Using pgRNA–Cas9 system to knockout MKL1 inhibited cell migration and proliferation

Abstract

As a transcription factor, megakaryoblastic leukemia 1 (MKL1) binds with serum response factor (SRF) to regulate targeted genes expression. Therefore, it involved in various cellular processes, such as cancer cells migration, proliferation and differentiation. However, the report about knockout MKL1 expression using CRISPR–Cas9 system in human genome is rare. Therefore, paired-guide RNA (pgRNA) library was constructed, and pgRNA–Cas9 system was used to delete MKL1 expression in HeLa cells. The result showed that the expression of MKL1 was decreased 95% by Western blotting. And wound healing assay and MTT assay indicated that depletion MKL1 inhibited cell migration and proliferation. Additionally, the protein expression levels of tumor suppressor factors p21, p53, pRB and DLC1 were changed after depletion MKL1. These data suggested that tumor suppressor factors p21, p53, pRB and DLC1 maybe played an important role in the cell growth and migration of MKL1-depletion cells.

Keywords:

• pgRNA
• Cas9
• MKL1
• cell migration
• proliferation

View retraction statement:

Retraction: Using pgRNA-Cas9 system to knockout MKL1 inhibited cell migration and proliferation

PUBLIC INTEREST STATEMENT

As a powerful tool for genomic editing, CRISPR system has played an important role in the study of molecular biology and therapy of genetic diseases. The CRISPR system can target the gene fragments that we are concerned on and change it, including insert, silence and knockout, to clear on and benefit from the gene. This research knockout the expression of transcriptional factor MKL1 in cervical cancer cell line HeLa using CRISPR/Cas9 system. It shows readers the outstanding performance of CRISPR/Cas9 system in genomic knockout. In addition, the study also suggested the application potential of the CRISPR system on investigating the function and mechanism of MKL1 in cervical cancer cells.

1. Introduction

Clustered regularly interspaced palindromic repeat (CRISPR) is first discovered in bacteria and considered as an adaptive immune system to degrade exogenous DNA (Bhaya, Davison, & Barrangou, 2011). According to the degradation modes of exogenous genetic material, CRISPR was classified to three systems (Terns & Terns, 2014). The works of type I and type III system need multiple protein complex cooperation. In contrast, the type II (CRISPR–Cas9), only with a Cas9 nuclease and a sgRNA (single-guide RNA), is more convenient and efficient (Gaj, Gersbach, & Barbas, 2013). The CRISPR–Cas9 system works by sgRNA–Cas9 complex, in which sgRNA guides Cas9 to target specific DNA site, as well as Cas9 nuclease cleaves DNA and produces a double stranded break (DSB). Then, with DNA repair pathways of non-homologous end joining and homology-directed repair, the mutation or insertion on genome would appear at the site around DSB (Mali et al., 2013). Two DSBs sites were digested using the paired-guide RNA (pgRNA) including two sgRNAs via cas9 system (Shalem et al., 2014). Because of these features, CRISPR–Cas9 system, as a very important and powerful genomic editing tool, is widely used. However, the CRISPR–Cas9 system also has off-target effect to induce DNA wrong cutting (Koo, Lee, & Kim, 2015). In order to eliminate the effect, it usually uses D10A (Cas9 Nickase) with pgRNA that target at DNA positive and negative strands to cut-off two nicks. Due to only two close enough nicks can come into a DSB, it can reduce by-product from excess DSBs (McCaffrey et al., 2016). Additionally, the pgRNA–Cas9 system, which uses pgRNA to guide wild Cas9 nuclease by pgRNA, can specifically cut-off a fragment from targeted DNA. At present, pgRNA–Cas9 system has been also applied in lncRNAs (Zhu et al., 2016). So, the pgRNA–Cas9 system was powerful and useful in the genomics edit.

Megakaryoblastic leukemia 1 (MKL1) expressed in almost all adult tissues, as a member of myocardin/MKL family, which can recruit serum response factor (SRF) to control targeted genes expression. Importantly, MKL1 can shuttle to various subcellular locations (Cen, Selvaraj, & Prywes, 2004). The activation of RhoA can facilitate release of MKL1 from G-actin-MKL1 complex and promotes it to enter the nucleus (Smith et al., 2013). It is reported that MKL1 is involved in cell proliferation, migration and differentiation (Scharenberg, Chiquet-Ehrismann, & Asparuhova, 2010). However, it is still controversial about the effect of MKL1 on cell proliferation. MKL1 promotes MIG6 (Descot et al., 2009) and p21 (Yang, Liu, Xu, & Yang, 2015) expression to induce cell apoptosis and inhibit cell growth. What is more, MKL1 induced the expression of Tenascin-C (Asparuhova, Ferralli, Chiquet, & Chiquet-Ehrismann, 2011) and CCN1 (Emre & Imhof, 2014) to facilitate cell migration and proliferation.

Tumor suppressor factors (p53, p21, pRB and DLC1 [deleted in liver cancer 1]) could affect cell cycle. An aberrant expression of p53 occurs in more than 50% of malignant tumors, it controls cell division and apoptosis (Meek, 2015). The p21, as a CDK inhibitor, can monitor the transformation from G1 phase to S phase. And, the expression of p21 was increased when DNA was damaged (Cazzalini, Scovassi, Savio, Stivala, & Prosperi, 2010). As a biological marker of cell proliferation (Schaal, Pillai, & Chellappan, 2014), pRB was first discovered in retinoblastoma (Hutcheson, Witkiewicz, & Knudsen, 2015) and regulated the proliferation of various cells by inhibiting progression of cell cycle, and it was considered. DLC1 was deleted or down-regulated in a variety of solid cancers and induces the activation of RHO family protein (RhoA, Cdc42, Rac) to inhibit cell growth (Wong et al., 2008).

For investigating the functions of MKL1, RNA interference (RNAi) technology was usually used to inhibit the protein expression in past. However, the problems are ineligible, which resulted in the disorder of all mRNA level, and the efficiency is short and temporary (Haussecker, 2014; Unniyampurath, Pilankatta, & Krishnan, 2016). Here, we tried to utilize pgRNA–Cas9 system to knockout MKL1 expression through deleting a fragment of MKL1 transcripts from the genome. Whereafter, the cell phenotype was analyzed using the MKL1-deleted cells lines, such as cells migration, proliferation.

2. Materials and methods

2.1. Cell culture, plasmids construction and sgRNAs design

The human cervical cancer cell line HeLa was purchased from ATCC (CCL2™). Cell was cultured in high-DMEM medium (Hyclone) in humidified air and 5% CO₂ at 37°C, supplemented with 10% FBS, 100 mg/ml streptomycin and 100 U/ml penicillin.

The plasmids CP-C9NU-01 and pCRISPR-SG01 were purchased from Gene Copoeia. CP-C9NU-01 was a Cas9 nuclease expression plasmid, which carried fluorescent protein mCherry and resistance gene Neo. pCRISPR-SG01 was a sgRNA expression vector carried resistance gene Hygro. Four sgRNAs were designed on exon 6 (M2, M3, M4) or exon 7 (M1), respectively. The detail sequences were listed as follow:

M1 5′-GGCCAAGGAGCTGAAGCCAAAGG-3′;

M2 5′-GGTTGTGTCTCAACTTCCGATGG-3′;

M3 5′-GGATAGTGGTTCCATTGGTGAGG-3′;

M4 5′-GGACTTGGCAGTGGGGATAGTGG-3′.

Plasmid was purified using the classic protocol of the rapid alkaline extraction procedure (Feliciello & Chinali, 1993). DNA concentration was measured by spectrophotometer.

2.2. Transfection and cell selection

The most appropriate concentration was confirmed by drug gradient screening. According to previous study, G418 (Biosharp) designed to 0, 200, 400, 600, 800 and 1000 μg/ml and Hygromycin B (MDBio, Inc) arranged to 0, 100, 200, 300 and 400 μg/ml were respectively added into wells cultured confluence 80% HeLa cells in 6-well plates. Under the pressure of drugs for 14 days, the minimum concentration which made cells completely die was the suitable one. Therefore, 800 μg/ml G418 was used to select after transfected plasmid CP-C9NU-01 in HeLa cells. And 300 μg/ml Hygromycin B was applied to separate cells co-transfected pgRNAs (data were no shown).

When the cell confluence was 80%, 2 μg DNA was transfected by the polyethylenimine (1 mg/ml, Polysciences) in 6-well plate. After 24 h, cell lines were selected using a rightly antibiotic for 14 days.

2.3. Western blotting

Proteins were extracted from HeLa cells by Western and IP Lysis Buffer (Beyotime) with 1% protease inhibitor cocktail (Thermo). The extraction was separated by 12% SDS-PAGE gel and transferred to PVDF transfer membrane (Millipore, Bedford) by electro-blotting. Blocking with 5% nonfat-milk, the membrane was incubated overnight at 4°C with a 1:1,000 dilution of primary antibodies GAPDH or MKL1 (Santa Cruze). Then, the HRP-conjugated second antibody (Goat anti-Mouse, Thermo) was used to incubation for 1 h at room temperature by a 1:2,000 dilution. Immunoblots were scanned using an Image Scanner (GE healthcare). Blot densitometry analysis was performed using ImageJ (National Institutes of Health).

2.4. Wound healing assay

For detecting the ability of cell migration, stably transfected HeLa cells grown to 70% confluence in 6-well plate were wounded using a 1-mm tip and then wash the cells with PBS to remove the debris. Cells were observed and photographed at 0, 24, 48 h after wounding. Cells migrating into wound surface and the average distance of migrating cells were determined under an inverted microscope at designated time points. The average distance cells migrated was measured using ImageJ.

2.5. MTT assay

To evaluate cell proliferation ability, 1 × 10³ cells per well were seeded in 96-well plates with complete medium and were determined per 24 h lasting for 7 days. First, 20 μl of MTT (5 μg/ml, Sigma) was added to each well, and cells continued to be incubated at 5% CO₂, 37°C for 4 h. Next, 100 μl of DMSO dissolved the formazan in cells at room temperature for 10 min. Then, the cell numbers were estimated by measuring the optical density at 570 nm with ChemiDOC™ XRS+ System (BIO-RAD). The absorbance of cell-free wells containing medium was set as a 0.

2.6. RNA analyses by qRT-PCR

Total RNA from MKL1-deleted cells was isolated using the Trizol Reagent (Invitrogen) according to a manufacturer’s protocol. The RNA quantitative analysis was determined by spectrophotometer and agarose gel electrophoresis. Then, RNA reverse transcription used the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa). The qRT-PCR with SYBR Green Mix (Thermo) was performed in CFX96™ Real-Time System (BIO-RAD) using a standard cycling profile. Each sample was analyzed in triplicate, and the GAPDH gene as an endogenous control was used to normalize the expression of targeted genes. Melting curve was detected to verify PCR specificity and the absence of primer dimers. All samples were run in triplicate. Data were analyzed by the ∆∆Ct method. The primers were as follows:

GAPDH-forward 5′-CGTGGAAGGACTCATGAC-3′;

GAPDH-reverse 5′-CAAATTCGTTGTCATACCAG-3′;

DLC1-forward 5′-CAGGCTGAGCTGTCCTATAAG-3′;

DLC1-reverse 5′-CTTTAAGATTTCCTCTGGCAC-3′;

p53-forward 5′-CCCTCCTCAGCATCTTATCCG-3′;

p53-reverse 5′-GGTGGTACAGTCAGAGCCAACCT-3′;

p21-forward 5′-TGTCTTGTACCCTTGTGCCTCG-3′;

p21-reverse 5′-AGTGGTAGAAATCTGTCATGCTGGT-3′;

pRB-forward 5′-TCAATTGGTGAATCATTCGGG-3ʹ;

pRB-reverse 5′-TCAGTGGTTTAGGAGGGTTGC-3ʹ.

2.7. Statistical analysis

Statistical analysis was done using SPSS standard version 13.0 software. The independent Student’s t-test was used to compare the continuous variables between two groups. The data were expressed as means ± SD from at least three independent determinations. Values of P < 0.05 (or P < 0.01) were considered statistically significant.

3. Results

3.1. Plasmids constructed

The two components of pgRNA–Cas9 system were expressed by different vectors, respectively. Plasmid CP-C9NU-01 carried fluorescent protein mCherry and resistance gene Neo (Figure 1A). Plasmid pCRISPR-SG01 is a sgRNA expression vector. The MKL1-targeted sequences designed on exon 6 (M2, M3, M4) and exon 7 (M1) were cloned into pCRISPR-SG01 by restriction sites BamHI and XbaI and through construction of pgRNA library pairing two different sgRNAs. Four pgRNAs (M1-3, M1-4, M2-3, M2-4) were formed to delete four different size fragments on MKL1 gene (Figure 1B and C).

Figure 1. The plasmid construction of pgRNA–Cas9 system. (A) Schematic of vector CP-C9NU-01. Labels of mCherry and neo were co-expressed with Cas9 nuclease. (B) Schematic of vector pCRISPR-SG01. Single-guide RNAs were cloned into pCRISPR-SG01 by BamHI and XbaI. (C) Schematic of pgRNAs designed on exon 6 (M2, M3, M4) and exon 7 (M1) of MKL1 gene, and the fragment cut off by pgRNAs.

3.2. MKL1 was significantly decreased by pgRNA–Cas9 system

For MKL1 knockout in HeLa cells, it was important to stably express the Cas9 nuclease in HeLa cells. Plasmid CP-C9NU-01 was transfected into HeLa cells by transfection reagent Lipo 2000. After 24 h, 800 μg/ml G418 was used to select transfected cells. It was observed of red fluorescence by inverted fluorescence microscope, and the cell line was named as HeLa-Cas9 (Figure 2A), which suggested Cas9 nuclease was expressed in HeLa cells. Then, pgRNAs were transfected into HeLa-Cas9, respectively. The monoclonal cell was separated by dilution culture, which made sure only 0 or 1 cell each well, and cultured in 96-well plates. By drug pressure at 800 μg/ml G418 and 300 μg/ml Hygromycin B, MKL-deletion cells were selected. The result showed that the expression level of MKL1 in the cell clones M144 (M1-4, 4), M145 (M1-4, 5) and M146 (M1-4, 6) was obviously decreased using Western blotting (Figure 2B). What is more, MKL1 expression was measured after third generation (Figure 2C) and fifth generation (Figure 2D) to ensure the expression level of MKL1 stably knockout. The result revealed that it was efficient to delete MKL1 by pgRNA–Cas9 system. Subsequently, the cell line M146 defined as HeLa-KO was chosen for the next research.

Figure 2. The Cas9 nuclease expression in HeLa cells and MKL1 knockout analyses by Western blot. (A) Fluorescence analysis in the HeLa cells after transfected CP-C9NU-01. The red fluorescence was from expression of mCherry. (B), (C) and (D) Western blot analysis the expression of MKL1. Figure part (B) is the result of first-generation cells; (C) is of third generation cells; (D) is of fifth-generation cells. HeLa-N.C., normal HeLa cells; HeLa-Cas9, HeLa cells transfected with CP-C9NU-01; M142, pgRNA-M1-4 2; M144, pgRNA-M1-4 4; M241, pgRNA-M2-4 1.

3.3. Depletion of MKL1 inhibits the migration in HeLa cells

For determining the migration activity in HeLa-KO cells, it was detected by scratch wound healing assay. The wound width of group M146 was obviously larger than the control at 24 or 48 h (Figure 3A and B). It suggested that the ability of migration was significantly decreased in MKL1-depletion cells compared with control.

Figure 3. Depletion of MKL1 suppressed the migration in HeLa cells. (A) Micrographs of MKL1-depletion cells at 0, 24, 48 h after wounding. (B) Column graph of wound width at 0, 24, 48 h. N.C., Ca9-expressed HeLa cells without pgRNAs; M146, pgRNA-M1-4 6; *p < 0.05; **p < 0.01.

3.4. Deficiency of MKL1 suppresses the proliferation in HeLa cells

Cells growth was measured by MTT assay. The value of absorbance at 570 nm of formazan dissolved in DMSO indirectly showed that the relative cell growth of HeLa-KO cells was obviously less after cultured third day (Figure 4A and B). It indicated that depletion MKL1 significantly decreased cell proliferation in HeLa cells.

Figure 4. The proliferation of MKL1-depletion cells was declined. (A) Histogram of relative cell quantity of MKL1-depletion cells by MTT assay tested for 7 days. (A) Line chart from (B). N.C., Ca9-expressed HeLa cells without pgRNAs; M146, pgRNA-M1-4 6; *p < 0.05; **p < 0.01.

3.5. Tumor suppressor factors were regulated with the absence of MKL1

It was already confirmed that MKL1 mediated cell growth and migration. However, the precise mechanism of the affection on cell growth and migration regulated by MKL1 is unclear. We tried to investigate the relationship between MKL1 and tumor suppressor (p21, p53, pRB and DLC1). qRT-PCR was used to measured the RNA expression level of these tumor suppressor in MKL1-deletion cells. As shown in Figure 5A, the transcription levels of p21 and pRB were significantly increased 1.5-folds; on the contrary, DLC1 was obviously decreased 80%. The expression level of p53 almost was not altered. Meanwhile, the protein expression level of p21, p53 and DLC1 was significantly decreased in MKL1-depletion cells (Figure 5B). These results suggested that MKL1 played a critical role in biological processes and it may affect the cell proliferation and migration by mediating the expression levels of p21, p53, pRB and DLC1.

Figure 5. Depleted MKL1 resulted in the change of tumor suppressor factors DLC1, p21, p53 and pRB. (A). qRT-PCR analysis the RNA expression level of tumor suppressor factors DLC1, p21, p53 and pRB. (B). Western blotting analysis the tumor suppressor factors expression level in MKL1-depletion cells. *p < 0.05; **p < 0.01.

4. Discussion

CRISPR–Cas9 system is different from RNAi. However, they can silence or knockout the expression level of proteins, RNAi exerts mainly on targeting RNA level and did not alter the genomics sequences and the phenotype could not stably heredity. In addition, the pgRNA–Cas9 system was widely used in various filed, and more flexible and specificity (Unniyampurath et al., 2016). Here, the pgRNA–Cas9 system which was used to delete MKL1 in HeLa cells is a new attempt. In the study, the pgRNA consisted of two sgRNA which were respectively promoted by two U6 promoters for better expression efficiency (Zhu et al., 2016). Through constructing pgRNA library of MKL1 gene, it was selected out for MKL1-deleted HeLa cell line HeLa-KO.

MKL1 is an important regulator, which involved in the differentiation (Nobusue et al., 2014), migration (Xing et al., 2015) and proliferation (Gurbuz, Ferralli, Roloff, Chiquet-Ehrismann, & Asparuhova, 2014). MKL1 binding with G-actin in cytoplasm is released by RhoA activated and then enters the nucleus where MKL1 recruits various proteins (co-activator) to target downstream gene (Bell et al., 2013). It is reported that MKL1 can increase cell migration by activating MMP9 transcription (Cheng et al., 2015), interacting with Filamin A to activate SRF (Kircher et al., 2015) and synergistic effects with STAT3 (Liao et al., 2014).

However, there is a dispute for MKL1 on cell proliferation. Previously, research reported that MKL1 promoted the expression of Tenascin-C (Berns et al., 2016) and CCN1 (Duggirala et al., 2015) to contribute to cell growth, and MKL1-depletion induced the expression of p16 and hypophosphorylation of pRB in DLC1-deficient HCC cells (Hampl et al., 2013). On the other hand, it was reckoned that MKL1 inhibited cells proliferation by arresting cell cycle via up-regulation of the expression level of p21 (Yang et al., 2015) and induced cell apoptosis through activating MIG6 (Descot et al., 2009; Hopkins et al., 2012).

Therefore, to confirm the function of MKL1 in HeLa cells, we used MKL1-delection HeLa cells to perform MTT assay and wound healing assay. The results showed cell proliferation and migration both are significantly suppressed in MKL1-depletion cells. In addition, the protein expression level of p21 was obviously decreased. It indicated that MKL1 may mediate cell growth via regulating p21 expression at least in partly.

Among all transcription changes of tumor suppressor factors, it is interesting that DLC1 was significantly decreased. Meanwhile, the ability of cell migration and proliferation was obviously decreased. So, it is hypothesis that there would be a negative feedback existing between MKL1 and DLC1. DLC1 was declined with depletion of MKL1. It was suggested that MKL1 could up-regulate DLC1 expression. However, as a Rho-GAP protein, DLC1 could decrease the RhoA activity and then blocked the nuclear localization of MKL1 (Wong et al., 2008), which down-regulated the expression of MKL1-depended genes again, including DLC1 gene. So, a negative feedback loop maybe exist between MKL1 and DLC1.

In conclusion, it is efficient to knockout MKL1 using pgRNA–Cas9 system. MKL1 mediated cell migration and proliferation via changing the expression of p21 and pRB in HeLa cells. Additionally, DLC1 was obviously decreased in MKL1-depletion cells which indicates that DLC1 may be a potential targeted gene of MKL1. However, the further investigation is required to confirm.

Competing interests

The authors declare no competing interests.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China: [Grant Number 31500622], the program of Wuhan University of Science and Technology: [Grant Number 25011401] and the Science and Technology Research Project of Hubei Province: [Grant Number B2016004].

Notes on contributors

Hong-Bo Liu

Hong-bo Liu is a master of College of life science and health, Wuhan University of Science and Technology. He focused on the development of CRISPR system and devoted to improve the knockout efficiency of CRISPR/Cas9 technology.

Peng Zheng

Peng Zheng, PhD of Institute of hydrobiology, Chinese Academy of Sciences, is interested in the network of non-coding RNA and protein interaction and studies on the molecular mechanism of long non-coding RNA HOTAIR regulating cervical cancer metastasis.

Tong-Cun Zhang

Tong-cun Zhang is the Dean of College of life science and health, Wuhan University of Science and Technology. He dedicated himself in the function and mechanism investigations of Myocardin family related transcription factor (MRTFs) in the pathophysiology of cardiovascular diseases and in the differentiation of stem cells and tumor cells.