Effect of lentiviral expression of circLIFR on the biological behavior of Hep3B hepatocellular carcinoma cells

LI Chen, LIU Lu-zheng, LU Han-yuan, CHEN Jia-cheng, CHEN Liang, WU Jin-cai,?

1.Clinical College of Hainan Medical University, Haikou 571199, China

25.7 D03e1p1a,r tCmheinnta of Hepatobiliary and Pancreatic Surgery, Hainan Affiliated Hospital of Hainan Medical University (Hainan General Hospital), Haikou

Keywords:

ABSTRACT Objective: To investigate the effect of lentiviral stable high expression of circLIFR on the biological behavior of Hep3B hepatocellular carcinoma (HCC) cells.Methods: Hep3B cell lines were infected with lentiviral packaging of circLIFR expression plasmids to construct stable circLIFR high expression HCC cells.The lentivirus infected with circLIFR high expression sequence was used as the circLIFR high expression group, the lentivirus infected with circLIFR high expression empty vector sequence was used as the negative control group,and the uninfected group was used as the blank control group.After that, circLIFR expression levels were detected by qPCR, and the back splice sites were identified by Sanger sequencing.The cell viability was examined by cell proliferation kit and invasive ability was determined by Transwell assay. Results: The qPCR and Sanger sequencing showed that the stable circLIFR expression of Hep3B cells was successfully established.The circLIFR high expression group had better cell proliferation viability than the negative control and blank control groups, and the differences were statistically significant (P ＜0.05).The number of cells crossing Matrigel gel in the negative control and blank control groups was (270.8±18.9) and (266.2±17.6), respectively,while the number of cells crossing Matrigel gel in the circLIFR high expression group was(396.6±32.9), and the differences were statistically significant (P＜0.05).Conclusion: High circLIFR expression considerably promotes the proliferation and invasive ability of Hep3B cells.

1.Introduction

Hepatocellular carcinoma (HCC) ranks sixth in incidence and third in mortality among common cancers worldwide, seriously endangering human health[1].Chronic hepatitis viral infections including hepatitis B virus (HBV), hepatitis C virus (HCV), longterm heavy alcohol intake, and aflatoxin are the main causative agents of HCC[2].Abnormalities such as persistent chronic inflammation, diffuse fibrosis, and abnormal liver proliferation can all evolve progressively into cirrhosis, which in combination with genetic or genetic regulatory abnormalities, develops into dysplastic liver nodules[2].Although currently, surgery-based combination therapy is the main treatment modality for HCC, patients still have a poor prognosis, high 5-year recurrence and metastasis rates, and a lack of effective molecular markers[3].Therefore, it is crucial to identify the key targets of tumor occurrence to improve early diagnosis of HCC or to develop new effective adjuvant therapies for mid to late-stage patients to delay tumor recurrence and metastasis to further improve the treatment outcome of HCC.

Circular RNAs (circRNAs) represent special circular RNA molecules generated by reverse splicing of the head and the tail during gene transcription.These genes were initially thought to be accidental "junk sequences", but recent studies have shown that they can be involved in tumor invasion and metastasis by acting as a sponge for microRNAs (miRNAs) and RNA binding proteins(RBPs)[4-5].It has been reported that m6A-modified circPOLR2A can interact with UBE3C and PEBP1 proteins to form a ternary complex and degrade PEBP1 protein via the ubiquitination pathway,thereby inhibiting Raf1/ERK pathway activation and participating in the progression of renal clear cell carcinoma[6].The circRNA EIF4G3 exerts oncogenic effects through a dual mechanism of promoting δ-catenin degradation and miR-4449/SIK1 axis, thereby curbing the proliferation and metastasis of gastric cancer (GC)cells[7].In HCC, silencing circCCNB1 can act as a miR-106b-5p sponge to promote tumor malignant process through DYNC1I1/AKT/ERK signaling pathway[8].Therefore, the study of circRNA supports the provision of a promising prognostic biomarker and molecular target for HCC.

Our previous study found that circLIFR (hsa_circ_0072309)showed low expression in HCC tissues.In this study, Hep3B stable strain with circLIFR overexpression was constructed by lentivirus infection.The cell proliferation kit and transwell assay were used to detect the effect of circLIFR high expression on the biological behavior of Hep3B cells.

2.Materials and methods

2.1 Experimental materials

Hep3B cells were purchased from the Institute of Cell Science,Shanghai Chinese Academy of Sciences, puromycin was purchased from Sigma company, and TAKARA reverse transcription kit was purchased from Beijing Baori Medical Co., LTD.GeneJET Gel purification kit was purchased from Thermo Fisher Co., LTD.,primers were synthesized by Shanghai Shengon Co., LTD.The cell counting kit (CCK-8 method) was purchased from Shanghai Heyuan Biotechnology Co., LTD.Transwell chamber was purchased from Corning Co., USA.All cells were cultured in eagle's complete medium (containing 10% fetal bovine serum) at 37 ℃ according to the instructions.

2.2 Construction of stable cell lines

First, the target virus circLIFR and control lentivirus (empty vector)were constructed.Hep3B cells were infected with the virus for 72 h, and a fresh medium (containing 1 μg/mL puromycin) was added every 3 d.Hep3B cells with stable and high expression of circLIFR(high expression group) and empty vector (negative control group)were finally screened and obtained, while the blank control group did not receive any treatment.About two weeks after the drug screening, photographs were taken under a fluorescence microscope to observe the effects of infection.

2.3 Quantitative real-time polymerase chain reaction (qPCR)

The total RNA was extracted according to the Trizol method,and the reaction system was prepared according to the TAKARA reverse transcription specification.After mixing, the reaction was centrifuged to complete the reverse transcription reaction.The qPCR reaction reagents were prepared according to the proportion of primers (primer information: circLIFR primer forward sequence:5'-ACCTAAAGTGGAACGACAGGG-3',reverse sequence:5'-GCAGTCAGTCTAATTTTACGAGC-3'; GAPDH primer forward sequence: 5'-GTCTTCACCACCATGGAGAA-3', reverse primer sequence: 5'-TAAGCAGTTGGTGGTGCAG-3') were mixed and tested on the machine.The reaction time was successively 35 s at 95℃ and 34 s at 60 ℃, a total of 40 cycles.

2.4 Sanger sequencing

The constructed circLIFR plasmid was amplified by PCR.After electrophoresis, the product was excised and recovered from the agarose gel.The DNA was purified according to the instructions of GeneJET Gel, and the purified DNA fragment was subjected to sanger sequencing according to the operation method to identify nucleosides.acid sequence.

2.5 Cell proliferation detection (CCK-8 method)

The cells of the blank control group, circLIFR high expression group, and negative control group were prepared into single-cell suspension with a concentration of 4×104cells/mL, and the number of cells in each well of the 96-well plate was about 4×103, with 3 replicates in each group.An appropriate amount of CCK-8 reagent was added at different periods (24 h intervals) after cell culture.After incubation at 37 ℃ for 2 h, the cells were moved one by one to the well plate of the microplate reader, and the absorbance value at 450 nm was read on the machine.

2.6 Transwell invasion assay

The samples were adjusted to a concentration of 5×105cells/mL using a 100 μL serum-free medium, and an appropriate amount of cells were placed into the upper chamber.Using a pipette gun, 500 μL of fresh medium was placed in each chamber and incubated in an incubator at 37 ℃.After 48 h, 500 μL of 4% paraformaldehyde reagent was added to each chamber and fixed at room temperature for 15 min.Finally, the crystal violet reagent was soaked for 30 min and washed 3-5 times with deionized water.Under the microscope(×100), check the number of cells in every 9 random fields and calculate the mean value.

2.7 Statistical processing

Graphpad Prism 8 was used to collate data and plot.Normally distributed measurement data were expressed as mean ± standard deviation (±s ).One-way analysis of variance was used for comparison between multiple groups, and LSD-T test was used for pairwise comparison.P＜0.05 indicates statistical significance.

3.Results

3.1 Construction of circLIFR high expression series HCC stable strains

At 14 days of lentiviral infection with puromycin screening of stable strains of Hep3B series, we observed under a fluorescence microscope that the cell fluorescence expression rate was over 90%in all groups (Figure 1A), suggesting that the lentivirus reached effective infection in all groups of Hep3B cells.Further, we applied qPCR to detect the expression of circLIFR in the cells of each group, and the results suggested that the expression of circLIFR in the cells of circLIFR high expression group was significantly higher than that in the negative control group and blank control group (F=4528, P＜0.001; where negative control group vs.blank control group, t =2.229, P=0.090; blank control group vs.circLIFR high expression group, t=67.29, P＜0.001; negative control vs.circLIFR high expression group, t=67.29, P＜0.001; n=3) (Figure 1B), Sanger sequencing to identify the circular splice sites of circLIFR (Figure 1C), and finally successfully constructed the high expression circLIFR series HCC cell lines.

3.2 Results of cell proliferation detection

Based on the stable construct of circLIFR high expression series of HCC cells, we applied CCK-8 cell proliferation kit to detect the effect of circLIFR on the change of value-added viability of Hep3B cells.The results showed that the overall proliferation viability of the cell lines in the blank control group, circLIFR high expression group and negative control group were significantly different(P＜0.05).CircLIFR high expression group was significantly better than the negative control group and blank control group, with significant differences (all P＜0.05).In contrast, no difference in cell proliferation viability was seen between the negative control group and the blank control group (P＞0.05, Table 1).This suggests that circLIFR high expression can significantly increase the proliferation viability of Hep3B cells.

3.3 Transwell invasion assay

Furthermore, we applied the transwell assay to identify the effect of circLIFR on the change of invasive ability of Hep3B HCC cells.The results suggested that the number of cells penetrating the Matrigel filter membrane in the blank control group, the circLIFR high expression group, and the negative control group were (266.2±17.6),(396.6±32.9), and (270.8±18.9), respectively, with an overall significant difference (F=84.47, P＜0.001).The number of cells in circLIFR high expression group that penetrated the matrigel filter membrane was higher than those of both groups (blank control vs.negative control, t=0.529, P=0.604; blank control vs.circLIFR high expression group, t=10.49, P ＜0.05; negative control vs.circLIFR high expression group, t=9.946, P＜0.05; n=9), and the differences were all statistically significant (Figure 2), which suggested that circLIFR high expression could significantly promote HCC cells invasion.

Fig 1 Construction of the circLIFR high expression series of HCC stable cell lines

Tab 1 Comparison of 450 nm absorbance values of Hep3B HCC cells in three groups (n=3,±s)

Tab 1 Comparison of 450 nm absorbance values of Hep3B HCC cells in three groups (n=3,±s)

Note: * indicates P＜0.05 with significantly different; t1=blank control vs.negative control, t2 = blank control vs.circLIFR high expression group, t3 = negative control vs.circLIFR high expression group

Time(h) Blank control group Negative control group CircLIFR high expression group F t 0 0.177±0.002 0.174±0.004 0.179±0.001 2.714 t1=1.162; t2=1.549; t3=2.100 24 0.233±0.004 0.231±0.005 0.371±0.006 752.900* t1=0.541; *t2=33.15; *t3=31.05 48 0.455±0.011 0.471±0.019 0.794±0.001 681.700* t1=1.262; *t2=53.16; *t3=29.40 72 0.802±0.006 0.812±0.022 0.978±0.029 64.620* t1=0.760; *t2=10.29; *t3=7.899

Fig 2 Transwell invasion assay of Hep3B HCC cell lines in three groups (×100)

4.Discussion

HCC is one of the "invisible killers" that endanger the health of our citizens, and even though clinical techniques have improved, patient prognosis is still poor.It has been reported that circRNA can regulate cell growth and differentiation, participate in HCC invasion and metastasis through various molecular pathways such as adsorption of miRNA, RBP, and encoded peptides, and can be used as a potential marker of tumor prognosis[4,5,9].Our previous study found that circLIFR (hsa_circ_0072309) exhibited low expression in HCC tissues.In this study, we further applied lentivirus to construct a stable strain of Hep3B cells and applied the cell proliferation kit and transwell assay to analyze the effect of circLIFR on the biological behavior of HCC Hep3B cells, respectively.The results suggested that high expression of circLIFR could significantly improve the proliferation viability of Hep3B cells and promote cell invasion.

According to the literature, leukemia inhibitory factor (LIF)is derived from the interleukin 6 family and is one of the most pleiotropic cytokines[10].LIF binds to the receptor LIFR, forms a high-affinity functional complex with the glycoprotein gp130,and is located at the cell membrane receptor mediating multiple signaling[11].Since LIFR does not have a fixed tyrosine kinase activity, it promotes downstream signaling mainly through the LIF/LIFR-JAK-STAT3 pathway[11].Studies have shown that the LIF/LIFR signaling axis regulates multiple aspects of cancer biological processes and is involved in tumor growth and development[12,13].For example, the LIF/LIFR/STAT axis maintains the stability of programmed death ligand 1 (PD-L1) protein, promotes the expression of metastasis-related genes, and may serve as a prognostic biomarker and a potential therapeutic target for prostate cancer[14].Secondly, LIF promotes YAP nuclear translocation and inhibits Hippo-YAP pathway activity by decreasing YAP phosphorylation,which ultimately promotes GC proliferation, migration, and invasion and inhibits tumor cell apoptosis[15].Another reported that LIFR could also act as a tumor metastasis suppressor through the Hippo-YAP pathway and confer a dormant phenotype of breast cancer(BC) cells spreading to bone[16,17].Thus, LIFR may play a dual role in the malignant process of tumors.In addition, in the tumor microenvironment, the LIF/LIFR signaling pathway regulates a variety of immune cell infiltrations, including effector T cells (Teff),macrophages, and regulatory T cells (Treg), and the combination of LIF-neutralizing antibodies and anti-PD1 inhibitors promotes tumor regression, enhanced immune memory, and overall survival[18,19].

CircLIFR (hsa_circ_0072309) is generated by exons 16, 17,18, and 19 being shed from LIF receptor subunit alpha during transcription and reverse spliced, located on human chromosome 5 (chr5: 38523520-38530768 strand: -).Studies have shown that circLIFR acts as an oncogenic repressor in the regulation of a variety of cancers, including bladder cancer, BC, and digestive malignancies such as GC[20-24].In bladder cancer, circLIFR acts synergistically with MSH2 to promote the interaction of MutS with ATM, thereby stabilizing p73, enhancing the sensitivity of bladder cancer cells to cisplatin in vitro and in vivo, and triggering apoptosis in tumor cells[20].In BC, circLIFR can act as a sponge for miR-492 and inhibit malignant processes such as cancer cell migration[23].CircLIFR also inhibits the PI3K/AKT signaling pathway by activating the PPARγ/PTEN pathway, thereby inhibiting the migration and metastasis of GC cells[24].Although circLIFR is lowly expressed in lung adenocarcinoma and serves as a biomarker for poor patient prognosis[25-26], recent experiments have shown that circLIFR promotes non-small cell lung carcinogenesis by interacting with miR-607 and upregulating the expression of its downstream target,m6A demethylase FTO[27].There is only one report in HCC, which showed that circLIFR exhibited tumor suppression by regulating miR-624-5p and easing the GSK-3β/β-catenin signaling pathway[28].In contrast, our study suggested that circLIFR high expression significantly increased the proliferation viability of Hep3B cells and promoted cell invasion.This is not quite consistent with our results,and the anti-tumor effects of circLIFR are controversial and deserve further exploration.

In response to the above phenomenon, we analyzed that due to the complex molecular mechanisms of tumorigenesis, there are some specific molecules that play dual roles in cancer regulation.p53 transcription factor, a recognized tumor suppressor, has been reported to promote tumor growth under certain circumstances, such as hypoxia, thus exhibiting its dual role[29].Although some circRNAs are similar to host genes in terms of pathogenic mechanisms such as pathways, due to their structural similarity they may also antagonize each other during molecular interactions and eventually exhibit opposite functions.Genes may also play different roles at the transcriptional and pre-translational levels, and at the protein level through binding to different target molecules, respectively.Since genes are differentially expressed in different individuals and some of them are organ-specific, they may play opposite roles in different systemic malignancies.

In conclusion, our preliminary experiments showed that circLIFR high expression promoted the proliferation and invasive ability of Hep3B cells, and we will continue to focus on and explore its potential mechanism of action in the future.

Authors' contribution:

LI Chen: experimental operation, data collation, manuscript writing; LIU Lu-zheng and LU Han-yuan: data collation, statistical analysis; CHEN Jia-cheng and CHEN Liang: experimental guidance,financial support; WU Jin-cai: research design, manuscript revision,financial support.

All authors declare no conflict of interest.