LncRNA PCGEM1 mediates oxaliplatin resistance in hepatocellular carcinoma via miR-129-5p/ETV1 axis in vitro

Background. Hepatocellular carcinoma (HCC) is one of the most severe malignant cancers that leads to high death rate worldwide. Recent research revealed that long non-coding RNAs (lncRNAs) exert a critical role regarding chemoresistance in numerous cancers, including HCC. Objectives. Our research aimed to explore the function and molecular mechanism of lncRNA PCGEM1 on oxaliplatin resistance of HCC in vitro. Materials and methods. Expression of the lncRNA PCGEM1 , together with miR-129-5p , and the mRNA level of ETV1 and drug resistance-related genes including LRPA , MDR1 and MDR3 were determined using quantitative real-time polymerase chain reaction (qRT-PCR) in an oxaliplatin-resistant HCC cell line (Hep3B/ OXA). Cell Counting Kit-8 (CCK-8) was employed to assess the viability and cell survival rate, and transwell assays were performed to measure the number of migrated or invaded cells. In addition, the relation among lncRNA PCGEM1 , miR-129-5p and ETV1 were determined using luciferase assay. Results. Our data indicated that PCGEM1 and ETV1 expression were enhanced in Hep3B/OXA cells. Furthermore, knockdown of lncRNA PCGEM1 significantly decreased the migration, invasion and mRNA expressions of LRPA , MDR1 and MDR3 , and the cell viability in Hep3B/OXA cells. The starBase online tool and luciferase assays verified that miR-129-5p targeted PCGEM1 and ETV1, signifying that PCGEM1 could enhance ETV1 expression via suppressing miR-129-5p . Conclusions. Our findings demonstrated that PCGEM1 modulated oxaliplatin resistance by targeting the miR-129-5p/ETV1 pathway in HCC in vitro, suggesting a potential strategy for the treatment of chemoresistant HCC.


Background
Hepatocellular carcinoma (HCC) is a severe type of malignant tumor, leading to a large number of cancer-related deaths worldwide. 1,2It is an invasive and fast-growing tumor that leads to higher recurrence and metastasis. 3Despite advancements in the therapeutic techniques for HCC such as radiotherapy, chemotherapy and surgery, the prognosis is still unsatisfactory, with an overall five-year survival rate of 15−40%. 4,5The underlying mechanism behind the pathogenesis of HCC is complicated and needs to be investigated for promising prognosis and early-stage detection. 6Therefore, novel targets and diagnostic indicators of HCC treatment are urgently needed.
Along with the progression of microarray analysis and sequencing, numerous novel non-coding RNAs have been discovered.Non-coding RNAs are sequences of nucleotides transcribed from chromatin and lack protein-coding capacity. 7Long non-coding RNAs (lncRNAs) are a group of non-coding RNAs having a length of >200 nucleotides. 8revious research showed that lncRNAs exert critical functions in the pathogenesis of HCC. 9 For example, lncRNA UBE2CP3 was overexpressed in HCC and enhanced tumor metastasis through the modulation of EMT and induced migration and invasion. 10Furthermore, other research revealed that overexpression of lncRNA UCA1 suppressed miR-216b expression and stimulated FGFR1/ ERK pathways to enhance the pathogenesis of HCC. 11oreover, lncRNA AK021443 mediated the migration and proliferation of HCC cells via EMT regulation in vitro. 12he lncRNA PCGEM1 was identified to be highly expressed in prostate cancer, and acts to enhance its proliferation. 13,14Also, PCGEM1 was highly expressed in ovarian cancer tissues and enhanced the invasion, migration and proliferation, while decreasing the apoptosis rate. 15urthermore, PCGEM1 induced metastasis and EMT via enhancing the SNAI1 expression in gastric cancer cells. 16owever, whether lncRNA PCGEM1 exerts its function in the development and tumorigenesis of HCC remains unknown.
It has been previously reported that oxaliplatin-mediated chemotherapy prolonged survival and reduced mortality during treatment of HCC patients in an advanced stage of the disease. 17Conversely, the resistance mechanism of oxaliplatin still needs to be elucidated.Numerous research has revealed that lncRNAs play a vital role in chemotherapy resistance in HCC.Several lncRNAs were highly expressed in chemoresistant HCC tissues and cells, and were shown to exert their function during oxaliplatin resistance and HCC pathogenesis. 18For example, lncRNA ARSR increased doxorubicin resistance in HCC by regulating PTEN. 2 Conversely, lncRNA HULC increased chemosensitivity by inhibiting autophagy in HCC cells. 19

Objectives
This study aimed to investigate the impact of lncRNA PCGEM1 on oxaliplatin resistance in vitro in an HCC cell line.Our findings elucidated that lncRNA PCGEM1 mediates oxaliplatin resistance in HCC via the miR-129-5p/ ETV1 axis, indicating that PCGEM1 might be a therapeutic target for HCC treatment.

Mycoplasma detection
Hep3B cells and Hep3B/OXA cells were kept in an RPMI-1640 medium containing 10% FBS and antibiotic-antimycotic solution (Spectrum Chemical Manufacturing Corp., New Brunswick, USA) and cultured for 3 years with occasional freezing as previously described. 21Blood agar plates and Mycoplasma IST2 kit (Biomereux Italia Spa, Florence, Italy) were used to perform titer of mycoplasmas as described previously. 21Twelve well plates were used to add mycoplasmas (1 × 10 5 CFU/mL) into the mycoplasma-free Hep3B cells (2 × 10 3 cells/well).Infected cells were analyzed using MycoGuard™ Mycoplasma PCR detection kit (GeneCopoeia) for the detection of mycoplasmas using the polymerase chain reaction (PCR) method, as described previously. 21Agarose gel electrophoresis was used to visualize amplified PCR products.

CCK-8 assay
Chemosensitivity and the rate of cell survival were examined using a CCK-8 assay.More specifically, 24-well plates were used to seed the cells at a density of 2 × 10 3 cells/well at 37°C.The CCK-8 solution (10 µL; Abcam, Cambridge, USA) was introduced into wells, and these plates were incubated for 1 h at 37°C.Proliferation was determined at 460 nm.The cell growth inhibition curve was detected by calculating the value of IC 50 as (1-OD/ OD 0 µM ) × 100%, as described previously. 22

Transwell invasion and migration assay
Transwell chambers (Becton Dickinson, Franklin Lakes, USA) were used to perform invasion and migration assay on Hep3B/OXA cells.For performing the invasion assay, Hep3B/OXA cells were passaged in a serum-free media (200 µL) and kept in the higher compartment, followed by 600 µL of a medium comprising 10% FBS into the lower compartment.The cells were fixed with polyoxymethylene after incubation for 48 h.For the migration assay, 24-well plates were used to seed Hep3B/OXA cells, and 1 × 10 6 cells were re-suspended in a serum-free media (200 µL) and kept in the higher compartment.Media (600 µL) with 10% FBS were introduced into the lower compartment, and cells were incubated with 5% CO 2 for 48 h at 37°C.Finally, the invasion and migration of cells was visualized after staining with 20% Giemsa solution (Sigma-Aldrich, St. Louis, USA) and counted from 5 random chambers using an inverted microscope (Nikon Corp., Tokyo, Japan).All the experimentations were repeated in triplicate.

qRT-PCR
Total RNA was extracted using Ribozol RNA extraction reagent (VWR) following the manufacturer's protocol (https://us.vwr.com/store/product/7437721/vwr-lifescience-ribozoltm-rna-extraction-reagent).A NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) was used to examine the purity and RNA concentration at 260/280 nm.Reverse transcription of total RNA (2 µg) into cDNA was performed using the SuperScript First-Stand Synthesis System.ABI PRISM 7500 and SYBR Premix ExTaq II kit (Thermo Fisher Scientific) was used to perform quantitative real-time PCR (qRT-PCR).Primers sequences are given below in Table 1.All the experiments were performed 3 times.GAPDH acted as the internal control, and the expressions of genes were measured using the 2 −ΔΔCt method. 23

Statistical analyses
Data are presented as the mean ± standard deviation (SD).IBM SPSS v. 19 (IBM Corp., Armonk, USA) and GraphPad Prism v. 8.0 (GraphPad Software, San Diego, USA) were used for statistical analyses of all data.The difference between the 2 groups was evaluated using Student's t-test while multiple-group comparisons were performed using analysis of variance (ANOVA) and Tukey's post hoc analysis.The general alpha value was set as 0.05.All the experimentations were performed 3 times.

Effect of lncRNA PCGEM1 and ETV1 on oxaliplatin-resistant HCC cells
Hep3B cells were treated with oxaliplatin, and the PC-GEM1 expression was examined using qRT-PCR.Results indicated that PCGEM1 expression was significantly enhanced in Hep3B/OXA cells compared to Hep3B cells without oxaliplatin treatment (Fig. 1A).The survival of Hep3B cells was assessed using a CCK-8 assay, and our data indicated that the percentage survival of Hep3B/ OXA cells was greater than Hep3B control cells (Fig. 1B).Furthermore, ETV1 mRNA expression was markedly enhanced in Hep3B/OXA cells compared to that of parental Hep3B cells (Fig. 1C).Next, oligonucleotides were prepared and Hep3B/OXA cells were transfected with them to reduce the expression of PCGEM1 (Fig. 1D).Moreover, our findings revealed that PCGEM1 knockdown considerably decreased the mRNA expression of drug resistance-related genes, including low-density lipoprotein receptor-related protein 1 (LRP1), multidrug resistance gene 1 (MDR1) and multidrug resistance gene 3 (MDR3) compared to that of the control group (Fig. 1E).
The CCK-8 assay was used to measure the cell growth inhibition curve by calculating the value of the inhibitory concentration (IC 50 ).Our findings elucidated that PCGEM1 knockdown markedly suppressed the IC 50 value in comparison to the control group (Fig. 1F).These data illustrated that PCGEM1 knockdown markedly reduced the oxaliplatin resistance of HCC cells.

Effect of lncRNA PCGEM1 on invasion and migration in the oxaliplatin-resistant HCC cells
Transwell assays indicated that PCGEM1 knockdown considerably reduced the number of invasive cells in Hep3B/OXA cells compared to that of the empty vector group (Fig. 2A).Our findings also revealed that PCGEM1 knockdown markedly reduced the number of migrating cells compared to the empty vector cells (Fig. 2B).These findings revealed that PCGEM1 knockdown inhibited the invasion and migration in Hep3B/OXA cells.

Discussion
The HCC is a hepatic malignant neoplasm. 24Despite great advancement in the therapeutic techniques for HCC, the overall outcomes remain unsatisfactory, 25 with a common obstacle being chemoresistance towards drugs such as oxaliplatin, doxorubicin and cisplatin, among others. 26ur study explored the regulatory functions of PCGEM1 in oxaliplatin resistance regarding miR-129-5p/ETV1 interactions using oxaliplatin-resistant HCC cells (Hep3B/ OXA) (Fig. 5).
The lncRNAs present a vital function in regulating tumorigenesis, as well as the chemoresistance of cancer cells. 27Previous research has suggested that dysregulated lncRNA expression might mediate potential chemoresistance. 28Our findings revealed that the PCGEM1 expression was enhanced in Hep3B/OXA cells compared to that of its parental cell line.Furthermore, knockdown of ln-cRNA PCGEM1 decreased the mRNA expression levels of LRP1, MDR1 and MDR3 in Hep3B/OXA cells, indicating that silencing of PCGEM1 might reduce the oxaliplatin resistance.Moreover, PCGEM1 knockdown significantly inhibited the proliferation, migration and invasiveness of Hep3B/OXA cells, which was in accordance with the hypothesis that downregulation of PCGEM1 might lead to the decrease in chemoresistance in vitro.Similar to our findings, it has been reported that downregulation of another lncRNA, lncRNA PVT1 was able to inhibit tumor progression and reduced chemoresistance to cisplatin in colorectal cancer patients. 29In HCC, lncRNA HULC was significantly upregulated in HCC cells after treatment with drugs including cisplatin and oxaliplatin, and   the additional knockdown of HULC enhanced chemoresistance of HCC cells through its interaction with SIRT1 19 .In this study, we found that the relative mRNA expression of ETV1 was decreased by the knockdown of lncRNA PCGEM1 in Hep3B/OXA cells.ETV1 has been reported to function as an oncogene in various human malignant tumors. 30,31A previous study indicated that ETV1 enhanced the invasion, migration and proliferation of breast cancer cells. 32Moreover, inhibitor of ETV1 enhanced the cellular sensitivity to docetaxel in prostate cancer. 33Similarly, ETV1 was reported to be modulated by Circ-ZNF609/ miR-1224-3p in lung cancer cells. 34We discovered that the expression of ETV1 was positively regulated by PC-GEM1 in Hep3B/OXA cells, suggesting that knockdown of PCGEM1 in HCC cells increased sensitivity to oxaliplatin, perhaps through the modulation of ETV1.
To predict the targeted sites between mRNA and ln-cRNA, bioinformatic prediction tools were used. 35By using these online tools, we established that miR-129-5p targeted PCGEM1.The miR-129-5p overexpression has been shown previously to attenuate the proliferation of PC-3 cells, 36 while in prostate cancer, miR-129-5p was downregulated in tissues and cells in comparison with normal counterparts, and it specifically inhibits ETV1. 36Furthermore, the upregulation of miR-129-5p was shown to curb the cell proliferation by suppressing ETV1 in prostate cancer cells. 36Finally, it was disclosed that miR-129-5p inhibited the development of colon cancer and osteosarcoma via regulating HMGB1. 37,38n conclusion, our data revealed the impact of lncRNA PCGEM1 in HCC oxaliplatin resistance in vitro.We have shown that PCGEM1 and miR-129-5p expression levels were negatively correlated to each other, representing the enrichment and antagonistic function.PCGEM1 downregulation led to an increase in miR-129-5p expression in Hep3B/OXA cells, while downregulation of miR-129-5p induced ETV1 mRNA expression.In our work, PCGEM1 downregulation reduced the oxaliplatin resistance in Hep3B/OXA cells via binding miR-129-5p and inhibiting ETV1.

Limitations
The limitation of this current study mainly exists in the absence of animal models.If respective animal models could be established to further validate the mechanism of the PCGEM1/miR-129-5p/ETV1 pathway on HCC oxaliplatin resistance, the research would be more valuable and complete.

Conclusions
Our findings demonstrated the potential mechanism of the PCGEM1/miR-129-5p/ETV1 pathway on HCC oxaliplatin resistance, suggesting the critical function of PC-GEM1 in HCC treatment and providing a new therapeutic target.

Fig. 5 .
Fig. 5.The illustration of the hypothetical mechanism of PCGEM1 in Hep3B/OXA cells

Table 1 .
The primer sequences for quantitative real-time polymerase chain reaction (qRT-PCR) analysis