miR-19-3p Targets PTEN to Regulate Cervical Cancer Cell Proliferation, Invasion, and Autophagy

Background Cervical cancer is the second most common cancer among women worldwide. Extensive studies have shown that microRNAs (miRNA/miR) can regulate the formation, progression, and metastasis of cancer. The purpose of this study was to investigate the effect of miR-19-3p on the proliferation, invasion, and autophagy of cervical cancer cells and to explore the underlying mechanism. Methods SiHa and HeLa cells were transfected with miR-19-3p mimic and inhibitor. miR-19-3p and PTEN expression were detected using real-time quantitative PCR and western blot, respectively. The binding between miR-19-3p and PTEN was predicted using Targetscan7.2 and verified by a dual-luciferase reporter gene assay. The effects of miR-19-3p on cell invasion and proliferation were evaluated by Transwell assays and MTT, respectively. The effect of miR-19-3p on autophagy was observed using fluorescence microscopy. Results The expression of miR-19-3p in cervical cancer tissues and SiHa and HeLa cells was significantly upregulated, whereas the expression of PTEN was significantly downregulated. PTEN was one of the direct targets of miR-19-3p. The miR-19-3p mimic significantly reduced the apoptosis rate and autophagy and promoted cell proliferation and invasion of the SiHa and HeLa cells. Conclusion In summary, miR-19b-3p can target PTEN to regulate the proliferation, invasion, and autophagy of cervical cancer cells. Our findings indicate the potential of miR-19-3p as a target for cervical cancer treatment in the future.


Introduction
Cervical cancer is the second leading cause of cancer-related deaths among women worldwide [1,2]. Persistent infection with high-risk human papillomavirus (HPV) is considered the main risk factor for cervical cancer [3]. About 50% of cervical cancers are related to persistent HPV16 infection, and about 20% are related to persistent HPV18 infection [4][5][6]. However, only ∼15% of women with persistent highrisk HPV infection will develop cervical cancer, which indicates that other factors are involved in the regulation of the occurrence and development of cervical cancer [7]. Te HPV vaccine has no protective efect on patients who are already infected with HPV or have cervical lesions. Terefore, understanding the pathogenesis of cervical cancer is important for developing therapeutic targets for this type of cancer.
MicroRNAs (miRNA/miR) are small noncoding singlestranded RNAs (19)(20)(21)(22)(23)(24)(25) nucleotides in length) [8]. miRNA silences its target genes via base-pairing with complementary sequences of 3′-untranslated region (3′-UTR) in the mRNAs of the target gene, thereby inhibiting translation or inducing mRNA degradation [9,10]. In this way, miRNA is involved in many biological processes, including cell proliferation, apoptosis, cell diferentiation, and development [11][12][13][14][15]. Accumulating evidence supports the importance of miRNA in cervical cancer. For example, Wang et al. reported that miRNA signatures can be useful for the screening, diagnosis, and prognosis of cervical cancer [10,16,17]. Hu et al. and Park et al. reported that miR-200a, miR-9, and miR-944 can be used to predict the survival rate of cervical cancer [18,19]. Te aberrant expression of miR-466 and miR-34a is closely associated with the occurrence and development of cervical cancer [20,21]. miR-21 can promote the proliferation of HeLa cells by targeting programmed cell death [22]. It has also been shown that miR-361 targets HSP90 to inhibit the invasion and epithelial-mesenchymal transition (EMT) of SiHa and HeLa cells [23]. Tere is increasing evidence about miRNA as a new biomarker and therapeutic target for cervical cancer. Upregulation and downregulation of miR-19b have been reported in various cancers, including rectal cancer [24], breast cancer [25], lung cancer [26], and pancreatic cancer [27], and miR-19b has been identifed as a key regulatory molecule in the mechanism of cancer development. However, the role of miR-19b-3p in cervical cancer remains unknown.
In this study, SiHa and HeLa cells were transfected with miR-19-3p mimics and inhibitors to detect cell proliferation, invasion, and autophagy. Te potential target of miR-19-3p was determined using the bioinformatics analysis tool TargetScan7.2 and verifed using a dual-luciferase reporter assay. Tis study will clarify the molecular mechanism of miR-19b-3p in regulating the occurrence and development of cervical cancer. We present the following article in accordance with the MDAR reporting checklist.

Tissue Samples.
In total, 20 pairs of cervical cancer tissues (tumour) and adjacent tissue (normal) samples were obtained from HPV16/HPV18-positive cervical cancer patients (age: 53.20 ± 10.21 years) who were admitted to Fuyang City People's Hospital from July 2019 to June 2020. For control, normal cervical exfoliated cells were collected from 10 healthy subjects who were HPV negative and without cervical lesions. Te study was approved by the Institutional Ethics Board of Fuyang City People's Hospital (no. (2020)0026), and informed consent was obtained from all individual participants.

Transwell Invasion Assay.
Te serum-free cell suspension (5 × 10 5 cells/ml; 200 μl) was added to the Transwell upper chamber, and 500 μl of DMEM medium containing 10% FBS was added to the lower chamber (Corning, USA). Te Transwell chambers were cultured at 37°C and 5% CO 2 for 24 h. Ten, the cells were fxed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet. Next, the cells were observed under an inverted fuorescence microscope (Axio Observer, Zeiss, Germany), and the migrated cells were counted.

Real-Time Quantitative PCR (RT-qPCR).
Total RNAs were extracted using TRIzol (10296010, Invitrogen, USA). Te reverse transcription of RNA into cDNA was performed using a PrimeScript ™ RT reagent kit (RR037A, Takara, Japan). We used a Mir-X miRNA frst-strand synthesis kit (638315, Takara, Japan) to detect the expression of miR-19-3p, and U6 was the internal reference gene. Using the TB Green ® Premix Ex Taq ™ II kit (RR820 B, Takara, Japan), we detected the expression of MUC4 mRNA, with GAPDH being the internal reference gene. Te primers were purchased from Sangon Biotech (Shanghai) Co., Ltd (Table 1). Te PCR procedure was 95°C for 5 min followed by 40 cycles at 95°C for 15 s and 60°C for 30 s. Te reaction was performed using a fuorescence quantitative PCR instrument (AFD4800, AGS, Hangzhou, China). Te relative expression levels were calculated using the 2 −ΔΔCt method [28].
2.6. Western Blot Analysis. RIPA Lysis Bufer (BL504A, Biosharp) was used to extract the total protein from the cervical cancer tissues and cells, and the protein concentration was determined by BCA (BL521A, Biosharp). Te protein sample (40 μg) was subjected to 10% SDS-PAGE. Te protein was then transferred to the PVDF membranes. After blocking with 5% BSA (4240GR025, Biofroxx) for 2 h at room temperature, the membranes were incubated with anti-PTEN (1 : 1000, ab32199, Abcam) and anti-GAPDH (1 : 1000. ab9485, Abcam) antibodies at 4°C overnight. After washing 3 times with TBST for 5 min each time, incubation with HRP-labelled secondary antibody (1 : 2000 dilution, ZB-2301, ASGB-BIO, China) was performed for 1 h at 37°C. Enhanced chemiluminescence (PE0010, Solarbio) was used   two-tailed t-test was used to compare the diferences between the two groups. A one-way ANOVA was used to compare diferences among multiple groups. Te correlation between two variables was analysed using Pearson correlation. P < 0.05 was considered to be signifcant [31].

Upregulation of miR-19-3p and downregulation of PTEN in Cervical Cancer. Te relative expressions of miR-19-3p
and PTEN mRNA were detected using RT-qPCR. Our results showed that the relative expression of miR-19-3p in the tumour samples was signifcantly higher than in the normal samples (P < 0.01) (Figure 1(a)), while the relative expression of PTEN mRNA in the tumour samples was signifcantly lower than in the normal samples (P < 0.01) (Figure 1(b)). For the expression of miR-19-3p and PTEN in SiHA, HeLa, and NC cells, the results showed that the relative expression of miR-19-3p in SiHa and HeLa cells was signifcantly higher than in the NC cells (P < 0.01) (Figure 1(c)). However, the relative expression of PTEN mRNA in the SiHa and HeLa cells was signifcantly lower than that in the NC cells (P < 0.01) (Figure 1(d)). Te expression of PTEN protein in tumour and normal samples was detected by western blot. Te results showed that the expression of PTEN protein in tumour samples was signifcantly lower than in normal samples (P < 0.01) (Figures 1(e) and 1(f )). Tese results suggest that miR-19-3p is upregulated and PTEN is downregulated in cervical cancer.

Te Proliferation and Invasion of Cervical Cancer Cells are
Regulated by miR- 19-3p. Te mimics and inhibitors were transfected into SiHa and HeLa cells. Te relative expression of miR-19-3p was detected using RT-qPCR. Cell proliferation was detected with an MTT assay, and cell invasion was detected with a Transwell assay. Te results showed that the miR-19-3p expression, cell proliferation, and invasion after transfection showed the same trend in the SiHa and HeLa cells. Te relative expression of miR-19-3p in the mimic group was signifcantly higher than that of the mimic-NC group (P < 0.01), while the relative expression of miR-19-3p in the inhibitor group was signifcantly lower than in the inhibitor-NC group (P < 0.01) (Figures 2(a) and 2(b)). Te proliferation of SiHa and Hela was signifcantly upregulated at 48 h after mimic transfection (P < 0.01) but was signifcantly downregulated at 48 h and 72 h after transfection with inhibitor (P < 0.01) (Figures 2(c) and 2(d)). Te invasion of the SiHa and HeLa cells was signifcantly inhibited at 48 h after transfection with the inhibitor (P < 0.05) (Figures 2(e) and 2(f )). Tese results suggest that miR-19-3p regulates the proliferation and invasion of cervical cancer cells. 19-3p. Te degree of autophagy was observed using fuorescence microscopy after miR-19-3p mimic transfection. Te results showed that SiHa and HeLa in the mimic, mimic-NC, and inhibitor-NC groups mainly exhibited green fuorescence, and there was little red fuorescence in the cytoplasm and nucleus, suggesting that there was virtually no autophagy. Te cells in the inhibitor group showed obvious red fuorescence, indicating that a large number of acidic autophagic lysosomal vacuoles had formed and that early apoptosis was induced ( Figure 3).

Discussion
Tere has been increasing evidence that dysregulated miRNAs are involved in the regulation of a variety of physiological processes in tumours, including cell proliferation, drug resistance, apoptosis, metastasis, and angiogenesis [10,32,33]. Research on miRNAs focuses on cancer-specifc up-or downregulated miRNAs and their target genes in order to clarify the pathogenesis of cancer [34,35]. Numerous reports have shown that miR-19 positively regulates tumorigenesis, EMT, cancer cell proliferation and invasion, and metastasis by targeting diferent targets in rectal cancer [24], breast cancer [25], pancreatic cancer [26], osteosarcoma [36], and lung cancer [27,37], indicating that miR-19 plays a carcinogenic role in cancer development. Our research confrmed that miR-19-3p is upregulated in cervical cancer and that miR-19-3p upregulation in vitro promotes the proliferation and invasion of SiHa and HeLa cells. Tese results indicate that miR-19-3p exerts a carcinogenic efect in cervical cancer. Te target of miR-19-3p was further predicted and analysed to determine Genetics Research 5 the potential mechanism of miR-19-3p in the occurrence and development of cervical cancer. Te PI3K/AKT pathway plays a vital role in cancer development. As a tumour suppressor gene, PTEN is a key negative regulator of the PI3K/AKT pathway, which is involved in regulating cell biological processes such as cell proliferation, invasion, and cycle arrest [38,39]. Downregulation of PTEN expression in a variety of cancers has been reported [40][41][42]  metastasis [45]. In this study, it was predicted that using TargetScan7.2 that PTEN is a target gene of miR-19-3p, and a dual-luciferase reporter gene assay further confrmed that miR-19-3p could bind specifcally to the 3′-UTR of PTEN. We further found that the miR-19-3p mimics upregulated miR-19-3p in SiHa and HeLa cells and inhibited the expression of PTEN protein. However, the miR-19-3p inhibitor reversed the inhibitory efect on PTEN. Tese results indicate that miR-19-3p can negatively regulate the expression of the target gene PTEN.
In summary, miR-19-3p can regulate the biological functions of cervical cancer cells by targeting PTEN. miR-19-3p is expected to become a potential therapeutic target for cervical cancer.

Conclusions
miR-19-3p downregulates the expression of PTEN by directly targeting the 3′-UTR of PTEN mRNA, thereby regulating the biological behaviours of cervical cancer cells, such as cell proliferation, invasion, and autophagy. Tis research provides new insights into the diagnosis and treatment strategies for cervical cancer.

Data Availability
Te data used to support the study are available from the corresponding author upon request.

Ethical Approval
All works were conducted in accordance with the Declaration of Helsinki (1964). Te study was approved by the Institutional Ethics Board of Fuyang City People's Hospital (no. [2020]0026).

Consent
Informed consent was obtained from all individual participants.