Introduction

Ovarian cancer is the deadliest malignancy of women’s reproductive systems and is referred to as the “silent killer”1. More than 70% of ovarian cancer patients are diagnosed with advanced stage, with a 5-year survival rate of less than 40%2,3,4. Tumor resection and chemotherapy combined with platinum are the main methods for clinical treatment of ovarian cancer5,6. However, most ovarian cancer patients develop resistance to chemotherapy drugs at the initial stage of treatment or during treatment, resulting in a high mortality rate and eventually treatment failure. Drug resistance is a key factor limiting drug treatment of ovarian cancer7,8. Therefore, it is of great significance to elucidate the molecular mechanism regulating drug resistance of ovarian cancer and to find new effective therapeutic targets.

Laminin gamma subunit 3 (LAMC3) is present on chromosome 9q34.1-2 and can play a role in tumor progression as an oncogene or tumor suppressor gene9. Previous reports have showed there were four potential tumor suppressor genes such as LAMC3 in colorectal cancer10. Additionally, LAMC3 was low expressed in breast cancer and its promoter was prone to methylation, which can be used as a reliable biomarker for breast cancer diagnosis11. To our knowledge, there have been few studies concerning LAMC3 and ovarian cancer. Only reported so far, LAMC3 has been shown to be significantly down-regulated in platinum-resistant ovarian cancer tissue in 20 patients12. Based on clinical tissue samples and open databases, our previous studies found that LAMC3 protein expression was significantly reduced in ovarian cancer tissues and correlated with ovarian cancer resistance. According to these research basis, the effects of LAMC3 protein expression on drug resistance of ovarian cancer cells needs to be undergone further study.

As far as we know, the effects of LAMC3 interference on drug resistance of carboplatin-resistant ovarian cancer cells have not been reported. Besides, the mechanism of action underlying the influences of LAMC3 interference on drug resistance in carboplatin-resistant ovarian cancer cells is likewise unclear. In the present study, the effects of LAMC3 interference on drug resistance of carboplatin-resistant ovarian cancer cells, including cell proliferation, apoptosis, cycle, and ultrastructure, were investigated. Additionally, using transcriptome sequencing, the molecular mechanism of LAMC3 interference regulating drug resistance in carboplatin-resistant ovarian cancer cells were studied. This study provides a theoretical basis for the application of LAMC3 in molecular targeted therapy of drug resistance in ovarian cancer patients.

Materials and methods

Reagents and culture medium

All of the chemical reagents were purchased from Sigma Aldrich, except for Roswell Park Memorial Institute 1640 (RPMI 1640) and fetal bovine serum (FBS), which were purchased from Wisent Corporation. The cell medium for ovarian cancer cells contained RPMI 1640 and was supplemented with 10% FBS.

Ovarian cancer cell lines

Ovarian cancer cells were collected from adult patients with ovarian serous adenocarcinoma who were treated at The Affiliated Tumor Hospital of Guangxi Medical University. Cell lines HeyA8 and SKOV3 were kind gifts from Prof. Fengxia Xue of Tianjin Medical University. Carboplatin-resistant human ovarian cancer cells used in this study were established by stepwise increased concentrations of carboplatin (range from 10 µmol/L to 95 µmol/L) for 15 months. During the establishment of carboplatin-resistant cells, the cells were incubated with gradual increasing concentrations of carboplatin for 24 h and then cultured without carboplatin until cells grew well. The cells of the control group (LAMC3 idle lentivirus Scramble served as a control) and the LAMC3 interference group were constructed by lentiviral transfection of carboplatin-resistant ovarian cancer cells, respectively.

Cell proliferation, cycle, and apoptosis assay

Plate colony formation assay was used to detect cell proliferation of ovarian cancer cells. Ovarian cancer cells of the control group and LAMC3 interference group were treated with a series of gradient concentration carboplatin drug, and the relative clone formation rate was analyzed by Image J software after 10 days. Propyridine iodide can be combined with DNA double strand to produce fluorescence, and the cell cycle is tested by flow cytometry. Ovarian cancer cells of the control group and LAMC3 interference group were treated with a series of gradient concentration carboplatin drug, the cell cycle pictures were analyzed by flow cytometry and ModFit, and then the proportion of G0/G1, S, G2/M phase cells were calculated. Cell apoptosis was detected by flow cytometry using BD Pharmingen’s PE Annexin V/7-AAD double staining apoptosis kit according to the manufacturer’s instructions.

CCK-8 assays

The effects of LAMC3 interference on cell viability of carboplatin-resistant ovarian cancer cells were analyzed by using the CCK-8 assay (Solarbio, Beijing, China) in six replicate cells. Cells were seeded into 96-well culture plates with 600 cells per well at 37°C with 5% CO2 in a humidified incubator. After 16 h of incubation, the cells were treated with varying concentrations of carboplatin (250, 125, 62.5, 31.25, 15.625, 7.8125, and 3.90625 nmol/L) for 72 h. The RPMI-1640 medium with treated cells was used as a negative control, and the RPMI-1640 medium without cells was used as a blank control. A total of 10 µL/well of CCK-8 (Solarbio) was added and the 96-well plates were incubated for 2 h at 37°C. The absorbance (at 450 nm) was calculated by GO Microplate Spectrophotometer (Thermo Fisher Scientific). Cell viability (%) = (ODtreated-ODblank)/(ODcontrol-ODblank) × 100%. The IC50 value was the concentration of the drug required when 50% of the cells were inhibited from proliferating.

Cell ultrastructure analysis

Ovarian cancer cells at 100% confluence were digested by 0.25% trypsin, harvested into tubes and washed with PBS for three times. Then, the cells were fixed with 2.5% glutaraldehyde at 4°C overnight. After refixation by 1% osmium tetroxide, gradient dehydration by ethaol and penetration, the cells were embedded in epon, stained with uranyl acetate. The sections (80 nm) were imaged under a HITACHI-7650 electron microscope at the core facility Guangxi Medical University.

Transcriptome sequencing analysis

Carboplatin-resistant ovarian cancer cells in the logarithmic growth phase were seeded at 5.5 × 105 cells/mL in the control group and 1.3 × 106 cells/mL in the LAMC3 interference group. The 32 mL cell suspension was inoculated in a 175-cm2 culture bottle. On the next day, after the cells were completely attached to the wall, the cells were treated with carboplatin with concentrations of 0 and 125 µmol/L for 72 h in RPMI-1640 medium. Subsequently, total RNA was extracted by Trizol method, and the data were analyzed based on Illumina NovaSeq 6000 sequencing platform.

Transcriptome sequenceing of was completed at Beijing Novogene Corporation. A total of 12 RNA samples were sequenced. Whole transcriptome data were collected and compared with the ribosome database to identify known RNA transcripts (mRNA), analysis differences between samples (at least 3 samples) and analysis differences between groups (at least 3 biological replicates per group). Differentially expressed genes were analysed using the DAVID database by identifying Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway maps.

Quantitative RT-PCR

Total RNA was isolated from ovarian cancer cells using Trizol reagent (Invitrogen, Carlsbad, CA) according to product instructions, and cDNA synthesis was performed with high purity RNA (total quantity 1 µg) using Prime Script TMRT reagent Kit (Osaka, Japan). The cDNA samples were then applied for qRT-PCR using SYBR Premix Ex Taq™ II (Osaka, Japan) and Real-Time PCR System (ABI7300, Applied Biosystems, Carlsbad, CA). Reaction conditions used for qRT-PCR were as follows: initial denaturation and enzyme activation at 95°C for 3 min, denaturation, amplication, and quantification at 95°C for 10 s, 60°C for 30 s, 40 cycles in total. The reactions were performed in triplicate. The primer sequences used in this study were displayed in Table 1, and the expression of genes was normalized to that of the internal control gene GAPDH. The 2−ΔΔCT method was used to calculate the expression level of target genes. All experiments were performed in triplicate.

Table 1 Primers for quantitative RT-PCR.
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Western blotting

Ovarian cancer cells at more than 80% confluency were harvested by trypsinization and lysed in protein lysis buffer with protein inhibitor cocktail on ice for 30 min, and then lysates were clarified by centrifugation at 13,000 × g for 10 min at 4°C. Centrifugal supernatant was then boiled in 100°C for 10 min. Proteins were separated in 12% precast gel running at 90 V. Gels were then transferred to nitrocellulose membranes at 60 mA for 30 min. Membranes were blocked in 5% skim milk/TBST prior to incubation in indicated primary antibodies (Cell Signaling Technology, USA) overnight. Secondary antibodies were then added for 1 h prior to detection either through chemiluminescence with ChemiDoc system.

Statistical analysis

All data were presented as the mean ± standard error. Statistical significance in two groups was analyzed by t-test and analysis of variance (ANOVA) with Duncan’s test was used if there were more than two groups. All statistical analyses were performed by using SPSS 22.0 software. p values < 0.05 were deemed significant, with * representing p < 0.05, **p < 0.01, and ***p < 0.001.

Results

LAMC3 expression is decreased in carboplatin-resistant ovarian cancer cells compared with parent cells

The expression levels of LAMC3 in ovarian cancer cells, including mRNA and protein levels, were investigated by qRT-PCR and WB. The results showed that, compared to the parent cells, the expression levels of LAMC3 mRNA and protein were significantly (p < 0.01) lowered in carboplatin-resistant ovarian cancer cells (Fig. 1). These results demonstrated that LAMC3 expression is down-regulated in carboplatin-resistant ovarian cancer cells compared with parent cells.

Fig. 1
figure 1

qRT-PCR and WB analyzed the expression levels of LAMC3 in the parental cells and carboplatin-resistant ovarian cancer cells. (a) qRT-PCR tested the mRNA levels of LAMC3 in the parental cells and carboplatin-resistant ovarian cancer cells. (b) WB detected the protein levels of LAMC3 in the parental cells and carboplatin-resistant ovarian cancer cells. (c) Relative band density of WB results. H: the parent cell HeyA8; H-C: the carboplatin-resistant ovarian cancer cell HeyA8-CBP. GAPDH expression was used as a standard for qRT-PCR. β-Tubulin expression was used as internal parameter for WB. The results were the mean ± SE of three independent experiments. p values < 0.05 were deemed significant, with * representing p < 0.05, **p < 0.01, and ***p < 0.001.

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Lentivirus transfection constructs the carboplatin-resistant ovarian cancer cell line that interferes with LAMC3 expression

The carboplatin-resistant ovarian cell line that interfered with LAMC3 expression was established by transfecting lentivirus (Fig. 2a). WB results showed that, compared with the control group (carboplatin-resistant ovarian cancer cells transfected with LAMC3 scramble), LAMC3 protein expression was significantly (p < 0.01) decreased in the carboplatin-resistant ovarian cells transfected with LAMC3 interference lentivirus (Fig. 2b, c). These results suggested that the carboplatin-resistant ovarian cell line with LAMC3 interference protein expression was successfully constructed.

Fig. 2
figure 2

Successful construction of carboplatin-resistant ovarian cancer cells with LAMC3 interference. (a) Scramble control group and LAMC3 interference group carboplatin-resistant ovarian cancer cells. (b) WB tested the protein levels of LAMC3 in scramble control group and LAMC3 interference group carboplatin-resistant ovarian cancer cells. (c) Relative band density of WB results. H-C-C3 Scramble: the carboplatin-resistant ovarian cancer cells transfected with LAMC3 scamble; H-C-C3 shRNA3: the carboplatin-resistant ovarian cells transfected with LAMC3 interference lentivirus. β-Tubulin expression was used as internal parameter for WB. Scale bars = 100 μm.

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LAMC3 interference reduces drug resistance of carboplatin-resistant ovarian cancer cells

The effects of LAMC3 interference on drug resistance of carboplatin-resistant ovarian cancer cells were evaluated through CCK-8 assays (Fig. 3a). The results showed that, when treated with carboplatin with appropriate gradient concentration, compared to the control group, the IC50 value in carboplatin-resistant ovarian cancer cells with LAMC3 interference was significantly lowered (49.4 ± 0.07 vs. 89.5 ± 1.30; p < 0.01) (Fig. 3b). These results demonstrated LAMC3 reduced drug resistance of carboplatin-resistant ovarian cancer cells.

Fig. 3
figure 3

CCK-8 assay detected the effects of LAMC3 interference on drug resistance of carboplatin-resistant ovarian cancer cells. (a) The cell viability curves of the control group and LAMC3 interference group that was treated with a series of gradient carboplatin for 72 h. (b) IC50 value of carboplatin drug on carboplatin-resistant ovarian cancer cells with LAMC3 interference. H-C-C3 Scramble: the carboplatin-resistant ovarian cancer cells transfected with LAMC3 scamble; H-C-C3 shRNA3: the carboplatin-resistant ovarian cells transfected with LAMC3 interference lentivirus. p values < 0.05 were deemed significant, with * representing p < 0.05, **p < 0.01, and ***p < 0.001.

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LAMC3 interference inhibits cell proliferation of carboplatin-resistant ovarian cancer cells

The effects of LAMC3 interference on cell proliferation of carboplatin-resistant ovarian cancer cells were investigated by plate clonal formation experiments (Fig. 4a). The results showed that, under the same concentration carboplatin drug treatment, the clonal formation ability of carboplatin-resistant ovarian cancer cells with LAMC3 interference was significantly (p < 0.05) reduced compared with the control group (Fig. 4b). These results suggested that LAMC3 interference inhibited cell proliferation of carboplatin-resistant ovarian cancer cells.

Fig. 4
figure 4

Plate cloning assay tested the effects of LAMC3 interference on clone formation of carboplatin-resistant ovarian cancer cells. (a) Clonal formation of carboplatin-resistant ovarian cancer cells from the control group and LAMC3 interference group for 10 day treatment with a series of gradient carboplatin. (b) Image J software analyzed the relative clone formation rate. H-C-C3 Scramble: the carboplatin-resistant ovarian cancer cells transfected with LAMC3 scamble; H-C-C3 shRNA3: the carboplatin-resistant ovarian cancer cells transfected with LAMC3 interference lentivirus. Control: the carboplatin-resistant ovarian cancer cells in H-C-C3 Scramble and H-C-C3 shRNA3 group were not treated with carboplatin. p values < 0.05 were deemed significant, with * representing p < 0.05, **p < 0.01, and ***p < 0.001. Scale bars = 100 μm.

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LAMC3 interference arrests cell cycle of carboplatin-resistant ovarian cancer cells

The effects of LAMC3 interference on cell cycle of carboplatin-resistant ovarian cancer cells were evaluated through flow cytometry (Fig. 5a, b). The results showed that, under carboplatin drug treatment with the same concentration, the proportion of G2/M phase was significantly (p < 0.05) increased in carboplatin-resistant ovarian cancer cells with LAMC3 interference compared to the control group (Fig. 5c). These results demonstrated that LAMC3 interference blocked cell cycle of carboplatin-resistant ovarian cancer cells in G2/M phase.

Fig. 5
figure 5

Flow cytometry detected the effects of LAMC3 interference on cell cycle of carboplatin-resistant ovarian cancer cells. (a) The cell viability curves of the control group and LAMC3 interference group that was treated with a series of gradient carboplatin for 72 h. (b) IC50 value of carboplatin drug on carboplatin-resistant ovarian cancer cells with LAMC3 interference. H-C-C3 Scramble: the carboplatin-resistant ovarian cancer cells transfected with LAMC3 scamble; H-C-C3 shRNA3: the carboplatin-resistant ovarian cells transfected with LAMC3 interference lentivirus. Control: the carboplatin-resistant ovarian cancer cells in H-C-C3 Scramble and H-C-C3 shRNA3 group were not treated with carboplatin. p values < 0.05 were deemed significant, with * representing p < 0.05, **p < 0.01, and ***p < 0.001. Scale bars = 100 μm.

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LAMC3 interference promotes cell apoptosis of carboplatin-resistant ovarian cancer cells

The effects of LAMC3 interference on cell apoptosis of carboplatin-resistant ovarian cancer cells were investigated by cell apoptosis detection kits (Fig. 6a, b). The results showed that, under the same concentration carboplatin drug treatment, compared with the control group, the cell apoptosis rate and cell death rate were significantly (p < 0.05) improved in carboplatin-resistant ovarian cancer cells with LAMC3 interference (Fig. 6c). These results suggested that LAMC3 interference enhanced cell apoptosis of carboplatin-resistant ovarian cancer cells.

Fig. 6
figure 6

Flow cytometry tested the effects of LAMC3 interference on cell apoptosis of carboplatin-resistant ovarian cancer cells. (a) A series of gradient concentration carboplatin drug treated carboplatin-resistant ovarian cancer cells of the control group and LAMC3 interference group for 72 h, respectively, and took bright field photographs under the microscope. (b) Flow cytometry analyzed cell apoptosis of carboplatin-resistant ovarian cancer cells from the control group and LAMC3 interference group. (c) The histograms of cell apoptosis distribution, including the proportion of living cells, early apoptotic cells, late apoptotic cells, total apoptotic cells, and dead cells. H-C-C3 Scramble: the carboplatin-resistant ovarian cancer cells transfected with LAMC3 scamble; H-C-C3 shRNA3: the carboplatin-resistant ovarian cells transfected with LAMC3 interference lentivirus. Control: the carboplatin-resistant ovarian cancer cells in H-C-C3 Scramble and H-C-C3 shRNA3 group were not treated with carboplatin. p values < 0.05 were deemed significant, with * representing p < 0.05, **p < 0.01, and ***p < 0.001. Scale bars = 100 μm.

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LAMC3 interference affects cell ultrastructure of carboplatin-resistant ovarian cancer cells

The effects of LAMC3 interference on cell ultrastructure of carboplatin-resistant ovarian cancer cells were evaluated through transmission electron microscope technique. The results showed that, under carboplatin drug treatment with the same concentration, compared to the control group, the cytoplasm of carboplatin-resistant ovarian cancer cells with LAMC3 interference became dark, the nuclear chromatin condensed and the edge shifted, and the apoptotic bodies shed outside the cells appeared (Fig. 7a). Additionally, the morphological characteristics of mitochondrial ridge reduction, iron death of mitochondrial outer membrane rupture, lysosomes of multiple damaged organelles wrapped in a single membrane, and autophagy lysosomes were also observed in carboplatin-resistant ovarian cancer cells with LAMC3 interference (Fig. 7b). These results demonstrated that LAMC3 interference changed cell ultrastructure of carboplatin-resistant ovarian cancer cells.

Fig. 7
figure 7

Transmission electron microscope detected the effects of LAMC3 interference on cell ultrastructure of carboplatin-resistant ovarian cancer cells. (a) The cell apoptotic morphology of carboplatin-resistant ovarian cancer cells from the control group and LAMC3 interference group were observed by transmission electron microscope after 24, 48, and 72 h treatment with carboplatin at a certain concentration. (b) The morphology of ferroptosis, lysosome, and autophagosome were observed by transmission electron microscope after 24, 48, and 72 h treatment with carboplatin in carboplatin-resistant ovarian cancer cells from the control group and LAMC3 interference group. H-C-C3 Scramble: the carboplatin-resistant ovarian cancer cells transfected with LAMC3 scamble; H-C-C3 shRNA3: the carboplatin-resistant ovarian cells transfected with LAMC3 interference lentivirus. Control: the carboplatin-resistant ovarian cancer cells in H-C-C3 Scramble and H-C-C3 shRNA3 group were not treated with carboplatin. Scale bars = 100 μm.

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Transcriptome sequencing analysis of carboplatin-resistant ovarian cancer cells with LAMC3 interference

To investigate the changes in potential differential genes caused by LAMC3 interference, transcriptome analysis of carboplatin-resistant ovarian cancer cells with LAMC3 interference and noninterference was performed using RNA sequencing. The results showed that, compared with carboplatin-resistant ovarian cancer cells with LAMC3 interference without carboplatin treatment, there were 5973 differentially expressed genes in carboplatin-resistant ovarian cancer cells with LAMC3 interference treated with carboplatin, of which 3029 genes were up-regulated and 2944 genes were down-regulated (Fig. 8a, b). GO functional analysis revealed that differentially expressed genes were significantly enriched into biological functions of DNA replication, autophagy, nuclear division, lysosomal membrane, ATPase activity, and cytokine receptor binding (Fig. 8c). KEGG signaling analysis showed that differentially expressed genes were significantly enriched into cell cycle, p53, ferroptosis, lysosome, DNA replication, and other signal pathways (Fig. 8d). These results suggested that LAMC3 interference may induce differential expression of a large number of genes, affected biological functions such as DNA replication, cell autophagy, and then influenced cell cycle, p53, ferroptosis, lysosome, and other signal pathways, ultimately reduced drug resistance of carboplatin-resistant ovarian cancer cells.

Fig. 8
figure 8

Transcriptome sequencing analyzed the effects of LAMC3 interference on carboplatin-resistant ovarian cancer cells. (a) The differentially expressed genes of sequencing samples were based on cluster analysis. (b) The differentially expressed genes of LAMC3 interference in drug resistance of carboplatin-resistant ovarian cancer cells before and after carboplatin treatment was based on volcanic map analysis. (c) The differentially expressed genes of LAMC3 interference in drug resistance of carboplatin-resistant ovarian cancer cells before and after carboplatin treatment by GO functional enrichment analysis. (d) The differentially expressed pathways of LAMC3 interference in drug resistance of carboplatin-resistant ovarian cancer cells before and after carboplatin treatment was based on KEGG pathway enrichment analysis. Scr_0 and shRNA3_0 represented H-C-C3 Scramble and H-C-C3 shRNA3 cells that were not treated with carboplatin, respectively; Scr_125 and shRNA3_125 represented H-C-C3 Scramble and H-C-C3 shRNA3 cells that were treated with 125 µmol/L carboplatin, respectively.

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LAMC3 interference changes cell cycle-related protein expression in carboplatin-resistant ovarian cancer cells

Combined with transcriptome sequencing results, the effects of LAMC3 interference on cell cycle-related protein expression, including G2/M phase associated proteins (p21 Waf1/Cip1, Cyclin B1, CDK7) and G0/G1 phase associated proteins (GSK-3β, CDK2, Cyclin D3, p-P53) in carboplatin-resistant ovarian cancer cells were investigated by WB. The results showed that, under the condition of gradient concentration carboplatin drug treatment, compared with the control group, the protein expression of p21 Waf1/Cip1 and p-P53 were significantly promoted, and the protein level of Cyclin B1, CDK2, and CDK7 were significantly (p < 0.05) lowered in carboplatin-resistant ovarian cancer cells with LAMC3 interference. However, the protein expression of GSK-3β and Cyclin D3 had no significant (p > 0.05) difference between the control group and the LAMC3 interference group (Fig. 9). These results demonstrated that LAMC3 interference affected G2/M and G0/G1 phase related protein expression in carboplatin-resistant ovarian cancer cells.

Fig. 9
figure 9

WB tested the effects of LAMC3 interference on the expression levels of cell cycle-related proteins in carboplatin-resistant ovarian cancer cells. H-C-C3 Scramble: the carboplatin-resistant ovarian cancer cells transfected with LAMC3 scamble; H-C-C3 shRNA3: the carboplatin-resistant ovarian cells transfected with LAMC3 interference lentivirus. Control: the carboplatin-resistant ovarian cancer cells in H-C-C3 Scramble and H-C-C3 shRNA3 group were not treated with carboplatin. GAPDH expression was used as internal parameter for WB.

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LAMC3 interference affects autophagy, ferroptosis, lysosome, and multidrug resistance related protein expression in carboplatin-resistant ovarian cancer cells

Combined with transcriptome sequencing results, the effects of LAMC3 interference on multidrug resistance protein (MDR1/ABCB1), autophagy associated proteins (LC3-I, LC3-II, SQSTM1/p62, mTOR), ferroptosis-related proteins (xCT/SLC7A11, GPX4), and lysosome protein (LAMP1) in carboplatin-resistant ovarian cancer cells were evaluated through WB. The results showed that, under gradient concentration carboplatin drug treatment, compared to the control group, the protein expression of LC3-I, LC3-II, SQSTM1/p62, and xCT/SLC7A11 were significantly enhanced, and the protein level of mTOR and LAMP1 were significantly (p < 0.05) reduced in carboplatin-resistant ovarian cancer cells with LAMC3 interference. However, the protein expression of GPX4 had no significant (p > 0.05) difference between the control group and the LAMC3 interference group. Furthermore, the expression level of multidrug resistance protein MDR1/ABCB1 was significantly (p < 0.05) lowered in carboplatin-resistant ovarian cancer cells compared with the control group (Fig. 10). These results suggested that LAMC3 interference changed autophagy, ferroptosis, lysosome, and multidrug resistance-related protein expression in carboplatin-resistant ovarian cancer cells, which indicating that the biological pathways of autophagy, ferroptosis, lysosome, and multidrug resistance may be involved in regulating drug resistance in carboplatin-resistant ovarian cancer cells.

Fig. 10
figure 10

WB detected the effects of LAMC3 interference on the expression levels of autophagy, ferroptosis, lysosome, and multidrug resistance-related proteins in carboplatin-resistant ovarian cancer cells. H-C-C3 Scramble: the carboplatin-resistant ovarian cancer cells transfected with LAMC3 scamble; H-C-C3 shRNA3: the carboplatin-resistant ovarian cells transfected with LAMC3 interference lentivirus. Control: the carboplatin-resistant ovarian cancer cells in H-C-C3 Scramble and H-C-C3 shRNA3 group were not treated with carboplatin. GAPDH expression was used as internal parameter for WB.

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Discussion

Ovarian cancer is the most common disease of the reproductive system and has the highest mortality rate among all gynecological malignancies13. Carboplatin is one of the adjuvant chemotherapy drugs for ovarian cancer patients after tumor cell reduction, and approximately 70% of patients relapse due to resistance to carboplatin after long-term drug treatment, showing shorter survival and poor quality of life14. The mechanism of resistance to chemotherapy drugs in ovarian cancer cells is complex, including multi-drug resistance, DNA damage repair, cell cycle regulation, apoptosis pathway, autophagy, and signaling pathway abnormalities15,16,17,18,19,20,21. Therefore, it is an urgent clinical need to explore the drug resistance mechanism of ovarian cancer and develop new methods for drug resistance treatment of ovarian cancer.

LAMC3 plays an important role in tumor progression as both an oncogene and a tumor suppressor gene. The published papers have shown that LAMC3 was low expressed in ovarian cancer, breast cancer, bladder cancer, and other malignant tumors11,12,22. Our previous study revealed that LAMC3 was down-regulated in ovarian cancer tissues and correlated with ovarian cancer resistance, but there was a lack of experimental verification, including cellular and molecular levels. In the present study, we investigated for the first time the effects of LAMC3 interference on drug resistance of carboplatin-resistant ovarian cancer cells. The results showed that LAMC3 interference reduced drug resistance of carboplatin-resistant ovarian cancer cells. These results were similar to the findings of other researchers who also reported the effects of LAMC2 on drug resistance in pancreatic ductal adenocarcinoma cells23. The difference is that LAMC2 regulates drug resistance of pancreatic ductal adenocarcinoma cells to gemcitabine through epithelial-mesenchymal transformation.

The cell cycle is a complex process that involves a large number of regulatory proteins, including p21 Waf1/Cip1, Cyclin B1, and CDK2, guiding the cell through a specific sequence of events that culminate in mitosis to produce two daughter cells24. The occurrence and development of tumor are closely related to cell cycle. In this study, the effects of LAMC3 interference on cell cycle and cycle-related proteins of carboplatin-resistant ovarian cancer cells were explored. The results showed that LAMC3 interference arrested cell cycle in G2/M phase and affected the expression levels of cell cycle-related proteins in carboplatin-resistant ovarian cancer cells. These results were in accordance with the published paper, which suggested that LAMA4 overexpression accelerated the fibroblast cell cycle and blocked more cells in G2/M phase25.

In tumor therapy, cell cycle arrest can lead to cell apoptosis, and morphological changes such as cell size reduction, dark cytoplasm color, chromatin aggregation and apoptotic bodies can be observed26. In the present study, the effects of LAMC3 interference on cell apoptosis of carboplatin-resistant ovarian cancer cells were investigated. The results showed that cell apoptosis level was enhanced in carboplatin-resistant ovarian cancer cells with LAMC3 interference. LAMC3 interference induced darkening of cytoplasm, aggregation of nuclear chromatin and edge shift, and the appearance of apoptotic bodies shed outside the carboplatin-resistant ovarian cancer cells. These results were consistent with the previous study that reported the effects of LAMC2 expression inhibition on cell apoptosis in pancreatic ductal adenocarcinoma cancer cells27. We speculate that the effects of LAMC3 interference on cell apoptosis increase may be caused by cell cycle arrest in carboplatin-resistant ovarian cancer cells.

In recent years, RNA-Seq technology has gradually become a powerful tool to elucidate the pathogenesis and search for potential biomarkers in ovarian cancer research28,29,30. In this study, transcriptome sequencing was used to analyze the differentially expressed genes of carboplatin-resistant ovarian cancer cells with LAMC3 interference before and after carboplatin treatment, and to explore the potential mechanism of LAMC3 interference in regulating drug resistance of ovarian cancer. The results showed that, compared to the control group, 3029 differentially genes were up-regulated and 2944 differentially genes were down-regulated in carboplatin-resistant ovarian cancer cells with LAMC3 interference. GO functional enrichment found that these differentially expressed genes mainly concentrated in biological processed such as DNA replication and cell autophagy. KEGG pathway enrichment revealed that differentially expressed genes were mainly enriched in cell cycle, p53 signaling, ferroptosis, lysosome pathways. Furthermore, WB results showed that, compared with the control group, the expression changes of key regulatory proteins such as cell autophagy, ferroptosis, and lysosome in the LAMC3 interference-group were consistent with the transcriptomic analysis results. These results indicated that biological process such as cell autophagy, ferroptosis, and lysosomal pathways mediated by these differentially expressed genes may play a crucial in LAMC3 interference reducing drug resistance of carboplatin-resistant ovarian cancer cells, and the detailed action mechanism needed to be further investigated. As we have not conducted the systematic verification experiments, the action mechanism of LAMC3 interference affecting drug resistance of carboplatin-resistant ovarian cancer cells, particularly concerning the GO and KEGG enrichment analysis result findings, such as p53 signaling, ferroptosis, remains unclear. Further studies will focus on these promising results from transcriptome sequencing in the present study.

Conclusions

In conclusion, LAMC3 interference lowered drug resistance of carboplatin-resistant ovarian cancer cells mainly through affecting cell cycle, autophagy, ferroptosis, and lysosome. The present study provides a novel idea for the application of LAMC3 in drug resistant treatment of ovarian cancer.