LMNA functions as an oncogene in hepatocellular carcinoma by regulating the proliferation and migration ability

Abstract The role of the LMNA gene in the development and progression of hepatocellular carcinoma (HCC) and the associated molecular mechanism is not yet clear. Therefore, the purpose of this study was to evaluate the relationship between LMNA and HCC. LMNA gene expression in normal tissues and corresponding tumours was evaluated and the Kaplan–Meier survival analysis was performed. Next, the LMNA gene was knocked out in the 293T and HepG2 cell lines using the CRISPR/Cas9 technique. Subsequently, the proliferation, migration and colony formation rate of the two LMNA knockout cell lines were analysed. Finally, the molecular mechanism affecting the tumorigenesis due to the loss of the LMNA gene was evaluated. The results showed that the LMNA gene was abnormally expressed in many tumours, and the survival rate of the HCC patients with a high expression of the LMNA gene was significantly reduced compared with the rate in patients with a low LMNA expression. The knockout of the LMNA gene in the HCC cell line HepG2 resulted in a decreased tumorigenicity, up‐regulation of the P16 expression and down‐regulation of the CDK1 expression. These findings suggested that LMNA might function as an oncogene in HCC and provided a potential new target for the diagnosis and treatment of HCC.

primary liver cancer. In the early stage of liver cancer, the clinical symptoms of the patients are not evident, although the disease develops quite rapidly. Generally, a correct diagnosis is reached when the liver cancer is in its late stage, thus becoming a greater threat to the life and health of patients, and resulting in a higher probability of death. 4,5 Due to its complexity, recurrence, metastasis and heterogeneity after surgical resection, HCC is one of the most deadly cancers even after surgical resection. 6 Like other cancers, HCC is also characterized by an abnormal gene expression. The LMNA gene encodes the two main isoforms lamin A and lamin C. Lamins are structural proteins forming the nuclear lamina, which is the inner nuclear membrane determining the nuclear shape and size. Three types of lamins have been previously described in mammal cells, such as A, B and C. 7 Lamin B1 and vimentin were the main overexpressed proteins in liver cancer tissues. 8 Thus, Lamin B1 and vimentin in the blood could be used as novel biomarkers for early HCC and can be detected by non-invasive methods. 9 Lamin A expression varies in a variety of tumour cells.
Its expression decreases in breast, prostate, colon, ovarian, gastric and endometrial cancer, leading to a reduction in overall survival and an increase in the number of metastatic sites and tumour recurrence. [10][11][12][13] In contrast, some studies revealed a link between increased lamin A expression and the development and progression of colorectal cancer, prostate cancer and ovarian cancer. [14][15][16] However, the role of the LMNA gene in the development and progression of HCC and the associated molecular mechanism remains unknown.
Therefore, the purpose of this study was to evaluate the relationship between LMNA and HCC. The LMNA gene was knocked out in 293T and HepG2 cell lines by the CRISPR/Cas9 technology.
Subsequently, the proliferation, migration and colony formation rate of the two LMNA knockout cell lines were analysed, and the tumorigenicity in vivo was tested in a subcutaneous tumour mouse model. Finally, the molecular mechanism affecting the tumorigenesis due to the loss of the LMNA gene in the 293T and HepG2 cells was explored.

| Ethical guidelines
All the experiments in this study were performed according to the guidelines of the Experimentation Ethics Committee of the Guilin Medical University, China (approval No. 2019-0008).

| Bioinformatic
Data related to the LMNA gene expression in various normal tissues and corresponding tumours were collected from the Proteomics DB, Max QB and MOPED databases. The Kaplan-Meier survival analysis was performed by the Kaplan-Meier plot (http://kmplot.com/ analysis).

| Cell lines and culture conditions
The human liver cancer cell line (HepG2) and HEK 293 kidney cell line expressing a mutant version of the SV40 large T antigen (293T) were stored in our laboratory. Cells were routinely cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, CA, USA) supplemented with 10% foetal calf serum (Gibco, CA, USA) and incubated at 37°C in a 5% CO 2 atmosphere. They were cultured until reaching 50% to 80% confluence before the next passage or further experiments.

| gRNA design
The gRNA was designed using the Massachusetts Institute of

| CRISPER/Cas9 technique
The resultant gRNA justice chain and antisense chain were renatured to form a double chain. The reaction system was the following: 1 μl of each positive and antisense chain, T4 linkase buffer (10×) 1 μl l, T4 polynucleotide kinase (PNK) 0.5 μl, double distilled water 6.5 μl, 95°C for 5 minutes, and left to cool down to 16°C for 10 minutes.
Then, BbsI endonuclease was used to shear PX459 at 4°C overnight, and the plasmid, after shearing, was recovered by electrophoresis.
The oligonucleotide double chain and the post-shear plasmid were recombined using T4 ligase. The reaction system was as follows: PX459 plasmid 50 ng, oligonucleotide double chain 2 μl, T4 connectase 1.5 μl, T4 connectase buffer solution 1.5 μl, double evaporation of the water until reaching 15 μl. The reaction procedure was as follows: after combining the oligonucleotide double chain and the T4 connectase at 16°C for 10 minutes, the reaction system was left overnight at 4°C. The recombined carrier suspension product of 5 ~ 10 μl was added to the 50 μl DH5a sensor cells, which were lightly blended and bathed in ice for 30 minutes, heated at 42°C for 90 seconds, left on ice for 2 minutes, and then directly coated on an ampicillin resistant LB plate. The reaction system was incubated overnight at 37°C, 3 ~ 5 white bacterial colonies were selected for culture, and plasmid DNA was extracted for sequencing verification and amplification. The amplified plasmid DNA was collected and stored at −20°C. The sequencing primers were the following: Two million cells were added to each well in a 6-well plate and washed with PBS twice after 12 hours. The Lipofectamine 3000 Transfection reagent (10 μl, Invitrogen, USA) was mixed with 250 μl bovine free media, and left at room temperature for 5 minutes.
Meanwhile, 5 μl recombinant plasmid (or exogenous LMNA expressing plasmid) was mixed with 250 μl bovine free media, and left at room temperature for 5 minutes. Then, the two liquids were thoroughly mixed and left at room temperature for 20 minutes. Finally, the mixed liquid was transferred into a culture plate. The complete culture medium was changed at 6-8 hours after transfection, fresh complete culture medium was replaced at 24 hours after transfection, and puromycin was added for screening (working concentration of 293T was 2.0 μg/mL, and that of HepG2 was 1.5 μg/mL). The screened cells were cultured for subsequent experiments.

| Immunofluorescence
The cells were immersed in PBS and seeded in glass slides at a concentration of 1 × 10 4 /ml. The slides were fixed in 4% paraformalde-

| Western blot
RIPA lysis buffer (plus PMSF) was used to lyse the cells to extract the total proteins, which were separated by a polyacrylamide gel electrophoresis and transferred into the PVDF membrane. After membrane blocking, the corresponding primary antibodies (1:1000, rabbit anti-human, anti-Lamin A/Lamin C/β-actin, Abcam, USA, and anti-P16/CDK1/MMP2/MMP9, BOSTER, China) were added to the membrane that was incubated overnight at 4°C. Next, the secondary antibody (1:8000, mouse anti-rabbit antibody, Abcam, USA) was added to the membrane that was incubated at room temperature for 1 hour. Finally, the bands were imaged using Gel imaging system.

| CCK-8 cell growth assay
The 293T and HepG2 wild-type and knockout cell lines were trypsinized, seeded into 96-well plates at a concentration of 1500 cells per well in triplicate, and cultured at 37°C. Cell counting kit-8 (KeyGen, China) was added after 18 hours and incubated for 1 hour. The absorbance was measured at 450 nm every other day until one of the cells reached the confluence of more than 70% to stop the cell proliferation experiment.

| Cell cycle detection
The cells in the logarithmic growth phase were seeded in a 6-well plate at a density of 1 × 10 6 cells/mL in 2 mL medium and in a 24-well plate in 1 mL medium, and the cells were collected after 24 hours. After centrifugation at 800 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was collected, washed twice with pre-cooled PBS, and with pre-cooled with 75% ethanol. Next, the cells were fixed at 4°C for more than 4 hours. After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded, the cells were washed once with 3 mL PBS, and finally 400 uL ethidium bromide (PI, 50 µg/mL), and 100 µl RNase A (100 µg/mL) (KeyGen, China) were added, and the cells were and incubated at 4°C for 30 minutes in the dark. Flow cytometry was used to detect 20 000-30 000 cells by a standard procedure, and the results were analysed by the cell cycle FACS software.

| Apoptosis detection
The cells were collected, washed once with PBS, centrifuged at 1000 r/ min for 5 minutes, and the supernatant was discarded. According to the kit instructions, each sample (293T-WT, 293T-KO, HepG2-WT, HepG2-KO) was resuspended in 100 μL binding buffer and then Annexin V-FITC (5 μL/tube) and PI staining solution (5 μL/tube) (KeyGen, China) were added and incubated for 10 minutes in the dark.
Next, 400 μL binding buffer was added to each tube, tubes were gently mixed, and flow cytometry analysis was performed within 1 hours.

| Wound closure assay
The cells were grown in 6-well plates until reaching 80-90% confluence. A wound was made using a plastic pipette tip across the cell surface. The remaining cells were washed three times to remove any cell debris and incubated at 37°C with serum-free DMEM. Six different areas of the wound per well were photographed at 0, 12, 24 and 36 hours and the migrating cells were compared. The cell migration distance was determined by measuring the width of the wound and subtracting half of this value from the initial half-width value of the wound. Each experiment was performed in triplicate and three separate experiments were performed.

| Cell transmigration assay
The transmigration assay was conducted in 24-well transwell cham- for each sample (as described above in Apoptosis detection).

| Plate clone formation
The cells were digested with 0.25% until obtaining single cells and the cell suspension was diluted at a concentration of 1 × 10 4 cells per mL. Fifteen hundred cells in their medium were added to each well of a six-well plate and incubated at 37°C under 5% CO 2 . When the clones could be distinguished by naked eye in the six-well plate, the cell culture stopped, and cells were fixed in 4% paraformaldehyde for 15 minutes. The crystal violet staining was performed and the number of clones of more than 50 cells was counted under the optical microscope. The colony formation rate was calculated as follows: (average number of clones/ number of inoculated cells) × 100%.

| Soft agar cloning
Cells were digested, centrifuged, counted and diluted to 1 × 10 4 cells/mL. Five hundred μL of the agar and 500 μL of the single-cell suspension was added to each well of a 6-well plate, and thoroughly mixed. The bottom of the 6-well plate was covered with a 1.2% agarose layer solidified at room temperature to form a double agar layer.
The incubation was performed for 4 weeks at 37°C under 5% CO 2 with 100 μl complete medium added at intervals to prevent drying.
The ell clone formation rate was calculated as follows: Cell clone formation rate = (number of cell clone formation/ number of inoculated cells) × 100%. ured with a calliper, and the volumes were calculated using the equation (length × width 2)/ 2. Thirty-one days after inoculation, mice were sacrificed and the tumours were excised and subjected to pathological examination. An analytical balance was used to measure the tumour weight, and a Vernier calliper to measure the long diameter as the length and the short diameter as the width, and then the formula (length × width 2)/ 2 was used to calculate the tumour volume.

| LMNA gene expression in different tumours from HCC patients and different cancer cells
To explore the specific changes in the expression of the LMNA gene in various tumours, a comparative analysis was performed between normal tissues and corresponding tumours as well as on different types of cancer cells using data obtained from the Proteomics DB, Max QB and MOPED databases. The data revealed that LMNA expression was significantly up-regulated in brain cancer cells (U251), bone cancer cells (U2OS), kidney cells (293T) and liver cancer cells (HepG2) ( Figure 1A). The Kaplan-Meier curve of patients with HCC showed a lower survival rate in patients with high LMNA expression ( Figure 1B).

| LMNA knockout cell lines from 293T and HepG2 by CRISPR/Cas9 technology
To further study the function of the LMNA gene, LMNA knockout cell lines were acquired by the CRISPR/Cas9 technology ( Figure 2A). Immunofluorescence and western blot were performed to verify whether the LMNA gene was successfully knocked out. The immunofluorescence results revealed that both cell lines did not express the LMNA protein, while the LMNB protein expression was unaffected ( Figure 2B). The WB results confirmed this result ( Figure 2C). Besides, the LMNC protein which is translated by the same LMNA gene was also down-regulated ( Figure 2D).

| Knockout of the LMNA gene in 293T and HepG2 cells led to a decreased cell migration and colony formation, and improved the transmigration ability
After the function tests, the tumorigenicity of the LMNA knockout cell lines was evaluated in vitro. The wound closure assay showed that the healing ability of both LMNA knockout cell lines was significantly decreased ( Figure 4A). In contrast, the transwell migration tests showed that the transmigration ability of both LMNA knockout cell lines was enhanced ( Figure 4B). Furthermore, the colony formation ability of each group of cells showed that the colony formation rate ( Figure 4C) and the average size of the clones ( Figure 4D) of the two LMNA knockout cell lines decreased

| WB analysis indicated that the ECM and cancer signalling pathway was changed after LMNA knockout
After concluding that the LMNA gene knockout resulted in a decrease in the tumorigenic capacity of tumour cells, the relevant molecular mechanism was investigated. RNA-seq analysis of the LMNA knockout cell lines and wild-type cells, which were obtained by the CRISPR/Cas9 technique described above, was performed, and the results showed that two different batches of four cells could be clustered ( Figure 6A). In addition, the GO enrichment map ( Figure 6B) and KEGG pathway ( Figure 6C) enrichment map showed the presence of many differentially expressed genes and regulatory pathways in LMNA knockout cells and wild-type cells. The expression of MMP2 and MMP9 in the ECM signalling pathway was then evaluated by WB, and the result showed that the expression of MMP2 and MMP9 was decreased (P < 0.01) in the two LMNA knockout cell lines ( Figure 6D), which was consistent with the RNAseq results.

| D ISCUSS I ON
The role of LMNA gene in tumours, in the development and progression of HCC and its molecular mechanism is still a challenge. In the current study, the relationship between LMNA and HCC was evalu- In this study, our hypothesis was that LMNA might play an oncogene role in HCC since HCC patients with higher LMNA expression showed a lower survival rate according to the Kaplan-Meier curve.
It is well known that the most important pathological type of HCC is the primary liver cancer, which accounts for approximately 90%. 17,18 LMNB1 expression (lamin B) is significantly up-regulated in HCC patients, thus, its expression may be used as a prognostic indicator in patients at an early-and late-stage HCC. 19 Lamin A, a nuclear lamina structural protein like lamin B, is critical for the stabilization of retinoblastoma tumour suppressor proteins pRb and p107. [20][21][22] These discoveries suggest that Lamin A/B might be closely related to the tumorigenesis.
In this work, LMNA protein expression in HepG2, and cells was significantly up-regulated suggesting that the LMNA gene might be relate to the malignant degree of tumour cells. In addition, the proliferation ability of HepG2 cells decreased after LMNA knockout and the cell cycle was arrested. Previous studies showed that the knock down of lamin A/C in human lung cancer cell lines leads to an increased tumour growth rate in vivo. 21,23 However, the knock down of lamin A/C in human primary diploid fibroblasts leads to G1 arrest and inhibits cell proliferation. 24 Thus, our conclusion was that the knockout of the LMNA gene in different cells has a different effect on cell proliferation and cell cycle, thus potentially explaining the different role of LMNA in different tumours.
In this study, we also found that P16 expression increased after knockout of the LMNA in HepG2 cells. P16 expression significantly decreased after the overexpression of LMNA, indicating that the LMNA gene could regulate the expression of P16.

F I G U R E 4
Influence of LMNA knockout on 293T and HepG2 cell migration, transmigration and ability of clone formation. Knockout of LMNA gene in 293T and HepG2 cells leads to (A) a decreased wound closure ability, and (B) increased cell transmigration ability (C) decreased formation of soft agar clones and (D) decreased platelet colony formation. Soft agar colony formation of the two LMNA knockout cells resulting in a decrease in the cloning rate and the size of the clone. Results were expressed as mean ± SD of triplicates (**P < .01) F I G U R E 5 Effect of LMNA knockout on the tumorigenicity in a nude mouse xenograft model. A, Tumorigenic analysis of 293T WT and knockout cells (2 × 10 6 cells for each group) in nude mice (n = 6) for 30 days. B, Analysis of the tumour formed by HepG2 WT and knockout cells (2 × 10 6 cells for each group) in nude mice (n = 6) for 30 days. C, Volume of the subcutaneous tumours by wild-type and LMNA knockout cell lines in nude mice (293T group *P < .05). D, Weight of the subcutaneous tumours by wild-type and LMNA knockout cell lines in nude mice. Results were expressed as mean ± SD of triplicates (*P < .05, **P < .01) F I G U R E 6 Differentially expressed genes in the LMNA knockout cell lines and their correspondent wild-type by RNA-seq. A, Heat map of 8 samples; the up-regulated genes are shown in red colour, and the down-regulated genes are shown in blue colour, the deeper the colour, the bigger the expression difference. The scale was by log 10 . B, GO analysis of the differential gene sets in the wild-type and LMNA knockout cell lines (WT vs KO). C, KEGG pathway analysis of differential gene sets in the wild-type and LMNA knockout cell lines (WT vs KO). D, Western blot results of MMP2/9 protein expression. Results were expressed as mean ± SD of triplicates (**P < . tissues. [33][34][35] The two LMNA knockout cell lines showed enhanced transmigration ability. The decrease in lamins potentiates cancer cell migration through narrow spaces, suggesting a potential role in metastasis. 23,[36][37][38] In our study, the migration ability of the two LMNA knockout cell lines was significantly lower than that of the wild-type cells, which is consistent with previous findings 23,36,38 .
On the contrary, the transwell migration ability increased in knockout cell lines. Our hypothesis was that the loss of lamin A/ C might result in a thinner nuclear membrane and an easier ability of deformation to pass through narrow gaps; furthermore, the loss of

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R S CO NTR I B UTI O N S
Heng Liu: Data curation (lead); Formal analysis (lead); Investigation

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Liping Wang
https://orcid.org/0000-0001-6372-5939 Ming Chen https://orcid.org/0000-0001-5935-7694 F I G U R E 7 Model of LMNA gene regulating the migration and proliferation of HCC cells HepG2. LMNA binding to the chromosome resulted in the downregulation of P16 and MMP2/9, then the collagen degradation was inhibited and the expression of CDK1 was up-regulated. Finally, cell proliferation was promoted and cell invasion was suppressed