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Silencing of lncRNA LINC00346 Inhibits the Proliferation and Promotes the Apoptosis of Colorectal Cancer Cells Through Inhibiting JAK1/STAT3 Signaling

Purpose The study was aimed to investigate the effect and mechanism of lncRNA LINC00346 on cell proliferation and apoptosis of colorectal cancer (CRC). Methods The expression of lncRNA LINC00346 in CRC tissues and cells was detected by qRT-PCR. LINC00346 was overexpressed and silenced in HT29 and LoVo cells by the transfection of pcDNA-LINC00346 and si-LINC00346. The proliferation of CRC cells was detected by CCK-8 and colony-formation assay. The apoptosis was detected by flow cytometry assay. The expression of apoptosis-associated proteins (Caspase-3, Bcl-2, Bax) and JAK1/STAT3 signaling-associated proteins (JAK1, STAT3, p-JAK1, p-STAT3) was detected by Western blot. The tumor growth was detected in mice subcutaneous injected with transfected HT29 cells. Results LINC00346 was significantly upregulated in CRC tissues and cells. Overexpression of LINC00346 significantly increased the OD450 values, number of colonies, decreased the apoptosis rate, upregulated Bcl-2, and downregulated Caspase-3 and Bax in HT29 and LoVo cells. Knockdown of LINC00346 exerted opposite results of proliferation and apoptosis on HT29 and LoVo cells. The expression levels of JAK1/JAK1 and p-STAT3/STAT3 were upregulated by LINC00346 overexpression. Tofacitinib (JAK1 inhibitor) reversed the tumor-promoting effect of LINC00346 overexpression on CRC cells. In vivo experiments further validated that LINC00346 overexpression promoted the growth of CRC xenograft tumors. Conclusion LncRNA LINC00346 promoted the proliferation and inhibited the apoptosis of CRC cells through activating JAK1/STAT3 signaling.


Introduction
Colorectal cancer (CRC) is a frequent malignancy globally and the leading cause of death in patients. 1 Most CRC patients have reportedly died from distant metastases, particularly liver metastases. 2 Surgery and chemotherapy are currently common treatments, but traditional chemotherapy for CRC has many limitations, including using highly toxic drugs caused adverse side effects. 3 Thus, it is necessary to investigate the mechanisms and targets associated with the treatment of CRC.
Long non-coding RNAs (lncRNAs) are a class of important non-coding RNA with limited or no protein-coding capacity. 4 LncRNAs have been proved to be a major regulator of gene expression, and they can play key roles in all kinds of biological functions and cancers processes. 5 Many lncRNAs including MALAT1, 6 UCA1, 7 and TUG1, 8 are upregulated in CRC, and play important roles in promoting CRC development and metastasis. LncRNA LINC00346, belongs to the intergenic lncRNA, has been found to be involved in many cancers. LINC00346 is upregulated in bladder cancer tissues, knockdown of LINC00346 inhibits the proliferation and migration of T24 and SW780 cells, and induces cell cycle arrest and apoptosis. 9 Shi et al, 10 have reported that LINC00346 overexpression remarkably enhances the proliferation and tumorigenesis of pancreatic cancer cells. However, the regulatory effects of LINC00346 in CRC are unclear.
JAK/STAT3 signaling pathway participates in various physiological processes, such as differentiation, cell growth, hematopoiesis and immune function. 11 More and more evidences indicate that abnormalities in the JAK1/ STAT3 signaling are crucial in tumorigenesis. For example, Xiong et al, 12 have demonstrated that STAT3, JAK1 and JAK2 are involved in CRC cell growth, invasion, survival and migration. JAK2, STAT1, STAT3 and STAT6 are related with colon cancer and STAT3, STAT4 and STAT6 are related with rectal cancer. 13 In addition, the expression of LINC00346 is increased in non-small cell lung cancer (NSCLC) cells and tissues, and LINC00346 promotes the proliferation and inhibits the apoptosis of NSCLC cells through regulating the JAK/STAT3 signaling pathway. 14 However, the relationship between JAK1/ STAT3 and LINC00346 in CRC is still unclear.
In this study, LINC00346 expression was detected in CRC tissues and cells. The regulatory effects of LINC00346 on the proliferation and apoptosis of CRC cells were analyzed. Then the mechanism of LINC00346 involving JAK1/STAT3 signaling was evaluated. Our findings may reveal a potential therapeutic target for CRC.

Tissue Samples
Tumor tissues and adjacent normal tissues were obtained from 52 CRC patients (22 males and 30 females, aged 35-53 years) from January 2017 to December 2018. This study was permitted by our hospital ethics committee, and informed consents were obtained from all patients.

Cell Culture
Human colon cancer cell lines (HT29 and LoVo) and normal human colon epithelial cell line FHC were obtained from American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured in DMEM (GIBCO, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS) (GIBCO), 100 IU/ mL penicillin and 100 mg/mL streptomycin (GIBCO). Cells were maintained at 37°C in a humidified incubator with a 5% CO 2 atmosphere.

Cell Transfection
SiRNA LINC00346 (si-LINC00346) and siRNA negative control (si-NC) were purchased by Invitrogen (Invitrogen, Carlsbad, CA, USA). Full length fragments of LINC00346 and negative control coding sequences were amplified by PCR and constructed into pcDNA3.1 vector (Invitrogen) to generate pcDNA-LINC00346 and pcDNA-NC. HT29 and LoVo cells were plated in 24-well plates (1 × 10 5 cells/well) and incubated at 37°C for 24 h. Then the above plasmids and si-RNAs were transfected into HT29 and LoVo cells using Lipofectamine 3000 (Invitrogen, USA). After 48 h of transfection, cells were used for further assays. In addition, transfected cells were treated with JAK1 inhibitor Tofacitinib (#14,703, Cell Signaling Technology, MA, USA) for 30 min before transfection.

qRT-PCR
Total RNA was extracted from tissues and cells using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). RNA was reverse transcribed to cDNA using Takara PrimeScript RT reagent kit gDNA Eraser. PCR was performed with the following conditions: an initial of 10 min at 95°C, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, 72°C for 34 s. GAPDH was used as an internal control. The relative expression level was calculated using the 2 −ΔΔCT method. The primer sequences are shown in Table 1.

Cell Viability Assay
Cells were seeded in 96-well plates (2 × 10 4 ), and cultured for 24, 48, 72 and 96 h. Then 10 μL CCK-8 solution (BD Biosciences, USA) was added to each well, and incubated for 2 h at 37°C. The optical density 450 nm (OD 450 ) was measured using a microplate reader.

Colony-Formation Assay
Cells were seeded in 6-well plates, and cultured for 14 days. After washed twice with PBS, the colonies were fixed with methanol for 15 min, and stained with crystal violet for 15 min. Positive stained colonies (more than 30 cells) were observed under an inverted microscope (Olympus Ckx53, Japan), and the number was counted randomly using Image J (1.48V).

Flow Cytometry Assay
Cells were seeded in 96-well plates, and cultured for 24 h. After washed 3 times with PBS, cells were resuspended and adjusted to 1 × 10 6 cells/mL. Then 500 μL cells were stained with 5 μL V-FITC and 10 μL of PI for 20 min in the dark. The apoptosis rate was detected by flow cytometry.

Statistical Analysis
Each assay was performed at least three times. Data statistical analysis was performed using SPSS 22.0 (SPSS Inc., Chicago, IL, USA). Data were presented as mean ± standard deviations. Comparison between two groups was determined by t-test, and comparison among more than two groups was determined by One-Way ANOVA, followed LSD test. Survival analysis was performed using Kaplan-Meier curve and analyzed using the Log rank test. A P value less than 0.05 was considered to be significant.

LINC00346 Is Overexpressed in CRC Tissues and Cell Lines
LINC00346 expression was detected in 52 cases of CRC tissues and normal tissues by using qRT-PCR. LINC00346 expression was significantly higher in CRC tissues compared with normal tissues (P < 0.001) ( Figure  1A). Simultaneously, LINC00346 expression was detected in CRC cell lines HT29 and LoVo, as well as normal human colon epithelial cell line FHC. The results showed that the expression of LINC00346 was upregulated in CRC cell lines (P < 0.01) ( Figure 1B). Kaplan-Meier survival analysis showed that the overall survival rate of CRC patients with high expression of lncRNA LINC00346 was significantly shortened compared with those with low expression (P = 0.0046) ( Figure 1C). Then the correlation between the expression of LINC00346 and clinicopathological features of CRC patients was further analyzed. As presented in Table 2, LINC00346   expression was positively related to TNM stage and lymphatic metastasis (P < 0.05), but not correlated with age, gender, or differentiation (P > 0.05).

Silencing of LINC00346 Inhibits CRC Cell Proliferation
To further confirm the effect of LINC00346 on the proliferation of CRC cells, lncRNA LINC00346 was overexpressed by the transfection of pcDNA-LINC00346, and silenced by the transfection of si-LINC00346 (P < 0.001) (Figure 2A). CCK-8 assay showed that silencing of LINC00346 significantly decreased the OD 450 values of HT29 and LoVo cells at 24, 48, 72, and 96 h post-culturing (P < 0.05). On the contrary, overexpression of LINC00346 significantly increased the OD 450 values of HT29 and LoVo cells (P < 0.05) ( Figure   2B). In addition, colony-formation assay showed that silencing of LINC00346 remarkably decreased the number of colonies of HT29 and LoVo cells, and overexpression of LINC00346 significantly increased the number of colonies (P < 0.05) ( Figure 2C).

Silencing of LINC00346 Promotes CRC Cell Apoptosis
To research the function of lncRNA LINC00346 on CRC cell apoptosis, the apoptosis rate was detected by flow cytometry assay. As shown in Figure 3A, the apoptosis rate of si-LINC00346 group was significantly increased in HT29 and LoVo cells (P < 0.01), and the apoptosis rate of pcDNA-LINC00346 group was significantly decreased (P < 0.01). In addition, the expression of apoptosis-related proteins, such as Caspase-3, Bax, and Bcl-2 was detected by Western blot. We found that the expression of Bcl-2 was elevated in pcDNA-LINC00346 group, but reduced in si-LINC00346 group. Furthermore, the expression of Caspase-3 and Bax were remarkably reduced in pcDNA-LINC00346 group, but elevated in si-LINC00346 group ( Figure 3B).

LINC00346 Promotes CRC Cell Proliferation and Inhibits Apoptosis by Activating JAK1/STAT3 Signaling
We further verified whether the regulatory effects of LINC00346 on CRC cells are associated with JAK1/ STAT3 signaling. CCK-8 and colony-formation assay showed that overexpression of LINC00346 remarkably increased the OD 450 values and number of colonies of HT29 cells (P < 0.05). Note worthily, Tofacitinib significantly reversed the promoting effect of pcDNA-LINC00346 on the proliferation of HT29 cells (P < 0.05) ( Figure 5A and B). In addition, flow cytometry assay showed that up-regulated LINC00346 remarkably decreased the apoptosis rate of HT29 cells (P < 0.001), while Tofacitinib significantly reversed the inhibiting effect of LINC00346 overexpression (P < 0.01) ( Figure  5C). Tofacitinib also significantly reversed the reducing effect of LINC00346 overexpression on the expression of Caspase-3 and Bax, and the promoting effect on Bcl-2 expression (P < 0.01) ( Figure 5D).

LINC00346 Promotes Tumor Growth of CRC in vivo
To further confirm the function of LINC00346 in CRC in vivo, HT29 cells were subcutaneously injected into nude mice. As shown in Figure 6A and B, the tumor volume in mice was increased with injection times. Overexpression of LINC00346 increased the tumor volume (P < 0.001) and Tofacitinib significantly reversed the promoting effect of LINC00346 on tumor volume (P < 0.001). Meanwhile, the tumor weight was remarkably increased in the pcDNA-LINC00346 group after 4 weeks, while reversed by Tofacitinib (P < 0.001) ( Figure 6C). As expected, p-JAK1/JAK1 and p-STAT3/STAT3 expression was significantly decreased with Tofacitinib in mice injected with pcDNA-LINC00346-transfected HT29 cells (P < 0.001) ( Figure 6D).

Discussion
LncRNAs are a class of non-coding RNAs involved in gene expression regulation and cancer pathogenesis. Emerging evidence has proved that some lncRNAs are upregulated in CRC, such as PANDAR, 15 MALAT1, 6 and ZFAS1. 16 LINC00346 has been found to be upregulated in CRC and increased the WBSCR22 expression via inhibiting miR-509-5p. 17 LINC00346 is recurrently amplified and high-expressed in gastric cancer, and its expression is positively correlated with poor prognosis. 18 Also, LINC00346 is upregulated in pancreatic tumor specimens and contributes to pancreatic cancer progression. 19 Here, we found that expression of LINC00346 was significantly elevated in CRC cells and tissues. Our research is in accordance with previous research, and indicates that LINC00346 may act as tumor-promoting factor in CRC. Guo et al, 20 have found that lncRNA FTX is significantly upregulated in CRC and significantly associated with differentiation grade, clinical stage, lymph vascular invasion, and poor survival. LncRNA ZFAS1 expression is upregulated in CRC, and upregulated ZFAS1 is correlated with advanced TNM stage, poor overall survival, and lymph nodes metastasis of CRC patients. 16 Likewise, we found that LINC00346 overexpression was closely correlated with a decreased survival rate in CRC patients, and was positive associated with TNM stage and lymphatic metastasis. These results suggest that LINC00346 may be a potential prognostic factor for CRC. Recent studies have suggested that LINC00346 plays vital role in cancer growth and apoptosis. LINC00346 silencing significantly suppresses cell viability, colony formation ability and DNA replication, and also downregulates the expression of cyclin D1, CDK 4 and CDK 6 in bladder cancer. 9 LINC00346 silencing promotes the apoptosis and inhibits the proliferation of NSCLC cells. 14 Furthermore, LINC00346 overexpression enhances the colony formation and proliferation of pancreatic cancer cells. 10 Here, knockdown of LINC00346 remarkably reduced the OD 450 value, number of colonies, increased the apoptosis rate, downregulated Bcl-2, and upregulated Caspase-3 and Bax in HT29 and LoVo cells. These results are consistent with previous studies, and indicate that silencing of LINC00346 inhibits the proliferation and promotes the apoptosis of CRC cells in vitro. To further research the role of LINC00346 in vivo, HT29 cells were injected into mice. We found that LINC00346 overexpression significantly increased the tumor volume and weight. The above phenomena illustrate that LINC00346 plays a tumor-promoting role in CRC, and LINC00346 silencing may be used as a potential therapeutic target for CRC. Recently, more and more evidence indicates that the JAK/STAT3 signaling is involved in the progression of CRC. 21 Blocking JAK/STAT3 signaling can not only inhibit CRC cell proliferation but also promote cell apoptosis. 12 RPTS significantly induces cell apoptosis in SW480 CRC cells through inhibiting the IL-6/JAK-STAT3 signaling. 22 Triptolide inhibits the colony formation, proliferation, and migration of colon cancer cells, also reduces the levels of JAK1 and phosphorylated STAT3. 23 LncRNA AB073614 induces epithelialmesenchymal transition of CRC cells by activation of the JAK/STAT3 pathway. 24 In our study, we found that LINC00346 overexpression increased the expression of p-JAK1/JAK1 and p-STAT3/STAT3 in HT29 and LoVo cells and Tofacitinib (JAK/STAT3 signaling inhibitor) reversed the effect of up-regulated LINC00346 on the promoting of proliferation and the inhibiting of apoptosis of CRC cells. To sum up, LINC00346 may promote the proliferation and inhibit the apoptosis of CRC cells through activating JAK1/STAT3 signaling.

Conclusions
In conclusion, LINC00346 was upregulated in CRC tissues and cells. Silencing of LINC00346 inhibited the proliferation, and promoted the apoptosis of CRC cells through blocking JAK1/STATS3 singling. In addition, overexpression of LINC00346 promoted the tumor growth in mice. LINC00346 may be used as a therapeutic target for CRC.

Ethics Approval and Consent to Participate
This study was approved by the ethics committee of The Friendship Hospital of Dalian. Written informed consent was obtained from all subjects.