The diagnostic value of lncRNA HOTAIR for cervical carcinoma in vaginal discharge and serum.

Cervical carcinoma was as one of most frequent tumor and early screening is an important way to detect cervical carcinoma in time, improve the cure rate and reduce mortality. [ 1 – 3 ] Long noncoding RNAs (lncRNAs) are a new class of RNAs with a length of more than 200 nucleotides, which are abnormally expressed in tumors and are directly or indirectly related to the occurrence and development of various tumors including cervical carcinoma. [ 4 ] In recent years, lncRNAs have received extensive attention in the field of tumor diagnosis and treatment. [ 5 ] LncRNAs have high tissue specificity and developmental stage specificity, so they are widely used as novel cervical carcinoma clinical diagnostic markers. more potential. [ 6 ] At present, several studies have systematically studied the correlation between HOX transcript antisense RNA (HOTAIR) and cervical carcinoma metastasis, and it has been confirmed that the expression of HOTAIR in cervical carcinoma primary and metastatic tissue samples is increased, and the expression level of HOTAIR in metastatic lesions is higher than that in normal epithelium, suggesting that HOTAIR is a great significance for the prediction of cervical carcinoma. [ 7 – 9 ] Clinical retrospective studies have shown that the expression level of HOTAIR in primary lesions is closely related to tumor metastasis and prognosis, and the prediction results are independent of clinically known risk factors such as tumor size, stage, and negative or positive expression of estrogen receptors. [ 10 – 12 ] It was reported that HOTAIR was as an indicator of cervical carcinoma metastasis which up-regulated of HOTAIR expression level can promote the invasive ability of cervical carcinoma cells and promote a large number of carcinoma cells to metastasize to the lung, while silencing HOTAIR expression can inhibit the invasive ability of tumor cells, suggesting that abnormal expression of HOTAIR may be an important reason for the metastasis of some cervical carcinoma cells. [ 13 , 14 ] This study aims to investigate the role of HOTAIR in the diagnosis of cervical carcinoma.

Data curation: Ming-Yan Liu. Conceptualization: Na Li.

A total of 150 cervical carcinoma patients (CC group) and 100 benign cervical disease patients (BCD group) from 2019 January to 2021 December in Tianjin Central Hospital of Gynecology Obstetrics were selected, all of which were diagnosed by pathology and imaging. 100 healthy women were the normal group with not cervical disease history. Vaginal discharge samples were collected from normal, BCD group and CC group by ductal lavage. The cervical carcinoma issue and paracancerous tissue were collected and stored in liquid nitrogen. Serum samples were collected from normal, BCD group and CC group by centrifuged at 2000 × g for 10 minutes. [ 15 ] The present study was approved by the Medical Ethics Committee of Tianjin Central Obstetrics and Gynecology Hospital, was performed according to the principles of the Declaration of Helsinki and all patients provided written informed consent prior to participation in the study. The total RNA from the issue, vaginal discharge and serum were extracted by Trizol reagent (Invitrogen). The cDNA was obtained by reverse transcription by BrightGreen 2 × qPCR MasterMix-Low ROX (ABM, Canada) and the expression of HOTAIR was determined by ABI 7500 realtime PCR instruments (Applied Biosystems, CA). The GAPDH was as the internal reference. The reaction conditions were as follows: 95 ºC for 15 seconds, 95 ºC for 10 seconds, 60 ºC for 20 seconds, and 72 ºC for 15 seconds, for 40 cycles. The gene expression was calculated using the 2 −∆∆ct method. The primer sequence was as Table 1 . The primer sequence. Data were expressed as x̄ ± s and analyzed using the SPSS 11.0 statistical software (IBM). The 1-way ANOVA for comparison among 3 groups and LSD t test for comparisons between 2 groups. Receiver operating characteristic (ROC) analysis was performed using GraphPad prism 8.0.1. P < .05 indicates a significant difference. [ 15 ]

We randomly selected 100 patients with cervical carcinoma, 50 benign cervical disease patients and 50 healthy women (Table 2 ), we found that the expression level of HOTAIR in cervical cancer tissue was significantly up-regulated than that in paired cervical paracancerous tissue (Fig. 1 A). Similarly, we found that there was correlated expression of HOTAIR both in vaginal discharge and serum of cervical carcinoma group with positively correlated with tumor malignancy, meanwhile, there was an significant up-regulated in metastasis group (CC-M group) than non-metastasis group (CC-NM group) (Fig. 1 B and C). We randomly selected 50 cervical carcinoma patient, and found the expression of HOTAIR was significantly reduced after surgery 3 months both in vaginal discharge and serum (Fig. 1 D and E). The characteristics for discovery cohort. Comparison of HOTAIR expression in cervical carcinoma, benign cervical disease patients and healthy women. (A) The expression of HOTAIR in cervical cancer tissues and paired paracancerous tissue. The expression of HOTAIR for cervical carcinoma, benign cervical disease and normal group in (B) serum and (C) vaginal discharge. The expression of HOTAIR for preoperative and postoperative cervical carcinoma in (D) serum and (E) vaginal discharge. *Compared with normal group, P < .05. # Compared with non-metastasis group, P < .05. HOTAIR = HOX transcript antisense RNA. The expression of HOTAIR from above-mentioned cervical carcinoma, cervical disease patients and healthy women was studied by ROC analysis. Meanwhile, the result showed that the area under curve of serum was 0.8518, sensitivity was 79%, and specificity was 94%, cutoff value was 1.34, meanwhile, the area under curve of vaginal discharge was 0.9723, sensitivity was 92%, specificity was 98%, cutoff value was 1.17 by ROC analysis (Fig. 2 ). We also randomly selected the expression of HOTAIR from 50 cervical carcinoma patients, 50 benign cervical disease patients and 50 healthy women (Table 3 ) to verify the ROC analysis, the result showed that the accuracy were 92.7% and 89.3% in vaginal discharge and serum, respectively (Table 4 ). The characteristics for validation cohort. The positive rate and accuracy for diagnosis. The ROC analysis of HOTAIR for cervical carcinoma diagnosis in serum and vaginal discharge. The ROC analysis of HOTAIR for cervical carcinoma diagnosis in (A) serum and (B) vaginal discharge. HOTAIR = HOX transcript antisense RNA, ROC = receiver operating characteristic.

Although progress has been made in the early diagnosis and treatment of cervical carcinoma, the rapid proliferation and high invasiveness of cervical carcinoma facilitate the infiltration of tumor cells into adjacent bladder, rectum and pelvic wall, leading to rapid deterioration of the patient’s condition, even death. [ 16 ] Therefore, it is imperative to deeply explore the mechanisms of cervical carcinoma formation and development, and to establish new diagnostic and therapeutic targets for optimal treatment of cervical carcinoma. Numerous studies have confirmed that lncRNAs such as HOTAIR were involved in a variety of biological processes, including proliferation, differentiation, apoptosis, cell migration, cell invasion, development, and immune responses. [ 17 ] In various tumor cells, the expression levels of certain specific lncRNAs will change, which is expected to be screened as markers for tumor diagnosis or potential drug targets. The expression of HOTAIR is up-regulated in gastric carcinoma, liver carcinoma, thyroid carcinoma, glioma, pancreatic ductal adenocarcinoma, cervical carcinoma, non-small cell lung carcinoma and other tumors, and has obvious correlation with tumor proliferation, invasion, metastasis and prognosis. [ 18 ] HOTAIR is a newly discovered star molecule that plays an important role in cervical carcinoma metastasis. At present, several studies have systematically studied the correlation between HOTAIR and cervical carcinoma metastasis, and it has been confirmed that the expression of HOTAIR in cervical carcinoma primary and metastatic tissue samples is increased, and up-regulation of HOTAIR expression level can promote the invasion of cervical carcinoma cells. However, silencing HOTAIR expression can inhibit the invasiveness of tumor cells, suggesting that abnormal expression of HOTAIR may be an important reason for the metastasis of some cervical carcinomas. Clinical retrospective studies have shown that the expression level of HOTAIR in primary lesions is closely related to tumor metastasis and prognosis, and the prediction results are independent of clinically known risk factors such as tumor size, stage, and negative or positive expression of estrogen receptors. [ 19 , 20 ] It is suggested that HOTAIR, as an indicator of cervical carcinoma metastasis, can make up for the deficiencies of existing predictive indicators. Therefore, HOTAIR not only participates in the process of tumor invasion and metastasis, but also is considered as a potential biological marker for the metastasis and prognosis of various carcinomas. This study found that the expression of HOTAIR in cervical cancer tissue, serum and vaginal discharge was several times higher than that in paracancerous tissue, healthy people and patients with benign cervical disease, and was higher in patients with metastatic cervical carcinoma than in patients with non-metastatic cervical carcinoma, confirming previous studies. the results of the study. At the same time, we found that HOTAIR was significantly reduced after surgery 3 months both in vaginal discharge and serum, indicating that HOTAIR in vaginal discharge and serum was a potential marker as development and prognostic for cervical carcinoma. In order to further explore the role of HOTAIR in the clinical diagnosis of cervical carcinoma, ROC analysis found that the sensitivity was 79% and the specificity was 94% with the expression of HOTAIR in serum. Validation with new samples of serum and vaginal discharge found that the accuracy were 92.7% and 89.3% in vaginal discharge and serum, respectively. There was a lower positive rate for cervical carcinoma diagnosis in serum compared with vaginal discharge, however, there also was a higher false positive for benign cervical disease diagnosis in vaginal discharge which may be correlation with higher sensitivity.