LINC00629 is increased by ER stress and suppresses cell apoptosis in osteosarcoma cell
To investigate the lncRNAs altered upon ER stress, the human osteosarcoma cell line MNNG/HOS was treated with 3 μM tunicamycin (TM) to induce pharmacological ER stress, and the cells were then subjected to lncRNA sequencing analysis. As shown in Fig. 1A-B, 567 upregulated and 570 downregulated lncRNAs were obtained (Supplementary Table 3). Among these altered lncRNAs, five significantly changed lncRNAs, LINC00629, LINC02591, LINC02604, LINC01733 and LINC00632, were selected (Fig. 1C). These lncRNAs were then confirmed by qRT–PCR. The result showed that LINC00629 was the most increased among the other significantly upregulated lncRNAs upon TM treatment in MNNG/HOS and 143B cells (Fig. 1D) and was gradually upregulated with increasing TM treatment time (Fig. 1E and Supplementary Fig. 1A).
To further verify that LINC00629 was a ER stress-induced lncRNA, we then treated MNNG/HOS cells with 1 μM thapsigargin (TG), a Ca2+-ATPase inhibitor, to induce ER stress, and the expression levels of LINC00629 were analysed by qRT–PCR. We found that LINC00629 was also remarkably increased in response to TG treatment (Fig. 1F). In addition, our subsequent data indicated that sarcoma patients with high LINC00629 expression had short survival times (Supplementary Fig. 1B).
To further investigate the role of LINC00629 in ER stress-induced apoptosis, we first stably knocked down LINC00629 in MNNG/HOS and 143B cells. Compared with those of control group, the expression of LINC00629 in shRNA LINC00629 groups were observably reduced (Supplementary Fig. 1C). These cells were then treated with 3 μM TM or 1 μM TG for 36 h. Western blot and CCK8 assays were performed to investigate the effects of LINC00629 on cell apoptosis and cell viability. Our data indicated that depletion of LINC00629 enhanced ER stress-induced cell apoptosis and promoted a decrease in cell viability (Fig. 1G-J and Supplementary Fig. 1D-E). Whereas, overexpression of LINC00629 decreased cell apoptosis and impaired ER stress-induced cell viability downregulation (Fig. 1K-N and Supplementary Fig. 1F). After that, to exclude off-target effects, the siRNA pool containing four different targeting sequences was used to knock down LINC00629 in MNNG/HOS cells, leading to significant inhibition of LINC00629 (Supplementary Fig. 1G). Consistently, inhibition of LINC00629 promoted ER stress-induced cell apoptosis and decreased cell viability (Supplementary Fig. 1H-I). Subsequently, we further detected the expression of LINC00629 in human osteosarcoma cell lines SJSA-1, U2OS, 143B, MNNG/HOS and MG63. We found that the expression levels of LINC00629 in SJSA-1 cells were lower than the other cells and SJSA-1 cells were sensitive to TM-induced apoptosis (Supplementary Fig. 1 J-K). Elevated LINC00629 inhibited TM-induced SJSA-1cell apoptosis (Supplementary Fig. 1 L).
Taken together, our data suggest that LINC00629 is an ER stress-induced lncRNA and contributes to the adaptation of osteosarcoma cell to ER stress.
LINC00629 promotes osteosarcoma cell tumorigenesis and metastasis in vitro and in vivo
To further explore the role of LINC00629 in osteosarcoma, colony formation and Transwell assays were performed to measure the effects of LINC00629 on cell clonogenicity and migration. As shown in Fig. 2A-F, depletion of LINC00629 markedly inhibited cell clonogenic potential and migration in MNNG/HOS and 143B cells. In contrast, forced expression of LINC00629 promoted cell clonogenicity and migration (Supplementary Fig. 2A-D). The subsequent sphere formation results suggested that inhibition of LINC00629 impaired the sphere formation capacity of MNNG/HOS and 143B cells (Fig. 2G-I).
To better understand the function of LINC00629 in osteosarcoma cell, we then determined the effects of LINC00629 on tumour growth and metastasis in vivo. To this end, MNNG/HOS cells with or without LINC00629 knockdown were implanted into nude mice. Compared with the control group, knockdown of LINC00629 significantly suppressed tumour formation, as indicated by reduced tumour weights and tumour sizes (Fig. 2J-L). Similarly, LINC00629-depleted cells exhibited significantly decreased lung metastasis abilities at approximately 3 weeks, as indicated by a notable decrease in the number of metastatic lesions and the average surface areas produced by LINC00629-depleted cells (Fig. 2M-O). Collectively, these data indicate that LINC00629 is an important oncogene in MNNG/HOS and 143B cells.
LINC00629 interacts with KLF4 and enhances its expression in MNNG/HOS and 143B cells
To elucidate the molecular mechanism whereby LINC00629 inhibited ER stress-induced apoptosis and promoted tumorigenesis in osteosarcoma, we used label-free quantitative proteomics to identify the differentially expressed proteins in MNNG/HOS cells with or without LINC00629 knockdown. Sixty-four downregulated and 56 upregulated genes were observed (Fig. 3A-B). Among these altered genes, we found that KLF4 was significantly downregulated in LINC00629-depleted cells (Fig. 3C). To further verify it, the expression levels of KLF4 were detected in MNNG/HOS and 143B cells with or without LINC00629 knockdown. Consistently, LINC00629 knockdown markedly decreased KLF4 protein levels but did not affect KLF4 mRNA levels (Fig. 3D and Supplementary Fig. 3A-B). Otherwise, elevated LINC00629 increased KLF4 expression (Fig. 3E).
Based the data that LINC00629 was increased in response to ER stress, we thus wanted to know whether LINC00629 contributed to KLF4 increase under ER stress. To this end, we detected the KLF4 expression in MNNG/HOS and 143B cells with or without LINC00629 knockdown under ER stress treatment and observed that KLF4 was significantly increased in response to TM treatment. However, the upregulation of KLF4 was impaired by LINC00629 knockdown (Fig. 3F and Supplementary Fig. 3C).
Considering that LINC00629 facilitated KLF4 protein expression, we wanted to determine whether LINC00629 interacts with KLF4. To test this hypothesis, we carried out an RNA pulldown assay in which biotin-labelled LINC00629 and antisense RNA were synthesized in vitro and incubated with whole-cell lysates of MNNG/HOS and 143B cells. As expected, KLF4 was precipitated by LINC00629 (Fig. 3G). In addition, an RNA immunoprecipitation (RIP) assay was performed and the results indicated that LINC00629 was enriched by the anti-KLF4 antibody relative to IgG, and the enrichment of LINC00629 in the KLF4 immunoprecipitate was increased under ER stress treatment (Fig. 3H-I and Supplementary Fig. 3D-E).
LINC00629 maintains KLF4 stability and inhibits its degradation
To investigate whether LINC00629 enhanced KLF4 expression in a proteasome-dependent manner, MNNG/HOS and 143B cells were first treated with the proteasome inhibitor MG132, and the expression of KLF4 was analysed by Western blot. As shown in Fig. 4A-B, MG132 reversed the downregulation of KLF4 induced by LINC00629 knockdown. Subsequently, we treated these cells with the protein synthesis inhibitor CHX to assess the alteration of KLF4 half-life in LINC00629-depleted cells and found that inhibition of LINC00629 decreased the stability of endogenous KLF4 protein (Fig. 4E-F and Supplementary Fig. 4A-B). Conversely, overexpression of LINC00629 elevated the stability of KLF4 (Fig. 4E-F). Similarly, our further data indicated that elevated LINC000629 decreased the ubiquitination of KLF4 (Fig. 4G). Conversely, knockdown of LINC00629 increased the ubiquitination of KLF4 (Fig. 4H and Supplementary Fig. 4C).
To further investigate whether LINC00629 decreased the polyubiquitination of KLF4 under ER stress, MNNG/HOS cells with or without LINC00629 knockdown were treated with 3 μM TM as indicated, and the ubiquitination of KLF4 was detected by Western blot. As shown in Fig. 4I, ER stress decreased the ubiquitination of KLF4. However, the decrease was abolished by LINC00629 knockdown. Taken together, these results indicate that LINC00629 elevates the stability of KLF4 and inhibits its degradation.
KLF4 transcriptionally upregulates LAMA4 expression
To identify the downstream genes of LINC00629 and KLF4, we used RNA sequencing to search for overmapping genes in MNNG/HOS cells with LINC00629 or KLF4 knockdown. We found 689 (277 upregulated and 412 downregulated) and 258 (106 upregulated and 152 downregulated) differentially expressed genes for LINC00629 and KLF4 knockdown, respectively (Fig. 5A-C and Supplementary Table 4 and 5). Not surprisingly, fifty genes exhibited overlapping expression (Supplementary Fig. 2A). Among these genes, we found that LAMA4 was significantly downregulated in LINC00629- and KLF4-depleted cells (Fig. 5D). To confirm this, the expression levels of LAMA4 were detected in LINC00629- or KLF4-depleted MNNG/HOS cells. Compared with the control cells, knockdown of LINC00629 and KLF4 notably suppressed LAMA4 expression (Fig. 5E-F). Similar results were obtained in 143B cells (Supplementary Fig. 5B-C). Additionally, we also found that inhibition of LINC00629 or KLF4 abolished ER stress-induced LAMA4 upregulation (Fig. 5G).
To investigate whether LINC00629 upregulated LAMA4 in a KLF4-dependent manner, we knocked down KLF4 in MNNG/HOS and 143B cells with or without LINC00629 overexpression. Then, the expression levels of LAMA4 were detected by Western blot and qRT–PCR. These results showed that depletion of KLF4 abolished the increase in LAMA4 by LINC00629 overexpression (Fig. 5H-I and Supplementary Fig. 5D-E). Collectively, our data indicate that LAMA4 is a downstream gene of the LINC00629-KLF4 pathway.
KLF4 directly binds to the promoter of LAMA4
To further confirm that KLF4 transcriptionally upregulated LAMA4, we assessed the effect of KLF4 on the promoter activity of LAMA4. The upstream sequence of LAMA4 and the truncations were cloned into pGL3-based luciferase reporter plasmids (named P1, P2, and P3), which were then transfected into 293 T cells with or without KLF4 overexpression (Fig. 6A). As shown in Fig. 6B, the luciferase activities of P1 and P2 but not P3 were increased in KLF4-overexpressing cells, suggesting that the region (− 800 to − 400 bp) was essential for KLF4-upregulated LAMA4 expression. Subsequently, we transfected P2 into MNNG/HOS and 143B cells with or without KLF4 or LINC00629 knockdown, and the luciferase activities of P2 were measured. As shown in Fig. 6C-E, depletion of KLF4 or LINC00629 decreased the luciferase activities of LAMA4 promoter (P2). Consistently, inhibition of KLF4 or LINC00629 abolished the ER stress-induced increase in the luciferase activities of LAMA4 promoter (Fig. 6F). To investigate whether LINC00629 affected the luciferase activity of P2 by upregulating KLF4, we then transfected P2 into LINC00629-overexpressing MNNG/HOS cells with or without KLF4 knockdown, and luciferase activity was measured. As expected, depletion of KLF4 eliminated the LAMA4 promoter luciferase activities increase by LINC00629 (Fig. 6G).
To further identify the potential binding sites of KLF4 on the LAMA4 promoter, we inspected the sequence of P2 using JASPAR software (https://jaspar.genereg.net), and one positive binding site was identified. To verify this hypothesis, we constructed two pGL3-based luciferase reporter plasmids containing a wild-type binding site (BS WT) and a mutant binding site (BS Mut) (Fig. 6H). These plasmids were individually transfected into 293 T cells with or without KLF4 overexpression, and the luciferase activity was measured. We found that the activity of BS WT but not BS Mut was significantly increased in response to KLF4 overexpression (Fig. 6I). Conversely, depletion of KLF4 decreased the luciferase activity of BS WT but not BS Mut (Fig. 6J).
Furthermore, the following chromatin immunoprecipitation (ChIP) assays showed that the chromatin fragment containing BS1 was specifically present in anti-KLF4 immunoprecipitation (Fig. 6K). The binding capacity of KLF4 to the LAMA4 promoter was enhanced in LINC00629-overexpressing MNNG/HOS cells (Fig. 6L). Taken together, these findings indicate that the BS region is of great significance for KLF4 to elevate LAMA4 expression and that LINC00629 upregulates LAMA4 in a KLF4-dependent manner.
Knockdown of LAMA4 suppresses the malignant behaviour of osteosarcoma cell and accelerates ER stress-induced apoptosis
LAMA4 belongs to the laminin family and plays an important role in many cancers [21]. However, its function in osteosarcoma is still unknown. To investigate this possibility, we first analysed the expression of LAMA4 from the TNMplot database and found that LAMA4 was significantly upregulated in osteosarcoma compared with normal tissues (Fig. 7A). To further evaluate the role of LAMA4 in osteosarcoma, we then knocked down LAMA4 in MNNG/HOS and 143B cells. As shown in Fig. 7B, compared with the control groups, the expression of LAMA4 in shRNA LAMA4 groups were substantially decreased. Then, Colony formation and Transwell assays were performed to measure the effects of LAMA4 on clonogenic potential and migration. As shown in Fig. 7C-F, depletion of LAMA4 markedly inhibited cell clonogenic potential and migration in MNNG/HOS and 143B cells, which was consistent with the role of KLF4 (Supplementary Fig. 6A-D). In addition, we also investigated the effects of LAMA4 on the cell adaptation to ER stress and found that knockdown of LAMA4 enhanced ER stress-induced apoptosis and promoted cell viability downregulation MNNG/HOS and 143B cells (Fig. 7G-J and Supplementary Fig. 6E-F).
To better understand the role of LAMA4 in osteosarcoma, a xenograft tumour formation assay was performed. MNNG/HOS cells with or without LAMA4 knockdown were injected into 4- to 6-week-old BALB/c (nu/nu) male nude mice. As shown in Fig. 7K-M, compared to the control group, inhibition of LAMA4 suppressed tumour growth, as indicated by a decrease in tumour weight, and accelerated cell apoptosis, as indicated by an increase in cleaved PARP. Taken together, our data indicate that LAMA4 plays an oncogenic role in MNNG/HOS and 143B cells.
LINC00629 elevates the osteosarcoma cell adaption to ER stress and facilitates tumorigenesis by activating the KLF4-LAMA4 axis
To determine whether LINC00629 elevates the osteosarcoma cell adaption to ER stress and facilitates tumorigenesis by regulating the KLF4-LAMA4 axis, we first overexpressed LINC00629 in KLF4- or LAMA4-depleted MNNG/HOS cells and these cells were treated with 3 μM TM for 36 h. Cell apoptosis was detected by Western blot. The result showed that elevated LINC00629 suppressed ER stress-induced cell apoptosis, as indicated by the cleaved PARP increase. However, the inhibitory effect of LINC00629 on cell apoptosis was abolished when KLF4 or LAMA4 was knocked down (Fig. 8A). Similarly, we also observed that inhibition of KLF4 or LAMA4 eliminated the increase in clonogenicity and migration induced by LINC00629 overexpression (Fig. 8B-E).
To further confirm this hypothesis, xenograft tumour formation and metastasis assays were performed. We found that the promoting effects of LINC00629 on tumour development and lung metastasis were abolished by KLF4 or LAMA4 depletion in nude mice (Fig. 8F-I). Collectively, these results suggest that LINC00629 depends on the KLF4-LAMA4 axis to elevate the adaption to ER stress and promote tumour growth and metastasis in MNNG/HOS and 143B cells.