Reduced KISS1 expression acts as a predictor for aggressive features of cervical carcinoma and promotes migration and invasion in cervical carcinoma cells

Background Cervical carcinoma causes high morbidity and mortality in patients largely due to its invasion and metastasis. Kisspeptin (KISS1) had been found as metastasis suppressor in many malignancies. But the clinical significance and biological functions of KISS1 in cervical carcinoma was not elaborated. Methods The expression levels of KISS1 were detected by quantitative real time polymerase chain reaction (qRT-PCR), Methylation specific PCR (MSP) analysis and western-blotting in cervical carcinoma tissues and cells. A eukaryotic expression plasmid was transfected into cervical epithelial cells in order to initiate KISS1 overexpression, and the endogenous was silenced in cervical epithelial cells using short hairpin RNA (shRNA) sequences. Transfection efficiency was validated by western blotting assays. To explore the effects of KISS1 on the metastatic phenotype of cervical carcinoma, the cell proliferation was examined by CCK8 assay, and the cell migration and invasion were detected by cell scratch and transwell invasion assays, respectively. We investigated the relevance between KISS1 expression with its methylation and clinicopathological features of cervical carcinoma. Low expression of KISS1 predicts a poor prognosis and was associated with lymph node metastasis and TNM stage. And methylation of KISS1 promoter, which was linked with KISS1 inactivation, was also found in most clinical samples of aggressive cervical carcinoma. In vitro cervical carcinoma C33A cells, KISS1 overexpression significantly inhibited cell proliferation, migration and invasion in cells. While KISS1 knockdown promoted proliferation, migration and invasion in cervical epithelial cell line CRL-2614. In summary, low-expression of KISS1 play a suppressive and invasion in cervical carcinoma cells. Its expression and methylation are associated with clinical progression of cervical carcinoma. KISS1 new target cervical


Introduction
Cervical cancer is characterized a malignant tumor originating from the cervical epithelium, which 4 mainly metastasizes to the surrounding tissues, organs and lymph nodes via local invasion and infiltration [1]. Although various comprehensive treatments including surgical resection, radiotherapy, and chemotherapy have been used at different stages of the disease, the incidence, and mortality of impaired quality of life are still rising [2,3]. Therefore, it is necessary to explore new biomarkers and therapeutic targets that will help develop targeted therapies for cervical carcinoma.
Metastasis suppressor genes play a key role in tumor metastasis [1]. A previous study demonstrated that the effect of metastasis suppressor genes on tumor metastasis was more critical than that of metastasis promoter genes, and the reduction in the expression of metastasis suppressor genes or gene loss might induce the invasion and metastasis of tumor cells [4]. KISS1 was initially identified as an important tumor metastasis suppressor gene in human melanoma cells [5]. KISS1 is located on the long arm of human chromosome-1. The protein-encoding gene acts as an endogenous ligand for Gprotein coupled receptor 54 (GPR54) and produces a variety of physiological effects, including inhibition of tumor cell proliferation, metastasis, invasion, and induction of tumor cell differentiation and apoptosis [6,7]. The decrease in KISS1 levels and its role in tumor invasion and metastasis has been evaluated in various tumors, such as the bladder[8], colorectum[9] and breast cancer [10].
However, the expression of KISS1 and its pathogenesis in cervical carcinoma remains to be elucidated.
This study examined the methylation status and expression level of KISS1 in cervical carcinoma tissues, and then assessed the association between KISS1 methylation, KISS1 expression and clinical clinicopathological features. The effect of KISS1 on the biological function of cervical carcinoma cell lines was also studied. Our goal was to investigate the role of KISS1 in the development and progression of cervical carcinoma and whether it could prevent and treat clinical cervical carcinoma.

Materials And Methods
Patients and specimens. 97 cases of human cervical carcinoma samples, 12 cases of intraepithelial neoplasia (IN) tissues and 92 cases of nonneoplastic normal tissues were collected at the time of surgical resection at the Jilin Cancer Hospital from July 2014 to July 2016. Samples were snap-frozen in liquid nitrogen and stored at -97˚C from 97 cervical carcinoma patients including with a median 5 age of 65 (range, 51-75 years); Paraffin-embedded tissues were prepared by the Department of Pathology at the same hospital from the 97 cervical carcinoma patients with a median age of 62 (range, 34-82 years). Informed consent was obtained from each patient before collection of tissues.
All diagnoses of cervical carcinoma were confirmed by histopathological examination. Information about patients and disease characteristics came from a review of the patient's medical records. The study was approved by the Institutional Ethical Review Committee of Jilin Cancer Hospital and adhered to the principles of the Declaration of Helsinki.
Genomic DNA was extracted using standard methods. At the time of running electrophoresis, methylation positive control (CpG methylation enzyme modification after fresh placenta tissues DNA as a template), the unmethylation positive control (fresh placenta tissues DNA as a template) and the negative control (H 2 O) were set. The reaction conditions: pre-denaturation at 95℃ for 10 min, denaturation at 95℃ for 45 sec, annealing for 45 sec (methylation-specific primer amplification annealing temperature 59℃, non-methylation specific primer amplification annealing temperature 55 ℃), 72℃ extension 50 sec, set the loop 34 times, and the final extension at 72℃ for 7 min. Agarose gel electrophoresis and staining were then performed. Data from the gel was collected using a under a UV transilluminator (ALPHA, San Leandro, USA) and subsequently analyzed.
Detection of the expression of KISS1 proteins by immunohistochemistry (IHC). Formalin-fixed, paraffin-embedded sections (4 µm) were placed into a 60-65℃ box overnight and deparaffinized in green transparent agent, rehydrated with an alcohol gradient, and washed briefly in distilled water.
To retrieve antigenicity, sections were boiled in 0.01 M citrate buffer (pH 6.0) for 20   Statistical analysis. All results were shown as mean ± SD and analyzed using GraphPad Prism 7 (GraphPad Software, San Diego, CA,USA). For the in vitro experiments, the differences were analyzed using the unpaired two-tailed t-test. For the experiments involving clinical tissues, the differences 8 were analyzed using Pearson's chi-square test. P < 0.05 was considered statistically significant.

Results
Methylation specific PCR (MSP) analysis of KISS1 gene promoter methylation. The MSP data obtained from 97 individuals showed that hypermethylation of the KISS1 gene promoter was observed in 71.1% (69/97) of a cervical carcinoma tissue, but only in 8.6% (8/92) of the nonneoplastic non-tumor tissues (Fig. 1A). Chi-square test showed that the hypermethylation of KISS1 in cervical carcinoma was significantly associated with lymph node metastasis and TNM stage (P < 0.05; Table 1). However, there was no statistically significant correlation between KISS1 promoter hypermethylation and other clinicopathological variables such as age, and histological grade (P > 0.05; Table 1). In addition, the mRNA expression levels of KISS1 were furtherly accessed in cervical carcinoma tissues and nonneoplastic cervical tissues. As expected, the mRNA expression levels of KISS1 was lower in cervical carcinoma tissues compared with non-neoplastic cervical tissues (Fig. 1B). The expression of KISS1 protein in cervical carcinoma tissues obtained from 97 individuals was assessed by western-blotting and immunohistochemical staining (Fig. 1C). As shown in Table 1, the expression of KISS1 was lower in cervical carcinoma tissues compared with that in non-neoplastic cervical tissues. In additionally, we found that KISS1 was expressed in the cytoplasm of cervical carcinoma cells (Fig. 1D and Table 2).
KISS1 staining was detected in 24.7% (24/97) of cervical carcinoma tissues, significantly fewer in nonneoplastic non-tumor tissues 88.0% (81/92). Then, we analyzed the correlation between KISS1 expression and clinicopathological features in cervical carcinoma patients. The low expression of KISS1 in cervical carcinoma was significantly associated with lymph node metastasis and TNM stage (P < 0.05). There was no statistical relationship with other variables such as age and histological grade (P > 0.05; Table II). In additionally, as shown in Fig. 2 lower levels in five cervical carcinoma cell lines (C33A, HeLa, Siha, Caski and HT3) compared with those in the human cervical epithelial cell line CRL2614 (Fig. 3A and B).

Overexpression of KISS1 on proliferation, migration, and invasion of cervical carcinoma cells.
We selected the cell lines C33A to investigate a gain-of-function of KISS1 in cervical carcinoma cell lines.
KISS1 expression was upregulated after overexpression in C33A cells. The transfection of KISS1 plasmid significantly upregulated the expression of KISS1 in both mRNA and protein levels in C33A ( Fig. 4A and B). CCK8 and colony formation assay revealed that overexpression of KISS1 promoted cell proliferation and viability in C33A cells (P < 0.01, Fig. 4C-E). Besides, cell OD values were significantly reduced at 36, 48, and 72 h in KISS1 overexpression group (Fig. 4E). The number of cells invasion through the chamber in KISS1 overexpression group was significantly lower than that of the vector group (P < 0.05) ( Fig. 4F and G). The scratch healing rate of KISS1 overexpressing transfected was significantly lower than that of the vector group (P < 0.05) (Fig. 4H). The results showed that overexpression of KISS1 inhibited cell migration, invasion and proliferation.  Fig. 5A and B). CCK8 and colony formation assay revealed that loss of KISS1 reduced cell proliferation and viability in CRL-2614 cells (P < 0.01, Fig. 5C-E). Matrigel invasion and wound healing assays were performed after transfection of KISS1 sh which reduced the transcription and production of KISS1 (P < 0.01, Fig. 5F-H). It can be seen that knockdown of KISS1 enhances cell migration and invasion in cervical epithelial cells.

Discussion
The signaling pathway in the initial control of tumor cells is activated and the primary tumor cells migrate into nonneoplastic tissues. After the tumor cells are in contact with the blood and lymphatic vessels, penetrate the basement membrane and the endothelial wall, and disperse through the lumen of the blood vessel to reach distant organs, metastasis occurs [13,14]. Therefore, exploring tumor metastasis suppressor genes to interfere with specific points in these steps to block the metastatic cascade and prevent metastatic tumor growth is critical for early diagnosis, treatment, and better clinical outcomes. The KISS1 gene was first reported as a novel metastasis suppressor gene in human melanoma and breast cancer cells [15,16]. The translation product of KISS1 is a protein of 145 amino acids, which is further cleaved into Kisspeptin-10, Kisspeptin-13, Kisspeptin-14, Kisspeptin-54 [17,18]. KISS1 proteins bind specifically to GPR54 (AXOR12 or hOT7T175) and induces the second messenger inositol trisphosphate (IP3) and diglycerides, which play a role in cell proliferation, differentiation and apoptosis[6, 7].
However, the expression of KISS1 and its pathogenesis in cervical carcinoma remains unclear. To investigate the mechanism of action of KISS1 in cervical carcinoma, promoter methylation and related clinicopathological data in the cervical carcinoma were analyzed. In this research, hypermethylation of KISS1 is significantly present in cervical carcinoma tissues compared to nonneoplastic tissues, indicating that methylation of KISS1 may contribute to the progression in cervical carcinoma.
Statistical analysis between the KISS1 promoter methylation and pathological parameters of patients showed that in cervical carcinoma patients with lymph node metastasis and TNM III + IV stage, the methylation positive rate of KISS1 promoter is higher, indicating the complexity of tumor pathogenesis is not only a reflection of genetic change by mutation or deletion but also a reflection of epigenetic alterations, such as DNA methylation. In addition to the deletion and mutation of related genes, aberrant changes in DNA methylation are considered as the third mechanism leading to antioncogene inactivation [19,20] which plays an essential role in tumor development. A previous study suggested that hypermethylation of KISS1 was occurred frequently in colorectal cancer [21]. Previous studies have also confirmed that KISS1 methylation is associated with tumor differentiation, depth of invasion, distant metastasis of lymph nodes, and predictive recurrence[9, 21,22]. These data indicated KISS1 methylation may be associated with invasion, metastasis and poor prognosis in cervical carcinoma. These results are similar to the results of our groups.
The KISS1 expression is substantially down-regulated in cervical carcinoma tissues compared to nonneoplastic non-tumor tissues according to immunohistochemical staining. This is consistent with the findings of Kostakis that found that KISS1 expression in nonneoplastic cervical mucosa was much higher than in malignant mucosa [23]. Then we statistically analyzed the pathological features of cervical carcinoma tissues, and found that low expression of KISS1 was significantly associated with lymph node metastasis and TNM phase. This is consistent with other studies that found that KISS1 has low expression in colorectal cancer and KISS1 is a putative metastasis suppressor in human colorectal cancer [24][25][26]. We hypothesized that KISS1 promoter methylation results in loss of expression and metastasis of cervical carcinoma. Previous studies have demonstrated that KISS1 hypermethylation is associated with loss of transcription and protein expression in colorectal cancer (CRC)[9].
Furthermore, promoter CpG island methylation reduced the expression of related tumor suppressor genes was the main tumor suppressor-inactivation mechanism in cervical carcinoma [27]. Therefore, it would be essential to examine whether KISS1 expression in cervical carcinoma tissues is also directly affected by its promoter methylation. This indicates that the KISS1 protein plays a role in inhibiting tumor metastasis during the development of cervical carcinoma, further confirming that the KISS1 gene is a metastasis suppressor gene in cervical carcinoma, and its down-regulation of expression has certain guiding significance for clinical development of individualized treatment plan and prognosis.
To verify the effect of KISS1 on cervical carcinoma transfer, we examined the biological function of  [28]. Chen pointed out KISS1 overexpression significantly decreased the invasiveness of olorectal cancer (CRC) cells [29]. Previous reports have also pointed out that the reduction of KISS1 expression promotes cell migration and invasion in pancreatic [30,31], ovarian [31], prostate [32], endometrial cancer [33]and nasopharyngeal carcinoma [34]. Considering the importance of migration and invasion ability to tumor progression and metastasis, the results demonstrate the therapeutic 12 potential of KISS1 by reducing the ability of tumor cell metastasis.
Data from proliferation experiments indicate that the overexpressed KISS1 has an inhibitory effect in C33A cells and the knockdown KISS1 has a promoting effect in knockdown CRL2614 cells. These effects appear 48 hours after treatment may because KISS1 takes more time to exert its effect on proliferation. Interestingly, the role of KISS1 in proliferation is various in different tumors.
Chen [29]found that the silencing of KISS1 gene had no influence on proliferation, and overexpression of KISS1 led to a significant decrease in the proliferation in HCT-116 colorectal cancer cells. However, knockdown of KISS1 in HT115 and HRT18 colorectal cancer cells has no effect on proliferation [25].
Inconsistent proliferation results may be related to the characteristics of different tumor cells. It has been reported that KISS1 inhibits growth of matrix-independent tumor but not of matrix-dependent tumor [34]. Therefore, the regulation of KISS1 in different tumor phenotypes is more complex than expected and requires further investigation.

Conclusion
Above all, the experiment demonstrated that KISS1, as a tumor metastasis suppressor gene, is closely associated with the development of cervical carcinoma and can inhibit the migration and invasion of cervical carcinoma to a certain extent. Consequently, KISS1 can be used as a new target for clinical treatment, which may not only eliminate local diseases, but also inhibit the systemic spread of cervical carcinoma cells, but requires further research to confirm.
Declarations YZ designed the study. ZW and MW performed the experiments. HY analyzed the data. HW and CW wrote the manuscript together. JQ and FM helped to revise the manuscript. All authors read and approved the final manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate
The study was approved by the Institutional Ethical Review Committee of Jilin Cancer Hospital and adhered to the principles of the Declaration of Helsinki. Informed consent was obtained from each patient before collection of tissues.

Patient consent for publication
Not applicable.