Proteins will be separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and electrophorectically transferred from the gel to nitrocellulose paper. Once the nitrocellulose paper has dried, it can be spotted with the primary antibodies against Bax and Caspase-9. Horseradish peroxidase-conjugated (HRP) antibodies will be used as secondary antibodies as they will fluoresce in the presence of each protein. The nitrocellulose paper will undergo chemiluminescent development using a Prometheus ProSignal Femto chemilumnescent HRP substrate kit. It will be visualized using an Odyssey FC. The presence and amount of each protein will be compared between non-modified and modified HCC cell lines. Western Blots will be prepared using cells that have been exposed to different microgram amounts of shikonin. b-actin will be used as the control for each sample, as its concentration should not change in the presence of shikonin, or due to the overexpression of miRNA-7.
The second method of protein quantitation will be an analytical protein microarray which will employ the “sandwich” assay format. This method uses two antibodies to detect the protein of interest. A capture antibody will immobilize the protein on the solid phase while the reporter (detection) antibody will generate a signal that is registered on the detection system18. This type of microarray allows for greater specificity to the target protein. Samples will be prepared from multiple HCC cell lines and each cell line will be exposed to varying amounts of shikonin.
The last protein quantitation method will be reverse-phased HPLC. The immobile phase, often composed of silica modified with a hydrocarbon molecule, will be highly hydrophobic and will attract and retain any hydrophobic areas of the target protein. The adsorption of the protein to the surface will remain until the organic solvent reaches a concentration at which the protein will desorb from the surface and elute from the column19. The amount of time that the protein is retained on the column will be measured and correspond to a specific protein.
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