The Regulatory Role And Its Mechanism Of MiR-612 On The Stemness Of Hepatocellular Carcinoma

Background and Purpose:Cancer stem cell theory has been proposed for about 30 years. Bonnet and Dick found that a specific subpopulation of tumor cells isolated from a Acute myeloid leukemia (AML) patient can cause the human-alike leukemia in NOD/SCID mice in 1997. Thereafter researchers successivefully separated cancer stem cells in a variety of solid tumors, such as breast cancer, brain cancer, lung cancer, prostate cancer, melanoma, ovarian cancer, colon cancer, head and neck cancer, pancreatic cancer, liver cancer, bladder cancer and skin cancer. Cancer stem cells possess some characteristics like self-renewal, anti-apoptosis, and resistance. Recent studies show that cancer stem cells not only contribute to initiate a tumor and promoting a tumor’s growth,but also contributes a lot in tumor’s metastasis. Studies suggest that EMT plays a very important part in the regulation of cancer stem cells, and they have cross-talk between each other. Researches show the form of cancer stem cells is regulated by multiple levels of genes, and abnormal microRNA expression is closely related to tumor cell EMT and the formation of cancer stem cells. Our early study suggest that miR-612 can inhibit the proliferation、invasion and metestasis of liver cancer by suppressing the EMT process. Therefor the present research is to study whether miR-612 can regulate the stemness of liver cancer and its role in the regulation of liver cancer invasive frontier, and try to find possible mechanisms within this rugulaton.Methods:We choose liver cancer cell lines MHCCLM3 (relatively low miR-612 expression) and HepG2 (relatively high miR-612 expression) for the study. Firstly transient transfection of miR-612 mimic and hairpin inhibitor was used to realize the purpose of over expression of miR-612 (miR-612-O) in HCCLM3 and the inhibitive expression of miR-612 (miR-612-i) in HepG2 cells. Then tumorsphere and clone formation in soft agar assay were used to culture the above treated cells. We used the methord of infection MHCCLM3/HepG2 with lentivirus to stablely overexpress or knock down miR-612. Then these cells were used to undergo cytotoxicity experiments in vitro and tumorigenicity in NOD/SCID mice. The MHCCLM3(HepG2) cells with stable overexpression of miR-612 and MHCCLM3 (HepG2) cells with stable knockdown of miR-612 were equally mixed and injected in nude mice to observe the role of miR-612 in the forefront of HCC invasion. The second part we investigate the role of the relationship between miR-612 and wnt/p-catenin by using Western blot, Real-time PCR, immunofluorescence and Luciferase reporter assays, adding wnt pathway agonists methords.Results:1) Transient transfection of miR-612 mimic into HCCLM3 cells significantly suppressed the ability to form tumorsphere and clone in sotft agar; and Transient transfection of miR-612 inhibitor into HepG2 cells can enhance the ability to form tumorsphere and clone in soft agar.2) HCCLM3 cells with stable overexpression of miR-612 exhibit increased sensitivity to chemotherapeutic drugs (cisplatin, fluorouracil) in vitro cytotoxicity assay, and furthermore the capacity to form subcutaneous tumors in NOD/SCID mice were suppressed too; HepG2 cells with stable knocked down miR-612 showed higher resistance to chemotherapy, furthermore the tumorigenicity in NOD/SCID mice were promoted.3) The miR-612-o-HCCLM3 cells (with green fluorescence) and the same number of the miR-612-i-HCCLM3 (with red fluorescence) cells were mixed together, and then were injected subcutaneously into nude mice. After four weeks, remove the tumor and made into flozen-section slices and were observed under fluorescence microscopy. The result revealed that cells distributed at the surrounding of tumors were mainly the miR-612-i-HCCLM3 cells. HepG2 cells were treated in the same way, and we observed the same result.4) Given the results of previous studies (miR-612 can inhibit proliferation, invasion and metastasis of liver cancer cells by suppressing the EMT process.), and inner-relationship between miR-612 and E-cad and β-catenin, thus we choose wnt/β-catenin signals as miR-612’s target genes in the regulation of liver cancer stem cells. Western blot results showed that miR-612 does not affect the overall protein level of intracellular β-catenin; further immunofluorescence experiments showed that miR-612 can reduce the transfer of β-catenin from cytoplasm to the nucleus; TOPflash luciferase experiments showed that miR-612 can reduce intracellular TCF/LEF activity; Real-time PCR and Western blot showed that miR-612 can reduce intracellular expression of c-Myc and cyclin-D1. Tumorsphere formation experiment showed miR-612 can reverse the enhanceed effect of wnt signaling pathway agonist wnt3α and BIO on the formation of tumorsphere.Conclusion:This study revealed miR-612 can suppress the tumor formation and clone formation of liver cancer cells; increase the sensitivity of liver cancer cells to chemotherapeutic drugs; inhibit tumorigenic capacity of liver cancer cells in NOD/ SCID mice; redistributed the liver cancer cells at the invasive frontier. And wnt/β-catenin signals was proved to plays an important role in the regulation of the stemness of live cancer cells by miR-612.The pleiotropic roles of miR-612 on HCC EMT and CSCs suggest that it could be an effective molecular target for HCC therapy.