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ORIGINAL ARTICLE
Year : 2019  |  Volume : 15  |  Issue : 1  |  Page : 126-131

Long noncoding RNA microvascular invasion in hepatocellular carcinoma is an indicator of poor prognosis and a potential therapeutic target in gastric cancer


1 Department of Gastroenterology, Linyi People's Hospital, Linyi, Shandong, China
2 Department of Oncology, Linyi People's Hospital, Linyi, Shandong, China

Date of Web Publication13-Mar-2019

Correspondence Address:
Prof. Luning Li
No. 27, Jiefang Road, Linyi, Shandong 276000
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.204882

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 > Abstract 


Background: Long noncoding RNAs (lncRNAs) have been shown to have a fundamental role in cancer initiation and development. LncRNA microvascular invasion in hepatocellular carcinoma (MVIH) has been identified as a potential prognostic marker in several cancers; however, its role in gastric cancer (GC) has not been elucidated.
Materials and Methods: A total of 152 tissue samples from patients underwent GC surgical resection in Linyi People's Hospital between 2007 and 2010 were collected. Quantitative real-time polymerase chain reaction was conducted to examine the expression level of lncRNA MVIH. The selection of clinically important cut-off scores for MVIH expression was based on receiver operating characteristic curve analysis. Then, the association between MVIH and GC clinicopathological parameters was analyzed. Moreover, univariate and multivariate Cox regression analysis were performed to reveal the relationship between MVIH and GC prognosis.
Results: GC tissues exhibited a higher lncRNA MVIH expression level than paired nontumoros tissues. High MVIH level was revealed to be associated with the T stage, tumor-node-metastasis (TNM) stage and lymphatic metastasis of GC. Specially, patients with high MVIH expression level showed significantly shorter overall survival rate and progression-free survival rate. Moreover, invasion depth, distant metastasis, TNM stage, and MVIH expression were identified as risk factors of GC poor prognosis on univariate Cox regression analyses. By further analyzing these factors with multivariate logistic regression, high MVIH, and distant metastasis were discovered to be independent risk factors of GC prognosis.
Conclusions: High MVIH is an independent risk factor of GC prognosis. LncRNA MVIH may serve as a potential therapeutic target and a prognostic marker of GC patients.

Keywords: Gastric cancer, long noncoding RNA, microvascular invasion in hepatocellular carcinoma, prognosis


How to cite this article:
Wang A, Du L, Jiang K, Kong Q, Zhang X, Li L. Long noncoding RNA microvascular invasion in hepatocellular carcinoma is an indicator of poor prognosis and a potential therapeutic target in gastric cancer. J Can Res Ther 2019;15:126-31

How to cite this URL:
Wang A, Du L, Jiang K, Kong Q, Zhang X, Li L. Long noncoding RNA microvascular invasion in hepatocellular carcinoma is an indicator of poor prognosis and a potential therapeutic target in gastric cancer. J Can Res Ther [serial online] 2019 [cited 2019 Aug 20];15:126-31. Available from: http://www.cancerjournal.net/text.asp?2019/15/1/126/204882




 > Introduction Top


Gastric cancer (GC) is the fourth most frequently diagnosed cancer and the second leading cause of cancer-related death around the world.[1] Particularly, the incidence rate and motility rate are relatively high in China.[2] Most GC patients have no specific symptoms at the early stage, and many patients are diagnosed at an advanced stage. Unfortunately, therapeutic approaches for advanced GC are limited at present, and the 5-year survival rate is only 10%–15% for GC patients with advanced stage.[3] Hence, it is of great importance to find novel biomarkers for GC early diagnosis and prognosis evaluation.

Long noncoding RNAs (lncRNAs) have attracted great attention in the past few years for its characteristics.[4] It has been discovered that lncRNAs are exquisitely regulated and are restricted to specific cell types to a greater degree than messenger RNA (mRNA).[5] In addition, lncRNAs were detected to have frequently evolutionarily conserved function, secondary structure and regions of microhomology.[6],[7] Furthermore, lncRNAs have been revealed to play a role in regulating proliferation, motility, immortality, angiogenesis, and viability in different cancer phenotypes.[4] Thus, lncRNAs are considered potentially early diagnostic marker and therapeutic target of cancer. LncRNA microvascular invasion in hepatocellular carcinoma (MVIH) was first investigated to be correlated with hepatic cellular carcinoma recurrence-free survival rate in 2012.[8] Subsequently, lncRNA MVIH was revealed as poor prognostic marker of breast cancer and nonsmall cell lung cancer patients.[9],[10] However, the clinical relevance and functional role of MVIH in carcinogenesis of GC have not been illuminated yet.

In this study, the expression of lncRNA MVIH was confirmed to be upregulated in GC tissues in comparison with paired adjacent normal tissues. Moreover, high expression of MVIH was demonstrated to be correlated with clinicopathological characteristics and poor prognosis of GC patients.


 > Materials And Methods Top


Tissue samples and clinical data collection

In this study, a total of 152 tissue samples from patients underwent GC surgical resection in Linyi People's Hospital between September 2007 and October 2010 were collected. The paired nontumoros tissues were isolated from at least 1 cm away from the tumor border and were shown to lack tumor cells by microscopy. The samples were immediately frozen in liquid nitrogen and stored at −80°C until analysis. Final diagnosis was made based on the pathological examination. None of the patients had received preoperative anticancer treatments. Patients with two or more different malignancies were also excluded.

The clinicopathological characteristics of the patients are shown in [Table 1]. Follow up were carried out primarily by out-patient examination or telephone. The mean follow up period was 51 months (ranges from 4 months to 100 months). Overall survival (OS) was defined as the interval between the dates of surgery and death or the last follow-up examination. Progression-free survival (PFS) was calculated from the date of tumor resection until recurrence occurs, death, or the last observation; if recurrence was not diagnosed, patients were censored on the date of death or the last follow-up. Tumor staging was defined according to the sixth edition of the tumor-node-metastasis (TNM) classification system published by the International Union against Cancer and the Barcelona Clinic Liver Cancer staging system. Written informed consent was obtained from all patients. This study was approved by the Ethics Committee of Linyi People's Hospital.
Table 1: Correlation Between MVIH Expression and Clinicopathological Characteristics of gastric cancer patients

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RNA isolation and quantitative real-time polymerase chain reaction

Trizol reagent (Invitrogen) was utilized to extract total RNAs from tumor tissues and paired nontumoros tissues following the instructions of the manufacturer. Total RNA was reversely transcribed into complementary DNA using the PrimeScript One Step Real-time Polymerase Chain Reaction (RT-PCR) Kit (Takara, Dalian, China). Quantitative RT-PCR (qRT-PCR) was performed with a SYBR PrimeScript RT-PCR Kit (Takara, Dalian, China) according to the manufacturer's protocol. The mRNA level of lncRNA MVIH was evaluated by qRT-PCR on an ABI7300HT instrument (Applied Biosystems, Foster City, CA, USA). The primer sequences for MVIH were 5′-GAGACAGGATTTAGCCGTGTTG-3′ (sense) and 5′-AGCACTTTGGAAGGCTTAGACA-3′ (antisense). The qRT-PCR assay was done in triplicate, and relative MVIH expression was normalized to glyceraldehyde 3-phosphate dehydrogenase using the comparative cycle threshold (2−ΔΔCT) method.

Selection of cut-off scores

The selection of clinically important cut-off scores for MVIH expression was based on receiver operating characteristic (ROC) curve analysis using the 0, 1-criterion. Briefly, the score with the shortest distance from the curve to the point with both maximum sensitivity and specificity, that is, the point (0.0, 1.0), was selected as the cut-off score leading to the largest number of tumors correctly classified as having or not having the clinical outcome. At MVIH score, the sensitivity and specificity for the OS and PFS under study was plotted, thus generating two ROC curves. The count was selected as the cut-off value, which was closest to the point with both maximum sensitivity and specificity. Cases defined as high MVIH expression were those with the scores above or equal to the cut-off value, whereas low MVIH expression represented those with scores below the value. To perform ROC curve analysis, clinicopathological features were dichotomized to death due to GC or censored (lost to follow-up, alive, or death from other causes).

Statistical analyses

All statistical analyses were performed using the SPSS 19.0 software (SPSS Inc., Chicago, USA). For statistical comparisons, the Chi-square test, the Fisher's exact test, the one-way analysis of variance, the two-tailed Student's t-test, or the Wilcoxon test were performed as appropriate. OS and PFS rates were calculated using the Kaplan–Meier method with the log-rank test applied for comparison. Univariate and multivariate Cox proportional hazards models were conducted to evaluate the survival data. Variables with the value of P < 0.05 in univariate analysis were used in subsequent multivariate analysis on the basis of Cox regression analyses. P < 0.05 was considered statistically significant.


 > Results Top


Long noncoding RNA microvascular invasion in hepatocellular carcinoma expression is up-regulated in gastric cancer tissues compared with paired adjacent nontumoros tissues

To identify the functional role of lncRNA MVIH in GC, its expression level was detected in 152 paired fresh GC tissues and paired adjacent nontumoros tissues by qRT-PCR assay. The results showed that lncRNA MVIH was upregulated in 112 (73.68%) GC tissues compared with paired nontumoros tissues. Moreover, the relative expression level of lncRNA MVIH in GC tissues was significantly higher than in paired nontumoros tissues (100% vs. 252% ±310%, P < 0.001) [Figure 1]a.
Figure 1: Long noncoding RNA microvascular invasion in hepatocellular carcinoma expression is increased in gastric cancer tissues and correlates with poor prognosis. (a) Quantitative real-time polymerase chain reaction was performed to analyze the relative expression of microvascular invasion in hepatocellular carcinoma in gastric cancer tissues and paired nontumoros tissues. (b and c) The cut-off points of microvascular invasion in hepatocellular carcinoma expression for overall survival and progression-free survival analyzed by receiver operating characteristic curve. (d and e) Kaplan–Meier analysis was adopted to investigate the overall survival and progression-free survival differences

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The association between microvascular invasion in hepatocellular carcinoma expression and gastric cancer clinicopathological characteristics

The selection of clinically important cut-off scores for MVIH expression was based on ROC curve analysis using the 0, 1-criterion. The sensitivity and specificity of MVIH were analyzed using variable cut-off values. The MVIH cut-off point was determined to be 3.85 for both OS and PFS in the cohort, which was selected as the uniform cut-off point to categorized GC patients as high or low MVIH expression [Figure 1]b and [Figure 1]c.

Furthermore, the correlation of MVIH expression with GC clinicopathological features was analyzed. As shown in [Table 1], high MVIH expression was discovered to be statistically correlated with a greater depth of invasion (P < 0.001), higher TNM stage (P < 0.001) and more frequent lymphatic metastasis (P = 0.045). However, there was no significant correlation between MVIH expression and other clinicopathological characteristics, such as age, gender, histologic differentiation, and distant metastasis (P > 0.05) [Table 1]. Taken together, these results implicated that MVIH up-regulation may be associated with tumor progression and development.

Microvascular invasion in hepatocellular carcinoma serves as a potential prognostic marker in gastric cancer patients

Kaplan–Meier analysis and log-rank test were performed to investigate the effects of MVIH expression on OS and PFS. The patients with high MVIH expression displayed shorter OS and PFS than those with low MVIH expression [Figure 1]d and [Figure 1]e.

Univariate Cox regression analyses identified 4 prognostic factors for OS: Invasion depth (T1, T2 vs. T3, T4) (P < 0.001, hazard ratio [HR] =2.018, 95% confidence interval [CI] =1.399–2.912), distant metastasis (yes vs. no) (P = 0.002, HR = 3.853, 95% CI = 1.651–8.991), TNM stage (I, II vs. III, IV) (P < 0.001, HR = 2.418, 95% CI = 1.555–3.760), and MVIH expression (low vs. high) (P < 0.001, HR = 2.809, 95% CI = 1.867–4.227). Four prognostic factors were identified on univariate Cox regression analyses for PFS: Invasion depth (T1, T2 vs. T3, T4) (P < 0.001, HR = 1.966, 95% CI = 1.365–2.831), distant metastasis (yes vs. no) (P = 0.005, HR = 3.307, 95% CI = 1.425–7.677), TNM stage (I, II vs. III, IV) (P < 0.001, HR = 2.321, 95% CI = 1.493–3.608), and MVIH expression (low vs. high) (P < 0.001, HR = 2.889, 95% CI = 1.921–4.346). Other clinicopathological characteristics, such as age, gender, location, histological differentiation, and lymph node invasion, were not statistically significant prognosis factors [Table 2]. Further identification with multivariate Cox regression model revealed that distant metastasis (P = 0.020, HR = 3.176, 95% CI = 1.203–8.388) and MVIH expression level (P < 0.001, HR = 2.522, 95% CI = 1.595–3.988) served as independent prognostic factors for poor OS. Similarly, we demonstrated that distant metastasis and MVIH expression level served as independent prognostic factors for PFS. Taken together, MVIH expression is an independent predictor for OS and PFS of GC patients [Table 2].
Table 2: Univariate and multivariate Cox regression analyses MVIH for PFS or OS of gastric cancer patients

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 > Discussion Top


Numerous studies have focused on lncRNAs in GC for their primary role in GC initiation and development.[4],[11],[12],[13],[14],[15] Yang et al.[15] have reviewed the potential mechanisms of lncRNAs mediating GC metastasis. They concluded that lncRNAs are involved in angiogenesis, cell-to-cell junction and adhesion, extracellular matrix degradation and thus regulating GC metastasis. In addition, some lncRNAs have been shown to be related with GC proliferation.[16],[17],[18] As is known to all, GC is asymptomatic in the early stage and most patients are diagnostic at the late stage for the lack of early diagnostic marker. It is necessary to find noninvasive cancer biomarkers for the early diagnosis of GC. Human plasma and gastric juice are relatively noninvasive tools for the early detection of cancer. However, the existing markers for GC diagnosis, such as carcinoembryonic antigen, carbohydrate antigen (CA) 19-9, and CA 72-4, are lack of enough specificity and sensitivity. LncRNAs could be detected in human plasma and gastric juice, which provides a much convenient way for GC early diagnosis prognostic predication. Some lncRNAs, such as H19, appear to have strong diagnostic potential as blood biomarkers for GC.[14] Interestingly, it was reported that higher lncRNA AA174084 postoperative plasma level was correlated with worse pathological results, besides, higher gastric juice AA174084 level was associated with greater tumor dimension and more advanced tumor stage. Therefore, AA174084 may be considered a potential marker for GC early diagnosis and prognostic evaluation.[19] Besides, some lncRNAs, including PVT1, JMJD1A, H19, PANDAR and so on, have been confirmed to be potential prognostic marker of GC.[20],[21],[22] In addition, lncRNAs are restricted to specific cell types to a greater degree than mRNA, and usually have evolutionarily conserved function and secondary structure, which make lncRNAs appropriate diagnostic markers and prognostic markers. Certainly, there are also lots of limitations in utilizing lncRNAs as diagnostic marker and prognostic marker, such as the detailed function of lncRNAs should be further illuminated and the specificity and sensitivity of lncRNAs should be improved. Together, lncRNAs can be promising biomarkers for clinical application for GC in the near future if we can solve the above questions with large-scale investigations.[23]

The functional role of lncRNA MVIH has been discovered in several cancers. Yuan et al.[8] reported that the deregulation of lncRNA-MVIH is a predictor for poor recurrence-free survival of hepatocellular cancer patients after hepatectomy and could be utilized as a potential target for new adjuvant therapies against active angiogenesis; mechanistically, they found MVIH could activate tumor-inducing angiogenesis by inhibiting the secretion of phosphoglycerate kinase 1. Recently, Shi et al.[24] demonstrated that MVIH could promote cell growth and inhibit cell apoptosis of hepatocellular cancer via inhibiting miR-199a expression. Furthermore, MVIH has been revealed to be a potential therapeutic target and prognostic marker in nonsmall cell lung cancer and breast cancer.[9],[10] However, there is no study about the function of MVIH in GC till now.

In this study, MVIH expression level was demonstrated to be significantly higher in GC tissue samples than in paired nontumorous tissues. Furthermore, the associations between MVIH expression and GC clinicopathological factors were investigated. Interestingly, patients with high MVIH expression levels exhibited a greater depth of invasion, higher TNM stage, and more frequent lymphatic metastasis. Besides, patients with high MVIH expression levels were revealed to have a significantly poorer OS and PFS compared with the low expression group. Moreover, high MVIH expression was confirmed to be a risk factor of GC prognosis both in univariate analysis and multivariate analysis, implicating MVIH is an independent prognostic factor of GC patients. Together, these observations indicated that MVIH may serve as a therapeutic target and prognostic marker of GC patients.


 > Conclusions Top


The present study demonstrated that high MVIH expression was associated with T stage, TNM stage, and lymphatic metastasis of GC patients; besides, the patients with high MVIH expression displayed much poor OS and PFS and high MVIH expression was revealed to be an independent risk factor for GC prognosis. Together, these results suggested that MVIH may serve as a potential therapeutic target and a prognostic marker of GC patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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