| Abstract |
Background: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death in men and the sixth in women. Those numbers reflect the aggressiveness of this disease while at the same time mirror the absence of effective therapeutic regimens. There have been conducted hundreds of clinical trials in which either combination chemotherapies or small molecule inhibitors and recently immune checkpoint inhibitors have been studied. However the only FDA approved drug for unresectable or metastatic HCC is sorafenib which is a tyrosine kinase inhibitor.
Our interest was to identify genes that play a role in liver oncogenesis and so we directed our focus to study a novel class of small non coding RNAs, the microRNAs.
MicroRNAs have been involved in the pathogenesis of different types of cancers, including liver cancer. Specific microRNA signatures have been identified to be deregulated in HCC patient tissues and also to correlate with different clinicopathological parameters including survival. Having all this in mind our mission was to identify microRNAs that have both functional and clinical relevance in HCC and examine their downstream signaling effectors.
Methods:
In this study, we have screened the human microRNAome, aiming to identify microRNAs that are potent regulators of HCC invasiveness. Initially we received 24 tissues from patients with liver cancer along with 14 from adjacent non tumor specimens. RNA extraction was performed and microRNA and gene expression levels were measured by quantitative real-time RT-PCR. We determined the expression levels of miR-9, miR-21 and miR-224 in those samples. Simultaneously a novel microRNA library screen was done after plating SNU-449 liver cancer cells in 96-well plates. Those were transfected with a microRNA library consisting of 316 microRNA mimics and 2 negative controls. 48hrs post transfection we evaluated the SNU-449 cell invasiveness. We also performed invasion assays in SNU-449 cells 24hrs after transfection specifically with miR-9 and anti-miR-9 and their respective controls. A TargetScan algorithm was used to identify miR-9 downstream direct targets based on bioinformatics. Those analyses were validated by performing 3’UTR luciferase assay. Precisely, we transfected SNU-449 cells with the reporter vectors carrying the 3’UTR of CDH1 or PPARA. The constructs harbored the seed sequence of miR-9 (wildtype) or had a deletion of this sequence (miR-9 mutant). At 24 hours, they were transfected with miR-9 or miR-control and at 48 hours luciferase activity was measured. We have also performed transfection with microRNA mimic for mir-9 overexpression as well as microRNA inihibitor to suppress the miR-9 activity and observed the expression levels of the downstream targets CDH1, PPARA, PDK4 and vimentin. Furthermore we performed cell proliferation as well as assessed sphere formation and colony assay in SNU-449 and HepG2 liver cancer cell lines, both of which were transfected with miR-9 and anti-miR-9 along with their respective controls.
Results: First we performed a high-throughput microRNA screen in SNU-449 liver cancer cells and assessed their invasiveness while secondly we evaluated in tissue the expression levels of the microRNAs derived from the above screen. Overexpression of miR-9 was found to be the top inducer of SNU-449 cell invasiveness, cell growth and their ability to form colonies in soft agar. Furthermore miR-9 levels were found in tissue to increase during HCC progression. Bioinformatics and 3’UTR luciferase analyses identified E-cadherin (CDH1) and peroxisome proliferator-activated receptor alpha (PPARA) as direct downstream effectors of miR-9 activity. Inhibition of PPARA suppressed CDH1 mRNA levels, suggesting that miR-9 regulates CDH1 expression directly through binding in its 3’UTR and indirectly through PPARA. On the other hand, miR-9 inhibition of overexpression suppressed HCC tumorigenicity and invasiveness. PPARA and CDH1 mRNA levels were decreased in HCC relative to controls and were inversely correlated with miR-9 levels.
Conclusions: Our study identified a novel microRNA signaling pathway, consisting of miR-9, PPARA and CDH1 that is deregulated in HCC patients affecting liver cancer cellular invasiveness and metastatic potential.
|
Identification of novel microRNAs involved in Hepatocellular carcinogenesis by high throughput screening
