Quan Rui ,Shi Jie ,Li Xiao ,Fu Shifeng ,Liu Wei

Abstract Objective:To investigate the effects of silencing neuropilin-1 (NRP-1) on the growth and autophagy of cervical squamous cell carcinoma SiHa cells and the survival of tumorbearing nude mice.Methods:NRP-1 lentiviral vector was constructed and transfected into SiHa cells.The cells were randomly divided into Control group,shRNA-NC group,NRP-1-shRNA1 group and NRP-1-shRNA2 group.RT-qPCR was used to detect the level of NRP-1 mRNA.Cell proliferation was detected by clone formation assay.Cell apoptosis was detected by flow cytometry.The protein expression levels of NRP-1,Bax,Bcl-2,Caspase-3,c-Myc,Beclin1,ATG7,LC3I and LC3II were detected by Western blotting.Tumor-bearing nude mice(injected subcutaneously with SiHa cells)were randomly divided into Control group and NRP-1-shRNA1 group.The tumor weight and survival rate of nude mice were detected.The apoptosis and autophagy in vivo were determined as well.Results:The NRP-1 mRNA and protein levels,c-Myc protein level and clone formation rate in NRP-1-shRNA1 and NRP-1-shRNA2 groups were significantly lower than those in Control group and shRNA-NC group,while the ATG7 protein level,cell apoptosis rate,Bax/Bcl-2 ratio,cleaved Caspase-3/Caspase-3 ratio,and LC3II/LC3I ratio were notably higher(P＜0.05).The in vivo experimental results showed a decrease in tumor weight and increases in the survival rate of nude mice,TUNEL-positive cells,Caspase-3 expression level,and LC3II/LC3I ratioin NRP-1-shRNA1 group (P＜0.05).Conclusion:Silencing NRP-1 can inhibit the proliferation of SiHa cells,promote apoptosis and autophagy,and promote the survival of the nude mice bearing the SiHa cervical squamous cell carcinoma xenograft.

Keywords cervical cancer;neuropilin-1;apoptosis;autophagy

Introduction

Cervical cancer is the fourth most common cancer among women in the world and is one of the main causes of cancer in women [1].Human papillomavirus (HPV) infection has been considered to be the main cause of cervical cancer,which is usually asymptomatic and transient [2].With the development of cervical cancer screening and prevention methods,such as HPV joint testing and HPV vaccination,cervical dysplasia and early screening for cancer can reduce the morbidity and mortality of cervical cancer[3].At present,the standard treatment guidelines for patients with cervical cancer are radiotherapy combined with cisplatin chemotherapy.Unfortunately,patients with advanced cervical cancer have a higher recurrence rate and poor survival rate in the first 5 years[4].Therefore,the identification of biomarkers that can be potentially used as indicators of normal biological process,pathogenic process or therapeutic response will have a direct and significant impact on the prognosis of patients.

NRP-1 is a member of neuropilins,which is mainly expressed on the membrane surface of nerve cells,tumor cells and vascular endothelial cells,and can promote angiogenesis and tumor development and metastasis[5].Previous studies have shown that the expression of NRP-1 is increased in cervical cancer,and its high expression is related to lymph node metastasis and clinical stage [6].The expression of silencing NRP-1 can reduce the viability of cervical cancer cells and the expression of immunosuppressive molecule TGF-β1,and induce the apoptosis of cervical cancer cells [7].However,the specific mechanism of NRP-1 on cervical cancer is still unclear.The purpose of this study was to explore the effects of silencing NRP-1 on the proliferation,apoptosis and autophagy of SiHa cells in cervical squamous cell carcinoma,and to further detect its effects on apoptosis,autophagy-related proteins and the survival of xenograft nude mice,so as to provide a new experimental basis for molecular targeted therapy of cervical cancer.

Materials and methods

Test reagents

Dulbecco’s modified Eagle’s medium,fetal bovine serum,trypsin and penicillin-streptomycin were purchased from Gibco Life Technologies.Cervical squamous cell carcinoma SiHa cell line was purchased from the cell bank of National Collection of Authenticated Cell Cultures;shRNA-NC,NRP-1-shRNA1 and NRP-1-shRNA2 vectors were constructed by Shanghai GenePharma Biotech Company.BCA protein concentration determination kit,Annexin V-FITC cell apoptosis detection kit,TUNEL cell apoptosis detection kit,horseradish peroxidase labeled goat antirabbit IgG were purchased from Shanghai Beyotime Biotechnology.Rabbit anti-NRP-1,Bax,Bcl-2,Caspase-3,cleaved Caspase-3,c-Myc,LC3I/LC3II,Beclin-1,ATG7,and β-actin monoclonal antibodies were purchased from Abcam.

Cell culture

SiHa cells were cultured in high glucose DMEM medium containing 10%fetal bovine serum and 1%penicillin-streptomycin and placed in 37 ℃,5%CO2incubator.The culture medium was changed every 3 days.The cells used in the experiment were logarithmic growth phase cells.

Cell processing and grouping

A total of 2 shRNA sequences were designed(NRP-1-shRNA1:5’-GATCCGCTACGACCGGCTAGAAATCTTTCAAGAGAAGATTTCTAGCCGGTCGTAGCTTTTTTG-3’,NRP-1-shRNA2:5’-GATCCGGGAAACTGGCATATCTATGATTCAAGAGATCATAGATATGCCAGTTTCCCTTTTTTG-3’) to interfered with the expression of NRP-1.ShRNA-NC,NRP-1-shRNA1 and NRP-1-shRNA2 were transfected into SiHa cells by LipofectamineTM2000.The cells were divided into Control group,shRNA-NC group,NRP-1-shRNA1 group and NRP-1-shRNA2 group.

RT-qPCR

The total RNA was extracted from SiHa cells of each group by Trizol method,and reverse transcribed into cDNA by PrimeScrip reverse transcription kit.The PCR reaction system was configured by SYBR Premix Ex Taq instructions.The upstream primer of NRP-1-shRNA1 was 5’-TACAGCATCCAATCAAGCCGACAG-3’,and the downstream primers 5’-ATCTTCTCATCTCCCAGGTCCACTT-3’.The upstream primer of NRP-1-shRNA2was 5’-CCACAGTGGAACAGGTGATG-3’,the primer of upstream primers was 5’-GCACGTGATTGTCATGTTCC-3’.The upstream primer of GAPDH was 5’-TGTGGGCATCAATGGATTTGG-3’,and the downstream primer of was 5’-ACACCATGTATTCCGGGTCAAT-3’.The reaction conditions were as follows:predenaturation at 95 ℃for 5 min,denaturation at 95 ℃for 30 s,annealing at 60 ℃for 30 s,extension at 72 ℃for 30 s,45 cycles.Taking GAPDH as internal reference,2-△△Ctmethod was used to calculate.

Detection of cell clone formation rate by clone formation assay

SiHa cells were cultured in a petri dish for 4 days and inoculated to a 6-well plate.About 500 cells in each well were cultured for 14 days.The cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.5%crystal violet.After PBS cleaning,the number of cell clones was observed and the cell clone formation rate was calculated.Cell clone formation rate(%)=number of cell clones/number of inoculated cells×100%.

Detection of apoptosis by flow cytometry

SiHa cells were collected into a 10 mL centrifuge tube,the number of cells per sample was 3×106/mL,1,500 r/min centrifuged for 5 min.The culture medium was discarded,washed once with incubation buffer,1,500 r/min centrifuged for 5 min.Resuscitated cells were incubated at room temperature without light for 15 min,incubated without light for 10 min after adding 10 μL Annexin V-FITC,and incubated without light for 30 min after adding 10 μL propidium iodide.The apoptosis of cells in each group was detected by flow cytometry.

Western blotting

The total protein was extracted from SiHa cells in logarithmic growth phase of each group,and the protein content was determined by BCA kit.The same amount of protein samples were extracted and denatured at 100 ℃for 5 min,then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane for 1 h.Primary antibodies NRP-1(1:1,000),Bax(1:1,000),Bcl-2(1:1,000),Caspase-3 (1:1,000),c-Myc (1:1,000),Beclin1 (1:1,000),ATG7 (1:1,000),LC3I (1:1,000),LC3II (1:1,000) and β-actin(1:1,000)were added into protein samples and incubated under 4 ℃overnight.The protein samples were cleaned,and added with horseradish peroxidase to label goat anti-rabbit IgG(1:10,000)at 4 ℃for 2 h incubation.The the ECL photoluminescence solution was added to the exposure process,and the gel imaging system was used to take pictures.The gray values of each band were counted by Image J.

Establishment of xenograft model in nude mice

Ten female 5-week-old Balb/c nude mice with weight(20±2) g were purchased from Beijing Vital River Laboratory Animal Technology Co.,Ltd.,license number:SCXK (Beijing) 2015-0001.The mice were raised under specific pathogen-free conditions.According to the method of Liang et al.[8],SiHa cells in logarithmic growth phase in Control group and NRP-1-shRNA1 group were selected and adjusted to 1×107cells/mL,which were injected subcutaneously on the back of nude mice near the armpit,0.2 mL per mice with 5 rats in each group.The mice reached tumor standard (tumor diameter≥0.5 cm).The survival of rats for 30 days was recorded and the survival curve for 30 days was drawn.The remaining rats were euthanized and obtained the tumor weight.

TUNEL staining

The tumor tissues of nude mice were made into paraffin sections and treated with dewaxing,hydration and transparency.TUNEL reaction mixture was added to the specimens,cover slides were added to react for 1 h,then rinsed with PBS solution for 3 times,DAB substrate was added for 10 min and rinsed for 3 times.The apoptosis rate of tumor cells was detected by microscope.Five visual fields were randomly selected from each section,and the total number of cells and the number of apoptotic cells were counted.The positive apoptotic cells showed brownish nucleus,and the apoptosis rate(%)=the number of apoptotic cells/the total number of tumor cells.

Immunohistochemistry

The tumor tissues of nude mice of each group were made into paraffin-embedded sections with a thickness of about 4 μm.The tumor tissues of each group were stained by immunohistochemistry according to the instructions of the immunohistochemical detection kit.The primary antibody was Caspase-3(1:200),the second antibody was horseradish peroxidase labeled goat anti-rabbit IgG,DAB chromogenic kit was used for coloration,hematoxylin for re-staining,and hydrochloric acid alcohol for color separation.Light microscope was used to observe the expression of Caspase-3 in the tumor tissue.Five visual fields were randomly selected,and the diffuse brown granules in the cytoplasm were considered as positive cells,and the percentage of positive cells was calculated.

Statistical analysis

GraphPad Prism 5 was used for statistical analysis.Data were expressed as mean±SD.LSD-ttest was used for inter-group comparison,and One-Way ANOVA was used for multi-group comparison.P＜0.05 was considered statistically significant.

Results

NRP-1 expression level

Compared with Control group and shRNA-NC group,the expression levels of NRP-1 mRNA and protein in NRP-1-shRNA1 and NRP-1-shRNA2 groups were decreased significantly(P＜0.05).Compared with NRP-1-shRNA1 group,the NRP-1 mRNA and protein levels in NRP-1-shRNA2 group were significantly increased(P＜0.05)(Figure 1).

Silencing NRP-1 reduces the clone formation rate of SiHa cells

Compared with Control group and shRNA-NC group,the clone formation rate of NRP-1-shRNA1 and NRP-1-shRNA2 groups decreased significantly (P＜0.05).Compared with NRP-1-shRNA1 group,the clone formation rate of NRP-1-shRNA2 group was significantly higher(P＜0.05)(Figure 2).

Figure 1 NRP-1 expression level.A:Detection of NRP-1 mRNA level by RT-qPCR.B:Detection of NRP-1 protein by Westernblotting.Compared with Control group,*P＜0.05;Compared with NRP-1-shRNA1 group,#P＜0.05.

Silencing NRP-1 promotes apoptosis of SiHa cells

Compared with Control group and shRNA-NC group,the apoptosis rate of NRP-1-shRNA1 and NRP-1-shRNA2 groups was significantly increased (P＜0.05).The apoptosis rate in NRP-1-shRNA2 group was significantly lower than that in NRP-1-shRNA1 group(P＜0.05).(Figure 3).

Silencing NRP-1 increases the ratio of Bax/Bcl-2 and cleaved Caspase-3/Caspase-3 and down-regulates the expression of c-Myc protein in SiHa cells.

Compared with Control group and shRNA-NC group,the ratio of Bax/Bcl-2 and cleaved Caspase-3/Caspase-3 in NRP-1-shRNA1 and NRP-1-shRNA2 groups increased significantly,while the level of c-Myc protein decreased significantly (P＜0.05).Compared with NRP-1-shRNA1 group,the ratio of Bax/Bcl-2 and cleaved Caspase-3/Caspase-3 was significantly lower and the level of c-Myc protein was significantly higher in NRP-1-shRNA2 group (P＜0.05)(Figure 4).

Silencing NRP-1 increases Beclin1,ATG7 protein levels and LC3II/LC3I ratio in SiHa cells

Compared with Control group and shRNA-NC group,the protein levels of Beclin1,ATG7 and LC3II/LC3I ratio in NRP-1-shRNA1 and NRP-1-shRNA2 group were significantly increased (P＜0.05).Compared with NRP-1-shRNA1 group,the protein levels of Beclin1,ATG7 and LC3II/LC3I ration in NRP-1-shRNA2 group were significantly lower (P＜0.05) (Figure 5).

Effect of silencing NRP-1 on xenograft in nude mice

Compared with Control group,the tumor weight of NRP-1-shRNA1 group decreased significantly,while the survival rate,apoptosis rate,Caspase-3 positive expression rate and LC3II/LC3I ratio of nude mice increased significantly(P＜0.05)(Figure 6).

Figure 2 Effect of silencing NRP-1 on the proliferation of SiHa cells.Compared with Control group,*P＜0.05;Compared with NRP-1-shRNA1 group,#P＜0.05.

Figure 3 Effect of silencing NRP-1 on apoptosis of SiHa cells.Compared with Control group,*P＜0.05;Compared with NRP-1-shRNA1 group,#P＜0.05.

Figure 4 Effect of silencing NRP-1 on the protein expression levels of Bax,Bcl-2,Caspase-3 and c-Myc in SiHa cells.Compared with Control group,*P＜0.05;Compared with NRP-1-shRNA1 group,#P＜0.05.

Figure 5 Effect of silencing NRP-1 on the protein expression levels of Beclin1,ATG7,LC3I and LC3II in SiHa cells.Compared with Control group,*P＜0.05;Compared with NRP-1-shRNA1 group,#P＜0.05.

Figure 6 Effect of silencing NRP-1 on xenograft nude mice.A:Tumor tissue of nude mice.B:Weight histogram of tumor tissue in nude mice.C:Line chart of survival rate of nude mice.D:Apoptosis of tumor tissue was detected by TUNEL staining and Caspase-3 was detected by immunohistochemistry.E:Histogram of apoptosis rate in tumor tissue.F:Histogram of positive expression rate of Caspase-3 in tumor tissue.G:Histogram of LC3II/LC3I ratio of tumor tissue.Compared with Control group,*P＜0.05.

Discussion

In many underdeveloped countries,the prognosis of patients with advanced cervical cancer is very poor due to the strong ability of proliferation,invasion and metastasis of cervical cancer [9].NRP-1 can participate in neuronal guidance,immune regulation,cardiovascular formation,cell migration,angiogenesis and tumorigenesis.Related studies have found that NRP-1 participates in the occurrence and development of cervical cancer by promoting angiogenesis,immunosuppression and other ways,and its expression level may be related to the prognosis of cervical cancer [10].In this study,by constructing the xenograft model of nude mice with cervical cancer,it was found that silencing NRP-1 could reduce the tumor weight,increase the survival rate and promote the apoptosis of tumor cells in nude mice.It is suggested that silencing NRP-1 can inhibit the growth of nude mice with cervical cancer.

The excessive proliferation and apoptosis imbalance of cancer cells is an important biological feature of cervical cancer.Inhibiting the proliferation of cervical cancer cells and regulating apoptosis is a necessary way to treat cervical cancer.Bax and Bcl-2 belong to Bcl-2 gene family.Bcl-2 is an inhibitor of apoptosis and Bax is a pro-apoptotic gene.The higher the ratio of Bax/Bcl-2 is,the more serious the apoptosis is[11].Caspase-3,a member of the Caspase family,belongs to apoptotic proteins.After activation,it can directly hydrolyze related proteins in cells and induce irreversible apoptosis [12].C-Myc protein can regulate cell proliferation and differentiation,and promote apoptosis.Cervical cancer is accompanied by overexpression and amplification of c-Myc[13].Li et al.[7]found that inhibition of NRP-1 gene expression can induce apoptosis of cervical cancer cells.The results in this paper is consistent with the above results,silencing NRP-1 expression can increase the ratio of Bax/Bcl-2 and cleaved Caspase-3/Caspase-3,and down-regulate the expression of c-Myc protein in Si-Ha cells.It is suggested that silencing NRP-1 can inhibit the proliferation of SiHa cells and promote apoptosis.

Autophagy is a major intracellular degradation process,which can inhibit the growth of some cancer cells when cells are subjected to certain stress conditions [14].Beclin1 gene,located on chromosome 17q21,can be used as a tumor suppressor gene to participate in the occurrence and development of most malignant tumors.Related studies have shown that down-regulation of Beclin1 expression in cervical cancer can reduce the cell’s ability of autophagy and increase the probability of tumorigenesis [15].ATG7 is an important protein in the regulation of autophagy,which can activate the process of autophagy and apoptosis by regulating the activity of many enzymes[16].Autophagy protein LC3 can be divided into LC3I and LC3II.LC3II locates on bilayer membrane and autophagosome and is a specific marker molecule of autophagosome.The ratio of LC3II/LC3I is positively correlated with the number of autophagy vesicles,which is the basis for judging the level of autophagy[17].In this study,it was found that silencing NRP-1 could increase the protein levels of Beclin1 and ATG7 and the ratio of LC3II/LC3I in SiHa cells.It is suggested that silencing NRP-1 can promote autophagy of SiHa cells.

In conclusion,silencing NRP-1 can inhibit the proliferation of SiHa cells,promote apoptosis and autophagy,inhibit the tumor formation ability of xenograft model of nude mice,and improve the survival ability of nude mice.NRP-1 is expected to become a target for molecular therapy of cervical cancer.

AcknowledgementsThis study was supported by the 2021-2022 Guiding Project of Hubei Healthcare Commission(No.WJ2021F013).