CN117976188A - Computer system and kit using serum lncRNA and HOTAIR as marker for assisting diagnosis of liver cancer - Google Patents

Abstract

The invention provides a computer system for diagnosing, detecting, monitoring and predicting prognosis of liver cancer, which is characterized by comprising the following components: i) An analysis module, the analysis module comprising: a test substance for determining the expression level of serum lncRNAHOTAIR of a subject, and; ii) an evaluation module comprising: judging the prognosis of liver cancer of the subject according to the serum lncRNAHOTAIR expression level determined in i). The system not only can be used for assisting in clinical screening diagnosis of liver cancer, but also can evaluate the treatment effect of surgery or non-surgery, and dynamically monitor the prognosis progress and recurrence of liver cancer.

Description

Computer system and kit using serum lncRNA and HOTAIR as marker for assisting diagnosis of liver cancer

Technical Field

The application relates to the technical fields of disease diagnosis and treatment and molecular biology, in particular to the value of serum LncRNA and HOTAIR in prognosis of liver cancer patients.

Background

Liver cancer may be caused by a variety of risk factors, including chronic infection of hepatitis b virus (HEPATITIS B VIRUS, HBV) or hepatitis c virus, cirrhosis (Liver Cirrhosis, LC), alcoholic liver disease, exposure to aflatoxin, and the like. The pathological types of primary liver cancer (PRIMARY LIVER CANCER, PLC) include three types of hepatocellular carcinoma (Hepatocellular Carcinoma, HCC), intrahepatic cholangiocarcinoma (INTRAHEPATIC CHOLANGIOCARCINOMA, ICC) and HCC-ICC mix, with the most common HCC accounting for about 85% -90%. According to domestic and foreign liver cancer diagnosis and treatment guidelines, surgical radical surgery is a basic stone for treatment selection of HCC patients, and liver transplantation provides an optimal long-term outcome for patients with liver function damage secondary to liver cirrhosis. However, since liver cancer is hidden, early symptoms are mostly absent, and many patients are at middle and late stages in diagnosis, the chance of operation is lost. And even the patients who receive radical resections of liver cancer have recurrence rate up to 70% after 5 years of operation. Local treatment methods of liver cancer such as selective hepatic angiography and embolic chemotherapy (TRANSCATHETER ARTERIAL Chemoembolization, TACE), radiofrequency ablation (Radiofrequency Ablation, RFA) and the like are often used for middle and late stage palliative treatment of the liver cancer or used as a bridge for liver transplantation, and can prolong the survival time of patients; however, the study shows that TACE treatment can only completely necrotize 25% of tumors, and the postoperative recurrence and metastasis probabilities are high. Thus, early diagnosis has become a key to improving survival in PLC patients; and identifying post-operative patients with potential relapse and determining who should receive adjuvant therapy based thereon is also critical to the prognosis of the patient.

Serum alpha fetoprotein (Alpha fetoprotein, AFP) is the most common method for clinical screening and prognosis dynamic monitoring of liver cancer at present, but the method has poor sensitivity and is easy to leak diagnosis. Elevated AFP can also occur in some benign liver diseases (e.g. hepatitis and cirrhosis), and even with low levels of threshold (i.e. 10-20 ng/mL), the sensitivity of AFP diagnosis is only 40-60% and the specificity is 80-90%. In addition, many liver cancer patients have negative AFP, and it is reported that 15% -30% of serum AFP levels of advanced liver cancer patients remain normal, so that AFP has defects in postoperative efficacy evaluation, recurrence monitoring and the like of liver cancer patients. The examination of ultrasound, CT and the like has important value for diagnosing liver cancer, but the two are not easy to find a tiny cancer focus, and the ultrasound is easy to be interfered by factors such as obesity, flatulence and the like of patients. The ultrasonic radiography, the enhancement CT, the magnetic resonance and other examination have certain advantages for diagnosing the small cancer focus, but also have the defects of large traumata, high cost, poor patient compliance and the like, so the method is not suitable for the early large-scale screening of the crowd. Therefore, it is important to find a blood tumor biomarker which can be diagnosed early and can monitor liver cancer prognosis after operation, has high sensitivity and specificity and small trauma, can diagnose or evaluate recurrence risk as early as possible and treat in time, and is beneficial to improving the prognosis life quality of PLC patients.

Long non-coding RNAs (Long noncoding RNAs, lncRNA) are non-protein coding RNAs that are more than 200 nucleotides in length. More and more studies have shown that lncRNA is involved in a wide range of biological processes and is associated with a variety of diseases such as cancer. Among various solid tumors or hematological malignancies, a large number of lncRNA are defined as oncogenes and tumor suppressor genes, involved in the cellular biological processes of tumorigenesis and metastasis. LncRNAs has abnormal expression in various tumor tissues and body fluids (such as blood, saliva, urine and the like), has been applied to early diagnosis of partial tumors, can well predict recurrence and metastasis of diseases, and is expected to play an important role in clinical diagnosis and treatment of diseases. For example, serum lncRNA-ATB can be used as a non-invasive diagnostic marker for early stage breast cancer and has the potential to monitor breast cancer progression and stage. Serum LNCRNA SNHG can be used as a survival predictor for short-term adverse events of gastric cancer patients.

HOX antisense transcribed RNA (HOX TRANSCRIPT ANTISENSE RNA, HOTAIR) is lncRNA which plays an oncogene in various tumors and plays an important role in tumor occurrence, metastasis and drug resistance. Gupta et al report that HOTAIR is highly expressed in metastatic breast cancer, and that its high expression in primary breast tumors is an important indicator for predicting subsequent metastasis and mortality. Furthermore, in epithelial ovarian and colon cancers, high expression of HOTAIR may also be predictive of poor prognosis and promote metastasis. Studies show that HOTAIR is also closely related to the occurrence and development of liver cancer, and high expression of HOTAIR can promote proliferation, survival and invasion of liver cancer cells. Zhong et al found that silencing HOTAIR promoted apoptosis of hepatoma cells and inhibited cell proliferation. Gao et al found that HOTAIR is highly expressed in liver cancer tissues, and that its mechanism of recurrence and metastasis to HCC is partly achieved by Wnt/β -catenin signaling. Yasuyuki et al found that the tumor size of HCC is related to high expression of HOTAIR, which promotes proliferation and diffusion of cancer cells by enhancing ATG7 and ATG3 expression to increase autophagy of hepatoma cells.

At present, clinical researches on diagnosis and prognosis of HOTAIR in liver cancer are focused on liver cancer histology, and no research on dynamic follow-up monitoring of liver cancer postoperative patients is available, so that whether the expression of HOTAIR in serum of liver cancer patients can be focused on clinical diagnosis and prognosis judgment is worth focusing on. In this section of the study, we evaluated whether or not it was involved in the development of malignant tumors by analyzing the relationship between serum HOTAIR expression of 70 liver cancer patients and their clinical pathological features. Potential diagnostic and prognostic value was then assessed by subject work signature (ROC) curves, kaplan-Meier and Cox regression analysis. And finally, tracking and follow-up 37 patients with liver cancer after operation, wherein the patients respectively have three different survival outcomes of good prognosis, recurrence and death, dynamically monitoring the change of serum HOTAIR of the patients, and judging whether the serum HOTAIR can be used as an index of postoperative progress and recurrence of the liver cancer patients. The method has important clinical value for early diagnosis and prognosis judgment of liver cancer and realization of early discovery, early intervention and early treatment of the liver cancer.

Disclosure of Invention

In this study, we found that preoperative serum HOTAIR expression levels in liver cancer patients were significantly higher than those in cirrhosis and healthy groups, consistent with previous study results. Subsequently, we evaluate the diagnostic ability of serum HOTAIR using ROC curve, and the results suggest that the combined detection of HOTAIR and AFP is optimal for diagnosing liver cancer, with AUC of 0.942, sensitivity and specificity of 84.3% and 90.8%, respectively, superior to the performance of HOTAIR or AFP as a single biomarker for detecting liver cancer. In addition, the combined detection of HOTAIR and AFP has a certain significance on the differential diagnosis capability of liver cancer stage, the AUC is 0.782, and the sensitivity and specificity are 86.7% and 65.0% respectively.

In the aspect of clinical pathological characteristics, the research result shows that the high HOTAIR expression level is obviously related to Cnlc stages, extrahepatic metastasis, vascular invasion, cancer thrombosis and tumor size, and also proves that the HOTAIR participates in the development of the PLC. These clinical pathological features are important factors affecting diagnosis and prognosis of liver cancer, are related to invasion and progress of liver cancer, and often indicate poor prognosis. The tumor size reflects the tumor load to a certain extent, and the larger the tumor, the more lncRNA is synthesized and released, and the more HOTAIR is; in contrast, after tumor destruction, tumor volume is reduced, and thus expression levels are reduced. In this study we found that serum HOTAIR expression levels were significantly reduced 1 month post-surgery in liver cancer patients compared to pre-surgery, suggesting that serum HOTAIR may be able to assess the effectiveness of surgical or non-surgical treatments and monitor recurrence during follow-up. Based on the above information, we speculate that HOTAIR may be a predictor of poor prognosis in liver cancer patients, and that serum expression levels may be helpful for clinical diagnosis and recurrence prediction of liver cancer.

Therefore, we follow the prognosis survival outcome within 3 years of the 51 liver cancer postoperative patients, and find out that HOTAIR, cnlc stage, vascular invasion and cancer thrombus formation are independent factors affecting survival of the liver cancer postoperative patients through COX regression multifactorial analysis, and HOTAIR is also an independent factor affecting recurrent metastasis of the liver cancer postoperative patients; and then, a survival and recurrence curve graph is drawn by a Kaplan-Meier method, and the survival rate and recurrence transfer rate of patients with low HOTAIR expression are found to be obviously superior to those of patients with high HOTAIR expression. This suggests that high HOTAIR expression has a significant negative impact on prognosis of patients, and has predictive value for prognosis of liver cancer patients.

Recurrence is a major complication after liver cancer surgery, and is generally classified into early recurrence and late recurrence by 2 years. Early recurrence (i.e., within 2 years after resection) accounts for more than 70% of tumor recurrence and is considered "true recurrence". Is generally considered to be pre-existing intrahepatic metastasis; and late recurrence after this period is thought to be caused primarily by "new born" tumors. Early recurrence is mainly determined by invasive features of the primary (resected and destroyed) tumor, such as tumor size, tumor diversity, vascular invasion, and higher serum alpha-fetoprotein levels. In BCLC 0 or stage a tumors, the survival rate without recurrence for 2 years was about 50% and 30%, respectively. Patients with high recurrence risk are identified after potential radical surgery, so that clinicians can provide proper monitoring to find recurrence at the earliest stage of liver cancer recurrence, measures are taken in time to improve the curative effect and prolong the life cycle of the patients.

Therefore, the 51 liver cancer postoperative patients are divided into three groups of prognosis good group, recurrence transfer group and death group according to different survival outcomes, the HOTAIR expression levels of four time points of preoperative, postoperative 1, postoperative 2 (recurrence) and postoperative 3 (retreatment) are monitored, and dynamic line diagrams are respectively made; and 37 patients in which the follow-up serum data were complete were compared pairwise using repeated measures of anova. The results show that the preoperative HOTAIR expression level of the death group is obviously higher than that of the prognosis-good group and the recurrence group; compared with preoperative patients, the serum hotapir of three groups of patients after operation is reduced, but hotapir is obviously increased when the patients in the recurrent group and the patients in the dead group recur, and hotapir is obviously reduced after retreatment. Surprisingly, we found that post-operative HOTAIR expression levels were higher in the recurrent group than in the dead group, considering that part of the reasons might be that the small sample size affected the statistics; another reason is that it is presumed that most of the patients in the recurrent group have longer survival time than those in the dead group, and the recurrent group patients have residual lesions remaining in a long period of time after TACE or RFA treatment, resulting in accumulation of the release of hotapir, thus making the expression level of hotapir higher; furthermore, all tumor characteristics (tumor size, pathology type and location) may affect HOTAIR serum expression levels, and further studies are needed to determine how tumors affect lncRNA expression levels. From the above data, we consider serum HOTAIR as an indicator for monitoring postoperative progression and recurrence in liver cancer patients.

In conclusion, the study shows that the serum LncRNA and the HOTAIR are highly expressed in liver cancer patients, can be used for assisting in clinical screening and diagnosis of liver cancer, can evaluate the curative effect of surgery or non-surgery, dynamically monitors the prognosis progress recurrence of liver cancer, and is a potential noninvasive serological marker for liver cancer.

Therefore, the invention provides the following technical scheme:

A computer system for diagnosing, detecting, monitoring, predicting prognosis of liver cancer, the system comprising:

i) An analysis module, the analysis module comprising: a detection substance for determining the expression level of LncRNA and HOTAIR in the subject's serum, and;

ii) an evaluation module comprising: determining the prognosis of liver cancer in said subject based on the serum LncRNA and HOTAIR expression levels determined in i).

A kit for assisting in diagnosing liver cancer adopts serum LncRNA and HOTAIR as markers for assisting in diagnosing liver cancer, and comprises substances for detecting LncRNA and HOTAIR.

Use of a substance for detecting LncRNA and HOTAIR in the preparation of a product for diagnosing, detecting, monitoring or predicting prognosis of liver cancer.

The invention also provides the following technical scheme:

A computer system for diagnosing, detecting, monitoring, predicting prognosis of liver cancer, the system comprising:

i) An analysis module, the analysis module comprising: detection substances for determining the levels of expression of LncRNA and HOTAIR and AFP levels in the serum of a subject, and;

ii) an evaluation module comprising: determining the prognosis of liver cancer in said subject based on the serum LncRNA and HOTAIR expression levels and AFP levels determined in i).

A kit for assisting in diagnosing liver cancer adopts the levels of serum LncRNA and HOTAIR and AFP as markers for assisting in diagnosing liver cancer, and comprises substances for detecting the levels of serum LncRNA and HOTAIR and AFP.

Use of a substance for detecting serum LncRNA and HOTAIR and AFP levels in the preparation of a product for diagnosing, detecting, monitoring or predicting prognosis of liver cancer.

The invention also provides the following technical scheme:

a combination marker consisting of LncRNA and HOTAIR and AFP.

Use of a combination marker consisting of LncRNA and HOTAIR and AFP in the preparation of a kit for prognosis risk assessment of liver cancer surgery.

The liver cancer is hepatocellular carcinoma.

The substances for detecting LncRNA and HOTAIR include substances used for nucleic acid extraction, reverse transcription and fluorescence measurement.

The substances for detecting LncRNA and HOTAIR comprise substances used by quantitative real-time polymerase chain reaction (qRT-PCR).

The substances for detecting LncRNA and HOTAIR comprise SYBR GREEN REALTIME PCR MASTER KIT, an RNA extraction and separation kit, REVERTRA ACE QPCR RT KIT, chloroform, absolute ethyl alcohol, deionized Water, DEPC water, 18s RNA primer and target gene primer.

The assessment module assesses potential diagnostic and prognostic value by subject work signature (ROC) curves, kaplan-Meier and Cox regression analysis.

Drawings

Fig. 1: chinese liver cancer clinical staging scheme (CNLC)

Fig. 2: comparing the serum HOTAIR expression level of the preoperative PLC group patient with that of the LC group and that of the HC group; * P < 0.01 between the two groups.

Fig. 3: A. serum HOTAIR and AFP diagnostic efficacy in PLC versus total control; B. serum HOTAIR was compared with AFP for diagnostic efficacy of liver cancer staging prediction.

Fig. 4: a survival curve of the patient after the liver cancer operation of the HOTAIR high expression group and the HOTAIR low expression group; and B, recurrence and transfer curves of patients after liver cancer operation in the HOTAIR high expression group and the HOTAIR low expression group.

Fig. 5: A. preoperative and postoperative serum HOTAIR expression changes in liver cancer patients for 1 month; b.14 patients with good prognosis after liver cancer operation had change in HOTAIR expression; c.17 cases of change in HOTAIR expression in post-operative death patients of liver cancer; HOTAIR expression changes in surviving patients after 15 liver cancer postoperative recurrence and retreatment.

Detailed Description

1. Data and method

1. Clinical data

1.1 Study object

The study is carried into 70 PLC patients (58 men and 12 women) with ages of 28-84 (56.20+/-11.81 years) in the clinic or ward of the Western stream hospital in Hangzhou and the people hospital in Yuhuan city, and the study is carried out in the period of 2 months in 2019 to 1 month in 2020; 35 patients with liver cirrhosis (men 26 and women 9) were aged 21-82 (54.31 + -14.47) and were LC group; 30 healthy physical examination persons (20 men and 10 women) are HC (Healthy control) groups with ages of 30-64 (51.23+/-7.16); the general data of three groups of cases are compared, and the difference has no statistical significance (P is more than 0.05). All patients were approved by the ethics committee, for whom the study was presented with the basic content prior to group entry, were approved for participation in the study and signed informed consent.

1.2 Inclusion criteria

(1) The PLC group inclusion standard refers to Chinese primary liver cancer diagnosis and treatment standard (2019 edition), and specifically comprises the following steps:

① Meets the pathological histology diagnosis of primary liver cancer; ② At least two of 4 examinations of dynamic enhancement MRI, dynamic enhancement CT, ultrasonic contrast or hepatocyte specific contrast agent Gd-EOB-DTPA enhancement MRI are found to show that the focus of arterial phase is obviously enhanced, the focus of intrahepatic focus in portal vein phase and/or balance phase is lower than liver parenchyma (namely, typical liver cancer image characteristics of 'fast-in and fast-out'), and can be diagnosed as liver cancer; ③ If the internal diameter of liver is more than 2cm, the 4 kinds of imaging examination can diagnose liver cancer if 1 characteristic liver cancer exists.

(2) LC group inclusion criteria reference "diagnosis and treatment of liver fibrosis consensus (2019), specifically:

① Meets the pathological histology diagnosis of liver cirrhosis; ② Imaging examination such as CT, B ultrasonic or liver hardness measurement prompts liver cirrhosis or portal hypertension; ③ Endoscopy shows the presence of esophageal gastric varices or gut ectopic varices. Any one of the above conditions is met, and liver cirrhosis can be diagnosed.

(3) Health group inclusion criteria: the health people with disease history such as malignant tumor, chronic medical diseases and infectious diseases are excluded by laboratory examination, ultrasound, CT and other auxiliary examination.

1.3 Exclusion criteria

(1) Other treatments such as surgery, radiofrequency ablation, interventional therapy (PEI, TACE, etc.), radiotherapy, chemotherapy, and immunotherapy have been performed previously in the outer hospital;

(2) Those suffering from primary malignancies other than PLC;

(3) Patients with cirrhosis with nodes of unknown nature;

(4) The patient or the family of the patient refuses to sign the informed consent;

1.4 general data

General case data includes name, sex, age, history of hepatitis B infection, whether it is accompanied with cirrhosis, tumor size, tumor number, whether there is vascular invasion, whether there is extrahepatic metastasis, whether there is cancer thrombosis, whether there is hepatic encephalopathy, ascites, past history of disease, family history.

The laboratory test result collection items include: alpha Fetoprotein (AFP), glutamic pyruvic transaminase (ALT), glutamic pyruvic transaminase (AST), albumin (Alb), total Bilirubin (TBIL), prothrombin Time (PT), triglyceride (TG), cholesterol (TC), and hepatitis B six.

Comprehensive data the PLC patients are staged according to the Chinese liver cancer staging scheme (CHINA LIVER CANCER STAGING, CNLC) of Chinese primary liver cancer diagnosis and treatment Specification (2019 edition) (figure 1).

1.5 Postoperative follow-up assessment

All patients received surgery (12 cases) or TACE (57 cases) or RFA (1 case) at the west stream hospital in hangzhou after the diagnosis of liver cancer, and subsequently received no or continued additional treatment (e.g., TACE, RFA, targeted and other proximity treatments) during follow-up. Blood samples were collected at multiple follow-up time points before, after, and after the recurrence and metastasis. The postoperative follow-up is based on imaging data (such as B ultrasonic, CT, MRI, etc.), liver function, serum AFP, etc., and is combined with hospitalization medical record to comprehensively evaluate postoperative curative effect, liver cancer recurrence and metastasis and death of patients. Once tumor recurrence or metastasis is confirmed, the time and location of recurrence or metastasis is recorded, and serum samples are collected. Liver cancer recurrence diagnosis criteria: the new nodule with the internal diameter of less than or equal to 2cm is found, and at least two of 4 examinations of dynamic enhancement MRI, dynamic enhancement CT, ultrasonic contrast or hepatocyte specific contrast agent Gd-EOB-DTPA enhancement MRI show that the focus of arterial phase is obviously enhanced, the focus of intrahepatic focus enhancement in portal vein phase and/or balance phase is lower than the liver parenchyma (namely, the typical liver cancer image characteristics of 'fast-in and fast-out') so as to judge that the liver cancer recurs; if the diameter of the liver is more than 2cm, the 4 kinds of imaging examination can judge recurrence if 1 characteristic liver cancer exists.

The post-operation follow-up time is 1 month after operation, the follow-up time is frequency every 3-6 months, and the follow-up end point is 2021 years 12 months. During which researchers follow-up the patient by querying the outpatient or inpatient medical history and telephone. Total survival time (OS) is from the beginning of surgery to death or last follow-up time, and median survival time after surgery is 23.0 (19.0-25.0) months; the total recurrence time is from the beginning of the operation to the first recurrence or transfer time, and the postoperative median recurrence time is 20.5 (13.5-25.0) months.

2. Experimental method

2.1 Sample collection

To reduce trauma to the patient, serum LncRNAs ml of all venous blood that was discarded after regular laboratory examination of the panelist was collected by serum LncRNAs (PLC patients were followed up 1 week before surgery, 1 week after surgery, 1 month after surgery, every 3-6 months later), centrifuged at a high speed of 3000r/min for 10min, and serum was drawn into a 1.5ml enzyme-free centrifuge tube and stored at-80 ℃. Serum AFP assay all members of the inclusion group were withdrawn 3ml venous blood in the early morning fasting state, centrifuged at 3000r/min for 10min at high speed, and the serum was isolated and stored at-80 ℃.

2.2 Experimental materials

2.2.1 Main instruments

2.2.2 Major reagents

2.3 Sample detection

All LncRNAs assays in this study were performed in the Hangzhou Aidic medicine laboratory and were performed using the qRT-PCR method. Total RNA is extracted from serum samples by using serum RNA extraction and separation reagent, and the purity and concentration of RNA are detected by a spectrophotometer (Nano). RNA was reverse transcribed according to the kit instructions and then qRT-PCR was performed with the S18 gene as an internal control. For each sample, 2 parallel tubes were used to calculate LncRNA and HOTAIR expression values in serum using the 2 ^-△△Ct method: Δct (experimental sample) =ct (gene of interest) -Ct (reference gene); Δct (control sample) =ct (gene of interest) -Ct (reference gene); ΔΔct= Δct (experimental sample) - Δct (control sample); thus, 2 ^-△△Ct was calculated.

Serum AFP assay AFP values were determined using chemiluminescent enzyme immunoassay (CLEIA) using standard instruments and kits of the present institute. Serum AFP is routinely detected by most of the patients admitted to the study group or reviewed in a follow-up manner, so that most of the patient AFP expression values are obtained by referring to outpatient and inpatient system case data and are not retested.

2.4 Detection method

2.4.1 Nucleic acid extraction

Reagent: serum/plasma RNA extraction and separation kit (DP 503, tiangen biotechnology Co., ltd., beijing, china)

(1) Sample treatment: adding 900 μl of lysate MZA into 200 μl of serum, shaking with a shaker, homogenizing for 30sec to completely homogenize, and mixing upside down;

(2) Standing at room temperature for 5min to completely separate nucleic acid protein complex;

(3) 200 μl chloroform was added, the tube was capped, vigorously shaken for 15sec, and left at room temperature for 5min.

(4) 12,000Rpm (-13,400 Xg), 4℃and centrifugation for 15min, the sample will be divided into three layers: yellow organic phase, white middle layer and colorless water phase, RNA is mainly in the water phase, transfer the water phase to new tube, go on the next operation.

(5) The volume of the transfer solution is measured, and absolute ethanol (for example, 500. Mu.l of transfer solution and 1ml of absolute ethanol) 2 times the volume of the transfer solution is slowly added, and the transfer solution are uniformly mixed (precipitation possibly occurs at the moment). The resulting solution was transferred to an adsorption column together with the precipitate, left at room temperature for 2min, centrifuged at 12,000rpm (13,400 Xg) for 30sec at room temperature, and the effluent was discarded after centrifugation, leaving the adsorption column.

(6) To the column, 700. Mu.l of deproteinized solution MRD was added, and the mixture was allowed to stand at room temperature for 2 minutes, and centrifuged at 12,000rpm (13,400Xg) at room temperature for 30sec, whereby the waste liquid was discarded.

(7) To the adsorption column, 500. Mu.l of a rinse solution RW was added, and the mixture was allowed to stand at room temperature for 2 minutes, centrifuged at 12,000rpm (13,400 Xg) at room temperature for 30sec, and the waste liquid was discarded.

(8) The step 7 is repeated once.

(9) Centrifuge at 12,000rpm (13,400Xg) for 2min and discard the collection tube.

(10) The adsorption column was transferred to a new RNase-Free 1.5ml centrifuge tube, 15-30. Mu. lRNase-Free ddH2O was added to the center of the adsorption membrane, and the mixture was left at room temperature for 2min and centrifuged at 12,000rpm (13,400 Xg) for 2min.

(11) Nano determines RNA concentration and purity.

2.4.2 Reverse transcription

Reagent: toyobo REVERTRA ACE QPCR MASTER Mix cDNA first Strand Synthesis kit (FSQ-101, toyobo biosciences, inc., shanghai, china).

The RT reaction solution is prepared in a 20 mu l system according to the following components by adopting a two-step method:

(1) The first step:

reverse transcription conditions: 65 ℃ for 5min; ice bath for 5min.

(2) And a second step of:

reverse transcription conditions were 37℃for 50min;98 ℃ for 5min; after the reaction is completed, the mixture is put at the temperature of minus 30 ℃ for standby.

2.4.3 Fluorescent quantitation

Reagent: as the measurement of the expression level of all genes, TOYOBO SYBR GREEN REALTIME PCR MASTER KIT (QPK-201, toyobo Biotechnology Co., ltd., shanghai, china) was used.

Instrument: PCR ABI 7500

Primer sequence:

(1) The PCR reaction liquid is prepared according to the following components:

S18, internal reference:

LncRNAs：

(2) Adding qRT-PCR reaction liquid into a 96-well plate, sequentially adding template cDNA, slightly blowing by a pipettor, uniformly mixing, sealing by a sealing film, and performing amplification reaction on the mixture by a qRT-PCR instrument after instantaneous centrifugation. The reverse transcription reaction conditions were as follows: a total of 40 cycles of 95℃1min,95℃15sec,60℃40 sec; then enter the dissolution profile procedure.

(3) And (3) judging an experimental result:

And (3) measuring the absorbance OD value of total RNA obtained by purifying each group of samples, wherein the concentration span of the samples is 5ng/ul to 60ng/ul, the OD (260)/OD (280) value is 1.7-2.0, the OD (260)/OD (230) value is basically about 2.0, and the total RNA and the quality meet the detection requirement of the LncRNA microarray.

3. Statistical method

Data analysis was processed using SPSS22.0 statistical software. The quantitative data are subjected to normal examination, and the data are subjected to normal distribution and are subjected to mean ± standard deviationThe non-normal distribution is represented by a median (25 quantiles, 75 quantiles). The single-factor analysis of variance is used for the comparison between groups, and the LSD method is adopted for the comparison between every two groups; the nonparametric rank sum test was used to analyze the relationship between HOTAIR and clinical pathology features; the ROC curve evaluates its diagnostic efficiency; calculating a risk Ratio (Hazard Ratio, HR) and a 95% confidence interval (Confidence interval, CI) using univariate and multivariate Cox proportional risk regression models to assess survival and recurrent effects of prognostic variables; the Kaplan-Meier method draws patient survival and recurrence curves; multiple sets of duplicate measurement anova were used to compare the HOTAIR level changes at different survival outcomes and different time points, with P <0.05 considered statistically significant.

2. Results

1. Comparison of the expression levels of three groups of serum HOTAIR

The qRT-PCR results show that the serum LncRNA and HOTAIR expression levels of the preoperative liver cancer patients are obviously higher than those of the liver cirrhosis patients and the healthy patients, the serum HOTAIR expression level of the liver cirrhosis patients is higher than that of the liver cirrhosis patients and the healthy patients, and the difference has statistical significance (P is less than 0.05), as shown in figure 2.

2. Analysis of correlation between serum HOTAIR and clinical characteristics of liver cancer patients

We analyzed the clinical characterization relationship between serum HOTAIR and 70 liver cancer patients. The results show that Cnlc stages, extrahepatic metastasis, vascular invasion, cancer thrombosis and tumor size of liver cancer patients are all related to high expression levels of serum LncRNA and HOTAIR (P < 0.05), while the results are not statistically significant in terms of age, sex, AFP, hepatitis B, cirrhosis and tumor number of the patients (P > 0.05), as shown in Table 1. Liver cancer Cnlc stage, extrahepatic metastasis, vascular invasion, cancer thrombosis, tumor size and other pathological features often suggest poor prognosis, so HOTAIR may be a predictor of poor prognosis for liver cancer patients, and deserves further study.

TABLE 1 clinical characterization relationship of serum HOTAIR in liver cancer patients

Analysis of serum HOTAIR diagnostic efficiency by roc Curve

Next, we analyzed ROC curves of the PLC group (70 persons) and the control group (LC group+healthy group, 65 persons) to investigate the diagnostic value of LncRNA and HOTAIR for liver cancer. The diagnostic ability of the serum marker AFP of the clinical common liver cancer is compared, and the result shows that the diagnostic efficiency of serum HOTAI on the PLC is superior to that of the AFP. Based on the above results, we further used binary Logistic regression analysis to evaluate the diagnostic efficacy of the HOTAIR and AFP combination test, which showed that the HOTAIR and AFP combination test had the best diagnostic efficacy on liver cancer, as shown in table 2 and fig. 3 (a).

In addition, we also analyzed the predictive ability of HOTAIR for stage Cnlc of liver cancer using ROC curves, comparing PLC stage I-II patients (40) with stage III-IV patients (30). The results show that the serum HOTAIR has a certain diagnostic ability for liver cancer stage prediction, and the combined detection of HOTAIR and AFP can further improve the diagnostic ability, as shown in table 2 and fig. 3B.

TABLE 2 comparison of serum LncRNA and HOTAIR with AFP in liver cancer diagnosis

4. Serum HOTAIR expression and prognosis for survival of liver cancer patients

By 2021, 12 months and 31 days, 19 out of 70 patients in the PLC group were out of visit, and 51 patients had post-operative follow-up serum and survival outcomes. The prognosis in 51 post-operative patients was good for 15 cases, 19 cases of recurrence and metastasis, and 17 cases of death. We took the median of preoperative serum HOTAIR (6.50) of 51 patients as the threshold and divided them into HOTAIR low expression (n=25) and high expression (n=26). As a result of comparing the total survival and total recurrence of the patients after liver cancer operation by Kaplan-Meier survival analysis, the survival rate of the patients after liver cancer operation in the HOTAIR low expression group is higher than that in the HOTAIR high expression group, and the recurrence and metastasis rate of the patients after liver cancer operation in the HOTAIR low expression group is lower than that in the HOTAIR high expression group, which is shown in fig. 4 (A.B).

In addition, we performed single-factor and multi-factor analysis of clinical factors related to liver cancer patients to further verify the relationship between serum HOTAIR expression and survival prognosis and recurrence and metastasis of liver cancer. Single factor COX analysis shows that HOTAIR, cnlc staging, extrahepatic metastasis, vascular invasion, cancer thrombosis, tumor size are relevant risk factors for overall survival of patients after liver cancer surgery; further multifactorial COX analysis showed that HOTAIR, cnlc staging, vascular invasion, and cancer plug formation are independent factors affecting survival of patients after liver cancer surgery, see Table 3. Single and multiple factor COX analyses showed HOTAIR to be an independent predictor of recurrence and metastasis in patients after liver cancer surgery, see table 4.

TABLE 3 single-and multifactor Cox regression analysis of Total survival in liver cancer patients

TABLE 4 single-and multi-factor Cox regression analysis of total recurrence and metastasis rates for liver cancer patients

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5. Preoperative and postoperative changes in serum HOTAIR expression levels in 1 month for liver cancer patients

The change of the serum HOTAIR expression level of 51 patients in the preoperative period and the postoperative period within 1 month is selected for the paired sample test statistical analysis, and the result shows that the serum HOTAIR expression level is obviously reduced in the postoperative period within 1 month, as shown in fig. 4A. This verifies that serum HOTAIR may be of value for liver cancer surgery efficacy or disease assessment.

6. Serum HOTAIR dynamic monitoring of prognosis of patients after liver cancer surgery

According to the previous analysis results, the high expression of serum HOATIR is positively related to the stage of liver cancer Cnlc, extrahepatic metastasis, vascular invasion and cancer thrombosis, and the pathological characteristics often indicate poor prognosis, and HOTAIR may be a predictor of poor prognosis of liver cancer patients. Our follow-up survival data showed good prognosis in 14 out of 51 post-operative patients, 19 with recurrent metastases, and 17 deaths. Of these, the 14 groups with good prognosis had 3 or more follow-up sera and 12; 3 or more follow-up sera in the 19 recurrent transfer groups (15 survivors after recurrent TACE or RFA treatment); there were 3 or more serous subjects 15 in the 17 deaths group (11 deaths after recurrence and treatment with TACE or RFA). We used the changes in the expression levels of the three serum HOTAIR groups as line graphs, respectively, as shown in FIG. 5 (A.B.C).

Three groups of pre-and post-operative serum total 4 patients (11 good prognosis, 15 recurrence, 11 death) were selected for each of the multiple duplicate measurement anova, see table 5. The result shows that the preoperative HOTAIR expression level of the death group is obviously higher than that of the prognosis-good group and the recurrence group; compared with preoperative patients, the serum hotapir of three groups of patients after operation is reduced, but hotapir is obviously increased when the patients in the recurrent group and the patients in the dead group recur, and hotapir is obviously reduced after retreatment. The data suggest that serum HOTAIR can be used as an index for monitoring postoperative progression and recurrence of liver cancer patients.

TABLE 5 serum HOTAIR dynamic monitoring three groups of patients repeated measurement analysis of variance results

^* P < 0.05 compared with T1; ^# P < 0.05 compared with T2; ^△ P < 0.05 compared with T3; ^● P < 0.05 compared to the good prognosis group; ^☆ P < 0.05 compared to the dead group.

To sum up: the results of this experiment are summarized as follows:

The prognosis value of serum LncRNA and HOTAIR on liver cancer patients is discussed, and whether the serum LncRNA and HOTAIR can be used as a serum marker for liver cancer prognosis dynamic monitoring is evaluated.

The method is used for quantitatively detecting the expression of lncRNA HOTAIR in serum of liver cancer, liver cirrhosis and healthy subjects by using a real-time polymerase chain reaction (qRT-PCR). The relationship between serum HOTAIR expression and the clinical pathology of PLC patients was analyzed using a rank-sum test to assess whether it was involved in the development of malignancy. The potential diagnostic and prognostic value was then assessed by subject work signature (ROC) curves, kaplan-Meier, and Cox regression analysis, respectively. And finally, dynamically monitoring the change of serum HOTAIR of the patients at 4 time points according to three different survival outcomes of good prognosis, recurrence and death of the patients after liver cancer operation, and further judging whether the serum HOTAIR can be used as an index of postoperative progress and recurrence of the patients after liver cancer operation by using repeated measurement variance analysis.

The result (1) the expression level of serum HOTAIR of liver cancer patients is obviously higher than that of liver cirrhosis and healthy groups (P is less than 0.01); (2) The expression level of the serum HOTAIR is obviously related to Cnlc stages of liver cancer patients, extrahepatic metastasis, vascular invasion, cancer thrombosis and tumor size (P is less than 0.01); (3) The ROC curve indicates that the optimal diagnosis efficiency can be obtained by combining HOTAIR and AFP, the AUC is 0.942, and the sensitivity and the specificity are respectively 84.3 percent and 90.8 percent; (4) COX regression multifactorial analysis suggests that HOTAIR, cnlc staging, vascular invasion, and cancer thrombosis are independent factors affecting survival of patients after liver cancer surgery, and HOTAIR is also independent factor affecting recurrent metastasis of patients after liver cancer surgery (P < 0.05); (5) Kaplan-Meier analysis shows that the survival rate and recurrence and metastasis rate of patients with low HOTAIR expression are obviously better than those of the patients with high HOTAIR expression (P=0.0127; P=0.033); (6) Repeated measurement of analysis of variance indicates that the preoperative HOTAIR expression level of the death group is obviously higher than that of the prognosis-good group and the recurrence group; compared with preoperative patients, the serum HOTAIR of three groups of patients after operation is reduced, while the HOTAIR of the recurrent group and the patient of the death group is obviously increased when the patients recur, and the HOTAIR of the patients after treatment is obviously reduced (P is less than 0.05).

Conclusion serum LncRNA and HOTAIR are highly expressed in liver cancer patients, can be used for identifying liver cirrhosis and liver cancer, assisting in clinical diagnosis of the liver cancer, evaluating the curative effect of surgery or non-surgery, dynamically monitoring the prognosis progress recurrence of the liver cancer, and is hopeful to become a serum marker for diagnosis and prognosis dynamic monitoring of the liver cancer.

Claims (10)

1. A computer system for diagnosing, detecting, monitoring, predicting prognosis of liver cancer, the system comprising:

i) An analysis module, the analysis module comprising: a test substance for determining the expression level of incrna HOTAIR in a subject, and;

ii) an evaluation module comprising: and (3) judging the prognosis of the liver cancer of the subject according to the determined expression level of the serum lncRNA HOTAIR in the i).

2. A kit for assisting in diagnosing liver cancer adopts serum lncRNA HOTAIR as a marker for assisting in diagnosing liver cancer, and comprises a substance for detecting the serum lncRNA HOTAIR.

3. The application of a substance for detecting the serum lncRNA HOTAIR in preparing a product for diagnosing, detecting, monitoring or predicting liver cancer prognosis.

4. The system of claim 1, the kit of claim 2, or the use of claim 3, wherein the liver cancer is hepatocellular carcinoma.

5. The system of claim 1, the kit of claim 2 or the use of claim 3, wherein the substance for detecting lncRNA HOTAIR comprises a substance used for nucleic acid extraction, reverse transcription and fluorescence measurement.

6. The system of claim 1, the kit of claim 2 or the use of claim 3, wherein the substance for detecting lncRNA HOTAIR comprises a substance for use with quantitative real-time polymerase chain reaction (qRT-PCR).

7. The system of claim 1, the kit of claim 2 or the use of claim 3, wherein the substance for detecting serum lncRNA HOTAIR comprises SYBR GREEN REALTIME PCR MASTER KIT, RNA extraction and separation kit, REVERTRA ACE QPCR RT KIT, chloroform, absolute ethanol, deionized Water, DEPC water, 18s RNA primer, and gene of interest primer.

8. The system of claim 1, wherein the assessment module assesses potential diagnostic and prognostic value by subject operating characteristics (ROC) curve, kaplan-Meier, and Cox regression analysis.

9. The system, kit or use of claim 6, wherein the substance that detects lncRNA HOTAIR comprises a primer sequence:

10. the system, kit or use of claim 6, wherein the substance for detecting lncRNA HOTAIR comprises preparing a PCR reaction solution according to the following components:

S18, internal reference:

LncRNAs：