Circ-MEG3 inhibits the malignant proliferation of human cervical cancer cells

doi:10.21203/rs.3.rs-4011205/v1

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Circ-MEG3 inhibits the malignant proliferation of human cervical cancer cells

https://doi.org/10.21203/rs.3.rs-4011205/v1

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Cervical cancer is one of the most common malignant cancers in the world and circular RNA plays an important role in the development of human cancer. However, Circ-MEG3 exact function is still unclear. Herein, our results indicate that circ-MEG3 inhibits the growth in vitro and in vivo of human cervical cancer cells. Moreover, we find that circ-MEG3 alters gene expression in human cervical cancer cells. In particular, there were differences in gene expression between rLV-circ group and rLV-circ-MEG3 group, among which 407 genes were up-regulated expression and 8562 genes were down-regulated expression. The important up-regulated genes mainly include CHIC2, EXT1, KDM6A, DDX5, PPARγ, CNBP, PRRX1, BTG1, FBXO11, SRGAP3, SET, PTEN, EIF4A2. The important down-regulated genes mainly include HSP90AB1, CALR, TPM4, HSP90AA1, MYH9, NPM1, HMGA1, CDKN2A, NDRG1, ATP1A1, SDHA, FUS, MYC, SEPT9, CCND3, DNAJB, SND1, CCND1, RPN1, H3F3A. Strikingly, our results show that circ-MEG3 alters cellular functions and cell signaling pathway. The important up-regulated KEGG mainly include p53signaling pathway, Ribosome biogenesis in eukaryotes. The important down-regulated KEGG mainly include cGMP-PKG signaling pathway, Wnt signaling pathway, mTOR signaling pathway, MAPK signaling pathway, Amphetamine addiction.

In conclusions, Circ-MEG3 inhibits the progression of human cervical cancer by altering gene expression and related signaling pathways, providing a theoretical basis for clinical diagnosis and treatment of cervical cancer. We will further explore how Circ-MEG3 functions during cervical carcinogenesis and screen its associated important targets.

Human cervical cancer

Circ-MEG3

Malignant proliferation

Cervical cancer is one of the most common malignant cancers in the world. The researches show human papillomavirus (HPV) is the most common sexually transmitted virus worldwide (1). Cervical cancer is the leading cause of death from gynecological cancer in developing countries such as India (2). At the same time, cervical cancer is the fourth most common cancer in women worldwide (3). Each year, more than 500,000 women worldwide are diagnosed with cervical cancer and more than 3 million die from the disease (4). Current standard treatments include radiation therapy, chemotherapy, and surgical resection. However, these treatments have significant side effects and limited efficacy in advanced disease (5). The only strategy that has led to human treatment is p53-targeted therapy, but it has not had much success (6).

Studies have shown that circular RNA plays an important role in the development of human cancer. Some circular RNA inhibit tumorigenesis, while others promote tumorigenesis. Some studies have discovered circRNAs with general oncogenic and tumor suppressor roles, with specific circRNAs changing gene expression in cancer cells (7). Growing evidence reveals that circRNAs contribute to various physiological and pathological processes, including the initiation and progression of cancer (8). A higher level of hsa_circ_001783 was significantly correlated with heavier tumor burden and poorer prognosis of patients with breast cancer (9). Circular RNA circRNA_0000285 promotes cervical cancer development by regulating FUS (10). Furthermore, the study shows circular RNA mediate gene expression at the transcriptional or post-transcriptional level, binding to microRNAs and other molecules and repressing their function (11). EML4-ALK 1 affects tumor growth, migration, invasion, apoptosis, cell cycle and resistance to the molecularly targeted drug crizotinib in H3122 cells. The expression of the EML4-ALK1 parental oncogene suppress the expression of EML4-ALK1 (12). Besides Chen et al. found that circ-0084927/miR-142-3p/ARL2 significantly blocked proliferation, migration and invasion of cervical cancer cells and induced cell cycle arrest by protein blotting (13). Circ-MEG3 is a circular structure formed by the long non-coding RNA MEG3 in the cell, and its exact function is still unclear.

At present, the exact molecular mechanism of human cervical carcinogenesis is unclear, so it is important to explore the molecular mechanism of cervical carcinogenesis. In this study, we will establish a stable overexpressing Circ-MEG3 cervical cancer cell line Hela and reveal the effect of Circ-MEG3 on cervical cancer cells.

Cell line the human cervical cancer cell line Hela (ATCC) was cultured in a dish containing DMEN medium(10%FBS BIOAGRIO) in a 5%CO2 cell incubator at 37℃.

Lentivirus infection the lentiviruses (rLV-circ and rLV-circ-MEG3) were prepared by Wuhan Viraltherapy Biotechnology Co., LTD. Ten microliters of 10⁸ titer lentivirus were added to a six-well plate containing cells. Purinomycin (5µg/ml) was added after 48 hours of cell culture.

Total RNA extraction the cells were digested with trypsin (0.25%) and cells were collected into a 1.5ml centrifuge tube, and then 1.5ml Trizol was added. Next, 200µl chloroform was added, violently shaken for 15s and centrifuged (12000rpm) at 4℃ for 10 minutes. 600µl isopropanol was added into the water phase and centrifuged (12000rpm) at 4℃ for 10 minutes. 1ml 75% alcohol was added to wash precipitates, and then centrifuge (8000rpm) at 4℃ for 5 minutes. Finally, 50–100µl DEPC water was added to dissolve RNA on ice for 30min.

RT-PCR the total RNA(1µg) was used for transcription reaction (RT) with RT reverse transcription kit according to the operating instructions (TIANGEN). That is, the RNA was reversely transcribed into cDNA. Next, the cDNA was amplified by the polymerase chain reaction (PCR) kit according to the operating instructions (TIANGEN). β-actin as the internal control.

Growth curve assay the cells were cultured in 96-well plates for cell growth assay. CCK8(10µl per well) was added and reacted for 4 h (37℃, 5%CO2) according to the operating instructions (Beyotime). The OD450 value was measured by microcoder SpectraMaxiD3 (Molecular Devices) according to the operating instructions for three consecutive days.

Determination of colony formation capacity A total of 500 cells were added to a six-well plate and cultured at 37℃ in an incubator with 5%CO2. The cells were fixed with methanol and then stained with 0.05% crystal violet (SANGON) for at least one hour after 7 days. The cellular clones were photographed and counted.

Tumor formation experiment in vivo Six four-week-old female Balb/c nude mice were selected and raised in the animal house of Tongji University. Hela cells (10⁷) were inoculated subcutaneously in the axils of each nude mouse. The subcutaneously transplanted tumor was dissected and weighed one month later, and the time of tumor occurrence in each mouse was recorded. The animal experiments were reviewed and approved by the Experimental Animal Ethics Committee of Tongji University.

RNA sequencing Total RNA was extracted from cells (rLV-circ group and rLV-circ-MEG3 group), and the RNA was quality-controlled and preformed RNA sequencing in Novogene, Beijing, including differential gene analysis, GO analysis and KEGG analysis.

Statistical analysis the mean value and SD value from the rLV-circ group and rLV-circ-MEG3 group were statistically calculated, and the P-value was calculated by T-test. P < 0.05 indicates a significant difference between the two groups, and P < 0.001 indicates an extremely significant difference between the two groups.

Circ-MEG3 inhibits the growth in vitro of human cervical cancer cells

To investigate the effect of Circ-MEG3 on human cervical cancer cells, the stable cell line Hela overexpressing Circ-MEG3 was constructed by lentivirus Infection. The Hela cells are infected with rLV-circ and rLV-circ-MEG3, respectively. Green fluorescent protein was expressed in rLV-circ group and rLV-circ-MEG3 group (Fig. 1Aa). Next, total RNA was extracted and the expression of Circ-MEG3 was detected by back-to-back RT-PCR. As shown in Fig. 1Ab, the expression of circ-MEG3 was significantly increased in the rLV-circ-MEG3 group compared with the rLV-circ group. Furthermore, the endogenous circ-MEG3 was not changed in both groups. Next, the cell proliferation capacity in vitro was measured using CCK8 in the two groups, and the results showed that the cell proliferation capacity was significantly decreased in the rLV-circ-MEG3 group compared with the rLV-circ group(24h:6.796 ± 0.063 vs 1.417 ± 0.102, P = 0.000000002; 48h: 6.918 ± 0.134 vs 2.718 ± 0.269, P = 0.000000015)(Figure1B)Then the colony formation ability of the two groups of cells was measured by crystal violet staining. The results showed that the colony formation rate was 72.8 ± 14.75% in rLV- circ group and 15.93 ± 8.12% in rLV-circ-MEG3 group. The colony formation rate was significantly decreased in the rLV-circ-MEG3 group compared with the rLV-circ group (72.8 ± 14.75% vs 15.93 ± 8.12%,P = 0.0233)(Fig. 1Ca&b).Collectively, these results suggest circ-MEG3 inhibits the growth in vitro of human cervical cancer cells.

Circ-MEG3 inhibits the growth in vivo of human cervical cancer cells

Given that Circ-MEG3 inhibits the proliferation ability in vitro, we will further determine whether Circ-MEG3 affects the growth ability in vivo of cervical cancer cell Hela, and perform tumor formation test in nude mice. Lentivirus-infected cells were inoculated subcutaneously into the armpits of nude mice, and the mice were dissected one month later and tumors were removed. As shown in Fig. 2A & B ,the tumor weight was significantly decreased in the rLV-circ-MEG3 group compared with the rLV-circ group (0.725 ± 0.095 gram vs 0.157 ± 0.042 gram, P = 0.000019).Furthermore the tumor appearance time was significantly increased in the rLV-circ-MEG3 group compared with the rLV-circ group (7.833 ± 0.753 days vs 15.33 ± 1.366 days, P = 0.000005)(Fig. 2C).Collectively, these results suggest circ-MEG3 inhibits the growth in vivo of human cervical cancer cells.

Circ-MEG3 alters gene expression in human cervical cancer cells

Given that Circ-MEG3 inhibits the proliferation ability in vitro and in vivo, we will further explore whether Circ-MEG3 affects the gene expression of cervical cancer cell Hela. The total RNA was extracted from the cells in rLV-circ group and rLV-circ-MEG3 group, respectively, and RNA sequencing was performed. As show in Fig. 3A-D, the total amount of gene expression was different between rLV-circ group and rLV-circ-MEG3 group. Of these, 11,753 genes were only in rLV-circ group, 167 genes were only in rLV-circ-MEG3 group, and 1,381 genes were in both groups. The columnar statistical map (Fig. 4A), the volcano map (Fig. 4B) and the cluster heat map (Fig. 4C) showed that there were differences in gene expression between rLV-circ group and rLV-circ-MEG3 group, among which 407 genes were up-regulated expression and 8562 genes were down-regulated expression. The important up-regulated genes mainly include CHIC2, EXT1, KDM6A, DDX5, PPARγ, CNBP, PRRX1, BTG1, FBXO11, SRGAP3, SET, PTEN, EIF4A2, MSI2, ZFHX3, FBXW7, ARID2, GPHN, MLLT10, SRSF2, HNRNPA2B1, ETV1, ARID1A, BCL9, MAX, BRAF, DDX6, CAMTA1. The important down-regulated genes mainly include HSP90AB1, RPL10, CALR, LMNA, TPM4, RPL5, HSP90AA1, MYH9, NPM1, HMGA1, CDKN2A, MSN, NDRG1, ATP1A1, EZR, SDHA, FUS, MYC, FSTL3, SEPT9, CCND3, DNAJB, SND1, CCND1, H3F3A.Collectively, these results suggest circ-MEG3 alters gene expression in human cervical cancer cells.

Circ-MEG3 alters cellular functions

Since circ-MEG3 regulates the level of gene transcription in human cervical cancer cells, we will consider whether circ-MEG3 affects the cellular functions. We adopted Gene ontology (GO) functional analysis, including three aspects Biological Process (BP), Cellular Component (CC) Molecular Function (MF). As shown in Fig. 5A&B,up-regulated BP mainly includes regulation of translation, regulation of mRNA metabolic process, negative regulation of translation, regulation of cellular amide metabolic process, negative regulation of cellular amide...,mRNA processing, positive regulation of mRNA catabolic process, RNA splicing, via transestenfication reactions...,mRNA splicing, via spliceosome, mRNA catabolic process; up-regulated CC mainly includes cytopiasmic ribonucleoprotein granule, cytoplasmic stress granule, ribonucleoprotein granule, spliceosomal complex, P-body, transcription factor complex, protein acetyltransferase complex, acetyltransferase complex,CCR4-NOT complex, histone acetyltransferase complex and up-regulated MF mainly includes mRNA binding, transcription cofactor activity, mRNA 3'-UTR binding, transcription coactivator activity, chromatin binding, single-stranded RNA binding, poly(A) binding, poly-purine tract binding, transcriptional activator activity, RNA polymerase..., transcription factor activity, RNA polymerase.... As shown in Fig. 5C&D, down-regulated BP mainly includes ribosome biogenesis, ncRNA processing, ribonucleoprotein complex biogenesis, rRNA metabolic process, rRNA processing, nucleoside monophosphate metabolic process, purine nucleoside triphosphate metabolic process, nucleoside triphosphate metabolic process, ribonucleoside triphosphate metabolic process, establishment of protein localization to...;down-regulated CC mainly includes mitochondrial inner membrane, mitochondrial protein complex, ribosome, ribosomal subunit, mitochondrial matrix,

mitochondrial membrane part, large ribosomal subunit, cytosolic part, cytosolic ribosome, focal adhesion and down-regulated MF mainly includes structural constituent of ribosome, catalytic activity, acting on RNA, cadherin binding, cell adhesion molecule binding, catalytic activity, acting on a tRNA, rRNA binding, ATPase activity, oxidoreductase activity, acting on NAD(P)H, ..., unfolded protein binding, isomerase activity. Together, these observations suggest that circ-MEG3 alters cellular functions in human cervical cancer cells.

Circ-MEG3 alters cell signaling pathway

Given that circ-MEG3 alters cellular functions in human cervical cancer cells, we will further explore whether circ-MEG3 alters the cells signaling pathway. We performed a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. As shown in Fig. 6A&B, the important down-regulated KEGG mainly include Ribosome, Protein processing in endoplasmic reticulum, Oxidative phosphorylation, Parkinson disease, Thermogenesis, Cell cycle, Non-alcoholic fatty liver disease (NAFLD), Huntington disease, Pyrimidine metabolism, p53signaling pathway, Ribosome biogenesis in eukaryotes, Alzheimer disease, Spliceosome, Glutathione metabolism, Lysosome, RNA transport. Collectively, these results suggest circ-MEG3 alters cell signaling pathway in human cervical cancer cells.

So far, we clearly verify that Circ-MEG3 significantly inhibits the growth ability in vitro and in vivo of human cervical cancer cells by altering the expression of a number of genes. In particular, RNA sequencing indicates that Circ-MEG3 achieves its anticancer effects by altering cell function and signaling pathways.

First, our results clearly show that Circ-MEG3 inhibits the growth in vitro of human cervical cancer cells. In fact, Circ-MEG3 is formed by cyclization of long strands of non-coding RNA. These results suggest that Circ-MEG3 plays the same role as MEG3 in cervical cancer. But the exact Molecular mechanism needs to be further explored. Secondly, it is worth noting that Circ-MEG3 affects the expression of some cervical cancer cells. For examples,Circ-MEG3 up-regulates CHIC2, EXT1, KDM6A, DDX5, PPARγ, CNBP, PRRX1, BTG1, FBXO11, SRGAP3, SET, PTEN, EIF4A2, MSI2, ZFHX3, FBXW7, ARID2, GPHN, MLLT10, SRSF2, HNRNPA2B1, ETV1, ARID1A, RBM15, BCL9, MAX, BRAF, DDX6, CAMTA1. The study shows that DDX6 is associated with the regulation of cancer cell growth and differentiation (14). The DDX6 gene down-regulated c-Myc in gastric cancer (GC) cells thereby inducing growth inhibition (15).It has also been shown that depletion of KDM16A in various cervical cancer line significantly inhibits cell activity. KDM6A expression is required for cervical cancer cell viability (16). And KDM16A deficiency activates cytokine/chemokine signaling and p53 haploinsufficiency enhances cell proliferation (17). Moreover, PPARγ is associated with the regulation of cancer cell growth and differentiation too. The downregulation of the PPARγ target Nf-kb is related to these tumors which were found to be immunologically abandoned (18). Inhibition of PPARγ suppressed tumor growth in vivo and downregulated the lipid synthesis program (19). High expression of PPARγ was significantly associated with improved prognosis (20). Besides, study shows miR-494 upregulation was significantly associated with PTEN downregulation and inhibition of miR-494 suppressed cell proliferation and growth by directly targeting the 3'-untranslated region (3'-UTR) of PTEN mRNA (21). Study also find that the expression of STXBP5-AS1 and PTEN was decreased and that of miR-96-5p was increased in CC(cervical cancer)(22). MiR-940 expression was negatively correlated with the expression levels of p27 and PTEN in cervical cancer specimens, inhibition of p27 and PTEN restored the inhibitory function of miR-940 silenced cells on cervical cancer cell growth and tumorigenic properties (23). The results of study suggest that overexpression of TUSC8 inhibits CC cell invasion and migration via upregulation of PTEN by miR-641 (24). Therefore, it is speculated that Circ-MEG3 exerts the anticancer function by inhibiting the above genes, but further proof is needed.

Also, Circ-MEG3 down-regulated genes HSP90AB1, RPL10, CALR, LMNA, TPM4,RPL5,HSP90AA1,MYH9,NPM1,HMGA1,CDKN2A,MSN,NDRG1,ATP1A1,EZR,SDHA,FUS,MYC,FSTL3,SEPT9,CCND3,DNAJB,SND1,CCND1,MALAT1,RPN1,SDC4,HLA-A,H3F3A. Study shows that CALR is associated with the regulation of cancer cell growth and differentiation. CALR was upregulated in most types of tumors compared to normal tissue. It indicates that most of the tumors with high expression of CALR have a higher risk of tumor mutation (25). Overexpression of CALR while ad-CALR and miR-206 were co-transfected attenuated the inhibitory effect of miR-206 on CALR expression (26). Upregulation of CALR expression in pan-cancer cohorts. Depletion of CALR reduced xenograft tumor growth (27). It has also been shown that expression of RPL11 and RPL5 in breast cancer tissues was significantly reduced, and further, overexpression of RPL11 / RPL5 significantly inhibited breast cancer cell growth (28). Besides, there are studies show the absence of BRCA1 and overexpression of MYC lead to the development of breast cancer (29). And CDKN2A expression levels were low in cervical cancer cell lines (30). CDKN2A methylation frequency tended to increase during cervical carcinogenesis (31). What’s more, data suggests that LINC01503 promotes cervical cancer cell progression through interaction with the miR-615-3p/CCND1 axis (32). The frequency of the CCND1 870A allele was significantly higher in cancer patients compared to controls (33). MiR-194 inhibited PTBP1 expression by binding to the 3'-UTR region of PTBP1 mRNA, resulting in decreased CCND3 levels (34). Knockdown of CCND3 induced cell cycle arrest and apoptosis (35). Therefore, it is speculated that Circ-MEG3 exerts the anticancer function by enhancing the above genes, but further proof is needed.

Thirdly, we are particularly concerned Circ-MEG3 alters cellular functions. For example, Circ-MEG3 up-regulates regulation of translation, regulation of mRNA metabolic process, cytopiasmic ribonucleoprotein granule, cytoplasmic stress granule, ribonucleoprotein granule, spliceosomal complex, P-body, transcription factor complex, protein acetyltransferase complex, acetyltransferase complex, mRNA binding, transcription cofactor activity, mRNA 3'-UTR binding, transcription coactivator activity, chromatin binding, single-stranded RNA binding. And down-regulated BP mainly includes ribosome biogenesis, ncRNA processing, ribonucleoprotein complex biogenesis, rRNA metabolic process, rRNA processing, nucleoside monophosphate metabolic process, purine nucleoside triphosphate metabolic process, nucleoside triphosphate metabolic process, ribonucleoside triphosphate metabolic process, establishment of protein localization to...; Circ-MEG3 down-regulated mitochondrial inner membrane, mitochondrial protein complex, mitochondrial membrane part, large ribosomal subunit, cytosolic part, cytosolic ribosome, focal adhesion, includes structural constituent of ribosome, catalytic activity, acting on RNA, unfolded protein binding, isomerase activity. Some study shows that the senescence response is widely recognized as a potent tumor suppressive mechanism (36). Cellular senescence is a potent tumor-suppressive process that halts the proliferation, essentially irreversibly, of cells at risk for malignant transformation (37). Cell senescence is induced by the p16/Rb pathway. p16 is specifically upregulated in senescent cells, suppressing CDK4/6 (38). Other studies Deviation from the strict control of MAPK signaling pathways has been implicated in the development of many human diseases including various types of cancers (39). To accomplish the transmission of a variety of signals, numbers of kinesins are closely associated with the MAPK cascade (40). The extracellular signal-regulating kinase ERK2 overactivation plays an important role in the development and progression of cancer (41). Aberrant activation of MAPK pathways is mostly the consequence of activating mutations in RAS and RAF proteins (42). Besides, in many human diseases, mTOR signaling pathway is deregulated (43). At the same time, mTOR, which is frequently activated in cancer, controls cell growth and metabolism (44). The mammalian target of rapamycin (mTOR) control cell growth and proliferation (45). Oncogenes and tumor suppressors mutated in these cancer types cause mTOR signaling dysregulated (46). What’s more, Wnt signaling is observed in many cancer entities. Wnt signaling affects maintenance of cancer stem cells, metastasis and immune control (47). The aberrant Wnt/β-catenin signaling pathway facilitates cancer stem cell renewal, cell proliferation and differentiation (48). Genetic or epigenetic events leading to hypo- or hyper-activation of the Wnt-β-catenin signaling cascade have also been associated with human diseases such as cancer (49). Wnt inhibition reduces proliferation (50). Therefore, we hypothesize that Circ-MEG3 works by altering cell function.

Strikingly, our results confirm Circ-MEG3 alters cell signaling pathway. Circ-MEG3 down-regulated protein processing in endoplasmic reticulum, oxidative phosphorylation, cell cycle, pyrimidine metabolism, ribosome biogenesis in eukaryotes, glutathione metabolism, RNA transport.

In conclusion, Circ-MEG3 inhibits the progression of human cervical cancer by altering gene expression and related signaling pathways, providing a theoretical basis for clinical diagnosis and treatment of cervical cancer. We will further explore how Circ-MEG3 functions during cervical carcinogenesis and screen its associated important targets.

Funding statement

This project is supported by the Innovation and Entrepreneurship Program for College

Author Contribution

T.P. and S.L. wrote the main manuscript text and Y.L. prepared figures1-6 and tables1-2. All authors reviewed the manuscript.

Data availability

Not applicable.

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Table 1 and 2 are available in the Supplementary Files section.

No competing interests reported.

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Circ-MEG3 inhibits the malignant proliferation of human cervical cancer cells

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