Research ArticleExperimental Studies

Expression of LncRNAs in HPV-induced Carcinogenesis and Cancer Cachexia: A Study in K14-HPV16 Mice

TÂNIA R. DIAS, JOANA M. O. SANTOS, DIOGO ESTÊVÃO, NATÁLIA R. COSTA, VERÓNICA F. MESTRE, BEATRIZ MEDEIROS-FONSECA, MARGARIDA M.S.M. BASTOS, PAULA A. OLIVEIRA, RUI M. GIL DA COSTA and RUI MEDEIROS
Anticancer Research May 2022, 42 (5) 2443-2460; DOI: https://doi.org/10.21873/anticanres.15723

Abstract

Aim: To evaluate the expression of lincRNA-p21, H19, EMX2OS, SNHG12 and MALAT1 in a mouse model of human papillomavirus 16 (HPV16)-induced carcinogenesis and cachexia. Materials and Methods: Chest skin, ear, tongue, penis and gastrocnemius muscle samples from wild-type mice (HPV−) and K14-HPV16 male mice (HPV+) were collected to evaluate the expression of the selected lncRNAs using real-time PCR (qPCR). Results: In chest skin and ear, H19, SNHG12, EMX2OS and lincRNA-p21 were down-regulated in HPV+ versus HPV– mice. In tongue and penile tissues, there was only down-regulation of lincRNA-p21 in HPV+ mice. Additionally, in penile tissue, lincRNA-p21 expression decreased in HPV-induced lesions comparing with normal tissues. In gastrocnemius muscle, MALAT1 was up-regulated and lincRNA-p21 was down-regulated in HPV+ versus HPV–mice. Conclusion: H19, SNHG12, EMX2OS and lincRNA-p21 may be involved in HPV-induced carcinogenesis. In addition, MALAT1 and lincRNA-p21 may play a role in muscle wasting and contribute to cancer cachexia.

Key Words:

High-risk human papillomavirus (HR-HPV) is characterised as an effective carcinogenic agent transmitted by sexual contact and a major cause of diverse types of cancer (1, 2). Besides the cervix, this infection is also associated with other anogenital cancers, such as cancer of anus, penis, vulva and vagina (3-6). Additionally, HR-HPV is also associated with head and neck cancers, including oropharynx, oral cavity and larynx (1, 3, 7-10).

Cancer patients often suffer from cancer cachexia, which is defined as a wasting syndrome and is characterised by systemic inflammation, loss of weight, and loss of adipose tissue and skeletal muscle (11-15). This syndrome conditionates therapeutic options of cancer patients, reducing their quality of life (16-18). Therefore, it is crucial to find new biomarkers and therapeutic targets for cancer cachexia (17, 19, 20).

Long non-coding RNAs (lncRNAs) have been demonstrated to be crucial mediators of numerous diseases (21). In cancer cachexia, the involvement of lncRNAs and their role have only been described in a few studies, however, it seems that they affect diverse molecular pathways of cachexia, including those involved in muscle wasting (20, 22, 23). Nevertheless, these non-coding RNAs seem to be potential biomarkers and therapeutic targets of cancer cachexia (20, 22, 23). Besides, lncRNAs are also significantly involved in hallmarks of malignancy associated with HPV-induced cancer development (24).

Various studies in different types of cancer have demonstrated an increase of the expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and a crucial role of this lncRNA in the regulation of several molecular pathways that lead to cancer progression (25-27). Besides, MALAT1 stimulates proliferation by increased levels of cyclin D1, cyclin E, and cyclin dependent kinase 6 (CDK6), which are crucial molecules in cell cycle regulation (28). Furthermore, MALAT1 was proved to be an important regulator of the skeletal muscle myogenesis being regulated by myostatin (29).

There are a few studies that have revealed differences in the H19 expression between HPV-positive and HPV-negative in cervical cancer as well as in head and neck squamous cell carcinoma, suggesting a potential role in HPV-related carcinogenesis (30). Although H19 is associated with the control of skeletal muscle differentiation and development, there are no studies related to cancer cachexia; therefore, it could be relevant to understand the role of this lncRNA in muscle wasting.

Moreover, an association between the HPV E6 and E7 and small nucleolar RNA host gene 12 (SNHG12) has been shown, demonstrating that this lncRNA is regulated by these oncoproteins (31). On the other side, it was also proved that this lncRNA controls the expression of the transcription factor signal transducer and activator of transcription 3 (STAT3) (32). Although STAT3 is crucial in cancer proliferation and invasion, it is also a key factor in muscle wasting (32-34).

The EMX2 opposite strand/antisense RNA (EMX2OS) is a lncRNA that seems to be a potential biomarker in the head and neck squamous cell carcinoma (35, 36). However, the role of EMX2OS in cancer progression is still controverse, since some studies have demonstrated that it acts as a tumour suppressor and others as an oncogene (37, 38). Its biological action in HPV-induced cancers remain scarce, while to best of our knowledge, there are no studies concerning muscle wasting.

LincRNA-p21 was described as a component of the p53 pathway, therefore, several studies identified this lncRNA as a key factor in suppression of carcinogenesis (39-41). Moreover, this lncRNA is able to bind with STAT3, suppressing the janus kinase 2 (JAK2)/STAT3 signalling and consequently acting as a tumour suppressor (42). Additionally, this signalling pathway is involved in one of the cachexia pathways, increasing its probable influence in triggering cachexia (33, 34).

K14-HPV16 is an useful in vivo animal model that mimics the HPV-induced carcinogenesis (43, 44). In these mice the expression of HPV16 early-region genes occurs in the epithelial basal cells, since these genes are under the control of the human keratin-14 (K14) promoter (44, 45). We have previously used these transgenic mice to study the role of diverse miRNAs in HPV-driven carcinogenesis (46-53). Moreover, this animal model is an effective model to study cancer cachexia, and muscle wasting has already been detected in these mice (53, 54). Thus, the aim of this study was to examine the expression of specific lncRNAs (MALAT1, H19, SNHG12, EMX2OS, LincRNA-p21) in skeletal muscle and target organs of HPV-driven carcinogenesis and to evaluate their potential role in multistep HPV-induced carcinogenesis and cancer cachexia.

Materials and Methods

Animal model. The generation of K14-HPV16 transgenic mice on a FVB/n background has been previously reported (44). These transgenic mice were kindly donated by Dr. Jeffrey Arbeit and Dr. Douglas Hanahan (University of California) through the USA National Cancer Institute Mouse Repository. The animal experiments were approved by the University of Trás-os-Montes and Alto Douro Ethics Committee (approval no. 10/2013) and the Portuguese General Veterinary Directorate (0421/000/000/2014). All the mice were housed and bred according to Portuguese (Decreto-Lei 113, August 7th) and European (EU Directive 2010/63/EU) legislation, under controlled temperature (23±2°C), light-dark cycle (12 h light/12 h dark) and relative humidity (50±10%). Food and water were provided ad libitum. In order to confirm the integration of the HPV16 early region, all mice were genotyped (55). Additionally, the mRNA expression of E6, E7 and E5 oncogenes was assessed in the studied organs by qPCR, using methods recently described by Neto et al., 2021 (56).

Experimental design, histological analysis, cachexia evaluation and genotyping. In this study, we used HPV 16-transgenic mice as previously described by Peixoto da Silva S et al., since their cachectic status and histological analysis were already established (55). Briefly, 19 male K14-HPV16 (HPV+) and wild-type (HPV-) were divided in 2 groups: Group 1 (9 male HPV–), group 2 (10 male HPV+). From 9-11 up to 31-33 weeks-old, mice were weekly weighted and food intake registered (55). At the time of the sacrifice chest skin, ear skin, tongue, penile and gastrocnemius muscle samples were weighted and collected into TripleXtractor reagent (Grisp®, Porto, Portugal), macerated and kept at –80°C until further use (55). Additionally, matched samples of chest skin, ear, penis and tongue samples were also collected for histological analysis. HPV+ mice showed a variety of lesions that were classified as previously described as hyperplasia, dysplasia and carcinoma, and also had higher food intake but lower body and gastrocnemius weights compared with HPV– mice (55).

RNA isolation. Total RNA from chest skin, ear skin, tongue, penile and gastrocnemius muscle samples preserved in TripleXtractor reagent (Grisp®) were extracted with chloroform (EMSURE®, Darmstadt, Germany) followed by centrifugation at 15.000 × g for 15 min at 4°C in order to separate the RNA phase. Then RNA purification was performed with a commercial kit GRS total RNA kit (Grisp®), according to the manufacturer’s instructions. DNAse I treatment was included for all the samples. RNA concentration and purity were measured using the NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA samples were kept at –80°C until further use.

Complementary DNA synthesis. Total RNA (300 ng) were used as template for cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). In a 20 μl reaction mix, 2.0 μl of 10× RT Buffer, 0.8 μl of 25× dNTP Mix (100 mM), 2.0 μl of 10× RT Random Primers, 1.0 μl of MultiScribe reverse transcriptase, 4.2 μl of nuclease-free water, and 10.0 μl of RNA were used. The reverse transcription was performed in a Biometra® Personal Cycler (Biometra) with the following conditions: 10 min at 25°C, 120 min at 37°C and 5 min at 85°C. A non-template negative control was included in all reactions. cDNA was further used as template for quantitative real-time PCR (qPCR).

Quantitative real-time PCR. The cDNA was then used as a template for quantitative real-time PCR (qPCR) using a StepOne qPCR real-time PCR device (Applied Biosystems, Waltham, MA, USA). Each reaction was performed with 5 μl of 2× TaqMan® Fast Advanced Master Mix (Applied Biosystems®, Waltham, MA, USA) with 0.5 μl of 20× TaqMan® Gene Expression Assays (β-actin: Mm01205647_g1; HPRT: Mm01545399_m1; Malat1: Mm03949341_s1; H19: Mm01156721_g1; SNHG12: Mm03930449_m1; EMX2OS: Mm01264773_m1; LincRNA-p21/Trp53cor1: Mm03976700_m1), 3.5 μl of nuclease-free water and 1 μl of cDNA sample, making a total volume of 10 μl. The quantification was performed in duplicate and samples with CT standard deviation values superior to 0.5 were excluded. Negative controls lacking cDNA were also included in all reactions. All target and endogenous controls for each sample were amplified in the same plate. The thermal cycling conditions for all assays were the following: 10 min at 95°C followed by 45 cycles of 15 s at 95°C and 1 min at 60°C. The amplification efficiency for all assays was confirmed using a 2-fold serial dilution with 6 dilution steps, using cDNA reverse transcribed from Universal Mouse Reference RNA (Invitrogen™, Waltham, MA, USA). The same baseline and threshold were set for each plate using the analysis software for qPCR from the Thermo Fisher Connect platform (Thermo Fisher Scientific), in order to generate threshold cycle (Ct) values for all the genes in each sample. HPRT and β-actin were tested as potential endogenous controls, and the combination of HPRT and β-actin was selected for chest skin, ear skin, tongue, penile and HPRT was selected for gastrocnemius muscle, since it showed the lowest standard deviation values using BestKeeper Software.

Statistical analysis. The expression of lncRNAs was analysed using the Livak method along with Mann-Whitney U-test using SPSS 27.0 software (IBM, Armonk, NY, USA). All the graphics were constructed using GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA, USA). The results were considered statistically significant when the p-values were ≤0.05.

Ethical approval. The animal experiments were approved by the University of Trás-os-Montes and Alto Douro Ethics Committee (approval no. 10/2013) and the Portuguese General Veterinary Directorate (0421/000/000/2014). All the mice were housed and bred according to Portuguese (Decreto-Lei 113, August 7th) and European (EU Directive 2010/63/EU) legislation.

Results

Expression of HPV16 E5, E6, and E7 in male mice. In order to confirm the expression of HPV16 oncogenes in the lesions of transgenic mice, we determined their mRNA expression at the different studied organs, namely chest skin, ear, tongue and penis. The mRNA expression of E6, E7 and E5 oncogenes was measured in these organs in the K14-HPV16 mice (Figure 1), whereas no expression was observed in the wild-type mice.

Figure 1.
Figure 1.

Normalised relative expression (−∆Ct) of oncogenes in K14-HPV16 mice. Expression of the oncogenes E5, E6, and E7 was examined in chest skin tissue (A), in ear tissue (B), in tongue tissue (C) and in penile tissue (D) *p<0.05; **p<0.01; ***p<0.001.

LncRNAs expression in chest skin. MALAT1 expression levels did not differ between HPV– and HPV+ mice (Figure 2A). The expression level of H19 was significantly higher in HPV– mice compared to HPV+ (3.32-fold, p=0.011) (Figure 2B). The expression levels of SNHG12 and EMX2OS were significantly higher in HPV– compared to the HPV+ mice (1.98-fold, p=0.034 and 3.92-fold, p=0.011, respectively) (Figure 2C, 2D). Concerning lincRNA-p21, a 9.33-fold higher expression level was observed in HPV– mice (p=0.001) in comparison with HPV+ (Figure 2E).

Figure 2.
Figure 2.

Relative expression of the lncRNAs in murine chest skin tissue. The expression levels of MALAT1 (A), H19 (B), SNHG12 (C), EMX2OS (D) and lincRNA-p21 (E) were compared between HPV– and HPV+ mice. *p<0.05; **p<0.01; ***p<0.001.

LncRNAs expression along with the histological chest skin analysis. We also decided to evaluate whether the expression of these lncRNAs was correlated with the type of lesion and severity. Consequently, we compared the lncRNAs expression among normal skin and hyperplastic skin. The expression of all the tested lncRNAs was significantly higher in normal skin than in hyperplastic skin. Specifically, H19 showed 3.32-fold change (p=0.011) (Figure 3A), SNHG12 expression showed 1.98-fold change (p=0.034) (Figure 3B), EMX2OS expression level was 3.92-fold higher (p=0.011) (Figure 3C), and lincRNA-p21 expression level was 9.33-fold higher (p=0.001) (Figure 3D).

Figure 3.
Figure 3.

Relative expression of the lncRNAs in murine chest skin tissue in the different types of histological skin classification. The expression levels of H19 (A), SNHG12 (B), EMX2OS (C) and lincRNA-p21 (D) were compared between hyperplastic skin and normal skin. *p<0.05; **p<0.01; ***p<0.001.

LncRNAs expression in ear. In the ear, MALAT1 expression did not show any changes. The H19 expression was 5.47-fold higher in HPV– than in HPV+ mice (p=0.005) (Figure 4B). Expression of SNHG12 was also significantly higher in HPV– mice (4.94-fold, p<0.001) (Figure 4C). In addition, EMX2OS and lincRNA-p21 showed 9.64-fold and 5.51-fold higher expression, respectively, in HPV– than in HPV+ mice (p<0.001 and p=0.001, respectively) (Figure 4D and Figure 4E).

Figure 4.
Figure 4.

Relative expression of the lncRNAs in murine ear tissue. The expression levels of MALAT1 (A), H19 (B), SNHG12 (C), EMX2OS (D) and lincRNA-p21 (E) were compared between HPV– and HPV+ mice. *p<0.05; **p<0.01; ***p<0.001.

LncRNAs expression along with the ear histological analysis. Next, we compared the expression of the lncRNAs in ear tissue with the histological analysis evaluation previously made. Accordingly, we compared the lncRNAs expression levels among normal tissue, hyperplastic and dysplastic lesions. The H19 expression level significantly differed between normal tissue and the hyperplastic lesion or the dysplastic lesion (p=0.011 and p=0.040, respectively) (Figure 5A). More specifically, normal tissue expressed H19 at a 5.46-fold higher level than hyperplasia and 1.01-fold higher than dysplasia. Similar patterns of expression levels were observed for SNHG12, EMX2OS, and lincRNA-p21. SNHG12 demonstrated 4.58-fold higher expression in normal tissue than in hyperplastic lesion (4.58-fold, p=0.001) or compared to dysplastic lesion (1.47-fold, p=0.040) (Figure 5B). Expression of EMX2OS was significantly higher in the normal tissue than in hyperplasia (8.41-fold, p=0.001), or in dysplasia (2.07-fold, p=0.040) (Figure 5C). The expression of lincRNA-p21 was 5.10-fold higher in normal tissue than in hyperplastic lesion and 1.41-fold higher in comparison with dysplastic lesion (p=0.003 and p=0.046, respectively) (Figure 5D).

Figure 5.
Figure 5.

Relative expression of the lncRNAs in murine ear tissue in the different types of histological classification. The expression levels of H19 (A), SNHG12 (B), EMX2OS (C) and lincRNA-p21 (D) were compared between hyperplasia, dysplasia and normal tissue. *p<0.05; **p<0.01; ***p<0.001.

LncRNAs expression in tongue. In tongue tissue, lincRNA-p21 was the only lncRNA that demonstrated a statistically significant difference between HPV– and HPV+ mice (p=0.045) (Figure 6D). Its expression level was 2.16-fold higher in HPV– than the HPV+ mice. Furthermore, we compared the expression of lincRNA-p21 in tongue tissue with the histological analysis evaluation previously made (normal, hyperplastic, dysplastic lesions). Nevertheless, we did not observe any statistically significant differences between the different types of histological classification (Figure 7). There was also one tongue tissue sample that was histologically classified as carcinoma; however, no statistical comparison could be performed since it was only one.

Figure 6.
Figure 6.

Relative expression of the lncRNAs in murine tongue tissue. The expression levels of MALAT1 (A), H19 (B), SNHG12 (C) and lincRNA-p21 (D) were compared between HPV– and HPV+ mice. *p<0.05; **p<0.01; ***p<0.001.

Figure 7.
Figure 7.

Relative expression of lincRNA-p21 in murine tongue tissue in the different types of histological classification. The expression levels of lincRNA-p21 were compared between hyperplasia, dysplasia and normal tissue. *p<0.05; **p<0.01; ***p<0.001.

LncRNAs expression in penile tissues. In penile tissues, the lncRNA lincRNA-p21 expression level had a statistically significant difference between HPV– and HPV+ mice (p=0.010) (Figure 8E). More specifically, its expression level was 2.91-fold higher in HPV– than in HPV+ mice penile tissues. We also evaluated the correlation between the expression of this lncRNA and the type of lesion (normal tissue and hyperplasia). There was also one penile tissue sample histologically classified as dysplasia, however, we did not perform statistical comparisons with this sample. LincRNA-p21 had significantly higher expression in normal penile tissue than in hyperplastic lesions (2.71-fold, p=0.042) (Figure 9).

Figure 8.
Figure 8.

Relative expression of the lncRNAs in murine penile tissue. The expression levels of MALAT1 (A), H19 (B), SNHG12 (C), EMX2OS (D) and lincRNA-p21 (E) were compared between HPV– and HPV+ mice. *p<0.05; **p<0.01; ***p<0.001.

Figure 9.
Figure 9.

Relative expression of lincRNA-p21 in murine penile tissue in the different types of histological classification. The expression levels of lincRNA-p21 were compared between hyperplasia, dysplasia and normal tissue. *p<0.05; **p<0.01; ***p<0.001.

LncRNAs expression in gastrocnemius muscle. The expression levels of MALAT1, H19, SNHG12, EMX2OS and lincRNA-p21 in gastrocnemius muscle were assessed by qPCR in all mice, in order to examine whether their expression is associated with muscle wasting. The MALAT1 expression levels were significantly increased in gastrocnemius from HPV+ mice (1.95-fold, p=0.050) when compared to control mice (Figure 10A). Concerning lincRNA-p21, a statistically significant difference was found between its expression levels in HPV– and HPV+ mice (2.18-fold higher in HPV–, p=0.003) (Figure 10E). Regarding H19, SNHG12, and EMX2OS, no significant difference was observed.

Figure 10.
Figure 10.

Relative expression of the lncRNAs in murine gastrocnemius muscle. The expression levels of MALAT1 (A), H19 (B), SNHG12 (C), EMX2OS (D) and lincRNA-p21 (E) were compared between HPV– and HPV+ mice. *p<0.05; **p<0.01; ***p<0.001.

Discussion

K14-HPV16 transgenic mice is a useful in vivo animal model that mimics the HPV-induced carcinogenesis (43, 44). Besides, this is also an effective animal model to study cancer cachexia since muscle wasting has already been detected in these mice (53, 54). Thus, this study examine the expression of specific lncRNAs (MALAT1, H19, SNHG12, EMX2OS, LincRNA-p21) in skeletal muscle and target organs of HPV-driven carcinogenesis and evaluate their potential role in multistep HPV-induced carcinogenesis and cancer cachexia. Therefore, H19, SNHG12, EMX2OS and lincRNA-p21 seems to be potential tumour suppressors during HPV-induced carcinogenesis and MALAT1 and lincRNA-p21 seem to play significant roles in muscle wasting, contributing to the pathophysiology of cancer cachexia.

LncRNAs expression and the HPV-induced carcinogenesis. Diverse lncRNAs are recognised as key factors in the regulation of gene expression in various biological functions and cellular contexts, being able to regulate several cell signalling pathways (57). Importantly, numerous lncRNAs seem to regulate the hallmarks of cancer and their deregulation has been related to cancer progression (58-60). As reviewed by our group, recent data have shown that lncRNAs are significantly involved in either promoting or countering the hallmarks of malignancy observed in HPV-induced cancers (24). In the present study, we found that specific lncRNAs were down-regulated in the different organs of HPV+ mice (Table I).

View this table:
Table I.

LncRNA down-regulation or up-regulation in HPV+ mice in all the organs.

MALAT1. Diverse studies described MALAT1 as an oncogene during the development of various cancers (61). This lncRNA was recognised as a potential promoter of cancer invasion and metastasis (62, 63). Besides, it was demonstrated that the expression of MALAT1 was positively correlated with HPV infection (63, 64). However, in our results, MALAT1 expression level had no statistically significant differences between wild-type and transgenic mice in any of the HPV-induced lesions. These results could be explained by the fact that, in our mice model, it is possible to observe several pre-malignant lesions and MALAT1 has been correlated with the later stages of the carcinogenesis process (27, 63).

H19. The function of the lncRNA H19 is still controversial since multiple studies demonstrated that H19 could act as either an oncogene or a tumour suppressor (65). So far, studies that correlated the role of H19 with HPV-related cancers remain scarce. Moreover, several studies have shown that the expression levels of H19 significantly differ between HPV-positive and HPV-negative head and neck cancers and cervical cancers; thus, demonstrating a potential role of this lncRNA in HPV-induced carcinogenesis (30, 66). Our results showed that H19 expression is decreased in the chest skin and in the ear of HPV+ mice when compared to HPV– mice. These results are in accordance with previous studies, which showed a down-regulation of H19 in HPV+ cells and lesions (30, 66). Moreover, in both chest skin and ear tissues, we demonstrated that H19 expression was lower in the lesions compared to normal tissue. Thus, we hypothesise that H19 may have an important role in HPV-induced carcinogenesis, acting as a tumour suppressor. Like it was previously described, IGF2 and H19 are two imprinted genes located adjacent to each other. H19 is regulated by genomic imprinting through methylation at the locus between H19 and IGF2 (67, 68). Abnormal imprinting of IGF2 and/or H19, commonly occurs in various types of cancer, including in HPV+ cervical cancer (69, 70). Therefore, in some diseases, including cancer, aberrant epigenetic modifications in biallelic H19 hypermethylation, leading to abnormal IGF2 expression and consequently uncontrolled cellular proliferation (69-71). The lower levels of H19 also inhibit the expression of HOTS which is another tumour suppressor gene, transcribed from the antisense sequence H19 (71). Therefore, we suggest that, in the chest skin and ear tissues of our mouse model, there may be an epigenetic dysregulation of H19, promoting a decrease of its expression and silencing its tumour suppressor function in HPV+ mice. Contrarily, in tongue and penile tissues, in which no statistically significant differences in H19 expression were observed between HPV– and HPV+ groups, H19 may have no crucial role in HPV-induced carcinogenesis.

SNHG12. Diverse studies demonstrated that the up-regulation of SNHG12 could trigger tumorigenesis, promoting proliferation, metastasis, invasion, and antiapoptotic signalling (72, 73). Data available concerning its role in HPV-associated cancers is limited, but there is some evidence showing that HPV16 E6 and E7 might regulate the expression level of SNHG12 (31). It was described that E6 and E7 target c-Myc, which promotes up-regulation of SNHG12. Therefore, it was proposed that oncoproteins control the expression of SNHG12 via c-Myc (31). Our results demonstrated that SNHG12 expression was decreased in the chest skin and in the ear of HPV+ mice when compared to HPV– mice. According to the type of lesions in the chest skin and ear samples, we observed a decrease of the SNHG12 expression level along with the severity of the lesions. Considering these results, we suggest that SNHG12 expression level decreases during the progression of HPV-carcinogenesis and with the severity of HPV-associated lesions in chest skin and ear tissues. In a previous study in cervical cancer, the authors showed that the oncoproteins E6 and E7 promote SNHG12 expression to enhance proliferation and invasion of the tumour (31). However, in our results, we obtained the contrary, SNHG12 expression was decreased in the presence of HPV. Therefore, in the studied mouse model and these different HPV-related lesions it could be interesting to explore if SNHG12 can act as a tumour suppressor regulating the miRNA-195-5p expression, as it has already been described in oesophagal squamous cell cancer (74). This miR-195-5p was described as able to inhibit the Wnt/β-catenin signalling pathway, decreasing the proliferation and migration of tumour cells (74, 75). For this reason, perhaps the oncoproteins E6 and E7 could regulate the SNHG12 expression, as in other studies, but in this case down-regulating and silencing its possible tumour suppressor functions. In tongue and penile samples, we did not observe any statistically differences in SNHG12 expression, between HPV– and HPV+ groups. Importantly, we have to take into account that we evaluate the relative expression of SNHG12 in different types of lesions from different organs; therefore, lncRNA may not act the same way in all these organs. Therefore, we can conclude that in tongue and penile tissues SNHG12 does not seem to have any role in the HPV-induced carcinogenesis.

EMX2OS. Studies related to the EMX2OS are very scarce; however, most of them describe this lncRNA as a tumour suppressor gene (76). Although it seems to be a potential biomarker in head and neck cancers, in other types of tumours there are no studies concerning the role of EMX2OS and HPV-related cancers (35, 36). Our results showed that EMX2OS expression is decreased in the chest skin and in the ear of HPV+ mice when compared to HPV– mice. We observed a statistically significant difference in the EMX2OS expression level in chest skin and ear tissue between the HPV– and HPV+ mice. Moreover, EMX2OS expression also decreased along with the severity of the lesions. Based on these results, we suggest a decrease in the EMX2OS expression level with the progression of the HPV-induced carcinogenesis process as well as with the severity of HPV-associated lesions. However, EMX2OS expression could not be quantified in the tongue tissue, supporting the tissue-specific expression of this lncRNA. Based in our results, we suggested that in the K14-HPV16 mouse model, the EMX2OS may act as a tumour suppressor in HPV-induced lesions. LncRNA EMX2OS is transcribed from the antisense strand of EMX2 and is located in its enhancer, therefore, recent studies suggested that EMX2OS might regulate the expression of EMX2 (37, 76). It was already proved that overexpression of EMX2 triggered apoptosis, inhibited cell migration and invasion, led to down-regulation of mesenchymal markers, and suppressed AKT and mTOR (77). Therefore, in HPV-induced cancers, the EMX2OS may act as a tumour suppressor since it is down-regulated. Consequently, this may lead to a down-regulation of EMX2, enhancing tumour development and progression. In penile tissues we did not observe any statistically differences in EMX2OS expression, between the different groups. Perhaps this lncRNA did not act the same way in all organs and its function as a tumour suppressor in HPV-induced carcinogenesis is not verified in penile tissues.

LincRNA-p21. Numerous studies have described this lncRNA as a potential suppressor of tumorigenesis in multiple cancers inhibiting the cell proliferation, cell survival, tumorigenesis, invasion, metastasis, and angiogenesis (41). To the best of our knowledge, there are no studies concerning HPV-related cancers. The results that we obtained demonstrated that lincRNA-p21 expression decreased in all organs of HPV+ mice when compared to those of HPV– mice. According to the type of lesion, we observed that normal chest skin, ear and penile tissues presents higher levels of lincRNA-p21 expression in comparison with the HPV-induced lesions. These results are in accordance with what we expected, once this lncRNA is a direct transcriptional target of p53 and seems to function as an element of p53 pathway (39). Therefore, lincRNA-p21 is a crucial tumour suppressor in numerous types of cancers controlling several processes including DNA damage response, apoptosis, and cell proliferation (40). Nevertheless, in K14-HPV16 mouse model certainly the p53 protein is depredating because the major function of the oncoprotein E6 is binding with the tumour suppressor p53, and induce its proteolysis (78, 79). The decrease of p53 protein levels leads to low levels of lincRNA-p21, inhibiting its tumour suppressor function in HPV-induced carcinogenesis. Concluding, the lincRNA-p21 is decreased in HPV+ mice, in chest skin, ear, tongue and penile tissues thus, demonstrating a crucial role in HPV-carcinogenesis process, acting as a tumour suppressor.

LncRNAs expression and cancer cachexia. Although the involvement of lncRNAs in cancer cachexia and the associated mechanism have only been described in a few studies, these RNAs seem to be crucial in the development of this syndrome (20, 23). Accumulating data shows that lncRNAs are associated with several biological processes through cachexia progression (20, 23). In Table II, the results of the MALAT1, H19, SNHG12, EMX2OS and lincRNA-p21 expression in gastrocnemius muscle are summarized. Diverse studies proved that MALAT1 has a critical role in myoblast differentiation and also in muscle regeneration. Although MALAT1 seems to be a crucial effector in muscle associated mechanisms, there are no studies that relate this lncRNA and muscle wasting. In our results, we verified that HPV+ group had a higher MALAT1 expression in comparison with HPV– group in gastrocnemius muscle. As previously described, chronic inflammation is one of the main characteristics of cachexia and affects several organs, including skeletal muscle. (11, 17, 80). Numerous studies showed that MALAT1 functions as a regulator of the inflammatory response in skeletal muscle (81, 82). Besides, studies demonstrate a positive correlation between MALAT1 expression and several inflammatory cytokines (TGF-β1, TNF-α, IL-6 and IL-10) (81). Since MALAT1 expression seems to be significantly correlated to inflammatory cytokines due to its important role in the inflammatory response of skeletal muscle, we expected a higher expression of this lncRNA, as was obtained (Figure 11). Hence, we suggest that MALAT1 up-regulation may promote higher levels of inflammatory cytokines enhancing the inflammatory activation and the degradation pathways (Figure 11). Therefore, the expression of MALAT1 seems to be correlated with muscle wasting and to the pathophysiology of cancer cachexia. Concerning lincRNA-p21, the HPV– group had a higher lincRNA-p21 expression than the HPV+ group. To the best of our knowledge, there are no studies that associate lincRNA-p21 expression with muscle wasting and pathophysiology of cancer cachexia. Nevertheless, the results that we obtained could be explained by the fact that this lncRNA direct binds STAT3 and consequently block the JAK2/STAT3 signalling (Figure 11) (42). Moreover, STAT3 seems to have a crucial role in muscle wasting (33, 34). This transcription factor, is a downstream effector of IL-6 and other pro-inflammatory mediators, that leads to the activation of caspases and myostatin conducting to apoptosis and muscle loss (Figure 11) (33, 34, 83). Considering our results, we can suggest that the down-regulation of lincRNA-p21 expression leads to an up-regulation of STAT3, which is an important effector in muscle wasting. Concerning the others lncRNAs, H19, SNHG12 and EMX2OS we did not find any statistically significant differences between the different groups. Therefore, we can suggest that in our mouse model, these lncRNAs do not play a crucial role in the muscle wasting process nor contribute to the pathophysiology of cancer cachexia.

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Table II.

LncRNA downregulation or upregulation in HPV+ mice in gastrocnemius muscle.

Figure 11.
Figure 11.

Hypothetical scheme of the roles of the studied lncRNAs, MALAT1, H19, SNHG12, EMX2OS and lincRNA-p21, in the muscle wasting pathway.

Conclusion

The lncRNAs H19, SNHG12 and EMX2OS seem to have tumor suppressor function in normal cells, since their low expression HPV-induced lesions may have a potential role in promoting HPV-induced carcinogenesis process in our mouse model. However, this potential role is only verified in chest skin and in the ear of the mice but not in penile and tongue tissues. On the other hand, lincRNA-p21 had concordant results for all the organs. Therefore, future studies should examine whether lncRNA-p21 is a potential biomarker for HPV-induced cancers and/or premalignant lesions. Moreover, in vitro and additional in vivo studies could access its potential applications as a therapeutic target. Concerning the muscle wasting evaluation, MALAT1 and lincRNA-p21 were shown to play a crucial role in muscle wasting process contributing to the pathophysiology of cancer cachexia.

Acknowledgements

This study was supported by the Portuguese League Against Cancer–Regional Nucleus of the North (Liga Portuguesa Contra o Cancro–Núcleo Regional do Norte) and by the Research Center of the Portuguese Oncology Institute of Porto (projects no. PI127-CI-IPOP-118-2019 and PI86-CI-IPOP-66-2017). Joana M.O. Santos is a PhD scholarship holder (SFRH/BD/135871/2018) supported by Fundação para a Ciência e Tecnologia (FCT), co-financed by European Social Funds (FSE) and national funds of MCTES.

Footnotes

  • Authors’ Contributions

    Conceptualisation, T.R.D., J.M.O.S., R.M.G.d.C. and R.M.; methodology, T.R.D., J.M.O.S., D.E., N.R.V., V.F.M., B.M.-F., P.A.O. and R.M.G.d.C.; validation, T.R.D., J.M.O.S.; formal analysis, T.R.D., J.M.O.S., R.M.G.d.C. and R.M.; investigation, T.R.D., J.M.O.S., D.E., N.R.V., V.F.M., B.M.-F., P.A.O. and R.M.G.d.C.; resources, P.A.O. and R.M.; writing—original draft preparation, T.R.D.; writing—review and editing, J.M.O.S. and R.M.G.d.C.; supervision R.M.G.d.C., P.A.O. and R.M.; funding acquisition, P.A.O., M.M.S.M.B. and R.M. All Authors have read and agreed to the published version of the manuscript.

  • Conflicts of Interest

    The Authors declare no conflicts of interest.

  • Received December 13, 2021.
  • Revision received March 11, 2022.
  • Accepted March 17, 2022.

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Expression of LncRNAs in HPV-induced Carcinogenesis and Cancer Cachexia: A Study in K14-HPV16 Mice
TÂNIA R. DIAS, JOANA M. O. SANTOS, DIOGO ESTÊVÃO, NATÁLIA R. COSTA, VERÓNICA F. MESTRE, BEATRIZ MEDEIROS-FONSECA, MARGARIDA M.S.M. BASTOS, PAULA A. OLIVEIRA, RUI M. GIL DA COSTA, RUI MEDEIROS
Anticancer Research May 2022, 42 (5) 2443-2460; DOI: 10.21873/anticanres.15723
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