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Significance of miR-141 and miR-340 in cervical squamous cell carcinoma

Abstract Background We investigated the expression and clinical significance of miR-141 and miR-340 in cervical squamous cell carcinoma (CSCC). Methods Expression of miR-141 and miR-340 in CSCC, high-grade squamous intraepithelial lesion (HSIL), and normal cervical squamous epithelium were detected by qRT-PCR. PTEN was assessed by immunohistochemistry. Their relationship with clinicopathological features was analyzed. Results The changes of miR-141 and miR-340 were different in CSCC, HSIL, and normal squamous epithelium (P = 0.030). miR-141 expression was statistically significant in gross type, differentiation, uterine corpus invasion, nerve invasion, vagina invasion, and FIGO stage in CSCC (P < 0.05). miR-340 expression was related to tumor size, differentiation, nerve invasion, lymph node metastasis, and FIGO stage in CSCC (P < 0.05). miR-141 and miR-340 expressions were statistically significant in different ages (P < 0.05) in HSIL. The AUC of miR-141 in CSCC diagnosis and that of miR-340 in HSIL diagnosis were 0.893 and 0.764, respectively. The sensitivity and the specificity of miR-141 for diagnosis of CSCC were 95.0% and 60.8%, respectively, while those of miR-340 for diagnosis of HSIL were 90.0 and 48.6%, respectively. miR-141 and miR-340 expressions are associated with PTEN expression (P = 0.002 and P < 0.001). Conclusion miR-141 and miR-340 may be associated with their target gene PTEN and involved in the carcinogenesis of cervical squamous epithelium.


Introduction
Cervical cancer is one of the most common malignancies in women. The latest statistics show there are more than half a million new cases and more than 300,000 deaths every year worldwide and about 100,000 new cases of cervical cancer and 30,000 deaths in China [1]. According to the 2018 data, the global incidence of cervical cancer is 13 per 100,000 people. Among the histological types of cervical cancer, cervical squamous cell carcinoma (CSCC) accounts for about 90% [2].
The most involved molecular mechanisms of CSCC development include mutations in tumor suppressor genes (such as p53, p16, and PTEN), genetic susceptibility, chromosomal translocation, and single nucleotide polymorphism [3]. The study of microRNA (miRNA) in the occurrence of malignant tumors has opened up new directions for elucidating the molecular mechanism of the cancerization from cervical epithelial lesions to CSCC. miRNA is a type of highly conserved noncoding small RNA (18-25 nucleotides) that can bind to the 3′ untranslated region of the corresponding mRNA, thus inhibiting translation or promoting degradation of the corresponding mRNA and silencing gene expression after transcription [4]. miRNAs have various types, and they are involved in the regulation of nearly one-third of protein-coding genes. They also have the characteristics of multiple targets and tissue specificity. miRNA can regulate multiple target genes, and one target gene can be regulated by multiple miRNAs. Currently, it is believed that miRNA regulates a variety of tumor-related genes and participates in tumorigenesis through regulating oncogenes and tumor suppressor genes [5]. Some miRNAs play a role similar to tumor suppressor genes, such as miR-125, miR-15a, miR-143, miR-145, and miR-340, while some function as oncogenes, such as miR-21, miR-17, miR-18a, miR141, miR-155, and miR-19a.
The tumor suppressor gene PTEN/MMAC1/TEP1 (phosphatase and tensin homolog deleted on chromosome Ten/ mutated in multiple advanced cancer/TGF beta regulated and epithelial cell-enriched phosphatase1) is located on human chromosome 10q23.3, with a total length of 200 kb. It has nine exosomes and eight introns. It is the first identified tumor suppressor gene with the dual specific phosphatase activity, and its structural and functional abnormalities are commonly found in many human tumors [6]. Our previous study [7] found that the PTEN gene was a predictive target gene for multiple miRNAs in endometrial cancer. We wonder if PTEN plays the same role in cervical cancer.
In this study, we first detected miR-141 and miR-340 expressions in CSCC by RT-PCR as well as PTEN expression by immunohistochemistry and then analyzed their relationship with clinicopathological parameters. Furthermore, we explored the mechanism and significance of miR-141 and miR-340 in the transformation process from cervical precancerous lesions (intraepithelial lesions) to cancerous malignancy. Our findings may provide important clues for exploring the key molecules of squamous intraepithelial lesion progression to CSCC and may lay the foundation for the risk prediction of CSCC, as well as the study of the prognosis, and cancerization of cervical intraepithelial lesions.

Tissues
In total, 104 patients with CSCC and 20 patients with highgrade squamous intraepithelial lesions (HSILs) who were admitted to Affiliated Tumor Hospital of Xinjiang Medical University were enrolled in this study. Surgical treatment was the primary therapeutic option in all cases. The cancer tissue specimens (n = 104) were obtained by surgical resection. The inclusion criteria were as follows: (1) patients were with CSCC or HSILs of the first onset diagnosed with cytology and/or histological biopsy and/or postoperative pathology. (2) Patients did not receive treatment before surgery, including chemotherapy, radiotherapy, and endocrine therapy. Patients with unqualified tissue specimens. For control, ten subjects who underwent total hysterectomy because of uterine fibroids were enrolled. Normal cervical squamous tissues were collected from these ten control subjects. One hundred and four CSCC tissue samples were used for the detection of PTEN expression. Genomic DNA was extracted from tissue samples of 20 CSCCs, 20 high-grade squamous intraepithelial lesions (HSILs), and 10 normal cervical squamous tissues (controls).
The clinical data of ethnic group, age, lymph node metastases, grade of cervical carcinomas, FIGO stage, histopathological type, gross type, tumor size, differentiation, invasion depth, myometrial invasion, uterine corpus invasion, vascular invasion, nerve invasion, and vagina invasion were collected ( Table 1). The 2009 International Federation of Gynecology and Obstetrics (FIGO) guidelines and the 2014 World Health Organization criteria [2] were used for the classification of clinical staging and histopathological type of cervical carcinoma. The local Ethics Committee approved this study.

qRT-PCR
Total RNA was extracted using the RNeasy FFPE kit (Qiagen, Beijing, China). The Nanodrop-2000 spectrophotometer (UV-2800H, UNICO, USA) was used to determine the RNA quality and concentration. Next, RNA was reverse transcribed into cDNA. The reaction system was (15 μL in total): 10 ng RNA sample (5 μL × 2 ng/mL), 3 μL reverse transcription primer, 1.5 μL 10 × RT buffer, 0.15 μL dNTPs (100 mmol/L), 1 μL Muhiscribe TM reverse transcriptase, 0.19 μL 20 U/μL RNase inhibitor, and 4.16 μL ribozyme-free water. The reaction conditions were as follows: 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min. The single-tube TaqMan miRNA assays were used to detect and quantify mature miRNAs. U6 small nuclear RNA (Ambion, Austin, TX, USA) was used as an internal normalization control. The relative quantity of each miRNA was calculated by the comparative CT (2 −ΔΔCt ) method, in which ΔΔCt was calculated as follows: Ct miR of interest-Ct U6 cancer -Ct miR of interest-Ct U6 control.

Immunohistochemical staining
Immunohistochemistry was performed using the PV-6000 kit (Beijing Zhong Shan-Golden Bridge Biological Technology CO., Ltd, Beijing, China) according to the instructions. The antibody was anti-PTEN (Cat# 138G6, 1:500, Cell Signaling, USA). The color development was performed with DAB. After counterstaining with hematoxylin, the sections were observed under the microscope. The relative PTEN expression level was presented as the immunoreactive score, which was evaluated according to the positive staining percentage and staining intensity. The positive staining percentage was defined as: 0-1%, 0 point; 1-10%, 1 point; 11-33%, 2 points; 33-66%, 3 points; and ≥66% positive cells, 4 points. The staining intensity was evaluated as follows: negative staining, 0 point; weak staining, 1 point; moderate staining, 2 points; strong staining, 3 points. The immunoreactive score was 0-12. When the immunoreactive score was ≤3, it was considered as PTEN loss [8]. The ones without incubation of primary antibodies were used as negative controls.

Statistical analysis
SPSS (version 17.0; SPSS Inc., IL, USA) was used for statistical analyses. Differences between the variables were statistically evaluated using the Student's t-test and Chi-square test. P (two tailed) <0.05 indicates a statistically significant difference.

Correlation and significance of miR-141/ miR-340 expression in the diagnosis of CSCC and HSIL
Pearson correlation analysis showed that miR-141 and miR-340 expression levels were negatively correlated in CSCC (R = −0.480), but the difference was not significant (P = 0.092). In addition, in HSIL, miR-141 and miR-340 expression levels were also negatively correlated (R = −0.466); however, the difference was also not statistically significant (P = 0.178; Table 5). At the optimal cut-off point, the sensitivity for the diagnosis of CSCC with miR-141 was 95.0%, and the specificity was 60.8%. The area under the curve was 0.893 ( Figure 1a). However, the sensitivity of miR-340 for the diagnosis of HSIL was 90.0%, and the specificity was 48.6%; and the area under the curve was 0.764 ( Figure  1b; Table 6).

PTEN expression in CSCC
As the target gene of miR-141 and miR-340, PTEN was expressed in the cytoplasm and the nucleus of CSCC (Figure 2). The positive rate of PTEN in CSCC was 42.3% (44/104).

Relationship between PTEN expression and clinicopathological features of CSCC
In 104 cases of CSCC tissues, we found that PTEN expression was different in different age groups. However, there was no significant difference (P = 0.083). Moreover, there was no significant correlation between PTEN and other clinicopathological parameters, including ethnic group  Table 7).

Discussion
Cervical cancer is one of the most common female malignant tumors in developing countries and is also one of the main causes of deaths in females [9]. Currently, the pathogenesis of CSCC has been extensively studied. However, the role of miRNA in the occurrence of malignant tumors has opened up new directions for elucidating the molecular mechanism of cervical cancer. Moreover, various abnormalities of miRNAs have been found in cervical cancer [10].
MicroRNA are small, single-stranded, noncoding RNAs that can act as oncogenes or tumor suppressor genes in the progression of tumors. Studies have shown that miRNA is differentially expressed in cervical cancer tissue, cervical intraepithelial neoplasia, and normal cervical tissue [11]. Moreover, miRNA is related to the occurrence, metastasis, and invasion of cervical cancer, which may be used as markers for the treatment and prognosis. Thus, miRNAs provide potential new targets for targeted tumor therapy. It is found that the relative expression of miR-141 mRNA in bladder cancer tissue was significantly higher than that in normal tissue adjacent to cancer [12]. However, the expression levels of miR-141 and miR-340 as well as their target gene PTEN in CSCC have been less studied.
miR-141, as a member of miR-200 family, is located at 12p13.3 and plays an important role in regulating tumor cell proliferation, migration, differentiation, and apoptosis [13]. Studies have shown that miR-141 is abnormally expressed in colorectal cancer, non-small-cell lung cancer, and gastric cancer and is increased in ovarian cancer, breast cancer, prostate cancer, renal cell cancer, and bladder cancer [14,15]. miR-340 is located at 5q35.3  and regulates the cell cycle by regulating various target proteins, which in turn affects tumor invasion and metastasis [16][17][18]. It functions as a tumor suppressor and is downregulated in solid tumors such as breast cancer, prostate cancer, gastric cancer, osteosarcoma, and melanoma [19,20]. In this study, we found that miR-141 expression was mostly upregulated in CSCC, and it showed a downward trend in tissues from CSCC to HSIL and normal squamous epithelium. In contrast, miR-340 expression was mostly downregulated in CSCC and had an upward trend in the tissues from CSCC to HSIL and normal squamous epithelium. These results indicate that miR-141 and miR-340 may play important regulatory roles in the development of normal cervical squamous epithelium into HSIL and even CSCC. We found that miR-141 was closely related to the gross type, differentiation, uterine corpus invasion, nerve invasion, vagina invasion, and FIGO stage in CSCC. At the same time, miR-340 was closely related to the tumor size, differentiation, nerve invasion, lymph node metastasis, and FIGO stage in CSCC. These results indicate that miR-141 and miR-340 are involved in the progression of CSCC and can be used as indicators for the progress evaluation of CSCC, but the specific mechanism is yet to be further studied.
In addition, we also found that miR-141 and miR-340 showed a negative correlation in CSCC and HSIL. But the difference was not statistically significant. These results suggest that miR-141 and miR-340 may have opposite regulatory functions in the process from HSIL to CSCC. However, this conclusion still needs to be verified by further expanding the sample size. The loss expression of PTEN, the common target gene of miR-141 and miR-340, is the molecular mechanism involved in various malignant tumors [21][22][23][24]. Our results showed that the PTEN-positive expression rate in CSCC was 42.3%. In addition, PTEN expression may be related to age, but not to the ethnic group, gross type, tumor size, differentiation, invasion depth, uterine corpus invasion, vascular invasion, nerve invasion, vagina invasion, lymph node metastasis, and FIGO stage. Therefore, we speculate that miR-141 and miR-340 might have not only common but also different regulatory effects on the pathogenesis from normal cervical squamous epithelium to HSILs and invasive CSCC.

Conclusion
In conclusion, miR-141 and miR-340 participated in the process of abnormal hyperplasia of cervical epithelium to HSIL and in the development of CSCC. However, the underlying mechanisms may be different. We found that miR-141 and miR-340 had higher sensitivity and specificity for the diagnosis of CSCC and the diagnosis of HSIL, respectively, suggesting that the combination of miR-141 and miR-340 could be used for the diagnosis of CSCC and HSIL. However, the regulatory mechanism of miR-141 and miR-340 on target gene PTEN still needs further research and verification in cell models or animal models. Moreover, our research has good clinical application prospects. In clinical practice, the detection of miR-141 and miR-340, as well as the immunohistochemical detection of PTEN, can be applied to the early diagnosis of cervical intraepithelial lesions and CSCC, which would provide a basis for diagnosis, treatment, and prognosis of CSCC and squamous intraepithelial lesions.
Funding information: This study was supported by Natural Science Foundation of Shenzhen University General Hospital (SUGH2019QD013) and Natural Science Foundation of Xinjiang Uygur Autonomous Region (2016D01C364).

Conflict of interest:
The authors report no conflict of interest.
Data availability statement: All data generated during this study are presented within the manuscript.