Effect of lncRNA LINC00324 on cervical cancer progression through down-regulation of miR-195-5p

Abstract Background Long non-coding RNAs (lncRNAs) have been widely used in the exploration of diseases in recent years. This paper introduced the significance of lncRNA LINC00324 (LINC00324) on the progression of cervical cancer and explored the mechanism of action and potential prognosis of LINC00324. Methods The cervical cancer tissues and adjacent normal tissues of 120 people were collected as research samples. The expression level of LINC00324 was assessed by RT-qPCR, as was miR-195-5p. Knockdown of LINC00324 on the proliferation ability of cervical cancer cells was determined with the help of cell counting kit-8 (CCK-8), and the number of cell migration and invasion was detected by the Transwell method. Luciferase reporter gene assay was used to analyse the correlation of LINC00324 and miR-195-5p. Kaplan–Meier survival curves and multivariate Cox analysis explained the potential prognostic significance of LINC00324 in cervical cancer. Results Significantly increased expression of LINC00324 and down-regulated miR-195-5p were negatively correlated in cervical cancer. Knockdown of LINC00324 inhibited the progression of cervical cancer, which was related to its mechanism of targeting and downregulating miR-195-5p. In addition, low expression of LINC00324 may prolong the survival period of patients with cervical cancer. Conclusions LINC00324 targets miR-195-5p to regulate the progression of cervical cancer and have potential as a prognostic molecular marker for cervical cancer. Plain Language Summary This paper introduced the mechanism and prognostic potential of LINC00324 in cervical cancer. The study found that LINC00324 expression was significantly elevated, while miR-195-5p level was down-regulated in cervical cancer. LINC00324 sponging miR-195-5p regulated the proliferation, migration and invasion of cervical cancer cells, thereby affecting the progression of cervical cancer. LINC00324 may be a prognostic biomarker for cervical cancer, providing a new direction for the treatment of patients.


Introduction
Cervical cancer is the only gynaecological malignant tumour with clear aetiology, which is associated with persistent infection of high-risk human papillomavirus (HPV) (Lan et al. 2020).The latest report claims that cervical cancer is the fourth most common malignancy among women worldwide, as well as the death rate.Meanwhile, the incidence of cervical cancer was higher in developing countries than in developed countries, with the second-highest incidence (Liu and Wang 2019, Yayla et al. 2019, Wang et al. 2022).Although great progress has been made in the prevention and treatment of cervical cancer, the five-year mortality rate of advanced patients is still high, and the molecular mechanism of malignant transformation of cells has not been fully revealed (Zhang and Fu 2021).Therefore, it is of positive significance for the treatment and prognosis of patients to understand the pathological mechanism of cervical cancer through molecular means.Long non-coding RNAs (lncRNAs) are related to a variety of biological processes such as development, metabolism and epigenetic regulation, with a length of more than 200 nucleotides (Lei et al. 2020, Li et al. 2022).A number of studies have stated that the abnormal expression of lncRNAs may lead to the disorder of various biological processes such as cell growth, proliferation and apoptosis, inducing the deterioration of cells and the formation of cancer (Zhang et al. 2022).LncRNAs have gradually developed into a broad and highly active research focus in tumourrelated aspects.LINC00324 is a lncRNA with a length of 2082 bp located on chromosome 17p13.1 (Xia et al. 2022).In previous studies, LINC00324 accelerated the apoptosis of papillary thyroid cancer cells by inhibiting Notch signalling, thereby delaying tumorigenesis (Wan et al. 2020).LINC00324 was reported in liver cancer (Gao et al. 2020), non-small cell lung cancer (Zhang et al. 2020) and gastric cancer (Wang et al. 2020a).Notably, the relevant mechanisms and functions of LINC00324 in cervical cancer remain to be studied.
Since there is a close correlation between molecules, lncRNAs have also been confirmed to participate in the regulation of post-transcriptional gene expression levels, which can act as molecular sponges for microRNAs (miRNAs) to mediate the levels of miRNAs and the regulation of miRNAs on their target genes, and ultimately regulate cell growth and tumour biological processes (He et al. 2020, Chu et al. 2021).For example, related studies in the field of cancer found that the lncRNA CASC11 affected the formation of bladder cancer, most likely through sponge miRNA-150 (Luo et al. 2019).For another example, lncRNA FEZF1-AS1 competitively bound miRNA-1254 to activate the biological behaviour of cervical cancer cells (Liang et al. 2021).Therefore, the regulation of the biological activity of LINC00324 targeting miRNAs in cervical cancer warrants further investigation.
This study explored whether LINC00324 plays a biological role in the generation and development of cervical cancer by targeting miR-195-5p, and focused on the expression level, molecular mechanism and prognostic value of LINC00324, providing a theoretical basis for the search of prognostic biomarkers and the exploration of clinical treatment methods for cervical cancer patients.

Acquisition of clinical samples
This study collected a group of independent tissue samples from cervical cancer patients, which were composed of 120 cervical cancer patients randomly selected from ShiJiaZhuang Maternity & Child Healthcare Hospital (approval number: 2016-03-21).Inclusion criteria for patients with cervical cancer: (1) all participants were diagnosed and confirmed by specialists.( 2 The obtained tissue samples were used to quantify the expression of LINC00324 and miR-195-5p in cervical cancer.LINC00324 expression and clinicopathological characteristics are recorded in Table 1.Moreover, the correlation between the expression of LINC00324 and the prognosis of cervical cancer patients is recorded in Table 2.

Cell culture and transfection
Cervical epithelial cell line H8 and cervical cancer cell lines (ME-180, Hela, SiHa and MS751) were cultured in DMEM medium (Hyclone, South Logan, UT) mixed with 10% foetal bovine serum (FBS) from American Type Culture Collection (ATCC).The CO 2 constant temperature incubator was set at 37 � C for the subculture of the cells mentioned above.

Gene expression identification
Cervical cancer samples were extracted with TRIzol reagent (Thermo Fisher, Waltham, MA) to obtain total RNA.RNA with qualified quality and concentration was used as a template, and Takara Prime Script TM RT reagent Kit was introduced to configure reverse transcription system to obtain corresponding cDNA, and then SYBR V R Premix Ex Taq TM II (Takara, Maebashi, Japan) was combined with specific primers to configure real-time quantitative PCR (RT-qPCR) system.The reaction was run on a Bio-Rad IQ-5 fluorescence quantitative PCR instrument (Hercules, CA) according to the program.For LINC00324 and miR-195-5p, GAPDH and U6 were selected as internal controls, respectively, and the data were processed through 2 -DDCt methods.

CCK-8 method to detect the cell proliferation ability
The transfected Hela and SiHa cells were prepared in DMEM complete medium to prepare cells suspension.The mixture was evenly mixed and inoculated on 96-well plates in 37 � C, 5% CO 2 constant temperature cell incubator.Cell counting kit-8 (CCK-8) reagent was pipetted into each well at fixed time points and the optical density (OD) at a wavelength of 450 nm was measured with a microplate reader after 2 h of incubation.The line chart of cell growth was drawn with the detection time as abscissa and OD 450 nm as ordinate.

Transwell assay to measure cell functionality
Based on the inoculum volume of 5 � 10 4 cells/200 mL, the Hela and SiHa cell suspensions were prepared in DMEM medium and added to the upper part of the Transwell chamber, while DMEM complete medium containing FBS was added to the bottom of the chamber.After 48 h culture, cells transferred to the bottom were stained with 0.1% crystal violet solution for 30 min.After washing with PBS, the number of cells that migrated was counted.For invasion assays, Matrigel (BD Biosciences, Franklin Lakes, NJ) was required to be moved to the upper Transwell chamber.

Luciferase reporter gene activity assay
Hela and SiHa cells were cultured in 24-well plates and cotransfected with luciferase reporter plasmid, wild-type LINC00324 (WT-LINC00324) and mutant-type LINC00324 (MUT-LINC00324) or mimic NC, inhibitor NC, miR-195-5p mimic and miR-195-5p inhibitor with Lipofectamine 2000 reagent.After 48 h, the cells were rinsed twice with PBS, and then transferred to the luciferase detection reagent for activity assay.

Statistical analyses
All statistical analysis and graphing were carried out by GraphPad 7.0 (La Jolla, CA) and SPSS 20.0 software (SPSS Inc., Chicago, IL).The differences between two groups were analysed by Student's t-test, while one-way analysis of variance (ANOVA) was used for multiple groups.The relationship between LINC00324 expression and clinical parameters of cervical cancer patients was detected by Chi-square test.The prognostic properties of LINC00324 were determined by Kaplan-Meier's survival curves and multivariate COX analysis.

Relationship of LINC00324 and miR-195-5p in cervical cancer tissue
LINC00324 and miR-195-5p levels in tissues of 120 patients were measured.Figure 1(A) suggests that the relative expression of LINC00324 in cancer tissues was higher than that in normal tissues.In Figure 1(B,C), miR-195-5p was relatively decreased, which showed a negative correlation with LINC00324 (r ¼ −0.6926, p < .001).

Expression of LINC00324 and clinicopathological features of cervical cancer patients
The division of LINC00324 low-expression and high-expression groups was based on the median expression of LINC00324, and Chi-square analysis was performed with the clinicopathological data of cervical cancer patients, and recorded in Table 1.It could be seen that LINC00324 expression was related to lymph node metastasis (p ¼ .013)and TNM stage (p ¼ .028),whereas the characteristics of cervical cancer patients such as age and tumour size were not statistically significant.

LINC00324 and miR-195-5p in cervical cancer cells
Through the incubation and subculture of cervical cancer cells, the relative expression of LINC00324 and miR-195-5p was evaluated.Figure 1(D) shows that LINC00324 was obviously upregulated in cervical cancer cells (ME-180, Hela, SiHa and MS751) compared with H8 cell lines.The expression of miR-195-5p in cervical cancer cells was consistent with its expression in tissues, showing a downward trend (Figure 1(E)).Hela and SiHa cells with sensitive expression were selected for in vitro assays, and si-LINC00324 was transfected into the cells.The transfection efficiency is shown in Figure 1(F).

LINC00324 interacted with miR-195-5p
Bioinformatics speculated that LINC00324 could be used as endogenous competitive RNA to bind miR-195-5p, and sequence alignment analysis found that LINC00324 had binding sites for miR-195-5p (Figure 2(A)).Figure 2(B,C) illustrates that transfection with miR-195-5p mimic inhibited the relative luciferase activity of WT-LINC00324 in Hela cells but did not weaken the luciferase activity of MUT-LINC00324.Meanwhile, miR-195-5p inhibitor markedly increased WT-LINC00324 luciferase activity, but there was no significant change in MUT-LINC00324, and the same was true in SiHa cells.This result demonstrated that LINC00324 directly targets miR-195-5p in cervical cancer.

Biological behaviour of LINC00324 in cervical cancer cells
The changes in the proliferation ability of the knockdown LINC00324 cells were detected by CCK-8 method, and the

Prognostic significance of LINC00324 in cervical cancer prognosis
Kaplan-Meier's survival curves and multivariate Cox regression analysis confirmed the prognostic potential of LINC00324 in patients with cervical cancer.In Figure 3, the lifetime of cervical cancer patients in the low-expression LINC00324 was higher than the high-expression LINC00324 (log-rank p ¼ .032).Multivariate Cox regression analysis further supported that LINC00324 was one of the notable factors associated with prognosis of cervical cancer patients (HR ¼ 3.830, 95% CI: 1.581-9.278,p ¼ .003;Table 2).

Discussion
Cervical cancer is a kind of gynaecological tumour with clear aetiology, which often occurs in women aged 20-50 who are sexually active (Szarewski 2012, Zhao et al. 2014).In addition to high-risk HPV infection, unhealthy sexual life, personal hygiene, obesity and drug abuse are also high-risk factors for cervical cancer (Farshbaf-Khalili et al. 2015, Tapera et al. 2019, Vargiu et al. 2022).In recent decades, the prevention and treatment of cervical cancer include HPV vaccination, regular gynaecological examination, resection, targeted and immunotherapy (Ishiguro et al. 2021).Nevertheless, the persistent infection of HPV, the spread of cervical cancer cells and the occurrence of complications still lead to poor prognosis of many patients in the middle and late stage, which threaten their life health (She et al. 2020, Giannini et al. 2023).Therefore, on the premise of preventing and screening the possible formation of cervical cancer (Giannini et al. 2022), identifying lncRNAs related to the occurrence of cervical cancer and explaining the mechanism of action play a key role in the prognosis of cervical cancer.
Studies related to LINC00324 have shown that the LIN00324 level was increased in colorectal cancer, while knockdown of LIN00324 negatively affected the biological effects of the cells (Ni et al. 2019).Pan et al. reported that LINC00324 was upregulated in lung adenocarcinoma and regulated progression by controlling levels of downstream miR-615-5p (Pan et al. 2018).Also, abnormal elevation of LINC00324 in oesophageal squamous cell carcinoma can be regarded as a liquid biopsy marker to monitor tumour occurrence (Sharma et al. 2022).As reported above, LINC00324 was increased compared to normal controls, both in cervical cancer tissues and cells.In vitro cell function assays indicated that the deletion of LINC00324 markedly inhibited the cells.Based on the scientific background of lncRNA as miRNA molecular sponge, this study investigated whether LINC00324 targets miR-195-5p to play a regulatory function in cervical cancer.Sequence alignment and online prediction found that LINC00324 had the basis for adsorbing miR-195-5p.The luciferase reporter gene assay demonstrated that miR-195-5p only affected the activity of WT-LINC00324 without changing the activity of MUT-LINC00324, that is, miR-195-5p was confirmed to be a target molecule of LINC00324.Some evidence has suggested miR-195-5p mediated the progression of colorectal cancer (Lin et al. 2019), lung cancer (Bu et al. 2021) and breast cancer (Yang et al. 2019).For instance, miR-195-5p targets YAP1 to function as a tumour suppressor in cervical cancer (Liu et al. 2020).The miR-195-5p/MMP14 axis suppressed the biological functions of cervical cancer cells and tumour progression (Li et al. 2018).Apart from that, ARL2 (Pan et al. 2019) and VEGF-A (Wang et al. 2020b) were considered to be direct targets of miR-195-5p as well.Afterwards, reversal experiments confirmed that the ability of silencing LINC00324 to inhibit the growth of cells was restored by miR-195-5p inhibitor, which further clarified that the interaction between LINC00324 and miR-195-5p slowed down the progression of cervical cancer.Clinical correlation analysis elucidated that LINC00324 and miR-195-5p level in tissues was negatively correlated, and the overall survival rate of cervical cancer patients with low LINC00324 expression was higher than that of the high expression group, indicating that LINC00324 was closely related to the prognosis of cervical cancer patients.
This study demonstrated for the first time that silencing LINC00324 suppressed the progression of cervical cancer by targeting and negatively regulating miR-195-5p, and pointed out the prognostic power of LINC00324 in the treatment of cervical cancer.Admittedly, there are still some shortcomings in our research, the mechanism of LINC00324 and miR-195-5p in cervical cancer needs to be further explored in detail.What is more, the biological function of LINC00324 was only understood at the cytological level in vitro in this study, which needs to be explained by in vivo assays.On this basis, combined with the existing treatment methods to explore the pathological pathway of cervical cancer, such as laparoscopic radical hysterectomy and immunotherapy (Monk et al. 2022, Di Donato et al. 2023), which is also a necessary choice to improve the prognosis and survival of patients.

Conclusions
In short, LINC00324 was elevated, while miR-195-5p was decreased in cervical cancer.LINC00324 sponging miR-195-5p was first proposed to regulate the growth, migration and invasion of cervical cancer cells, suggesting that LINC00324 has the potential as a prognostic marker for cervical cancer patients.It provided valuable ideas for the mechanism research and prognosis evaluation of cervical cancer patients.
) All participants had clear clinical information recorded.(3) Patients did not receive other treatment before surgery.Exclusion criteria: (1) Cervical cancer patients with previous radiotherapy or chemotherapy.(2) Patients with other diseases or pregnancy.(3) Patients with uncertain diagnostic results.This study was approved by the hospital ethics committee and the consent of the patients, which conformed to the Enhancing the QUAlity and Transparency Of health Research (EQUATOR) network guidelines.
results are shown in Figure2(D,E).Knockdown of LINC00324 had an inhibitory effect on the growth of Hela and SiHa cells.In addition, recovery experiment was carried out to verify the sponge effect of LINC00324 on miR-195-5p, which suggested that the cellular effects of silencing LINC00324 were restored by miR-195-5p inhibitors compared to the control group.Transwell assay examined the influences of knockdown LINC00324 on the behaviour of Hela and SiHa cells.Silencing LINC00324 slowed the migration (Figure2(F)) and invasion (Figure2(G)) abilities of cervical cancer cells.Similarly, recovery experiments were performed to verify the mechanism of cell migration and invasion ability, and it was found that miR-195-5p inhibitor eliminated the inhibitory effect of silencing LINC00324.The above in vitro assays demonstrated that LINC00324 directly targeted and down-regulated miR-195-5p, which may play a role in inhibiting cervical cancer metastasis.

Figure 3 .
Figure 3. Prognostic significance of LINC00324 in cervical cancer prognosis.Kaplan-Meier's curves for cervical cancer patients with low and high expression of LINC00324 (log-rank p ¼ .032).

Table 2 .
Multivariate Cox analyses of prognosis of cervical cancer patients.