Investigation of Mono-allelic RACK1 Deletion Effects on Cell Growth and PKC mRNA Expression

The µ-opioid receptor is a primary target of clinically used opioids, and RACK1 regulates its transcription by physically interacting with MOR transcription regulator PCBP1 in the cytosol to prevent its nucleus translocation. Two mono-allelic RACK1 knockout cell lines named RACKA and RACKB were generated by the CRISPR-Cas9 technique using WT NMB neuroblastoma cells. As RACK1 also contains a PKC-binding domain, this study aims to evaluate the impacts of mono-allelic deletion on cell growth rates and PKC isoform expression. Although a slight decreased tendency of growth rate in RACKA and RACKB cells was observed when compared to that of WT NMB cells, statistical analysis shows that no significant differences were found in cell growth rates over a 4-day period among NMB, RACKA, and RACKB. In addition, cell viabilities of RACKA and RACKB were consistent with our previous observation that mono-allelic RACK1 knockout cells were viable. RT-PCR analysis detected the presence of PKC Alpha, Gamma, Delta, Epsilon, Eta, Theta, and Lambda, and the absence of PKC Beta and Zeta in all three cell lines. Semi-quantitative analysis showed a slight increase in PKC Alpha in RACKA (85.42%) and RACKB (96.41%) cells compared with WT NMB (63.03%) cells, and a slight decrease in PKC Gamma 1 was also observed in RACKA (15.52%) and RACKB (18.13%) cells when compared to WT NMB (41.25%) cells. However, the statistical analysis further showed that there was no significant difference in the expression level of PKC Alpha, Gamma 1, Gamma 2, Delta, Epsilon, Eta, Theta, and Lambda among NMB, RACKA, and RACKB, suggesting that mono-allelic RACK1 deletion did not significantly alter PKC mRNA expression. Bioinformatics analysis was performed to investigate the interactions of PCBP1-RACK1, RACK1-PKC Alpha, and PKC Alpha-PKC gamma. Collectively, this study observed that mono-allelic RACK1 deletion slightly affected cell growth rates, slightly increased PKC Alpha mRNA levels, and slightly decreased PKC Gamma 1 mRNA levels. Statistical analyses revealed that mono-allelic RACK1 knockouts had no statistically significantly effect on cell growth or mRNA expression of various PKC isoforms. Bioinformatics analysis implicated the competitive interactions between PCBP1-RACK1 and RACK1-PKC Alpha, as well as RACK1-PKC Alpha and PKC Alpha-PKC gamma. Future research should continue investigating the linkage and impacts among PCBP1, RACK1, PKC, and MOR.