Illumina HiSeq 2500 sequencing; NUDT21 Negatively Regulates PSMB2 and CXXC5 by Alternative Polyadenylation and Contributes to Hepatocellular Carcinoma Suppression
NUDT21 Negatively Regulates PSMB2 and CXXC5 by Alternative Polyadenylation and Contributes to Hepatocellular Carcinoma Suppression
Human liver cells and liver cancer cells were purchased from the Shanghai Cell Collection (Shanghai, China). All cells were cultured in Dulbecco's modified Eagle's medium with 10% feta calf serum (FCS), penicillin (50 U/ml), streptomycin (50 mg/ml), and glutamine (2mM) at 37°C in a 5% CO2 humidified atmosphere. Given the functional involvement of NUDT21 in APA formation and its tumor suppression activities mentioned above, we hypothesized that NUDT21 exert its tumor suppression, at least in part, by influencing APA and 3'UTR. To test this hypothesis, we aimed to examine all instances of NDUT21-related 3'UTR alteration in HCC cells. To this end, we generated NUDT21 overexpression line in Hep3B and NUDT21 knockout line in HL7702, and identified poly(A) sites with 3T-seq.  RNA was isolated from using Trizol (Invitrogen), reverse-transcribed using RevertAid™ First Strand cDNA Synthesis Kit (Fermentas); Quantitative PCR was performed using gene-specific primer pairs and SYBR Green.  1µg total RNA was used to generate cDNA with Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions using 3'RACE-RT as a primer. The ﬁrst PCR was done with a PSMB2 or CXXC5-speciﬁc forward primer and Outer as reverse primer. Nested PCR was done with a nested PSMB2 or CXXC5-speciﬁc forward primer and inner as reverse primer. The PCR product from the nested PCR was separated on an agarose gel, cloned and sequenced.
Experimental Factor: cell line
HL7702
Experimental Factor: genotype
NUDT21 knockdown by shRNA