Abstract
Purpose: To explore the molecular function of miR-196b-5p in hepatocellular carcinoma (HCC).Methods: MiR-196b-5p expression levels in HCC tissue samples were assessed by qRT-PCR. MiR-196b-5p was knocked-down or over-expressed in HepG2 cells by transfecting the cells with plasmids expressing either a miR-196b-5p inhibitor or mimic, respectively, while cell proliferation was assessed by MTT assay. The interaction of miR-196b-5p with target molecules was confirmed using luciferase reporter assay. Cell cycle was investigated by flow cytometry, while NFκBIA expression was assessed by western blotting.Results: MiR-196b-5p was over-expressed in HCC, and miR-196b-5p expression levels in patients with HCC were related to tumor grade. MiR-196b-5p over-expression promoted cell proliferation and colony formation and suppressed cell cycle arrest and apoptosis. The results of luciferase reporter assay showed that miR-196b-5p reduced NFκBIA expression in HepG2 cells by binding to a response element in the 3′ UTR of NFκBIA. Further investigation showed that NFκBIA interacts with NFκB1 and reduces the concentration of NFκB1 in HepG2 cells. The promoter of ATP-binding cassette sub-family B member 1 (ABCB1) was also targeted and bound by NFκB1, which altered the expression of ABCB1 in HepG2 cells.Conclusion: MiR-196b-5p regulates cell proliferation in drug-resistant HCC cell lines via activation of the NFκB/ABCB1 signaling pathway.
 Keywords: Hepatocellular carcinoma, miR-196b-5p, NFκBIA, NFκB1, ABCB1
Highlights
90 % of primary liver cancers are hepatocellular carcinoma (HCC)
HepG2 cells were collected and lysed in RIPA buffer (Thermo Fisher) to generate crude extracts, and the proteins in the extracts were separated by 15% SDS-PAGE
Drug resistance in colorectal tumor cells was promoted by introduction of miR-196b-5p, and the sensitivity of colorectal cancer cells to chemotherapeutics was increased by knocking down miR-196b-5p [11]
Summary
90 % of primary liver cancers are hepatocellular carcinoma (HCC). The morbidity of HCC is among the highest of all cancers [1], and many cases of HCC (50 %) are diagnosed at an advanced stage [2]. Various treatments have been developed for HCC, the prognosis is still poor [3,4]. HCC is a highly angiogenic, solid tumor that is mainly induced by dysregulation of angiogenesis and cell proliferation and evasion of apoptosis. -©---2-0--2--0--T--h--e--a--u-t-h--o--r-s-.--T--h-i-s--w---o-r-k---is---li-c-e--n--s-e--d--u--n--d-e--r--t-h-e---C--r-e--a-t-i-v-e---C--o--m--m---oT-n-r-so--p-A-Jt-t-r-Pi-b-hu--at-ir-om-n---R4-.-e0-s--,I-nJ-t-ae-n-r-nu--aa-tr-i-yo-n-2-a-0-l2-L-0-ic-;-e-1-n9-s-(-e1--)-:--3-9 chromosomal aberrations, and molecular pathway alterations have been shown to be involved in the molecular pathogenesis of HCC [5]. All cells were cultured in DMEM containing 10% fetal bovine serum
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