Multifaceted investigation underlies diverse mechanisms contributing to the downregulation of Hedgehog pathway-associated genes INTU and IFT88 in lung adenocarcinoma and uterine corpus endometrial carcinoma

Results

Investigations on the prognostic values of INTU in multiple cancer types

The Kaplan-Meier plotter was initially used to assess the prognostic significance of INTU expression in 21 types of cancer. We found that INTU expression was significantly associated with the overall survival (OS) probabilities in patients with nine different cancer types. In liver hepatocellular carcinoma (LIHC) and lung squamous cell carcinoma (LUSC), patients with an increased level of INTU had poor OS probabilities. However, in the remaining cancer types, including esophageal adenocarcinoma (EAC), esophageal squamous cell carcinoma (ESCC), kidney renal papillary cell carcinoma (KIRP), LUAD, pancreatic ductal adenocarcinoma (PDAC), sarcoma (SARC) and UCEC, reduced levels of INTU were correlated with decreased OS probabilities in patients (Figure 1).

Figure 1. Evaluation of the prognostic significance of INTU mRNA level in different cancer types. Pan-cancer survival analysis was carried out to determine the relationship between INTU mRNA level and OS probabilities in 21 different types of cancer. Decreased INTU expression was found associated with poor prognosis in EAC, ESCC, KIRP, LUAD, PDAC, SARC and UCEC patients, whilst high level of INTU correlated with poor prognosis in LIHC and LUSC patients.

Hh pathway-related genes were enriched in LUAD and UCEC tumor samples

We next sought to evaluate the expression of INTU in tumor samples from different cancer types. The expression of INTU in tumor samples from The Cancer Genome Atlas (TCGA) were compared with that from normal samples from TCGA and The Genotype-Tissue Expression project (GTEx). The results showed that the INTU transcription level was significantly downregulated in LUAD, LUSC and UCEC samples, whereas in esophageal carcinoma, KIRP, LIHC and SARC, no significant change of INTU expression between tumor and normal samples was detected (Figure 2A and Supplementary Figure 1A). No significant changes in the expression of housekeeping genes, including beta-actin (ACTB), beta-2-microglobulin (B2M) and ubiquitin C (UBC), were detected in LUAD and UCEC tumor samples when compared to their respective normal samples (Supplementary Figure 1B). In line with our prognostic analysis, the lower levels of INTU in LUAD and UCEC tumor samples (Figure 2A) coincided with poor OS probabilities in cancer patients (Figure 1). We thus decided to focus on LUAD and UCEC in our subsequent studies.

Figure 2. The Hh pathway-associated genes were enriched in LUAD and UCEC tumor samples. (A) The expression of INTU was significantly downregulated in LUAD and UCEC tumor samples. ^* denotes p < 0.05. (B) The GO and Reactome enrichment analysis on the top 100 genes that showed similar expression pattern as INTU from LUAD tumor samples. BP indicates biological process, and CC indicates cellular compartment. (C) The GO and Reactome enrichment analysis on the top 100 genes that showed similar expression pattern as INTU from UCEC tumor samples. (D) Construction of the PPI network using genes that showed similar expression pattern as INTU in LUAD tumor samples. Two enriched Reactome pathways “Anchoring of the basal body to the plasma membrane” and “Hedgehog ‘off’ state” were highlighted. (E) Construction of the PPI network using genes that showed similar expression pattern as INTU in UCEC tumor samples. Three enriched Reactome pathways “Anchoring of the basal body to the plasma membrane”, “Hedgehog ‘off’ state” and “Intraflagellar transport” were highlighted.

The Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database was used to select the top 100 genes with similar expression patterns as INTU from LUAD and UCEC tumor samples (Supplementary Table 1). We then performed gene enrichment analysis to investigate whether certain enriched Gene Ontology (GO) terms and Reactome pathways could be identified from the LUAD and UCEC gene sets. We found that in LUAD, genes were enriched in cilium-associated biological processes, including cilium morphogenesis, assembly and movement. The most enriched subcellular localization pattern was found in association with ciliary compartments. Meanwhile, two Reactome pathways, “Anchoring of the basal body to the plasma membrane” and “Hedgehog ‘off’ state,” were highlighted (Figure 2B). Similar enriched biological processes and cellular compartment terms were identified from the UCEC gene set, where genes were found enriched in three Reactome pathways, including the “Hedgehog ‘off’ state”, “Intraflagellar transport” and “Anchoring of the basal body to the plasma membrane” pathways (Figure 2C).

Protein–protein interaction networks were subsequently constructed. In the LUAD gene set, the centrosomal genes centrosomal protein 83 (CEP83), centrosomal protein 162 (CEP162), centrosomal protein 290 (CEP290), centriolin (CNTRL), nephrocystin 4 (NPHP4) and MKS transition zone complex subunit 1 (MKS1) were associated with the “Anchoring of the basal body to the plasma membrane” pathway, while INTU, IFT88, MKS1 and WD repeat domain 90 (WDR90) were involved in the “Hedgehog ‘off’ state” pathway (Figure 2D). In the UCEC gene set, coiled-coil and C2 domain containing 2A (CC2D2A), CEP83 and nephrocystin 1 (NPHP1) were found in the “Anchoring of the basal body to the plasma membrane” pathway. The protein transport-associated genes dynein cytoplasmic 2 heavy chain 1 (DYNC2H1), IFT88, intraflagellar transport 140 (IFT140) and WD repeat domain 19 (WDR19) were involved in the “Intraflagellar transport” pathway, while DYNC2H1, INTU, IFT88, IFT140 and WDR19 were involved in the “Hedgehog ‘off’ state” pathway (Figure 2E).

IFT88 downregulation was associated with the poor prognosis of LUAD and UCEC patients

Similar to INTU, IFT88 was implicated in the “Hedgehog ‘off’ state” pathway in both the LUAD and UCEC gene sets (Figure 2D, 2E). In addition, INTU expression significantly correlated with the expression of IFT88 from LUAD and UCEC tumor samples (Figure 3A, 3B). Similar to INTU (Figure 1), we found that LUAD and UCEC patients with lowered IFT88 levels also showed decreased OS probabilities (Figure 3C, 3D). Taken together, these results highlight the significance of the downregulation of the Hh pathway-associated genes INTU and IFT88 in the prognosis of LUAD and UCEC patients. We therefore sought to delineate the underlying mechanisms of INTU and IFT88 downregulation in LUAD and UCEC tumor samples.

Figure 3. The expression of IFT88 correlated with INTU expression in LUAD and UCEC samples, and hypermethylation of INTU^CpG was detected in LUAD and UCEC samples. (A, B) The expression of IFT88 positively correlated with the expression of INTU in LUAD (A) and UCEC (B) tumor samples. (C, D) Decreased expression of IFT88 was found associated with reduced OS probabilities in LUAD (C) and UCEC (D) patients. (E) Two putative CpG islands were identified in INTU gene promoter. (F) Two CpG islands were predicted in IFT88 gene promoter. (G, H) Treatment of 5-azacytidine induced INTU expression in NCI-H1975 (G) and AN3 CA (H) cells. n = 3 biological replicates. Each n represents an independent preparation of cell RNA samples. Error bars represent S.E.M. Statistical analysis was performed using two-tailed unpaired Student’s t-test. ^* denotes p < 0.05. (I, J) Treatment of 5-azacytidine induced IFT88 expression in NCI-H1975 (I) and AN3 CA (J) cells. n = 3 biological replicates. Each n represents an independent preparation of cell RNA samples. Error bars represent S.E.M. Statistical analysis was performed using two-tailed unpaired Student’s t-test. ^* denotes p < 0.05. (K) Hypermethylation of the INTU^CpG was detected in LUAD and UCEC tumor samples. (L) No difference in IFT88^CpG methylation levels were detected in LUAD and UCEC samples when compared to their respective normal control samples.

The methylation level of INTU gene promoter CpG islands was upregulated in LUAD and UCEC tumor samples

DNA methylation is a typical epigenetic modification through which gene expression is modulated [13]. In gene promoter region, CpG islands are regions with densely-accumulated CG dinucleotides, and methylation of CpG islands leads to gene silencing [14]. In each of the INTU and IFT88 gene promoter regions, two putative CpG islands were identified (Figure 3E, 3F). To investigate the association between DNA methylation and INTU and IFT88 gene expression, the LUAD (NCI-H1975) and UCEC (AN3 CA) cell lines were treated with 5-azacytidine, a DNA methyltransferase inhibitor [15], the INTU and IFT88 levels were subsequently detected. We found that upon treatment of 5-azacytidine, both INTU and IFT88 levels were upregulated in NCI-H1975 and AN3 CA cells, suggesting a negative correlation between DNA methylation and INTU and IFT88 gene expression (Figure 3G–3J). The methylation levels of INTU^CpG and IFT88^CpG were further examined in LUAD and UCEC tumor samples. We found that the methylation level of INTU^CpG was significantly upregulated in both LUAD and UCEC tumor samples (Figure 3K). When compared with normal tissues, no significant change in IFT88^CpG methylation levels was detected in LUAD and UCEC tumor samples (Figure 3L). This suggests that hypermethylation of INTU^CpG potentially contributes to the reduction of INTU levels in LUAD and UCEC samples.

Involvement of the transcriptional factor TCF4 in the modulation of INTU and IFT88 levels

Transcription factors are a set of regulatory proteins that bind to gene promoter DNA sequences and modulate gene transcription [16]. A total of nine communal transcription factors, including core-binding factor subunit beta (CBFB), histone deacetylase 2 (HDAC2), transcription factor jun-D (JUND), serum response factor (SRF), small ubiquitin-like modifier 2 (SUMO2), TATA-box binding protein associated factor 1 (TAF1), TATA box binding protein (TBP), transcription factor 4 (TCF4) and yin yang 1 (YY1), were identified in the INTU and IFT88 gene promoters (Figure 4A). Next, we evaluated the expression of these transcription factors in LUAD and UCEC samples. Among these nine transcription factors, we found that only TCF4 (Figure 4B) expression was significantly downregulated in both LUAD and UCEC tumor samples (Figure 4C and Supplementary Figure 2). The TCF4 protein level was further found to be significantly reduced in both LUAD (Figure 4D) and UCEC (Figure 4E) samples. More importantly, the expression of TCF4 was positively correlated with the expression of INTU and IFT88 in LUAD and UCEC samples (Figure 4F, 4G). Similar to INTU (Figure 1) and IFT88 (Figure 3C, 3D), the reduced levels of TCF4 were associated with decreased OS probabilities in LUAD and UCEC patients (Figure 4H, 4I). Altogether, TCF4 was identified as a putative upstream regulator in controlling the expression of INTU and IFT88 in LUAD and UCEC tumor samples.

Figure 4. TCF4 was identified as a potential transcription factor that mediates INTU and IFT88 downregulation in LUAD and UCEC tumor samples. (A) Nine common transcription factors, including CBFB, HDAC2, JUND, SRF, SUMO2, TAF1, TBP, TCF4 and YY1 were predicted in INTU and IFT88 gene promoters. (B) Illustration of TCF4 binding consensus sequence and the putative TCF4 binding sites in INTU and IFT88 promoter sequence. (C) The TCF4 transcript level was downregulated in LUAD and UCEC tumor samples. ^* denotes p < 0.05. (D, E) The protein level of TCF4 was downregulated in LUAD (D) and UCEC (E) tumor samples. (F, G) The expression of TCF4 positively correlated with the expression of INTU and IFT88 in LUAD (F) and UCEC (G) tumor samples. (H, I) The LUAD (H) and UCEC (I) patients with lowered level of TCF4 showed reduced OS probabilities.

We further provided experimental evidence in support of our findings. Chromatin immunoprecipitation was performed to investigate the interaction between TCF4 protein and INTU and IFT88 gene promoters. We found that in both the NCI-H1975 and AN3 CA cells, the binding of TCF4 to INTU and IFT88 gene promoters was detected (Figure 5A, 5B). The transcriptional regulatory function of TCF4 on INTU and IFT88 gene expression was subsequently determined. When endogenous TCF4 was knocked down in NCI-H1975 and AN3 CA cells, the transcript levels of INTU and IFT88 were downregulated (Figure 5C, 5D). These data further support the positive correlation between TCF4 and INTU and IFT88 levels in LUAD and UCEC samples (Figure 4F, 4G).

Figure 5. TCF4 interacted with INTU and IFT88 promoters and mediated their gene expression. (A, B) The binding between TCF4 and INTU and IFT88 promoters was detected in NCI-H1975 (A) and AN3 CA (B) cells. n = 3 biological replicates. Each n represents an independent preparation of ChIP samples. Error bars represent S.E.M. Statistical analysis was performed using two-tailed unpaired Student’s t-test. ^* denotes p < 0.05 and ^** denotes p < 0.01. (C, D) Knockdown of TCF4 downregulated the transcript levels of INTU and IFT88 in NCI-H1975 (C) and AN3 CA (D) cells. n = 3 biological replicates. Each n represents an independent preparation of RNA and protein samples. Error bars represent S.E.M. Statistical analysis was performed using two-tailed unpaired Student’s t-test. ^*denotes p < 0.05, ^**denotes p < 0.01 and ^***denotes p < 0.001.

Identification of hsa-miR-212-3p as the upstream microRNA targeting Hh-related genes INTU and IFT88

In addition to gene silencing, post-transcriptional regulation of gene expression mediated by non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), has been reported [17, 18]. The DIANA-TarBase v8 database was used to select potential miRNAs that target INTU or IFT88 based on experimental evidence [19]. We identified 23 miRNAs that target the INTU transcript and four miRNAs that target the IFT88 transcript (Figure 6A). Interestingly, two miRNAs, hsa-miR-210-3p [20] and hsa-miR-212-3p [21], were shown to target both INTU and IFT88 (Figure 6A, 6B). We next evaluated the correlation between the miRNA level and INTU or IFT88 expression in LUAD and UCEC tumor samples. The expression of hsa-miR-212-3p, but not hsa-miR-210-3p, was found to be negatively correlated with the expression of INTU and IFT88 in LUAD and UCEC samples (Figure 6C–6F and Supplementary Figure 3). Moreover, we found that when hsa-miR-212 was overexpressed in NCI-H1975 and AN3 CA cells, downregulation of INTU and IFT88 protein levels were detected (Figure 6G, 6H). This further suggests the negative regulatory function of hsa-miR-212 on the expression of INTU and IFT88.

Figure 6. Identification of hsa-miR-212-3p as a communal miRNA against INTU and IFT88 in LUAD and UCEC samples. (A) Identification of hsa-miR-212-3p and hsa-miR-210-3p miRNAs that target both INTU and IFT88 transcripts. (B) Construction of the miRNA-target gene regulatory network. (C, D) The expression of hsa-miR-212-3p negatively correlated with the expression of INTU (C) and IFT88 (D) in LUAD samples. (E, F) The expression of hsa-miR-212-3p negatively correlated with the expression of INTU (E) and IFT88 (F) in UCEC samples. (G, H) Overexpression of hsa-miR-212 led to the downregulation of INTU and IFT88 protein levels in NCI-H1975 (G) and AN3 CA (H) cells. The MALAT1 levels were not affected. n = 3 biological replicates. Each n represents an independent preparation of protein or RNA samples. Error bars represent S.E.M. Statistical analysis was performed using two-tailed unpaired Student’s t-test. ns indicates no significant difference. ^* denotes p < 0.05.

Identification of MALAT1 as an upstream lncRNA

We next examined upstream lncRNAs using the DIANA-LncBase v3 database. A total of 63 lncRNAs were obtained, and their expression levels in LUAD and UCEC samples were evaluated (Figure 7A). The levels of four of the 63 lncRNAs, including HOXA transcript antisense RNA, myeloid-specific 1 (HOTAIRM1), KMT2E antisense RNA 1 (KMT2E-AS1), metastasis associated lung adenocarcinoma transcript 1 (MALAT1) and nuclear paraspeckle assembly transcript 1 (NEAT1), were significantly downregulated in LUAD and UCEC tumor samples when compared with their respective normal tissues (Figure 7B and Supplementary Figure 4A). The prognostic significance of these four lncRNAs were further evaluated. We found that MALAT1, but not HOTAIRM1 and NEAT1, showed prognostic significance in both LUAD and UCEC patients (Figure 7C, 7D and Supplementary Figure 4B). Similar to what was detected regarding INTU (Figure 1) and IFT88 (Figure 3C, 3D), the reduced level of MALAT1 contributed to poor OS probabilities in LUAD and UCEC patients (Figure 7C, 7D). Overexpression of hsa-miR-212 did not modulate MALAT1 levels in NCI-H1975 and AN3 CA cells (Figure 6G, 6H). In contrast, MALAT1 has been shown to target hsa-miR-212-3p in two independent studies (Figure 7E) [22, 23]. Moreover, the expression of MALAT1 was found to be positively associated with the mRNA levels of INTU and IFT88 in LUAD and UCEC tumor samples (Figure 7F, 7G). To further validate the regulatory function of MALAT1 on the expression of INTU and IFT88, MALAT1 was overexpressed in NCI-H1975 or AN3 CA cells (Figure 7H, 7I). We found that in MALAT1-overexpressing cells, the protein levels of INTU and IFT88 were simultaneously increased (Figure 7H, 7I). These results therefore confirm the role of MALAT1 in regulating the expression of INTU and IFT88 in lung and endometrial cancer cells.

Figure 7. Identification of MALAT1 as a communal lncRNA mediating INTU and IFT88 expression in LUAD and UCEC samples. (A) Identification of the lncRNAs against hsa-miR-212-3p and construction of the lncRNA-miRNA regulatory network. (B) The expression of MALAT1 was significantly downregulated in LUAD and UCEC tumor samples. (C, D) Decreased level of MALAT1 was found associated with poor prognosis in LUAD (C) and UCEC (D) patients. (E) The MALAT1 was identified as targeted lncRNA against hsa-miR-212-3p from two independent studies. (F, G) The expression of MALAT1 positively associated with the expression of INTU and IFT88 in LUAD (F) and UCEC (G) tumor samples. (H, I) Overexpression of MALAT1 caused the upregulation of INTU and IFT88 protein levels in NCI-H1975 (H) and AN3 CA (I) cells. n = 3 biological replicates. Each n represents an independent preparation of RNA and protein samples. Error bars represent S.E.M. Statistical analysis was performed using two-tailed unpaired Student’s t-test. ^* denotes p < 0.05.

Discussion

In this study, we demonstrated that the downregulation of the INTU and IFT88 was correlated with reduced survival probabilities in LUAD and UCEC patients (Figures 1, 3C, 3D). We next sought to explore the driving forces causing this downregulation in LUAD and UCEC tumor samples, and identified multifaceted mechanisms in the DNA, RNA and protein levels contributing to INTU and IFT88 downregulation (Figures 3–7). This study provides a comprehensive mechanistic investigation regarding INTU and IFT88 downregulation in cancer, and further highlights the involvement of Hh signaling in carcinogenesis.

The dysregulation of Hh signaling has been documented in multiple types of cancer. In BCC, medulloblastoma and rhabdomyosarcoma, mutations in Hh-related genes activate Hh signaling in support of the over-proliferation and tissue invasion of cancer cells [24–26]. In addition to genetic mutation, epigenetic modification also contributes to aberrant Hh signaling in tumor samples. For example, the hypermethylation of hedgehog-interacting protein (HHIP), a gene encodes for a negative regulator of Hh signaling, was determined in pancreatic cancer samples. This leads to the reduced expression of HHIP, followed by the upregulation of Hh signaling [27]. In the majority of solid tumors, including colorectal cancer (CRC), the mutation in Hh-related genes was rarely detected [28]. Interestingly, in CRC, the stromal Hh pathway targets were found downregulated despite the increased expression of Hh ligand. This might be due to the insensitivity of stromal cells to epithelial Hh ligand, or the impairment of tissue architecture in tumor stroma [29, 30]. In addition, the restoration of stromal Hh signaling markedly alleviated tumorigenesis, whereas inhibition of Hh signaling exacerbated tumor progression [31]. These findings taken together suggest multiple mechanisms in contributing to the dysregulation of Hh signaling in different cancers, and also highlight the oncogenic role of Hh signaling. Here, we reported two subsets of enriched Hh pathway-associated genes with similar downregulation expression patterns in LUAD and UCEC tumor samples (Figure 2D, 2E). We further showed that lowered levels of INTU, IFT88 or MKS1 were correlated with decreased OS probabilities in LUAD patients, while UCEC patients with reduced INTU, IFT88 or IFT140 levels had a poor prognosis (Figures 1, 3C, 3D, Supplementary Figure 5A, 5B). INTU was found to be necessary for the ciliary recruitment of IFT-A proteins, and MKS1 functionally associates with IFT complexes in mediating the transport of cargo proteins to support ciliary outgrowth [8, 32]. Depletion of INTU or IFT machinery components leads to ciliogenesis defects [12, 33, 34]. Importantly, the loss of ciliary structures has been recorded in lung and endometrial cancer patient samples [35, 36]. Moreover, stimulation of ciliogenesis has been reported to combat against lung cancer cell proliferation, invasion and epithelial-mesenchymal transition [37]. These findings therefore indicate that disruption of Hh signaling components might cause ciliogenesis defects, which in favour of oncogenesis in LUAD and UCEC tissues.

TCF4 belongs to the helix–loop–helix (HLH) family of proteins ubiquitously expressed throughout different human tissues [38]. A basic residue group on the TCF4 N-terminus is critical for its DNA-binding function, while the C-terminal HLH domain mediates the dimerization of TCF4 when it binds to DNA [39]. Several studies have demonstrated the association between TCF4 function and DNA methylation [40, 41]. Specifically, TCF4 was correlated with DNA hypomethylation in mammalian epithelial stem cells. Upon conditional knockout of TCF4, the TCF4-bound differentially methylated DNA sequence was found to be strongly methylated [41]. Interestingly, when looking into the TCF4 binding site in the INTU promoter sequence, we found that the TCF4 site resides in the INTU^CpG (highlighted in Supplementary Table 2). The methylation level of INTU^CpG was upregulated in LUAD and UCEC samples (Figure 3G). Reduction of the TCF4 protein level was recorded in both LUAD and UCEC tumor samples (Figure 4D). Such attenuation of the TCF4 level might result in INTU^CpG hypermethylation, which in turn leads to the downregulation of INTU expression in LUAD and UCEC samples. Meanwhile, TCF4 did not associate with IFT88^CpG (Supplementary Table 2), and no significant change in IFT88^CpG methylation level was detected in LUAD and UCEC samples (Figure 3H).

Genetic mutations in TCF4 have been reported in neurological disorders, including Fuchs’s corneal dystrophy [42], Pitt–Hopkins syndrome [43], and schizophrenia [44], as well as non-neurological diseases, including primary sclerosing cholangitis [45] and sporadic Sonic Hedgehog-associated medulloblastoma (SHH MB) [46]. Functional analysis was carried out on mutant TCF4 proteins harboring the mutations identified from SHH MB patients. Experimental findings highlighted the loss-of-function behind these TCF4 mutations, as exemplified by the fact that mutant TCF4 proteins failed to inhibit the proliferation of medulloblastoma cells, unlike the wild-type TCF4 protein [40, 47]. We also found a nonsense mutation at the arginine 174 residue (R174^*) on the TCF4 protein in eight UCEC patient samples (Supplementary Figure 6A, 6B), and the R174 site was conserved across different species (Supplementary Figure 6C). This nonsense mutation generates a truncated TCF4 protein which lacks the C-terminal HLH motif that is crucial for mediating gene transcription, suggesting the loss of TCF4 transactivating function due to the presence of such a mutation. This could serve as another mechanism leading to the downregulation of Hh-related genes in UCEC tumor samples. Interestingly, a similar TCF4^R174* mutation was previously reported in patients with SHH MB and Pitt–Hopkins syndrome [46, 47], suggesting that the communal loss-of-function mechanism is involved in a broad spectrum of human disorders.

miRNAs and lncRNAs are two major subtypes of ncRNAs associated with the well-documented ceRNA mechanism that is essential for controlling gene expression at a post-transcriptional level [18, 48]. We identified a novel and communal MALAT1-hsa-miR-212-3p regulatory network that downregulated INTU and IFT88 expression in LUAD and UCEC samples (Figure 5). The recurrent fusion of MALAT1 with the GLI1 gene was reported in patients with gastroblastoma and plexiform fibromyxoma [49, 50]. This MALAT1-GLI1 fusion mutation activated Hh signaling and consequently led to malignant tumor formation, suggesting a relationship between MALAT1 function and Hh signaling activity [49, 51, 52]. In this study, we further highlighted the involvement of MALAT1 in regulating Hh pathway-associated genes. We found that in addition to INTU and IFT88, other Hh-related genes with similar expression profiles as INTU were also enriched in LUAD and UCEC samples (Figure 2D, 2E). Interestingly, the expression of hsa-miR-212-3p was negatively associated with the expression of MKS1 and WDR90 in LUAD samples (Supplementary Figure 7A) and DYNC2H1, IFT140 and WDR19 in UCEC samples (Supplementary Figure 7B). Positive correlations were determined between MALAT1, MKS1 and WDR90 in LUAD samples and MALAT1, DYNC2H1 and WDR19 in UCEC samples (Supplementary Figure 7C, 7D). Taken together, these findings emphasize the MALAT1-hsa-miR-212-3p network as a master upstream regulator targeting downstream Hh pathway-associated genes in LUAD and UCEC tumor samples. In addition, MALAT1 binds to active chromatin sites and regulates gene transcription by recruiting chromatin modifiers or transcription regulators to specific genomic loci [53, 54]. This might be an alternative mechanism that accounts for MALAT1’s regulation on Hh pathway-associated gene expression and is worthy to be further investigated.

Several recent studies have reported the anti-tumorigenesis role of TCF4 in colorectal carcinoma and SHH MB [47, 55]. TCF4 is capable of attenuating the proliferation of colon cancer and medulloblastoma cells, whereas loss of TCF4 exerts the opposite effect, favoring tumorigenesis [47, 56, 57]. The effect of MALAT1 on tumor cell growth and invasion is controversial. Although the oncogenic functions of MALAT1 have been reported in malignancies such as colorectal and liver cancer [58, 59], more recent studies have highlighted the tumor suppressive role of MALAT1 against the growth and metastasis of glioma and breast cancer cells [60, 61]. These findings taken together suggest a cancer type-dependent role of MALAT1 in tumorigenesis. Given the functions of TCF4 and MALAT1 as tumor suppressors, targeting the functional elevation of TCF4 and MALAT1 could be therapeutically beneficial against tumorigenesis. In fact, the identification of small molecule drugs aimed at stimulating TCF4 function is now under investigation (Pitt Hopkins Research Foundation; https://pitthopkins.org/portfolio-item/pilot-study-to-identify-small-molecule-activators-of-tcf4-as-a-treatment-for-pitt-hopkins-syndrome/). Meanwhile, different small molecule activators for MALAT1 have been reported (Table 1). A combinatorial drug treatment has been demonstrated as an effective therapeutic strategy in combating carcinogenesis [62–64]. The use of both TCF4 and MALAT1 activators in the treatment against LUAD and UCEC would be an interesting topic worthy of further exploration.

Table 1. Summary of MALAT1 small molecule activators.

lncRNA	Small molecules	Effect on lncRNA expression	Approved by FDA	Validated by experiments	Validation method	Experimental material	References
MALAT1	Carboplatin + Docetaxel	Up-regulation	Yes	Yes	Quantitative real-time PCR	Ovarian cancer cell line	[81]
MALAT1	Quercetin	Up-regulation	Yes	Yes	Quantitative real-time PCR	Rheumatoid arthritis fibroblast-like synoviocytes	[82]

In summary, we showed that the downregulation of the Hh pathway-associated genes INTU and IFT88 was correlated with poor prognosis in LUAD and UCEC patients. Moreover, we demonstrated novel TCF4 and ncRNA-involved mechanisms that contribute to the downregulation of INTU and IFT88 in LUAD and UCEC tumor samples (Figure 8). We further propose that a treatment strategy that simultaneously targets TCF4 and MALAT1 to enrich INTU and IFT88 might be a promising therapeutic intervention against LUAD and UCEC.

Figure 8. Illustration of the underlying mechanisms that contribute to INTU and IFT88 downregulation in the lung and endometrial cancers.

Author Contributions

Z.S.C. designed research, performed experiments and analyzed the data, H.Y.E.C. and Z.S.C. wrote and revised the paper.

Acknowledgments

The human endometrial adenocarcinoma cell line AN3 CA was a kind gift from Prof. Chi Chiu Wang (Department of Obstetrics and Gynecology, The Chinese University of Hong Kong, China). The pcDNA-MALAT1 plasmid was a kind gift from Prof. Huating Wang (Department of Orthopaedics and Traumatology, The Chinese University of Hong Kong, China).

Conflicts of Interest

The authors declare no conflicts of interest related to this study.

Ethical Statement

Biological and chemical safety approval for this study (14122815) was obtained from the Chinese University of Hong Kong.

Funding

Zhefan Stephen Chen was supported by a Postdoctoral Fellowship in Clinical Neurosciences program between The Chinese University of Hong Kong and University of Oxford (Nuffield Department of Clinical Neurosciences and Pembroke College).

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