1 Background

Liver cancer is one of the most common six malignant tumors with high malignancy and a poor prognosis around the world. Although the genetic information and the risk factors of liver cancer formation have been increasingly identified, the exact mechanisms underlying the initiation, development, and metastasis of liver cancer are poorly understood [1].

The Klf6 gene, which belongs to Krüppel-like family (containing C2H2 zinc finger transcription factors), is greatly related to tumorigenesis via a high rate of somatic mutation in the carcinomas of prostate, colon, stomach, liver, lung, neck, pituitary, nervous system, etc. [2, 3]. The functional role of dysregulated alternative splicing in cancers is now recognized in a range of human cancers including liver, lung, stomach, prostate, colon, etc. It has been reported that many genes associated with cancer can be alternatively spliced, and their expression can lead to the production of multiple splice variants with antagonistic function. Generally, the Klf6 gene, which is known to be an important gene in the initiation, development, and metastasis of tumors, can be alternatively spliced into biologically active isoforms called Klf6-SV1, -SV2, and -SV3 [4, 5]. In particular, the SV1 variant has been shown to accelerate the progression and metastasis of prostate cancer [6, 7]. The functional role of Klf6-SV1 in tumorigenesis was most well-defined among three Klf6 spliced variants because it can be highly expressed in many human malignant tumors, and its over-expression is observed in cancer patients with poor prognosis and low survival rate [8,9,10]. Growing results have shown that elevated expression of Klf-SV1 variant partially promotes the metastasis, progression, and mortality of cancer through its antagonism to Klf6 [11]. Furthermore, elevated Klf6-SV1/Klf6 mRNA ratios have been demonstrated with their functional characteristics in liver cancer samples. On the other hand, over-expression of SV2 variants in IHH and HepG2 cells was observed to significantly reduce cell proliferation by apoptosis [12,13,14]. Although the functional outcome of Klf6 splice variant has become clear, the pathway regulating expression of Klf6 splice variant remains ambiguous.

Recently, a large cluster of transcriptional enhancers called super enhancers (SEs) have been identified. Furthermore, SEs have been shown to specifically regulate cell identity in many tumors, including liver, lung, stomach, and prostate, as genomic domains that are large non-coding enhancers [15,16,17,18,19,20]. We have already identified a Klf6-related super enhancer in human HepG2 cells using genomic editing techniques and demonstrated that Klf6-related SE regulates cellular proliferation as a potent regulator of Klf6 gene expression [21]. We assumed that regulation of cell proliferation by Klf6-related super enhancer may be associated with Klf6 variants in deeper study.

In this study, we obtained the cell clones of a Klf6-related super enhancer deleted by means of the CRISPR/Cas9 system-mediated genetic engineering as previously described [21] and also found that inhibition of cell proliferation by deletion of Klf6-related super enhancer is caused by Klf6 variants in human hepatoma (HepG2) cells. At first, it was demonstrated that deletion of Klf6-related super enhancer induces the upregulation of SV2 variant and leads to the inhibition of cell proliferation in HepG2 cells. In addition, it was shown that Klf6-related super enhancer leads to the induction of p21 (cell-cycle-controlling gene) and Bax (pro-apoptotic gene) genes mediated by upregulation of SV2 variant. The results provide the functional significance of Klf6-related super enhancer in understanding the transcriptional regulation mechanism of Klf6.

2 Methods and materials

2.1 Cell culture and transfection experiments

HepG2 was derived from the Chinese Academy of Science. The cell culture and transfection were carried out according to cell culture and transformation protocols as previously described [21].

2.2 RNA extraction, reverse transcription, and real-time PCR

Total RNA isolation was performed using TRIzol (Life Tech) extraction for the target clones.

A total of RNA (1 μg) was reverse transcribed using the first-strand complementary DNA synthesis with random primers. RT-PCR was performed with KLF6 5′ and 3′ untranslated region-specific primers on 10 ng cDNA derived from the target clones as previously described [9]. qRT-PCR was carried out with SYBR Green PCR Master Mix (Applied Biosystems). The relative expression ratio of genes in the cells was quantified by the 2−△△CT method.

Primers selected for the experiments are as following: 5′-CGGACGCACACAGGAGAAAA-3′/5′-CGGTGTGCTTTCGGAAGTG-3′ (Klf6), 5′-CCTCGCCAGGGAAGGAGAA-3′/5′-CGGTGTGCTTTCGGAAGTG-3′ (Klf6-SV1), 5′-TCGGGGAAGCCAGGAGAA-3′/5′-CGGTGTGCTTTCGGAAGTG-3′ (Klf6-SV2), 5′-CGGACGCACACAGGTGTT-3′/5′-TCTGCTCCCTCAGAGGTGCC-3′ (Klf6-SV3), 5′-ACTCTCAGGGTCGAAAACGG-3′/5′-CCTCGCGCTTCCAGGACTG-3′ (P21), 5′-ATCCAGGATCGAGCAGGGCG-3′/5′-ACTCGCTCAGCTTCTTGGTG-3′ (Bax), 5′-GTGTGGCGACATATGCAGCT-3′/5′-CAAGATCAGCAGTCTCATTC-3′ (beta-actin).

The analysis proceeded in triplicate, and the relative level of gene expression was assessed as beta-actin.

2.3 Cell proliferation assay and Western blot assay

Proliferation was determined by estimating [3H] thymidine incorporation as previously described [9]. Separation of proteins for Western blot assays proceeded on 10–15% SDS-PAGE gel as previously described [21]. The beta-actin protein was used as a loading control.

2.4 Plasmid construction and transfection

The pSUPER-siSV1, -siSV2, and -siSV3 plasmids used to downregulate Klf6-SV1, -SV2 and -VS3 expression were constructed according to the previously described method, and the pSUPER-Luc construct was used as a control [9]. The pSUPER-si-wtKLF6 construct was generated as previously described using the pSUPER vector [22]. To insert the targeting sequence, DNA oligonucleotides were designed and cloned into the BglII-HindIII sites of the pSUPER vector. The characteristics of the designed DNA oligonucleotides are shown in Table 1.

Table 1 DNA oligonucleotides for pSUPER vector
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Generation of cell clones over-expressing Klf6-SV variants was performed as previously described [12]. The coding sequences of Klf6 and Klf6-SV variants were obtained by PCR amplification and inserted into p3XFLAG-CMV-10 vector (Sigma).

3 Results

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.

Fig. 1
figure 1

Klf6 gene expression and cell proliferation assay. a Klf6 gene expression assay. The Klf6 expression level was measured by qRT-PCR. The results represent the mean of five amplifications, ***p < 0.001. b Klf6 proteins expression assay. Klf6 proteins were determined by western blot. c Cell proliferation assay. [3H] thymidine incorporation was determined at 72 h. The results represent the mean of eight experiments, **p < 0.01

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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.

Fig. 2
figure 2

Expression of Klf6-SV isoforms in the target clones. a RT-PCR of cDNAs with Klf6 variant-specific primers [9]. PCR products were visualized after agarose gel electrophoresis and ethidium bromide staining. b SV/Klf6 mRNA ratio analysis by qRT-PCR. The figure shows Klf6-SV expression relatively to Klf6 expression in the target clones. The results are the mean of three different quantifications, *p < 0.05, **p < 0.01

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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.

Fig. 3
figure 3

Expression of Klf6-SV isoforms and cell proliferation assay in the target clones. a Expression assay of Klf6-SV1 and -SV2. The SE-del clone cells were transfected with pSuper-siSV1, 2 or control pSuper-siLuc. b Cell proliferation assay. [3H] thymidine incorporation was determined at 72 h. Each experiment was performed in triplicate, **p < 0.01. c Expression assay of Klf6-SV isoforms: The SE-non-del clone cells were transfected with pSuper-siSV1,2,3 or control pSuper-siLuc. The SE-non-del clone cells were transfected with pSuper-si-wtKlf6 or control pSuper-siLuc. The results represent the mean of three amplifications, **p < 0.01,*p < 0.05. d Cell proliferation assay. Each experiment was performed in triplicate, **p < 0.01,*p < 0.05,#p > 0.05

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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.

Fig. 4
figure 4

Over-expression of Klf6-SV2 triggers increased expression of p21 and Bax. a Expression of p21 mRNA in SE-del clone transfected with siSV2. The results represent the mean of three amplifications, **p < 0.01. b Expression of p21 and Bax proteins in SE-del clone transfected with siSV2. The gels are representative of three analyses. c Expression of Bax mRNA in SE-del clone transfected with siSV2. The results represent the mean of three amplifications, *p < 0.05

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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.

Fig. 5
figure 5

The effect of Klf6 variants on the expression of P21 and Bax genes. a Expression of p21 mRNA in SE-non-del clone transfected with various siRNA. The results represent the mean of three amplifications, ♯p > 0.05, *p < 0.05. b Expression of Bax mRNA in SE-non-del clone transfected with various siRNA. The results represent the mean of three amplifications, **p < 0.01, *p < 0.05,♯p > 0.05. c Expression of p21 and Bax proteins in SE-non-del clone transfected with various siRNA. d Expression of p21 mRNA in SE-non-del clone with over-expression of various Klf6 variants. The results represent the mean of three amplifications, ♯p > 0.05, *p < 0.05. e Expression of Bax mRNA in SE-non-del clone with over-expression of various Klf6 variants. The results represent the mean of three amplifications, **p < 0.01, *p < 0.05,♯p > 0.05. f Expression of p21 and Bax proteins in SE-non-del clone with over-expression of various Klf6 variants

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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.

4 Discussion

The Klf6 transcription factor is closely related to tumorigenesis through high somatic mutation rates in multiple carcinomas including prostate, colon, stomach, liver, lung, and neck [2, 3]. The role of dysregulated alternative splicing in cancer is now being studied in a variety of cancers, and it is confirmed that many cancer-related genes are alternatively spliced and their expression leads to the production of multi-splice variants with antagonistic function. Klf6 is alternatively spliced into biologically active isoform called Klf6-SV1, -SV2, and -SV3 [4, 5]. Although the functional outcomes of Klf6 splice variants have become clearer, the pathways regulating expression of Klf6 splice variants remain obscure. Therefore, the observation of expression levels of Klf6 variants and their respective functions in human HepG2 cells is essential for further understanding of their mechanisms. Disruption of Klf6-related SE induces inhibition of proliferation in human HepG2 cells. We hypothesized that inhibition of cell proliferation induced by the disruption of Klf6-related super enhancer may be associated with Klf6 variants. In our study, the results show that Klf6-related super enhancer regulates expression of Klf6 variants in human HepG2 cells. As shown in Fig. 2b, the expression of Klf6-SV2 was three times higher than one of the control, but the expression of Klf6-SV1 was increased very little. Specifically, the functional modeling of findings using siRNA specific to Klf6-SV2 in human HepG2 cells revealed that changes in expression level directly affect cell proliferation. We conducted an experiment to determine whether the over-expression of Klf6-SV2 induced by disruption of Klf6-related SE is actually associated with cell proliferation in human HepG2 cells. As shown in Fig. 3, down-expression of Klf6-SV1, -SV2, and -SV3 inhibited cell proliferation in siSV1 cells and promoted in siSV2 cells, but not in siSV3 cells. Furthermore, cell proliferation was shown to be directly associated with Klf6-SV2 levels. These results show that over-expression of Klf6-SV2 induced by disruption of Klf6-related SE is an important factor associated with inhibition of cell proliferation in human HepG2 cells. The above results are further supported by literature [1, 7, 9, 12, 13, 23] that over-expression of Klf6-SV1 promotes cell proliferation, while over-expression of Klf6-SV2 inhibits cell proliferation. It is also consistent with data that downregulation of wtKlf6 promotes cell proliferation in hepatocellular carcinoma (HCC) [24]. We demonstrated that over-expression of Klf6-SV2 induces transcriptional activation of p21 (CIP/WAF1) and Bax genes (Fig. 5). The results show that over-expression of Klf6-SV2 exerts partially mediated anti-proliferative and pro-apoptotic effect by induction of p21 (CIP / WAF1) and Bax. The results are consistent with the literature [1, 9, 12, 13, 23] showing that over-expression of Klf6-SV2 induces anti-proliferation and apoptosis.

We proposed a novel model regulating cell proliferation via modulation of Klf6-SV2 expression into the growth-relating isoforms (Fig. 6). Klf6-related super enhancer regulates expression of Klf6-SV2, thereby altering the relative ratio of Klf6 to Klf6-SV2, which leads to inhibition of cell proliferation. This model requires further studies, such as what is the signaling pathway between the super enhancer and biologically active isoforms, and how it is regulated [25,26,27,28,29].

Fig. 6
figure 6

The model regulating cell proliferation by super enhancer. a Pre-disruption model of Klf6-related SE. When Klf6-related SE is not disrupted, it upregulates Klf6 expression and induces cellular proliferation. b Post-disruption model of Klf6-related SE. When Klf6-related SE is disrupted, it upregulates Klf6-SV2 expression which leads to p21-mediated growth inhibition. The size of the arrow in the figure represents the relative expression intensity of the target gene. In other words, an arrow indicated by a thick line means strong gene expression compared to a thinly marked arrow

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5 Conclusions

Several studies have identified new cancer therapeutics that target super-enhancers of various tumor types. In our study, we have elucidated the functional properties of the Klf6-related super enhancer on the expression of Klf6 splice variants which may be helpful in the target treatment of Klf6-related super enhancer. the disruption of Klf6-related super enhancer showed not only induced the up-regulation of Klf6-SV2 but also led to a significant reduction of proliferation in HepG2 cells. It was demonstrated that the disruption of Klf6-related super enhancer led to the induction of p21 and Bax genes mediated by the up-regulation of Klf6-SV2. Therefore, it showed that Klf6-related super enhancer modulates cell proliferation via the regulation of Klf6-SV2 expression in human hepatoma (HepG2) cells. The results provide the functional significance of Klf6-related super enhancer in understanding the transcriptional regulation mechanism of Klf6.

But this result raises an interesting question of how cellular signals converge to regulate splicing events in promoting tumor progression. Targeted analysis of molecular pathways of Klf6-related super enhancer that regulates Klf6-SV2 expression will provide further insight into the role of this gene in the progression of human cancer.