3.1 Deletion of Klf6-related SE induces inhibition of cell proliferation in human HepG2 cells
A clone of Klf6-related SE deletion (SE-del) clone was obtained using the CRISPR/Cas9 system to evaluate the effect of Klf6-related SE on the expression of Klf6 gene in HepG2 cells. The experimental procedure for obtaining SE-del clones in HepG2 cells has been detailed in previously published literature [21]. Expression of Klf6 gene in SE-del clone using qRT-PCR was decreased by 80% or more, and strong inhibition of Klf6 gene expression was observed as shown in Fig. 1a.
Reduced expression of Klf6 gene in SE-del clone was confirmed at the protein level by using Western blot, and expression of Klf6 protein was also significantly inhibited in SE-del clone as compared with the control (Fig. 1b). After deletion of Klf6-related SE, the effect of cell proliferation was evaluated using [3H] thymidine incorporation assay, concurrent with the result of decreased Klf6 expression, it observed a markedly inhibited proliferation (Fig. 1c).
3.2 Deletion of Klf6-related SE regulates expression of Klf6-SV variants in human HepG2 cells
As shown in the above results, since significant inhibition of cell proliferation as well as expression of Klf6 after deletion of Klf6-related SE was observed, therefore, it attempted to analyze the expression characteristics of Klf6 variants by RT-PCR.
The SE-del clone expression pattern through RT-PCR of cDNA derived from the target clone containing the SE-del clone, and the control clone revealed a strong inhibition of the full-length tumor suppressor Klf6 concurrently with a severe change in the relative expression level of the Klf6 splice variants Klf6-SV1, -SV2, and -SV3 (Fig. 2a). Quantitative real-time PCR (qRT-PCR) analysis of cDNA derived from the target clones showed that the SV/Klf6 mRNA ratio was increased in Klf6-SV1 and Klf6-SV2, but decreased in Klf6-SV3. Especially, the expression of Klf6-SV2 was three times higher than that of the control, but the expression of Klf6-SV1 was increased very little. These results show that expression of Klf6 splice variants in human HepG2 cells is strongly regulated by Klf6-related super enhancer, and in particular, inhibition of cell proliferation after deletion of SE is likely to be associated with over-expression of Klf6-SV2.
3.3 Deletion of Klf6-related SE induces inhibition of proliferation through over-expression of Klf6-SV2 in human HepG2 cells
On the basis of the above findings, we conducted an experiment to determine whether over-expression of Klf6-SV2 induced by the disruption of the Klf6-related SE is actually associated with cell proliferation in human HepG2 cells. Firstly, we specifically inhibited the expression of Klf6-SV1 and -SV2, which showed increased expression after the disruption of the Klf6-related SE using target-specific siRNAs in SE-del clone (Fig. 3). Targeted reduction of Klf6-SV1 and -SV2 expression in SE-del clone was confirmed at the mRNA level by qRT-PCR (Fig. 3a). Comparing to the siLuc control cells, the expression level of Klf6-SV1 in the siSV1 cells was decreased by at least 45% (p < 0.01), whereas the expression level of Klf6-SV2 in the siSV2 cells was decreased by at least 55% (p < 0.05). The effects of Klf6-SV1 and -SV2 inhibition were determined by measuring cell proliferation (Fig. 3b). Cell proliferation was increased twofold in siSV2 cells (p < 0.01) but decreased by half in siSV1 cells (p < 0.01). The results indicate that over-expression of Klf6-SV1 promotes cell proliferation, but its downregulation induces inhibition. On the other hand, over-expression of Klf6-SV2 inhibits cell proliferation, but its down-expression promotes cell proliferation and thus appears to be responsible for inhibition of cell proliferation induced by deletion of the Klf6-related SE.
Secondly, we inhibited the expression of Klf6-SV1, -SV2, -SV3, and wtKlf6 using target-specific siRNAs in SE-non-del clone (Fig. 3). As shown in Fig. 3c, d, downregulation of Klf6-SV1 (35%, p < 0.05) induces inhibition of cell proliferation, but down- regulation of Klf6-SV2 (35%, p < 0.05) promotes cell proliferation. Expression of Klf6-SV3 in siSV3 cells was decreased by at least 72% (p < 0.05) compared to the siLuc control cells, but there was no significant difference in cell proliferation analysis (#p > 0.05). However, expression of the wtKlf6 in si-wtKlf6 cells was decreased by 55% (p < 0.01) but a significant increases in cell proliferation (p < 0.01). As shown above, the results show that over-expression of Klf6-SV2 induced by the disruption of the Klf6-related SE is an important factor associated with cell proliferation in human HepG2 cells, especially inhibition of cell proliferation.
3.4 Over-expression of Klf6-SV2 induces an increased expression of p21 and Bax in human HepG2 cells
The expression of cell cycle-regulated genes were analyzed to elucidate the proliferation-related mechanism induced by Klf6-SV2. In response to over-expression of Klf6-SV2, cell cycle-regulating p21 (CIP/WAF1) expression was higher about two-fold in SE-del clone than that of control clone (Fig. 4a). Elevated expression in p21 (CIP / WAF1) protein was also identified by Western blot (Fig. 4b). A similar method was used to elucidate the apoptotic mechanism induced by over-expression of Klf6-SV2. Among the proteins involved in apoptosis, elevated expression of the pro-apoptotic Bcl-2-associated Bax gene was demonstrated (Fig. 4c). Increased expression in Bax was also confirmed by Western blotting (Fig. 4b), and the results suggested that Klf6-SV2-induced apoptosis is associated with the mitochondrial apoptotic pathway. Knockdown of Klf6-SV2 expression by siRNA (Fig. 4a (+), c (+)) induces inhibition of p21 and Bax at the protein (Fig. 4b) and mRNA (Fig. 4a, c). As a result, Klf6-SV2 exhibits partially mediated anti-proliferative and pro-apoptotic effects through induction of p21 (CIP / WAF1) and Bax.
Knockdown of Klf6 variants by various siRNAs in SE-non-del clone can be observed in which over-expression of Klf6-SV2 increases the expression of p21 and Bax at the protein level (Fig. 5c) and mRNA (Fig. 5a, b). In addition, over-expression of various variants in SE-non-del clone also demonstrated that increased expression of p21 and Bax are dependent on over-expression of Klf6-SV2 (Fig. 5d–f]. As shown in Fig. 5, unlike the other variants, Klf6-SV3 did not affect the expression of P21 and Bax genes (♯p > 0.05). Upregulation of Klf6-SV1 reduces the expression of P21 and Bax genes, but its downregulation increases expression of P21 and Bax genes.
The results show that Klf6-related super enhancer leads to an induction of the cell cycle-regulating p21 and the pro-apoptotic Bax genes mediated by up-regulation of SV2 variant.